This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Protein Tyrosine Phosphorylation in Haematopoietic
Cancers and the Functional Significance of Phospho-
Lyn SH2 Domain
By
Lily Li Jin
A thesis submitted in conformity with the requirements for the degree of Ph.D. in
Molecular Genetics, Graduate Department of Molecular Genetics, in the University of
Dependencies on FGFR3 activation have been demonstrated in MM cell lines carrying
activating mutations of this RTK, where a small molecule inhibitor of FGFR3 was able to
induce apoptosis and differentiation in the MM cell lines Kawasaki Medical School 11
12
(KMS11) and KMS18, which contain the respective Y373C and G384D substitutions in
FGFR3 (85). Y373C is located in the extracellular region and mediates ligand-
independent receptor dimerization, leading to the constitutive activation of FGFR3;
whereas, G384D FGFR3 was only activated when stimulated by a ligand, but was able to
induce aberrant MAPK, STAT1, and STAT3 phosphorylations (86). Moreover, Y373C,
but not G384D, mutant induced transformation in the NIH3T3 mouse embryo fibroblast
cell line (86). These data show that mutations in FGFR3 have different grades of
activation capabilities. Furthermore, inhibiting FGFR3 activation in a KMS11 xenograft
mouse model impeded tumor growth (85), suggesting FGFR3 as a “driving” kinase for
this type of MM. Consistent with the prominent role of FGFR3 in MM cell lines and
murine model, t(4;14) translocation is associated with more aggressive diseases and poor
prognosis in MM patients (84).
Cytokines in the blood and bone marrow microenvironment can activate TK
pathways and induce MM pathogenesis. The most prominently implicated cytokines are
interleukin 6 (IL-6) and insulin-like growth factor 1 (IGF-1). IL-6 binds and activates the
IL-6 receptor, which triggers the JAK-STAT pathway causing alterations in gene
transcription. Interestingly, a key player of the JAK-STAT pathway, STAT3, is
constitutively active in primary CD138-positive MM cells, and inhibition of STAT3 leads
to apoptosis in these cells (87). Additionally, IL-6 knock-out mice failed to develop B
cell cancers, suggesting an essential role of IL-6 in B cell neoplasms (88). On the other
hand, IGF-1 binds and activates the RTK IGF-1 receptor (IGF1R), which is ubiquitously
expressed in MM and the MM-related monoclonal gammopathy of undetermined
significance (MGUS), a condition that usually preceeds MM. One study reported the
13
observation of aberrantly high expressions of IGF1R in approximately 10% of MGUS
and MM (89). Moreover, a number of studies linked the over-expression of IGF-1 or
IGF1R to poor-prognostic subgroups (89-91). In addition, cultured MM cell lines are
sensitive to the inhibition of IGF1R and its related insulin receptor (INSR) by a small
molecule inhibitor, suggesting a dependency on these RTKs for myeloma cell survival
(92). Therefore, cytokine dysregulation may induce abnormalities of pY signaling and
contribute to MM pathology.
In addition to dysregulated TKs, two PTPs are implicated in MM: Ptpn6 (a.k.a.
Shp1) and Ptp4a3 (a.k.a. Prl-3). In 79.4% of primary MM samples, the expression of
Ptpn6 is suppressed by promoter hypermethylation. Although this was not linked to
patient survival, the restoration of Ptpn6 expression by a DNA methyltransferase
inhibitor was accompanied by decreased STAT3 phosphorylation in a cultured MM cell
line, implying a role of Ptpn6 in down-regulating the JAK-STAT pathway (93).
Moreover, the Ptpn6 protein is established as a negative regulator of growth factor
signaling and oncoproteins (94-98), and the PTPN6 gene is widely described as a tumor
suppressor gene (99, 100). As a result, loss of Ptpn6 function may encourage proliferation
and promote MM pathogenesis. In contrast, amplification of Ptp4a3 mRNA and protein
levels was observed in primary myeloma cells. This is associated with tumor cell
migration, invasion, and metastasis in other types of human cancers (e.g. gastric, colon,
rectal cancers, and melanoma) (101-103). However, the function of this amplification in
MM still needs clarification.
1.1.3.2 Acute Myeloid Leukemia
14
AML is the most frequent form of leukemia and comprises approximately 25% of
leukemic cases in Western society, with an incidence of 3.7 per 100,000 persons (104). It
is a cancer of the myeloid cells, marked by the presence of at least 20% maturation-
arrested myeloblasts in the blood stream and bone marrow (105). As a result of the
abnormal accumulation of myeloblasts, AML patients experience symptoms such as
fatigue, bone pain, fever, shortness of breath, easy bruising, and unusual bleeding.
Treatment for AML traditionally includes intensive chemotherapy and haematopoietic
stem cell transplantation, but, with a low success rate. Only approximately 35-40%
younger patients (< 60 years) and 5-10% older adults (> 60 years) are cured 5 years post-
diagnosis (106). Encouragingly, TK inhibitors are undergoing extensive clinical
development for AML; and some have demonstrated promising efficacy in clinical trials
(107, 108), providing a potential alternative for patients who fail to respond to
conventional therapy.
Dysregulation of RTKs (i.e. Flt3, Kit, Met, Mer, FGFR1) is extremely prevalent in
AML (109-112), with an estimated 40-60% of AML patients harbouring abnormalities in
RTK and another 15-25% with mutations in RTK downstream effectors (i.e. Ras, Jak2)
(109, 113). Among these, activating mutations in Fms-like tyrosine kinase 3 (Flt3) have
received highest attention for their role as an important molecular marker and prognostic
factor (114-117).
Flt3 is a transmembrane receptor primarily expressed in myeloid and lymphoid
progenitor cells; and involved in growth factor signaling and hematopoiesis (118-120).
Normally, Flt3 exists as an inactive, unphosphorylated, and monomeric protein. When
stimulated, Flt3 dimerizes through its juxtamembrane region and becomes
15
phosphorylated and active. Genomic alterations leading to the constitutive activation of
Flt3 have been described in its A-loop and the juxtamembrane regions, with the most
common type of mutation being the segmental duplication of several amino acids, or
internal tandem duplication (ITD), in the juxtamembrane domain, presenting in 15-35%
of AML cases (114-117). Flt3-ITD allows ligand-independent receptor dimerization and
activation. This mutation is associated with rapid disease progression, resistance to
therapy, and poorer patient outcomes compared to the patients without Flt3-ITD (121-
123).
Compared to TKs, dysregulation of PTP in AML is less frequent. The most
commonly observed abnormal PTP is the gain-of-function mutant of Ptpn11, primarily
through genetic alterations in its SH2 domain (Section 1.1.1.4.2), which was found in
about 5% of AML patients (62). Ptpn11 is described as an oncoprotein in many cancers
(67) and is associated with Flt3-ITD-induced proliferation in bone marrow progenitors
and primary AML samples (124). Besides Ptpn11, Ptpn7 is amplified in the blasts of
some AML patients, but the functional impact of this amplification has not been clarified
(125).
1.2 Mass Spectrometry Based Proteomics
Mass spectrometry (MS) based proteomics has been developed as a sensitive
method for the large-scale characterization of sample-derived peptides (126). Due to the
nature of MS, the identification and quantification of peptides can be achieved
simultaneously. MS analysis combined with advances in separation and purification
techniques of pY-containing or PTP-derived peptides has enabled global comprehensive
analysis on pY-mediated signaling networks.
16
1.2.1 Mass Spectrometry for Proteomics Studies
MS is an analytical method that measures the mass-to-charge ratio (m/z) of the
ionized analyte in its gas phase. In proteomics research, solutions containing peptides are
first resolved on a reverse-phase chromatography column (commonly packed with C18
resin) and volatilized by an ionization source, for example, electrospray ionization (ESI)
(127). The gas-phase analyte is then transferred into a coupled mass analyzer, which
alternates between a full MS scan that performs comprehensive analysis on the analyte
(MS1 analysis) and up to a set number of MS/MS scans (or MS2 analysis), which records
the fragmentation pattern of a small subset of the analyte ions automatically determined
by the computer. The fragmentation pattern (or MS2 spectrum) can be used to infer the
peptide identity. The recorded data is then searched automatically using one or a
combination of search algorithm(s), such as MASCOT (128), SEQUEST (129), and
X!Tandem (130), against a reference database to assign amino acid sequences to
fragmented ions (reviewed in 131). A typical experiment can generate a list with
thousands of peptides. This approach of coupling liquid chromatography separation to
mass spectrometry (LC-MS/MS) is a powerful technique in proteomics for analyzing
complex biological samples (126, 132, 133).
1.2.2 Label-Free Quantification by Mass Spectrometry
Quantification by MS can be achieved through either label-based (by incorporating
chemical labels or heavy isotopes) (134-136) or label-free approaches (137). The label-
free method is relatively low-cost and can be applied to analyzing primary tissue samples,
in contrast to the label-based method such as stable isotope labelling by amino acids in
17
cell culture (SILAC) (138). In both types of approaches, MS is relatively quantitative
because the extracted quantification information can only be used to compare the same
peptide across different samples, but not different peptides within the same sample. This
is because molecularly-distinct peptides respond differently to MS detection; as a result,
the extracted quantification/molar quantity ratio is characteristic for each peptide.
However, one can overcome this difficulty by applying pre-determined correction factors
to correlate the quantification information of two distinct peptides and compare their
relative molar quantities within the same sample, as described in my previous publication
(139).
1.2.2.1 Measurement by Intensity of MS1 Ion Current
One popular approach in label-free quantification is measurement by MS1 ion
current intensity (reviewed in 137). Specifically, the height or area under the peak of the
extracted ion current chromatography (XIC) of a peptide, generated as the peptide elutes
off the reverse-phase chromatography column, is used as a quantitative measure, due to
the observation that it is linearly related to the peptide quantity (140, 141). This is a
measurement of relative quantities and has a linear range of over three orders of
magnitude (138, 139). However, since the MS2 analysis samples a subset of ions in the
analyte, frequently, a peptide with an XIC is not associated with an MS2 spectrum such
that the peptide can not be positively identified. In this case, the MS1 chromatograph of
multiple samples may be aligned by elution time and cross-referenced to combine the
MS2 spectra libraries, assigning identifications to previously unidentified
chromatographic peaks, allowing the quantification of more peptide ions. This can be
18
done in proteomics software such as MaxQuant that automatically identifies and retrieves
quantification information for peptide ions (142).
1.2.2.2 Selected Reaction Monitoring
Selected reaction monitoring (SRM) is a method used in MS in which a precursor
ion, usually a peptide, with a specific m/z is selectively isolated from a complex sample,
fragmented, and monitored for a defined fragmentation reaction product (143, 144). The
precursor and fragment ion pair is collectively called a transition. Typically more than
one transition is monitored for each targeted peptide, and the co-presence of most of the
transition-defined fragments is required to positively identify the peptide. Moreover,
quantification by SRM MS can be achieved by measuring the intensities of the ion
currents of the product ions (143, 144). This type of MS analysis is usually carried out
with a triple-quadrupole mass spectrometer (e.g. Thermo Scientific TSQ Vantage) and
has the advantage that it eliminates signals form other ion species and increases detection
sensitivity (143, 144). I previously showed that low-level (< 1%) tyrosine
phosphorylations on the SFK Lyn can be sensitively and reproducibly quantified by SRM
MS, demonstrating the utilization of SRM in monitoring protein phosphorylations (139).
1.2.3 Peptide Enrichment
1.2.3.1 Enrichment of Tyrosyl Phosphorylated Peptides
Tyrosyl phosphorylated peptides are low abundance compared to other peptide
species in a biological sample due to three reasons: 1) phosphorylated proteins merely
compose 1-2% of total proteins in a whole cell lysate extract (3); 2) only a few sites are
phosphorylated on a phosphoprotein, leaving the majority of the protein-derived peptides
19
non-phosphorylated; 3) phosphorylation usually occurs at low stoichiometry (139), such
that the non-phosphorylated peptide is in excess of its phosphorylated counterpart. As a
result, pY-specific enrichment must be performed prior to MS analysis to facilitate the
complete sampling of the collection of pY-containing molecules. The most commonly
used methods for phosphopeptide enrichment are antibody-based affinity purification,
immobilized metal affinity chromatography (IMAC), and titanium dioxide
chromatography (145). For purification of tyrosyl phosphorylated peptides, antibody-
based affinity purification has been proven to be very effective (146). An experiment
using this approach may identify up to hundreds of pY sites in a biological system.
Examples of such studies include the identification of activated RTKs and their
phosphorylated substrates without the prior knowledge of the activated pathways (147),
and characterization of downstream pY sites in induced systems (148-150).
1.2.3.2 Enrichment of Oxidized Cysteine-Containing Peptides (qPTPome)
A method was recently developed by our lab in collaboration with the group of Dr.
Benjamin Neel (Ontario Cancer Institute, Toronto) for the purpose of quantifying the
expressed PTPome in a biological system (151). This method exploits the property that
the cysteine-containing catalytic site of all classical PTPs is highly conserved, such that
an antibody developed against the signature motif of Ptpn1, with the critical cysteine
irreversibly “hyperoxidized” to sulfonic acid (VHCSO3HSAG) (152), may effectively
isolate most, if not all, classical PTPs. In particular, cellular PTPs were converted to the
hyperoxidized state, protease-digested, and subjected to immunoprecipitation with the
antibody. This process was coupled to LC MS/MS or SRM to facilitate the quantitative
profiling of isolated peptides as a measure of PTP expression (the qPTPome method). As
20
demonstrated, this approach reliably identified and quantified classical PTPs in a number
of cell lines and tissues (151). This method, combined with the pY profiling techniques,
enables a novel and comprehensive type of analysis of pY regulation in biological
systems.
1.3 Key Statistical Analysis Used in this Thesis
1.3.1 Partial Least Squares Regression
Partial least squares regression (PLSR) is a statistical method to model a set of
response variables based on a large number of predictor variables (153). It attempts to
predict response variables from predictors by simultaneously decomposing the predictor
and response variables into a shared set of orthogonal factors, or components, such that
the amount of variation in the response variables explained by the variation in the
predictor variables is maximized. The underlying model of a PLSR analysis is: X=TPT+E;
Y=UQT+F, where X is an n by m matrix of predictors, Y is an n by p matrix of responses,
T and U are n by l matrices of projections of X and Y, respectively, and P and Q are the
respective m by l and p by l orthogonal loading matrices of X and Y. E and F are matrices
of error terms, assumed to be independent random normal variables that are identically
distributed. X and Y are decomposed so as to maximize the covariance between T and U.
A popular alternative to PLSR is principal component regression (PCR), in which,
instead of decomposing both the predictor and the response variables, the principal
components of the predictor variables are used as predicting factors (154). PCR creates
models that describe the variability in the predictor variables without considering the
variability of the responses. PLSR, however, takes into account the variability of both the
21
response and the predictor variables, therefore, may create models that can fit the
response variables with fewer components. For the purpose of this thesis, PLSR was used
because a specific relationship between the predictors (TKs, PTPs) and the responses
(cellular pY) was sought after, and it was essential to consider the variability of the
responses.
1.4 Specific Aim for this Thesis
The aim of this thesis is to evaluate the hypotheses that 1) the cellular pY state is a
quantitative output of the activities of TKs and PTPs in biological systems (Chapters 2 &
3); and 2) the SH2 domains of SFKs, acting as downstream effectors of pY signaling, are
functionally modulated by phosphorylation (Chapter 4). The model being examined here
is depicted in Figure 1.1. Two human cancer models, MM and AML, were used.
Figure 1.1. A proposed model for the regulation and functional effect of cellular pY.
The cellular pY in a biological system is regulated by the concerted action of tyrosine
kinases (TKs) and protein tyrosine phosphatases (PTPs); and SH2 domains are involved
as a class of downstream effectors/readers of pY signaling, whose function in cells can
generate phenotypic output.
Y pY TK PTP
Effectors/Readers (i.e. SH2 Domains) Phenotype
22
Chapter 2 Comprehensive Analysis of Protein Phosphotyrosine
in Multiple Myeloma
A part of the work described in this chapter has been published as:
Robert Karisch,1,2,* Minerva Fernandez,2 Paul Taylor,3 Carl Virtanen,2 Jonathan R. St-
Germain,3 Lily L. Jin,3 Isaac S. Harris,2 Jun Mori,4 Tak W. Mak,2 Yotis A. Senis,4 Arne
O¨ stman,5 Michael F. Moran,3 and Benjamin G. Neel1,2 (2011) Global proteomic
assessment of the classical protein-tyrosine phosphatome and “redoxome”. Cell. 146,
826-840
The published work include pY and PTP profiling data in MM cell lines (parts of Figure
2.3 A and 2.5 A that show data for cell lines) and the PLSR analysis in MM cell lines
using PTP expression as a predictor for cellular pY (Figure 2.4 A), as Figure 7 A-C in the
publication. The mouse xenograft tumors used in this study were raised by Zhihua Li1,2
and Dr. Suzanne Trudel1,2. The PTP profiling data was provided by Robert Karisch1,2 and
Jonathan St-Germain3, and the PLSR analysis shown in Figure 2.4 A was done by Dr.
Carl Virtanen2. All other biochemical experiments and bioinformatics analyses described
in this chapter were performed by Lily Jin.
1Department of Medical Biophysics, University of Toronto, Toronto M5G 2M9, ON,
Canada 2Campbell Family Cancer Research Institute, Ontario Cancer Institute and Princess
Margaret Hospital, University Health Network, Toronto, ON M5G 1L7, Canada 3Program in Molecular Structure and Function, Hospital For Sick Children, and
Department of Molecular Genetics and McLaughlin Centre for Molecular Medicine and
Banting and Best Department of Medical Research, University of Toronto, Toronto, ON
M5G 1L7, Canada 4Centre for Cardiovascular Sciences, Institute of Biomedical Research, School of Clinical
and Experimental Medicine, College of Medical and Dental Sciences, University of
Birmingham, Birmingham B15 2TT, UK 5Cancer Center Karolinska, Department of Pathology and Oncology, Karolinska Institute,
Stockholm 17176, Sweden
23
2.1 Abstract
The cellular pY and PTP expression profiles in five MM cell lines and their
corresponding mouse xenograft tumors were examined by MS in order to assess the
effect of the total activated TKs and expressed PTPs on the systemic pY output in the
MM cancer model. Biochemical and bioinformatics analyses revealed that distinctive
physiologies were associated with MM cultured cell lines and xenograft tumors, with
different sets of TKs and PTPs implicated in cells and tumors. These results also
demonstrate an association between the activity/expression of the enzymes (i.e.
TKs/PTPs) and the cellular pY profile, supporting a model wherein the global pY output
is dependent on the TK and PTP states in the biological system.
2.2 Introduction
MM is a fatal B cell cancer that arises from transformation during the development
of blood stem cells into normal plasma B cells. The pathogenesis of MM is
heterogeneous (83, 155). One of the most important prognostic factor and oncogenic
driving mechanism in MM is the upregulation and aberrant activation of FGFR3, which
results from a t(4;14)(p16;q32) chromosomal translocation seen in approximately 15% of
MM cases. Inhibiting FGFR3 in vitro and in pre-clinical models have demonstrated
promising anti-proliferative/antitumor effects (85, 156, 157), supporting the development
of FGFR3-targeted therapy for t(4;14)-positive MM. In the recent past, another RTK
IGF1R also emerged as a potential driving kinase in MM. IGF1R was shown as an
important autocrine and paracrine factor that affected MM growth, survival, and drug
resistance (92, 158, 159), which was also aberrantly expressed in MM with its expression
24
significantly linked to disease severity (89, 90). These data, combined with the
observation that the MAPK and PI3K/Akt signaling pathways downstream of RTKs are
frequently altered and aberrantly stimulated in MM (160-162), suggest a likely role of
disrupted pY signaling in MM pathogenesis. Given that MM is still largely incurable (83),
elucidating the regulation of protein pY signaling in MM is of great importance and will
likely reveal novel oncogenic drivers for therapeutic targeting.
Five established human primary tumor-derived MM cell lines representing
heterogeneous subtypes of MM have been selected for a comprehensive study on cellular
protein pY regulation. Three cell lines (KMS11, KMS18, LP1) contain t(4;14), among
which, two (KMS11, KMS18) harbour activating FGFR3 mutations. The FGFR3
mutations carried by KMS11 and KMS18 (Y373C and K650E, respectively) have
different grades of activation capabilities with Y373C producing a more active form of
FGFR3 (Section 1.1.3.1). Correspondingly, the growth rates of KMS11 and KMS18 cells
were reduced by a FGFR3 inhibitor, while the other cell lines were insensitive to the
inhibition (85). Comparatively, the proliferation of KMS11, KMS12, and RPMI8226 cell
lines were inhibited by an IGF1R/INSR inhibitor in vitro, while that of the other cell lines
were not (92). Interestingly, LP1 cells were not sensitive to either inhibitor, thus
representing a subtype with unknown oncogenic driving TKs. These varying responses to
TK inhibitions indicate that diverse pY-mediated mechanisms are associated with the
subtypes represented by the five cell lines. The molecular characteristics described here
are summarized in Table 2.1.
Additional molecular abnormalities have been reported for these cell lines. Somatic
mutations on the oncoproteins p53 (E285K) (163) and K-Ras (G12A) (164) were
25
detected in PRMI8226 with uncharacterized functional implications. Moreover, Mdm2
was up-regulated and promotes proliferation and survival in RPMI8226 cells (165).
Furthermore, gene expression studies identified mRNA over-expression of c-maf in
KMS11, LP1, and RPMI8226 (166); c-myc in KMS11, KMS12, and LP1 (167); and
cyclin d1 in KMS12 (166). These abnormalities provide another layer of dysregulation
that may contribute to MM pathogenesis, and were also summarized in Table 2.1.
MM cells, grown as suspending cells in tissue culture, develop into solid tumors
when injected into mice, suggesting different physiologies are associated with cultured
cells and xenografts. To gain a comprehensive understanding of MM cell and tumor
biology, the five selected cell lines were inoculated into mice to produce xenograft
tumors (by Zhihua Li and Suzanne Trudel, Ontario Cancer Institute, Toronto). The cell
lines and their corresponding tumors were analyzed by Western blots (Section 2.3) and
MS, which generated quantitative profiles of 109 unique pY-containing peptides,
encompassing 106 pY sites, and 36 PTPs across ten samples using label-free
quantification. A comparison of the proteomic signatures showed distinctive differences
between the tumors and cells. Integrated analysis revealed novel relationships between
the activated TKs, total expressed PTPs, and the cellular protein pY (Sections 2.4 - 2.6).
26
Table 2.1. Molecular Characteristics of MM Cell Lines
27
2.3 Western Analysis Showed Distinct Expression, Glycosylation,
Phosphorylation of pY Signaling Molecules in MM Samples
To characterize the MM samples, the expression/phosphorylation of key pY
signaling molecules were examined by Western blots (Figure 2.1), which revealed
distinctive molecular differences among the samples: the expression of FGFR3 and Lyn
(a SFK highly implicated in B cell disorders (25)), as well as the phosphorylation of Lyn
Y508 (inhibitory) and SFK A-loop (activating), were highly varied across MM cultured
cell lines and tumors. In particular, FGFR3 expression were consistent with the genotype,
where elevated levels were seen in the t(4;14)-positive samples with the highest
expressions coincided with FGFR3 activating mutations. Whereas, Lyn Y508 and SFK A-
loop phosphorylations, while showing fluctuations across different MM subtypes, was
generally decreased and increased, respectively, in tumors compared to the cultured cells,
suggesting increased SFK activities in the tumors. Moreover, although its expression was
uniform across the samples, the activating phosphorylation of Erk 1/2 was drastically
increased in the tumors. Because Erk 1/2 is a downstream effector of MAPK signaling
that controls growth and proliferation, this data suggests a tumor-specific up-regulation of
MAPK pathways. Unlike FGFR3/Lyn and like Erk 1/2, the IGF1R/INSR expression was
ubiquitous and mostly uniform across the MM samples. However, like FGFR3, IGF1R
was heavily asparagine (N)-glycosylated in MM (Figure 2.1 A). The fact that
glycosylated-IGF1R was resistant to Endo H but not PGNase F suggests that only hybrid
and complex, but not high-mannose, oligosaccharides were attached to IGF1R.
Interestingly, IGF1R appeared under-glycosylated in KMS18 (Figure 2.1 A right, note
the downshift of the 100 kDa band) and hyper-glycosylated in the tumors compared to
28
the cell lines (Figure 2.1 A right, note the disappearance of 75 kDa bands in Lanes 6-10
compared to Lanes 1-5). Glycosylation protects proteins from degradation and impacts
localization (168), therefore, altered glycosylation implies changes in stability and
localization of the RTKs. Finally, the global protein pY profile varied across the samples,
potentially as an output of the differentially regulated pY signaling molecules (Figure 2.1
B, pY blot). Taken together, these Western data show diverse features in pY signaling
molecules/effectors not only across the cell lines, but also between cultured cells and
their cognate tumors, suggesting that distinctive protein pY regulations are at play in
these MM samples.
29
Figure 2.1. MM cell lines have distinctive molecular features
A, Western blots showing bands of FGFR3 and IGF1R before and after deglycosylation
by the glycosidase endoglycosidase H (Endo H) (removes asparagine-linked high-
mannose oligosaccharides) or PNGase F (removes asparagine-linked high-mannose,
hybrid, and complex oligosaccharides from glycoproteins). B, Western blots showing the
total pY, expressions of INSR, Lyn, and Erk 1/2, as well as the phosphorylations of A-
loop in SFKs, Y508 in Lyn, and T202/Y204 in Erk 1/2. Beta-actin was used as a loading
control.
A
B
30
2.4 pY Profiling Revealed Distinctive Differences between MM
Cultured Cell lines and Xenograft Tumors
To gain an in-depth understanding of the pY regulation in MM, comprehensive
quantitative pY profiles were generated for the ten MM samples. To this end, cell- or
tumor-derived proteolytic peptides, equalized by total protein amounts, were enriched for
pY-containing molecules by immunoprecipitation. The enriched fractions were analyzed
by MS. The MS results were searched automatically against the human International
Protein Index (IPI) database (ftp://ftp.ebi.ac.uk/pub/databases/IPI/last_release/; v3.68;
87,061 FASTA entries) for peptide identifications (see flowchart in Figure 2.2 A). This
experiment was repeated once. A total of 109 unique pY peptides, corresponding to 106
pY sites, were identified across two replicates of ten MM samples.
The total numbers of MS/MS spectra for pY peptides, as well as unique pY peptides,
unique pY sites, proteins associated with pY peptides, and kinases identified in each MM
sample are shown in Table 2.2. In the case that the identified peptide sequence is
conserved in more than one protein, only one protein is counted to represent the group. A
comparison across the cell lines showed that KMS12 had the lowest incidences of
identifications, consistent with a phenotype that lacked an association with aberrant TK
activation, while the t(4;14)-positive cell lines contained significantly more incidences of
pY identifications. Curiously, the number of pY identifications in RPMI8226 were
comparable with that of the t(4;14)-positive cell lines. Since KRAS mutation was
observed in RPMI8226 cells (164), up-regulation of the MAPK pathways, which have
been implicated in MM (162), could be one potential mechanism that gives rise to the
high count of pY identifications. A comparison between the cell and their cognate tumor
31
samples revealed only 36-54% overlap between the unique pY sites identified (Figure 2.2
B), with generally more identifications observed in cultured cells than in tumors. Motif
analysis of the 106 total pY sites revealed an enrichment of the signature A-loop-
bounding sequences DFG…APE (Figure 2.2 C). Since phosphorylation of Y within the
A-loop suggests increased catalytic activity of the parent kinase (Section 1.1.1.1), this
observation implies an abundance of activated kinases in MM. Indeed, 22 out of 109
peptides correspond to phosphorylations within the A-loop of kinases (including TKs,
Serine/Threonine kinases, and dual-specific kinases). Among these, nine peptides,
representing seven proteins, were derived from TKs (refer to Figure 2.3 B).
32
Figure 2.2. pY profile reveals difference between cell and tumor and an abundance
of TK A-loop phosphorylations
A, a flowchart showing schematic overview of experimental procedures. MM cells were
injected into mice to produce xenograft tumors by Zhihua Li (Ontario Cancer Institute)
and all other experiments were performed by Lily Jin. B, Venn diagrams of unique pY
sites identified in MM cell and tumor samples for each cell line. C, sequence logos
showing the abundance of surrounding residues (corresponding to the sizes of letters) of
106 pY sites identified in MM. The conserved A-loop-bounding sequences are
highlighted in red.
A B
C
33
Table 2.2. Number of proteins, peptides, and pY sites identified in MM cells and
tumors
34
Next, I examined the quantitative profile of the identified pY peptides (raw
quantification data in Appendix Table 1). A heatmap based on the quantification levels of
the pY peptides was created. Unsupervised hierarchical clustering separated the cell and
tumor samples into two distinct groups according to the pY profiles (Figure 2.3 A),
suggesting distinguishable differences between the cells and tumors. A closer
examination revealed that a total of ten peptides were significantly different between the
cells and tumors, with paired Student’s t-test p value < 0.05 (Figure 2.3 B), which include
the MAPK1 pY187-containing peptide that is on average 2.8-fold higher in tumors and the
tyrosine kinase 2 (Tyk2) pY292-containing peptide that is on average 3.2-fold more
abundant in cells. Moreover, the inhibitory phosphorylation (Y15) in Cdk 1/2/3 was
significantly higher (on average by 4.1-fold; p < 0.01) in cells than in tumors. Since the
Cdk’s are essential for driving each phase of a cell cycle (169), this data suggests
enhanced cell cycle progression in the tumors. Together, these data indicate that pY
signaling may be differentially regulated in MM cells and tumors through the action of
different kinases, which may be reflected on downstream molecules (e.g. Cdk 1/2/3) as
effectors of the pY regulation.
Because of the noted abundance of A-loop pY in MM, and phosphorylated A-loop
represents the activations of its parent kinase, I hypothesized that the activated TKs in
MM dictate the overall cellular pY. If this were true, unsupervised hierarchical clustering
based on the levels of the A-loop pY along should separate the cells from tumors. To this
end, I generated a heatmap based on the quantitative profiles of TK A-loop pY in MM
samples (Figure 2.3 C). As the results indicate, the samples were clustered into two
distinct groups, mostly separating the cells from tumors, suggesting that the activation of
35
subsets of TKs was responsible for the different cell and tumor pY signature. However,
KMS12 tumor and RPMI8226 cells were clustered reciprocally with the cells and tumors,
respectively. The reason may be that high-level phosphorylation in the A-loops of FGFR3
and Lyn (or Hck, which contains the same A-loop-derived tryptic peptide sequence as
Lyn) in KMS12 tumor confers a cell-like signature, while the activation of Src (or Yes,
Fyn, Lck, which contain the same A-loop-derived tryptic peptide sequence as Src) and
low phosphorylation of FGFR3 in RPMI8226 cell render it more similar to tumors.
36
Gene Site
Y Kinase Site
A B
C
Gene Site Cell/Tumor
Figure 2.3. Quantitative pY profile
distinguishes cell from tumor. A, a heatmap
showing unsupervised hierarchical clustering
based on the quantitative pY profile in MM. B,
a heatmap showing unsupervised hierarchical
clustering based on ten peptides whose levels
were found to be significantly altered between
cell and tumor samples. The gene, site, and the
cell to tumor fold change are given on the right.
C, a heatmap showing unsupervised
hierarchical clustering based on TK A-loop
phosphorylations. Alternative proteins that
contain the same A-loop derived tryptic peptide
sequence are shown in brackets.
37
To further dissect the relationship between activated TKs and cellular pY, I
analyzed the co-variation between cellular pY levels and TK A-loop phosphorylations by
PLSR (153). This analysis produced mathematical models based on the assumption that
the variations in cellular pY levels can be predicted based on the variations in TK A-loop
phosphorylations. Because my previous analysis showed distinctive pY features
associated with MM cells and tumors, the cell and tumor samples were analyzed
separately. The PLSR algorithm automatically determined three-component models for
both cells and tumors wherein 99.8% and 99.9% of variations in cellular protein pY could
be predicted based on TK A-loop phosphorylation levels in cells and tumors, respectively.
The first and second components in the model for cells were able to predict 86.5% and
9.8% of pY variations (Figure 2.4 A) and the first two components in the model for
tumors were able to predict 74% and 14% of pY variations (Figure 2.4 B), respectively.
Among all the tested TKs, FGFR3 and Ptk2 correlated best with Component 1 in cells,
and Src (or Yes, Fyn, Lck) correlated best with both Component 2 in cells and
Component 1 in tumors. Abelson Tyrosine-Protein Kinase 1 (Abl1) and FGFR3
correlated best with Component 2 in tumors, while also contributing to Component 1.
These TKs were implicated by this analysis as key modulators of pY signaling in their
perspective MM system. A comparison between the models revealed a reduced influence
of FGFR3 and a increased influence of Src (or Yes, Fyn, Lck) on the cellular protein pY
in the tumors than in the cells, consistent with the prominent role of SFKs downstream of
signaling of growth factors and cytokines that may present in the tumor
microenvironment of the animal model. These results also suggest Ptk2, Abl1, and Src
38
(or Yes, Fyn, Lck), in addition to the well-established oncogenic driver FGFR3, as
candidate cancer drivers.
Figure 2.4. Cellular pY variation can be predicted based on TK activation levels
A and B, correlation plots of the A-loop phosphorylation of TKs and the predicting
components computed by the partial least squares regression (PLSR) algorithm.
Alternative proteins containing the same A-loop derived tryptic peptide sequence are
given in brackets. Percentage in brackets indicates the portion of variation in cellular pY
predicted by the associated component.
A B
39
2.5 Co-Variance Analysis of PTP and pY Profiles Implicated a Subset
of PTP in pY Regulation
In order to assess the influence of negative enzymatic regulation on cellular pY and
develop a comprehensive understanding of pY regulation in MM, PTP expression
profiles in MM samples were produced in our lab through collaboration with Robert
Karisch, a former graduate student in Dr. Benjamin Neel’s lab (Ontario Cancer Institute,
Toronto), and by using the qPTPome method (151). The expression levels of a
complement of 36 classical PTPs were examined by SRM MS, among which, 24 were
found to be expressed in our samples (Appendix Table 2). Using the PTP expression
profile, I performed unsupervised hierarchical clustering to seek relationships among the
MM samples (Figure 2.5 A). Interesting, this analysis generally separated the cell and
tumor samples into two clusters, showing that, in addition to cellular pY and TK
activation, the regulation of PTP expressions was also different between cells and tumors.
The exceptions were LP1 tumor and KMS12 cell, which were clustered reciprocally with
the cell and tumor groups, respectively. Moreover, the cognate cell and tumor samples of
KMS12 and LP1 cell lines were paired up respectively (p < 0.05), indicating that the PTP
expressions in these cell lines were influenced more by the genotype than the growth
environment. This observation was not seen in the clusters based on pY or TK A-loop
phosphorylations, and may suggest that genotype exerts a heavier influence on PTP
expressions than on pY, which is more dynamic and responds quicker to environmental
stimuli.
To further dissect the relationship between PTPs and cellular pY, PLSR analysis
was applied to generate models wherein PTP expression levels were used as predictors
40
for cellular pY levels. Again, the cell and tumor samples were analyzed separately. The
analysis on the five cell samples was done by Dr. Carl Virtanen (Ontario Cancer Institute).
A three-component model was generated, which showed that 99.3% of variations in pY
levels were predictable based on PTP variation. In particular, Component 1 predicted
97.2% and Component 2 predicted 1.7% of variations (Figure 2.5 B). Moreover, the
fluctuation in Ptpn1, Ptpn6, and Ptpn11 expressions (correlated best with Component 1)
collectively predicted 97.2% of the variations in the pY sites analyzed. Notably, this was
a positive correlation, where increased PTP expression corresponded to increased pY
levels, which was consistent with the prominent role of Ptpn1 in RTK signaling (96, 170,
171) and Ptpn11 as a highly implicated oncoprotein in haematopoietic cancers (64, 67).
Moreover, this was the first time a correlation between total pY and PTP expression was
demonstrated in a biological system, and was published as a part of a paper in Cell (151).
Next, to understand the role of PTPs in the regulation of pY in MM tumors, I
performed a PLSR analysis and discovered that, with a three-component model, 95.5% of
pY variations in the five MM tumor samples could be predicted based on PTP expression.
In particular, Component 1 predicted 27% and Component 2 predicted 58% of variations
(Figure 2.5 C). Moreover, Ptpn9 and Ptpn11 correlated best with Component 1, and
Ptpn7 and Ptprg correlated best with Component 2, which collectively predicting a
combined 85% of variations in total pY levels. This analysis implicated these PTPs in pY
regulation in MM tumors. The fact that different sets of PTPs were implicated for cells
and tumors implies that the dephosphorylation of pY was differentially regulated, further
confirming that distinct molecular biologies were associated with xenograft tumors and
Germain3,8, Robert Karisch4,7, Paul Taylor1,8, Mark Minden7, Michael D. Taylor2,5,8,
Benjamin G. Neel4,7, Gary D. Bader3,6, and Michael F. Moran1,3,7,8
The pY profile in AML samples was generated by Dr. Jiefei Tong1,8 (Figure 3.1 A) and
the PTP profile was generated by Rob Karisch4,7 and Dr. Jiefei Tong1,8 (Figure 3.2 A).
The bioinformatics analyses in this chapter were performed independently by Lily Jin.
1Program in Molecular Structure & Function, Hospital for Sick Children, Toronto. 2Program in Developmental & Stem Cell Biology and Arthur and Sonia Labatt Brain
Tumour Research Centre, Hospital for Sick Children, Toronto. 3Department of Molecular Genetics, University of Toronto. 4Departmet of Medical Biophysics, University of Toronto. 5Department of Laboratory Medicine and Pathobiology, University of Toronto. 6The Donnelly Centre, University of Toronto. 7Princess Margaret Cancer Centre, Toronto. 8Peter Gilgan Centre for Research and Learning, Hospital For Sick Children, 686 Bay
Street, Toronto, ON, M5G 0A4, Canada.
*Equal contributions were made by these authors.
63
3.1 Abstract
To further dissect the relationship between activated TKs, expressed PTPs, and the
cellular pY, 12 human primary AML samples were obtained. MS-based proteomics
analysis generated a quantitative pY profile for the 12 samples and a PTP expression
profile for a subset (eight out of 12) of the samples. A collection of 219 unique pY sites
and 16 PTPs were quantified. Co-variance analysis showed a high degree of
correspondence between TK activation/PTP expression levels and the cellular pY, which
also implicated a subset of TKs and PTPs in pY regulation in AML.
3.2 Introduction
Dysregulation of pY signaling networks is common in cancer cells and is frequently
linked to somatic mutations and altered enzymatic functions. This is exemplified by AML,
a myeloid cancer with heterogeneous disease pathology often relating to mutations in pY
signaling molecules (Section 1.1.3.2). Inhibiting aberrantly activated TKs in AML have
demonstrated promising therapeutic potentials, implying the importance of pY signaling
in AML (213). Moreover, profiling of phosphoproteins in AML by reverse-phase protein
arrays defined protein signature groups that correlated with response and survival,
suggesting that AML may be stratified into prognostic groups with distinct
phosphorylation networks (214). Monitoring the signaling responses of single AML
cancer cells by multiparameter flow cytometry suggests extensive remodeling of the
signaling network at the single-cell level (215). These data provide a rationale for
performing proteomics studies and elucidating the molecular wirings of pY signaling in
AML.
64
The reciprocal actions of TKs and PTPs constitute important steps of pY regulation.
Given the critical relationships between pY signaling and cancer (7, 8, 24, 200) (Section
1.1), it is unsurprising that a number of TKs and PTPs are highly implicated in human
malignancies: TKs are well-established targets in neoplastic disorders and 37 PTPs were
linked to cancer with approximately half described as oncoproteins and the other half as
tumor suppressors (31). Flt3 with ITD mutation and Pptn11 are well-known positive
drivers and prognostic indicators in AML (Section 1.1.3.2). Additionally, distinct pY
patterns in primary samples and cell lines (214-216) and changes in PTP expressions (217)
have been reported for AML. These data combined with the demonstration that PTP
expression profiles affect cellular pY (151) make a curious case that how the activated
TKs and expressed PTPs impact cellular pY in AML. A model depicted in Figure 1.1 was
examined in this study using AML primary samples.
Twelve human AML primary samples were obtained and analyzed by MS (patient
information given in Table 3.1). Based on the MS analysis, quantitative pY profiles were
produced for the 12 samples; due to sample limitation, PTP profiles were only generated
for eight of the 12 samples. Integrated analysis of the pY and PTP profiles revealed
interesting insights into the pY-TK-PTP interplay and implicated a number of PTPs and
TKs in AML.
65
Table 3.1. AML patient information corresponding to primary samples
FAB is abbreviated for French-American-British classification; FLT3-ITD denotes Flt3
internal tandem duplication; NR, No Tx, and CR denote no response, no test, and
complete remission. Patient data was provided by Dr. Mark Minden (Princess Margaret
Cancer Centre, Toronto).
66
3.3 pY Profiling Revealed Distinctive Features in Patient Subgroups
and Implicated a Subset of TKs in pY Regulation
To produce the pY profiles, whole cell lysate proteins extracted from AML samples
were digested by trypsin and enriched for pY-containing tryptic peptides through
immunoprecipitation by Dr. Jiefei Tong in our lab. The enriched peptides were analyzed
by MS, and the result files were given to me for computational analysis. The files were
searched against the UniProtKB/Swiss-Prot human protein FASTA database (2013
September release; 540,958 sequence entries) for peptide identifications. The phospho-
modified peptides were then quantified by label-free quantification in the MaxQuant
software (142). This work produced the quantitative profile for 219 pY sites on 159
proteins across 12 AML primary samples (Figure 3.1 A; Appendix Table 3). Some TKs
highly implicated for AML (e.g. Flt3, Kit, Syk) were identified: activating mutations of
Flt3 and Kit were frequently observed in AML (Section 1.1.3.2) (218) and Syk was
recently identified as an AML target (219). A sequence logo based on the 219 pY sites
suggested a prevalence of the TK A-loop phosphorylations by showing an enrichment of
the conserved A-loop-bounding signature sequences DFG…APE (Figure 3.1 B). A close
examination of the dataset revealed a collection of eight TKs whose A-loops were
tyrosine-phosphorylated, indicating stimulated kinase activities for these TKs. A heatmap
of the A-loop phosphorylations is shown in Figure 3.1 C.
67
Figure 3.1. Characterization of pY in AML primary samples
A, a heatmap showing unsupervised hierarchical clustering based on the quantitative pY
profile in AML, with sample IDs labelled on top. B, sequence logo showing the
abundance of surrounding residues (corresponding to the sizes of letters) of 219 pY sites
identified in AML. The conserved A-loop-bounding sequences are highlighted in red. B,
a heatmap showing unsupervised hierarchical clustering based on TK A-loop
phosphorylations. The z-scores of the quantification values of each peptide across the
AML samples were used. Alternative proteins containing the same tryptic peptide
sequence are given in brackets. D, Bar graphs of the summed MS signal intensities of the
complement of cellular pY-containing peptides (top) and TK A-loop-derived
phosphopeptides (bottom). A Pearson’s correlation coefficient summarizing the
relationship between the top and bottom graphs is shown. ****p<0.0001.
A
Kinase Site
B
C
Group 1 Group 2
D Pearson’s coefficient=0.94****
68
Unsupervised hierarchical clustering based on either the total pY or TK A-loop
phosphorylations separated the AML samples into two groups (p < 0.01): generally a
group with high phosphorylations and a group with low phosphorylations. For pY-based
clusters (Figure 3.1 A), the low-phosphorylation group (Group 1) contained seven
samples, all of which had low TK A-loop phosphorylations (Figure 3.1 C and D; sample
IDs: 240, 385, 347, 286, 454, 395, 118). Comparatively, the high-phosphorylation group
(Group 2), which comprised five samples, had relatively highly phosphorylated TK A-
loops (Figure 3.1 C and D; sample IDs: 9217, 215, 272, 135, 212). Consistent with this, a
correlation heatmap of TK phosphorylation levels against 219 cellular pY sites showed a
largely positive relationship (Figure 3.2 B; judged by the overwhelming red color in TK-
associated lanes). Moreover, the Pearson correlation coefficient between the total MS
signals generated by the complement of pY peptides (containing 219 pY sites) and TK A-
loop-derived phosphopeptides was calculated to be 0.94 (p < 0.00001) (Figure 3.1 D).
These data show that TK A-loop phosphorylations positively correspond with the levels
of cellular pY.
The eight TKs identified in the AML samples were clustered into two groups based
on their A-loop phosphorylation levels (Figure 3.1 C): a group of five TKs (Btk, Fes, Tec,
Syk, and Fgr) that are generally highly phosphorylated in the high phosphorylation group
and a group of three TKs (Abl2, Lyn, and Lck) whose A-loop phosphorylation is
generally low except in one patient (sample ID: 9127). This type of grouping scheme was
reproduced in the correlation-based cluster where the groups of five and three TKs were
included in Groups B and A (Figure 3.2 B) of the TK-associated clusters, respectively.
These data further demonstrate a correspondence between cellular pY and TKs:
69
subgroups of TKs may be upstream of distinctive sets of pY sites and modulate pY
through distinct pathways. Boxes 4 and 5 in Figure 8 B highlight regions of intense
positive correlation where pY sites (listed in Table 3.2) are positively correlated with
clusters of phosphorylated TKs. These regions suggest a relationship in which the
implicated TKs are potential upstream regulators of the associated pY sites.
Taken together, these data indicate that TK activation/phosphorylation may
positively regulate the cellular pY levels. They also showed that distinctive pY features,
namely high or low overall phosphorylations, were associated with subgroups of AML
patients.
70
Figure 3.2. PTP expression and TK activation are correlated with cellular pY
A, a heatmap showing unsupervised hierarchical clustering based on PTP expression
profile in eight AML samples. The z-scores of the quantification values of each PTP-
derived peptide across the AML samples were used. B, a correlation heatmap showing
unsupervised hierarchical clustering based on Pearson correlation coefficients of PTP
expression or TK phosphorylation with the levels of 219 pY sites. Boxes highlight
regions of high-density correlations. Representative pY site positively correlates with
PTP and negatively correlates with TK (MAPK pY187), or negatively correlates with PTP
and positively correlates with TK (Syk pY526), is indicated.
B A Group 1 Group 2
71
Table 3.2. Proteins and sites associated with boxed regions in Figure 3.2 B
72
Next, to quantitatively define the relationship between activated TKs and the
cellular pY, a PLSR analysis was performed. The algorithm automatically determined an
eight-component model that was able to predict 100% of variations of cellular pY based
on TK A-loop phosphorylation levels (as an indicator of TK activation), with the first
three components predicting 85.6% of the variations collectively. Figure 3.3 A shows the
correlation plot of TK A-loop phosphorylations with Components 1 and 2. As shown,
Syk, Fes, and Fgr correlated best with Component 1, predicting 46% of cellular pY
variations, and Abl2 and Lck (or Src, Yes, or Fyn that share the same A-loop tryptic
peptide sequence as Lck) correlated best with Component 2, predicting 29% of pY
variations. Notably, Syk, Fes, and Fgr belong to the collection of five TKs whose A-loops
are generally highly phosphorylated in the high phosphorylation group of patient samples;
and Abl2 and Lck belong to the class of three TKs that generally have low
phosphorylations in all the samples. These data indicate that two major axis of pY
regulation by different sets of TKs may be present; therefore, different pY regulation
schemes may possibly define AML subtypes. This analysis also implicates the TKs Syk,
Fes, Fgr, Abl2, and Lck (or Src, Yes, Fyn) as major pY modulators in AML, whose
activation levels were able to collectively predict 75% of pY variations.
73
Figure 3.3. Cellular pY variation can be predicted based on PTP expression or TK
activation levels
A and B, correlation plots of TK A-loop phosphorylation (A) or PTP expression (B) and
the predicting components computed by the partial least squares regression (PLSR)
algorithm.
A B
74
3.4 Integrated Analysis of PTP Expression and Cellular pY Implicated
PTPs in AML pY Regulation
To gain a comprehensive overview of pY regulation in AML, the TK antagonist
enzyme PTPs were quantitatively analyzed in eight AML samples (a subset of the 12
primary samples). An expression profile of the entire collection of classical PTPs (the
expressed PTPome) was generated by the MS-based qPTPome method (151), through the
combined effort of Rob Karisch (Ontario Cancer Institute, Toronto) and Dr. Jiefei Tong
in our lab. A total of 16 PTPs were shown to be expressed, for which a quantitative
profile was generated across the eight samples by label-free quantification in MS
(Appendix Table 4). The generated profiles were passed on to me for bioinformatics
analysis. Unsupervised hierarchical clustering based on the quantitative PTP expression
profiles distinguished two patient subgroups, with Group 1 containing three samples that
expressed relatively higher levels of PTP compared to Group 2, which comprised five
samples (Figure 3.2 A). The fact that this grouping of patients share little commonality
with the clustering of patients based on pY or TK A-loop phosphorylations (compare
Figure 3.2 A to Figure 3.1 A and C) shows that PTPs are not simply negative regulators
of cellular pY, but whose regulation of cellular pY involves a more complicated inter-
relation. To visualize the relationship between PTP expression and cellular pY levels, a
correlation heatmap was created. An integrated heatmap showing correlations between
TK phosphorylation/PTP expression and the levels of 219 pY sites is presented in Figure
3.2 B. Clusters of positive and negative correlations are highlighted by Boxes 1 to 5 and
the list of associated pY sites are given in Table 3.2. The pY sites showing positive
correlations with TKs and negative correlations with PTPs may be subjects of the net
75
antagonistic regulation of phosphorylation by TK and dephosphorylation by PTP. An
example of this is the A-loop phosphorylation of Syk, which was positively correlated
with TK phosphorylations but negatively correlated with the expression of PTPs such as
Ptpn11, Ptpra, and Ptpn1 (Figure 3.2 B, as indicated; Box 3). Conversely, pY sites that
negatively correlate with TK and positively correlate with PTPs may demonstrate indirect
regulation by these enzymes. An example of this is the A-loop phosphorylation of
MAPK1 (Figure 3.2 B, as indicated; Box 1). Syk is a target in AML (219) and MAPK1 is
downstream of mitogen-activated protein kinase kinase kinase 1 (MEK), another AML
target (220). These data show that AML targets/their related pathways may be
differentially modulated by TKs and PTPs. Together, these observations suggest diverse
mechanisms associated with PTP-mediated regulation of pY-containing polypeptides in
AML.
To further elucidate the relationship between PTP expression and the cellular pY, a
PLSR analysis was performed, which showed a high degree of predictability of pY levels
based on PTP expression. Only 11 out of the 16 expressed PTPs were quantified in at
least three of the AML samples and were used for this analysis. Using an automatically
determined six-component model, the fluctuations in PTP expression levels were able to
predict 100% of pY variations, with Component 1 along predicting 98.88% of pY
variations (Figure 3.3 B). Out of 11 PTPs tested by the algorithm, ten PTPs, namely
were conducted by Cluster 3.0 and visualized by Java Treeview (Version 1.1.5r2;
http://jtreeview.sourceforge.net/).
Partial Least Squares Regression (PLSR) Analysis
For descriptions of the PLSR analysis, refer to Section 2.8 under “Partial Least
Squares Regression (PLSR) Analysis”.
84
Chapter 4 Determination of the Functional Consequences of
Tyrosine-Phosphorylation of the Lyn SH2 Domain
The work described in this chapter has been published as:
Lily Jin,1,4,* Leanne Wybenga-Groot,2,3,* Jiefei Tong,1 Paul Taylor,1 Mark Minden,5,6
Suzanne Trudel,5,6 C. Jane McGlade,2,3,5 and Michael F. Moran1,4,6 (2015) Tyrosine
phosphorylation of the Lyn SH2 domain modulates its binding affinity and specificity.
Mol Cell Proteomics. 14(3):695-706.
The pY profiles in primary AML and chronic lymphocytic leukemia samples were
generated by Dr. Jiefei Tong1,4,6. The protocol for purification of phosphorylated or
unphosphorylated Lyn SH2 domain was developed with the help of Dr. Jiefei Tong1,4,6
and Dr. Leanne Wybenga-Groot,2,3. All other experiments and computational analyses in
this chapter were performed by Lily Jin.
Programs in 1Molecular Structure and Function and 2Cell Biology, and 3The Arthur and
Sonia Labatt Brain Tumour Research Centre, The Hospital For Sick Children, 686 Bay
Street, Toronto, M5G 0A4, Canada
Departments of 4Molecular Genetics and 5Medical Biophysics, University of Toronto 6Princess Margaret Cancer Center, 610 University Avenue, M5G 2M9, Toronto, Canada
85
4.1 Abstract
SH2 domains bind pY-containing polypeptides and modulate cellular functions by
directing protein-protein interactions. Proteomics analysis conducted in our lab showed
frequent phosphorylation of SFK SH2 domains in blood system cancers such as AML,
chronic lymphocytic leukemia (CLL), and MM. To understand the functional impact of
SH2 domain phosphorylation, I carried out biochemical analysis using the SFK Lyn SH2
domain as a model and found that phosphorylation of the conserved residue Y194 alters
the affinity and specificity of Lyn SH2 domain to pY-containing proteins/peptides.
Analysis of the preferred binding motifs revealed a change in specificity of the SH2
domain for the pY+2/+3 residue of the ligand. These results present another layer of
regulation wherein protein-protein binding through SH2 domains is regulated by
upstream SH2 TKs and PTPs.
4.2 Introduction
SH2 domains are modular functional units of a protein that recognize specific pY-
containing motifs. They are found in various signaling molecules and are important for
signal transduction due to their ability to mediate protein-protein interactions (Section
1.1.1.4). Given the reversible nature of protein tyrosine phosphorylation (3), the
interactions of SH2 domains to pY-containing molecules are inherently dynamic,
comprising key steps through which the signaling networks within a cell are controlled
and regulated. In addition to binding proteins in trans, SH2 domains participate in
intramolecular interactions, with one example being the auto-regulation of SFKs (Section
1.1.1.2). Therefore, SH2 domains regulate signaling enzymes and are indispensible for
86
mediating signal transduction in response to extracellular stimuli. The structure of SH2
domains consists of conserved antiparallel β-sheets and two α-helices, which form
variable binding surfaces that recognize different pY motifs (Section 1.1.1.4.1). For SFKs,
the SH2 domains were found to bind most tightly to sequences containing the pYEEI
motif, in which the hydrophobic pY+3 residue inserts into a hydrophobic pocket bounded
by residues of the EF and BG loops; altering these residues can lead to changes in
substrate selectivity for the SFK SH2 domains (51-53). In addition to binding pY-
containing polypeptides, SH2 domains themselves can be phosphorylated (54-56), which
appeared to be a general mechanism for modulating the binding properties of SH2
domains. Here, I report the frequent phosphorylations of a conserved tyrosine within SFK
SH2 domains in the haematopoietic cancers AML, CLL, and MM, as well as other
cancers. In vitro binding studies using Lyn SH2 domain as a model indicate that this
phosphorylation affects phosphoprotein and phosphopeptide binding to Lyn SH2 domain.
These results suggest that SH2 domain phosphorylation may be a general mechanism for
modulating SFK functions and may constitute an additional layer in the regulation of pY-
mediated signaling.
4.3 A Conserved Tyrosine in SFK SH2 Domains Is Phosphorylated in
Cancer Samples and Cancer-Derived Cell Lines
To identify sites of protein tyrosine phosphorylations, the pY profiles for 12
primary AML (See Chapter 3) and five Revlimid-treated CLL patient samples were
generated. From these data, I observed that Lyn Y194, or the equivalent Hck Y209 and Lck
Y192, were phosphorylated in 11 out of the 12 AML samples (Figure 4.1 A). Moreover,
Lyn Y194, Lck Y192, or their equivalents Fgr Y209 or Blk Y188, were phosphorylated in five
87
CLL samples (Figure 4.1 B). Consistent with these results, published phosphoproteomic
studies reported the phosphorylation of Lyn Y194 or its equivalent SFK residues in non-
small cell lung cancer (NSCLC) cell lines and tumors, breast cancer specimens, and in
the cell lines of chronic myeloid leukemia (175, 254, 255). Together, these results show
that phosphorylation of Y194 in the SH2 domain of Lyn, or its equivalent in SFK family
members, is frequent in cancer and cancer-derived cell lines.
88
Figure 4.1. SFK SH2 domain phosphorylation was detected in AML, CLL, MM and
is regulated by phosphatase
A and B, relative MS signal intensities of SFK SH2 domain phosphorylations in the
peripheral blood mononuclear cell samples harvested from 12 newly diagnosed AML (A)
and five Revlimid-treated CLL (B) patients. C, Bar graphs representing the measured Lyn
Y194 phosphorylation stoichiometry in human MM tumor-derived cell lines. The cells
were either harvested without treatment, or after treatment with pervanadate. D, Sequence
alignment of human SFK SH2 domains, with secondary structure elements corresponding
to Lyn kinase indicated on top. The residues are numbered based on Lyn. Lyn Y194 and
residues within the pY or pY+3 binding pockets are marked with a red, orange, or green
box, respectively.
D
89
4.4 SFK SH2 Domain Phosphorylation Is Regulated
To determine if SFK SH2 domain phosphorylation is regulated in cells, I measured
the absolute phosphorylation stoichiometry of Lyn Y194 in four MM cell lines, untreated
or treated with pervanadate, a pan PTP inhibitor. The measurement was performed using
an SRM-based method that I developed previously (139). Particularly, Lyn Y194 was
phosphorylated at 0.7% and 0.4% in untreated KMS18 and LP1 cells, which increased to
4.7% and 0.9% in pervanadate-treated KMS18 and LP1 cells, respectively. Consistently,
Lyn Y194 phosphorylation was not detected in untreated KMS11 and RPMI8226 cells, but
was increased to 4.3% and 2.0% in pervanadate-treated KMS11 and RPMI 8226 cells,
respectively (Figure 4.1 C). The fact that Lyn Y194 phosphorylation was increased in
pervanadate-treated cells suggests that Lyn pY194 is regulated by TK and PTP activities in
vivo.
4.5 Lyn SH2 Domain Phosphorylation Modulates Its Binding to pY
Peptides
To understand the impact of phosphorylation of the Lyn SH2 domain on its binding
to phosphorylated ligands, the binding profiles of the Lyn SH2 domain for pY peptides
was compared to that of phosphorylated Lyn SH2 domain. To this end, I purified
unphosphorylated and phosphorylated Lyn SH2 as shown in Figure 4.2 A. Specifically,
Lyn SH2 was purified from bacteria, phosphorylated in vitro, and subjected to either
IMAC or cation exchange chromatography to resolve phosphorylated and
unphosphorylated proteins. The IMAC eluate was shown to be tyrosyl phosphorylated by
Western blot analysis (Figure 4.2 B). Samples corresponding to the three cation exchange
90
chromatographic peaks were analyzed by SRM and the fractions corresponding to peak 1
was estimated to contain > 95% pY194-Lyn SH2 (Figure 4.2 C). Unphosphorylated Lyn
SH2 was obtained either by omitting the addition of Ephrin A4 (EphA4) kinase during
purification, or by collecting the appropriate chromatographic fractions. Streptavidin (SA)
immobilized phospho- or unphosphorylated Lyn SH2 domain (hereafter referred to as
pSH2 and SH2, respectively) was mixed with a pool of highly phosphorylated peptides
produced from pervanadate-treated Mv4-11 cells, an AML cell line containing the Flt3
ITD mutation, which expresses a constitutively active Flt3 protein that facilitates the
accumulation of pY. Next, bound peptides were eluted into acidic solutions for MS
analysis. To assess the enrichment potential of the SH2 domains, I also generated a
quantitative profile of cellular pY in Mv4-11 cells as background, which serves as a basis
for comparison. Three technical replicates of the pY profiling experiment identified a
total of 504 pY-containing peptides.
91
Figure 4.2. Purification of phosphorylated Lyn SH2 domain
A, a flow chart showing the schematic overview of the steps involved in purifying
phospho-Lyn SH2. B, Western blots analysis of IMAC-based enrichment of phospho-
SH2. FT indicates flow-through. C, the UV 280 nm chromatography showing the
separation of phospho- and unphospho-Lyn SH2 by cation exchange column (top); and
the SRM ion currents associated with Y194- or pY194-containing peptide in 3
chromatographic peaks (bottom). D, Western blot of purified phospho- (pSH2) and
unphospho-Lyn SH2 (SH2) demonstrating the SH2 and pSH2 aliquots contain
comparable amounts of proteins.
A B D
mAu
500
400
300
200
100
0
C
92
A collection of 66 pY peptides, encompassing 69 pY sites, were identified to bind
to either SH2 or pSH2. Among these, 18 singly phosphorylated peptides bound to both
SH2 and pSH2, while the remaining 48 peptides, encompassing 51 pY sites, bound to
SH2 only (Figure 4.3 A; Table 4.1). A total of 27 SH2/pSH2-binding pY sites were not
identified by the anti-pY immunoprecipitation (IP) approach that generated the total
cellular pY profile in Mv4-11 cells. Sequence logos were generated (WebLogo (256),
http://weblogo.berkeley.edu/logo.cgi) for the 69 SH2-binding and 18 pSH2-binding pY
sites, as well as the cellular collection of 531 (i.e. 504+27) pY sites identified in Mv4-11
cells (Figure 4.3 B). The sequence logos for the SH2 or pSH2 bound pY sites
demonstrate an enrichment of the canonical SFK SH2-binding motif with the sequence
pYEE[V/I/L] (47, 49, 51), while the sequence logo for the cellular pY did not show any
distinct motif. To further understand the selectivity of SH2/pSH2 domains, I calculated
the fold-enrichment of specific motifs surrounding the SH2- or pSH2-bound pY sites by
comparing to the total pY background in Mv4-11 cells, using the motif-x online statistical
software (257). This analysis showed that the SH2-bound motifs pYExI and pYExV were
enriched 28-fold and 14-fold respectively, and the pSH2-bound motif pYE was enriched
four-fold (p-values < 0.001). These data demonstrate that, while both SH2 and pSH2
were able to select for defined binding motifs, the preference of pSH2 for pYE, but not
pYExI or pYExV, shows that the specificity of the SH2 domain towards the pY+3
residue is diminished by SH2 domain phosphorylation. On the other hand, these data
suggest that SH2 phosphorylation does not influence the specificity at the pY-binding
pocket for the pY+1 residue.
93
Table 4.1. Phosphopeptides bound to Lyn SH2 or pSH2 identified by AP-MS
94
To examine the impact of Lyn SH2 phosphorylation on binding selectivity, the
relative amounts of the 18 pY peptides bound to both SH2 and pSH2 were extracted by
MaxQuant software (142) based on their MS XIC signal intensities. The average
quantification values across three replicates were used to generate a binding heatmap
(Figure 4.3 C), which showed increased bindings for SH2 compared to pSH2 (3 to 779-
fold increase) for all 18 pY peptides. This data indicates that these pY peptides have
preferences for unphosphorylated Lyn SH2. Moreover, it suggests that phosphorylation
of the SH2 domain reduces its affinity for pY peptides.
95
Figure 4.3. Lyn pSH2 shows reduced affinity for pY peptides compared to SH2
A, a Venn diagram of pY sites affinity-bound to Lyn SH2, pSH2, or enriched by anti-pY
IP from Mv4-11 whole cell lysate derived peptides. B, sequence logos based on the 69
SH2-binding (top) and the 18 pSH2-binding (middle) pY sites, and the collection of 531
pY sites identified in Mv4-11 cells (bottom). C, a heatmap based on the quantitative
profiles of the pY sites bound to SH2 and pSH2, ordered top to bottom by descending
fold-changes, labelled on the right by the gene name, pY site, surrounding sequences, and
fold-change. D, bar graphs showing dissociation constants (Kd) of pY peptides binding to
Lyn SH2 or pSH2. **p < 0.01.
A
B
C
D SH2
pSH2
Cellular pY
96
To further assess the effect of SH2 phosphorylation, the dissociation constants (Kd)
of isolated Lyn SH2 and pSH2 for four pY-containing peptides were determined in a set
of in vitro fluorescence polarization binding assays. Two of the peptides, pYEEI and
pYEEL, contain the canonical pYEE[V/I/L] SH2-binding motif. The other two, although
do not contain the preferred motif, were derived from proteins functionally linked to Lyn
SH2 domain. These include peptides modeled after the Lyn autoinhibitory C-terminal tail
(pYQQQ) (20) and the Fc gamma receptor IIb (FcγRIIb) immunoreceptor tyrosine-based
inhibition motif (pYSLL) (258). The Kd values of isolated Lyn SH2 domain for the
pYEEI and pYEEL peptides were determined to be 0.75 (±0.2) µM and 0.94 (±0.2) µM,
respectively. This value is consistent with the 0.58 µM Kd value reported for binding a
pYEEI motif-containing peptide to Lyn SH2 (259), and the 0.3-0.6 µM Kd values
reported for binding high-affinity peptides to other SFK SH2 domains (260). As expected,
the affinity of Lyn SH2 for pYQQQ and pYSLL appeared significantly lower, with Kd
measuring 4.9 (±0.6) µM and 3 (±0.9) µM, respectively (Figure 4.4). This is consistent
with the 4 µM Kd value reported for Lck SH2 binding to a low affinity peptide
containing the pYQPG motif (260). Interestingly, the Kd values measured for all four
peptides generally decreased for pSH2 compared to SH2, with the affinity of pYEEI and
pYEEL decreasing to 1.9 ±0.2 and 2.8 ±0.5 fold less, respectively, and the affinity of
pYQQQ and pYSLL decreasing to 1.5 ±0.1 and 2.1 ±0.3 fold less, respectively (Figure
4.3 D). Significantly, all four peptides were found to have lower affinities for pSH2 than
SH2, regardless of whether they had relatively high or low affinities in binding to SH2.
These results also demonstrate that phosphorylation of the Lyn SH2 domain reduces its
affinity for binding pY-containing peptides.
97
Figure 4.4. Binding curves of Lyn SH2/pSH2 with phospho-peptide probes
Representative curve from five replicates is shown with Kd for SH2 and pSH2 indicated.
Trendline was plotted automatically in the software Prism 4 (GraphPad Software:
http://www.graphpad.com/scientific-software/prism/). The average and standard
deviation of Kd across five replicates are given in Section 4.5 and shown in Figure 4.3 D.
98
4.6 Lyn SH2 Domain Phosphorylation Modulates Its Binding to pY-
Containing Proteins
To see if phosphorylation impacts Lyn SH2 binding to phosphoproteins, the binding
profiles of the Lyn SH2 domain and the phosphorylated Lyn SH2 domain for pY-
containing proteins was compared. Pervanadate-treated Mv4-11 whole cell lysates were
incubated with immobilized Lyn SH2 or pSH2 to affinity-purify pY-containing proteins.
The bound proteins were either analyzed by anti-pY Western or protease-digested for MS
analysis. Remarkably, Western blot analysis showed dramatic reduction in
phosphoprotein binding for pSH2 compared to SH2 (Figure 4.5 A; compare lanes 4 and
5). Moreover, the quantitative profiles generated by MS analysis identified 539 proteins
that were significantly enriched through affinity-binding to Lyn SH2 or pSH2, compared
to the streptavidin (SA)-only negative control; among which, 36 known Lyn interactors
(GeneCards (261), www.genecards.org) were included. The MS analysis also detected
166 pY sites on these proteins. The fact that a lower number of pY sites were identified
compared to the number of bound proteins implies the affinity-enrichment of protein
complexes, such that indirect SH2-binders were also recovered by this method.
Consistent with the peptide binding experiments described in Section 4.5, fewer proteins
bound pSH2 compared to SH2 (Figure 4.5 B), suggesting that the ability of Lyn SH2 to
bind native proteins was decreased by phosphorylation at Y194. This is consistent with
previous findings that phosphorylation of the equivalent tyrosine in the SH2 domain of
Lck reduced its ability to bind proteins derived from pervanadate-treated Jurkat T cells
(54).
99
Out of the 539 bound proteins, 394 bound selectively better (≥ two-fold) to either
SH2 or pSH2, exhibiting binding preferences (Appendix Table 5). Among which, 352
bound two-fold or better to SH2 and the other 42 bound two-fold or better to pSH2. I
used a hidden Markov model method (262) to predict direct binders of Lyn SH2 based on
their theoretical pY motifs. The database for theoretical pY motifs include the entire
collection of pY sites on the 394 differential proteins identified in this experiment and
catalogued in the PhosphoSitePlus online database (263) (www.phosphosite.org/),
amounting to a total of 1988 pY sites. As a result of this analysis, 66 of the 394 proteins
were predicted to bind directly to Lyn SH2, with 62 bind preferentially to SH2 and four
bind preferentially to pSH2. These 66 proteins were significantly enriched for proteins in
immune system process (g:Profiler (264) p value<0.001), including pathways in cellular
response to stimulus (42 proteins, p<0.01), signaling (41 proteins, p<0.001), and
regulation of immune system process (17 proteins, p<0.01). These data are consistent
with the prominent role of Lyn in B cell signaling (25). Among the predicted direct
binders, 16 were known Lyn interactors (Figure 4.5 C). This is a 15.8-fold enrichment (p
value<0.001) of Lyn interactors compared to a similar search against the complement of
catalogued human protein pY (PhosphoSitePlus, March 2015 download; 10240 pY
proteins) background. Comparison of the proteins and phosphopeptides captured by the
affinity purification method revealed 27 instances of overlap (20 predicted direct binders
including five know Lyn interactors), wherein a phosphopeptide and its parent native
protein were both enriched by binding SH2 or pSH2 (Table 4.2). These data suggest that
the 66 predicted interactors possibly interact with Lyn in vivo, likely through pY-
100
mediated binding to Lyn SH2 domain. However, these predictions still require
experimental validation.
Conceptually, proteins containing the pY moiety-binding SH2 or PTB domains
might bind pY194 of the pSH2 domain, thus demonstrating a preferred selectivity toward
pSH2 versus SH2. However, results of this experiment indicate that all SH2/PTB
domain-containing phosphoproteins bound Lyn SH2 at least 1.7-fold better than pSH2
(Table 4.3), implying that the major mode of interaction is mediated through the pY
motifs on the phosphoproteins and the Lyn SH2 domain, and that this interaction can be
disrupted by phosphorylation of Lyn SH2.
101
A B
C
Fold change Prefer SH2 Prefer pSH2
102
Figure 4.5. Lyn SH2 phosphorylation modulates its binding affinity for
phosphoproteins
A, an anti-pY Western blot showing differential binding of phosphoproteins to Lyn SH2
or pSH2. Untreated (Lane 1) or pervanadate-treated (Lane 2) Mv4-11 cells were lysed
and the whole cell lysate proteins were mixed with empty streptavidin (SA) beads (Lanes
3, 6), Lyn SH2 (Lanes 4, 7), or pSH2 (Lanes 5, 8) for affinity purification. The flow-
through (Lanes 6-8) and the bound proteins (Lanes 3-5) were indicated. Streptavidin blot
for biotin-labelled SH2 and pSH2 confirms equal loading of the proteins. B, a volcano
plot of log 10 Student’s t-test p-value (based on 6 replicates) against log 2 fold change of
abundances of protein bound to SH2 compared to pSH2. Horizontal line indicates
statistical significance cut-off. C, a schematic overview of proteins bound differentially
(2-fold or higher) to Lyn SH2 (blue) or pSH2 (green), with the darkness of color
indicating fold changes. Predicted direct Lyn-binders are in diamond-shaped nodes and
connected to Lyn SH2 by a line. Reported Lyn interactors are labelled in red. Asterisks
indicate the Student’s t-test significance levels: *p<0.05; **p<0.01; ***p<0.001.
103
Table 4.2. SH2/pSH2-binding phosphoproteins identified by peptide AP
The expression “(ph)” denotes phosphorylation of the preceding residue.
Table 4.3. Fold change of SH2 or PTB domain-containing proteins bound to Lyn
SH2 and pSH2
104
4.7 Discussion and Conclusion
Through proteomics approaches, I discovered that a conserved tyrosine in the SH2
domain of Lyn and other SFKs was prevalently phosphorylated in AML/CLL primary
samples and MM cell lines. Phosphorylation of the equivalent tyrosine on SFKs was
observed in other cancer systems such as non-small cell lung cancer cell lines and tumors
(175) and human epidermal growth factor receptor 2 (HER2)-overexpressing breast
tumor specimens (254), suggesting potential important roles for this phosphorylation. For
this reason, I investigated the impact of pY on the binding behaviour of SH2 domain
using Lyn SH2 domain as a model. As a result, I found that Y194 phosphorylation within
the Lyn SH2 domain reduced its binding affinity and modulated its selectivity for
phosphopeptides and phosphoproteins.
I observed increased Lyn SH2 (Y194) phosphorylation in MM cells treated with
pervanadate, a phosphatase inhibitor, indicating that Lyn pY194 was regulated.
Consistently, other groups also observed the regulation of SH2 phosphorylation in SFKs;
and this was usually associated with functional consequences. In particular, induction of
Src phospho-Y213 was observed in vivo following platelet derived growth factor (PDGF)
or HER2/heregulin (HRG) stimulation (21, 254). While phosphorylation of Src Y213 by
PDGFR in vitro potentially abolished the binding of Src C-terminal autoinhibitory motif
and led to Src activation (21), HER2/HRG stimulated Src Y213 phosphorylation promoted
Src kinase activity as well as Fak Y861 phosphorylation (254). Moreover, pY213 in Src was
found to affect its localization, building a link between Y213 phosphorylation and breast
cancer metastasis (254). Additionally, stimulation of Jurkat T cells with anti-CD3
antibodies induced phosphorylation of Lck Y192 (265). Together, these observations with
105
my results propose a model where SFK SH2 phosphorylation influences substrate
selection, protein localization, and allosteric interactions with the kinase domain or the C-
terminal tail. In agreement with this, my results show altered affinity and selectivity of
Lyn SH2 for pY-containing molecules due to SH2 phosphorylation, suggesting a role of
SH2 phosphorylation in regulating the protein-protein interaction and downstream
signalling of SFKs.
Lyn Y194 is located on the EF loop, within the consensus sequence
G[G/W][Y/F/L]YI[S/T][P/T/S]R, which is conserved in SFK SH2 domains, but not in all
SH2 domains (40). The EF loop controls the accessibility and shape of the pY+3 pocket
(51), thus, to a modest degree, regulates substrate specificity (42). Therefore, it is
reasonable to speculate that Y194 phosphorylation modulates substrate recognition for the
pY+3 residue. Indeed, my results showed that phosphorylated Lyn SH2 had a preference
for pYE but not pYExI or pYExV motif, suggesting that phospho-Y194 abolished the
selectivity for pY+3 residue binding. The fact that phosphorylation only reduced the
affinity of Lyn SH2 binding to synthetic pY peptide by 1.5-2.8 fold may reflect that pY
contributes most energy to binding, and the C-terminal flanking residues of pY (the pY+
sites) contribute less. Combined, these data suggest that Lyn Y194 phosphorylation within
the EF loop modulates substrate selectivity for the pY+3 residue while modestly reducing
the binding affinity of pY-containing ligands.
Although my data showed a generally reduced binding of pSH2 to pY-containing
molecules, for a small subset of protein ligands, the binding was increased. This subset of
42 proteins includes significantly enriched biomolecules that are involved in degenerative
1 Machida, K., Mayer, B. J., and Nollau, P. (2003) Profiling the global tyrosine phosphorylation state. Mol Cell Proteomics. 2, 215-233 2 Blume-Jensen, P., Hunter, T. (2001) Oncogenic kinase signalling. Nature. 411, 355-365 3 Hunter, T. (1989) Protein modification: phosphorylation on tyrosine residues. Current Opinion in Cell Biology. 1, 1168-1181 4 Pawson, T., and Nash, P. (2003) Assembly of cell regulatory systems through protein interaction domains. Science. 300, 445-452 5 Pawson, T., and Kofler, M. (2009) Kinome signaling through regulated protein-protein interactions in normal and cancer cells. Curr Opin Cell Biol. 21, 147-153 6 Hunter, T. (2009) Tyrosine phosphorylation: thirty years and counting. Curr Opin Cell Biol. 21, 140-146 7 Arora, A., and Scholar, E. M. (2005) Role of tyrosine kinase inhibitors in cancer therapy. J Pharmacol Exp Ther. 315, 971-979 8 Zhang, J., Yang, P. L., and Gray, N. S. (2009) Targeting cancer with small molecule kinase inhibitors. Nat Rev Cancer. 9, 28-39 9 Robinson, D. R., Wu, Y. M., and Lin, S. F. (2000) The protein tyrosine kinase family of the human genome. Oncogene. 19, 5548-5557 10 Parsons, J. T., and Weber, M. J. (1989) Genetics of src: structure and functional organization of a protein tyrosine kinase. Curr Top Microbiol Immunol. 147, 79-127 11 Hubbard, S. R., and Till, J. H. (2000) Protein tyrosine kinase structure and function. Annu Rev Biochem. 69, 373-398 12 Rawlings, J. S., Rosler, K. M., and Harrison, D. A. (2004) The JAK/STAT signaling pathway. J Cell Sci. 117, 1281-1283 13 Nishizumi, H., Horikawa, K., Mlinaric-Rascan, I., and Yamamoto, T. (1998) A double-edged kinase Lyn: a positive and negative regulator for antigen receptor-mediated signals. J Exp Med. 187, 1343-1348 14 Sada, K., Minami, Y., and Yamamura, H. (1997) Relocation of Syk protein-tyrosine kinase to the actin filament network and subsequent association with Fak. Eur J Biochem. 248, 827-833 15 Brickell, P. M. (1992) The p60c-src family of protein-tyrosine kinases: structure, regulation, and function. Crit Rev Oncog. 3, 401-446 16 Boggon, T. J., and Eck, M. J. (2004) Structure and regulation of Src family kinases. Oncogene. 23, 7918-7927 17 Yamaguchi, H., and Hendrickson, W. A. (1996) Structural basis for activation of human lymphocyte kinase Lck upon tyrosine phosphorylation. Nature. 384, 484-489 18 Xu, W., Doshi, A., Lei, M., Eck, M. J., and Harrison, S. C. (1999) Crystal structures of c-Src reveal features of its autoinhibitory mechanism. Mol Cell. 3, 629-638 19 Xu, W., Harrison, S.C. and Eck , M.J. . (1997) Three-dimensional structure of the tyrosine kinase c-Src. Nature. 385, 595-602 20 Sicheri, F., and Kuriyan, J. (1997) Structures of Src-family tyrosine kinases. Curr Opin Struct Biol. 7, 777-785
143
21 Stover, D. R., Furet, P., and Lydon, N. B. (1996) Modulation of the SH2 binding specificity and kinase activity of Src by tyrosine phosphorylation within its SH2 domain. J Biol Chem. 271, 12481-12487 22 Ingley, E. (2008) Src family kinases: regulation of their activities, levels and identification of new pathways. Biochim Biophys Acta. 1784, 56-65 23 Parsons, S. J., and Parsons, J. T. (2004) Src family kinases, key regulators of signal transduction. Oncogene. 23, 7906-7909 24 Sen, B., and Johnson, F. M. (2011) Regulation of SRC family kinases in human cancers. J Signal Transduct. 2011, 865819 25 Xu, Y., Harder, K. W., Huntington, N. D., Hibbs, M. L., and Tarlinton, D. M. (2005) Lyn tyrosine kinase: accentuating the positive and the negative. Immunity. 22, 9-18 26 Hallek, M., Neumann, C., Schaffer, M., Danhauser-Riedl, S., von Bubnoff, N., de Vos, G., Druker, B. J., Yasukawa, K., Griffin, J. D., and Emmerich, B. (1997) Signal transduction of interleukin-6 involves tyrosine phosphorylation of multiple cytosolic proteins and activation of Src-family kinases Fyn, Hck, and Lyn in multiple myeloma cell lines. Exp Hematol. 25, 1367-1377 27 Ishikawa, H., Tsuyama, N., Abroun, S., Liu, S., Li, F. J., Taniguchi, O., and Kawano, M. M. (2002) Requirements of src family kinase activity associated with CD45 for myeloma cell proliferation by interleukin-6. Blood. 99, 2172-2178 28 Iqbal, M. S., Tsuyama, N., Obata, M., and Ishikawa, H. (2010) A novel signaling pathway associated with Lyn, PI 3-kinase and Akt supports the proliferation of myeloma cells. Biochem Biophys Res Commun. 392, 415-420 29 Dos Santos, C., Demur, C., Bardet, V., Prade-Houdellier, N., Payrastre, B., and Recher, C. (2008) A critical role for Lyn in acute myeloid leukemia. Blood. 111, 2269-2279 30 Tonks, N. K. (2006) Protein tyrosine phosphatases: from genes, to function, to disease. Nat Rev Mol Cell Biol. 7, 833-846 31 Julien, S. G., Dube, N., Hardy, S., and Tremblay, M. L. (2011) Inside the human cancer tyrosine phosphatome. Nat Rev Cancer. 11, 35-49 32 Alonso, A., Sasin, J., Bottini, N., Friedberg, I., Osterman, A., Godzik, A., Hunter, T., Dixon, J., and Mustelin, T. (2004) Protein tyrosine phosphatases in the human genome. Cell. 117, 699-711 33 Zhang, Z. Y. (2003) Mechanistic studies on protein tyrosine phosphatases. Prog Nucleic Acid Res Mol Biol. 73, 171-220 34 Meng, T. C., Fukada, T., and Tonks, N. K. (2002) Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo. Mol Cell. 9, 387-399 35 Sundaresan, M., Yu, Z. X., Ferrans, V. J., Irani, K., and Finkel, T. (1995) Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science. 270, 296-299 36 Meng, T. C., Buckley, D. A., Galic, S., Tiganis, T., and Tonks, N. K. (2004) Regulation of insulin signaling through reversible oxidation of the protein-tyrosine phosphatases TC45 and PTP1B. J Biol Chem. 279, 37716-37725 37 Singh, D. K., Kumar, D., Siddiqui, Z., Basu, S. K., Kumar, V., and Rao, K. V. (2005) The strength of receptor signaling is centrally controlled through a cooperative loop between Ca2+ and an oxidant signal. Cell. 121, 281-293
144
38 Songyang, Z., Shoelson, S. E., McGlade, J., Olivier, P., Pawson, T., Bustelo, X. R., Barbacid, M., Sabe, H., Hanafusa, H., Yi, T., and et al. (1994) Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav. Mol Cell Biol. 14, 2777-2785 39 Li, W., Young, S. L., King, N., and Miller, W. T. (2008) Signaling properties of a non-metazoan Src kinase and the evolutionary history of Src negative regulation. J Biol Chem. 283, 15491-15501 40 Liu, B. A., Jablonowski, K., Raina, M., Arce, M., Pawson, T., and Nash, P. D. (2006) The human and mouse complement of SH2 domain proteins-establishing the boundaries of phosphotyrosine signaling. Mol Cell. 22, 851-868 41 Waksman, G., Kominos, D., Robertson, S. C., Pant, N., Baltimore, D., Birge, R. B., Cowburn, D., Hanafusa, H., Mayer, B. J., Overduin, M., Resh, M. D., Rios, C. B., Silverman, L., and Kuriyan, J. (1992) Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides. Nature. 358, 646-653 42 Waksman, G., Kumaran, S., and Lubman, O. (2004) SH2 domains: role, structure and implications for molecular medicine. Expert Rev Mol Med. 6, 1-18 43 Waksman, G., Shoelson, S. E., Pant, N., Cowburn, D., and Kuriyan, J. (1993) Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms. Cell. 72, 779-790 44 Bibbins, K. B., Boeuf, H., and Varmus, H. E. (1993) Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides. Mol Cell Biol. 13, 7278-7287 45 Mayer, B. J., Jackson, P. K., Van Etten, R. A., and Baltimore, D. (1992) Point mutations in the abl SH2 domain coordinately impair phosphotyrosine binding in vitro and transforming activity in vivo. Mol Cell Biol. 12, 609-618 46 Tinti, M., Kiemer, L., Costa, S., Miller, M. L., Sacco, F., Olsen, J. V., Carducci, M., Paoluzi, S., Langone, F., Workman, C. T., Blom, N., Machida, K., Thompson, C. M., Schutkowski, M., Brunak, S., Mann, M., Mayer, B. J., Castagnoli, L., and Cesareni, G. (2013) The SH2 domain interaction landscape. Cell Rep. 3, 1293-1305 47 Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., and et al. (1993) SH2 domains recognize specific phosphopeptide sequences. Cell. 72, 767-778 48 Fantl, W. J., Escobedo, J. A., Martin, G. A., Turck, C. W., del Rosario, M., McCormick, F., and Williams, L. T. (1992) Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways. Cell. 69, 413-423 49 Huang, H., Li, L., Wu, C., Schibli, D., Colwill, K., Ma, S., Li, C., Roy, P., Ho, K., Songyang, Z., Pawson, T., Gao, Y., and Li, S. S. (2008) Defining the specificity space of the human SRC homology 2 domain. Mol Cell Proteomics. 7, 768-784 50 Eck, M. J., Shoelson, S. E., and Harrison, S. C. (1993) Recognition of a high-affinity phosphotyrosyl peptide by the Src homology-2 domain of p56lck. Nature. 362, 87-91 51 Kaneko, T., Huang, H., Zhao, B., Li, L., Liu, H., Voss, C. K., Wu, C., Schiller, M. R., and Li, S. S. (2010) Loops govern SH2 domain specificity by controlling access to binding pockets. Sci Signal. 3, ra34
145
52 Kimber, M. S., Nachman, J., Cunningham, A. M., Gish, G. D., Pawson, T., and Pai, E. F. (2000) Structural basis for specificity switching of the Src SH2 domain. Mol Cell. 5, 1043-1049 53 Marengere, L. E., Songyang, Z., Gish, G. D., Schaller, M. D., Parsons, J. T., Stern, M. J., Cantley, L. C., and Pawson, T. (1994) SH2 domain specificity and activity modified by a single residue. Nature. 369, 502-505 54 Couture, C., Songyang, Z., Jascur, T., Williams, S., Tailor, P., Cantley, L. C., and Mustelin, T. (1996) Regulation of the Lck SH2 domain by tyrosine phosphorylation. J Biol Chem. 271, 24880-24884 55 Comb, W. C., Hutti, J. E., Cogswell, P., Cantley, L. C., and Baldwin, A. S. (2012) p85alpha SH2 domain phosphorylation by IKK promotes feedback inhibition of PI3K and Akt in response to cellular starvation. Mol Cell. 45, 719-730 56 Qian, X., Li, G., Vass, W. C., Papageorge, A., Walker, R. C., Asnaghi, L., Steinbach, P. J., Tosato, G., Hunter, K., and Lowy, D. R. (2009) The Tensin-3 protein, including its SH2 domain, is phosphorylated by Src and contributes to tumorigenesis and metastasis. Cancer Cell. 16, 246-258 57 Lappalainen, I., Thusberg, J., Shen, B., and Vihinen, M. (2008) Genome wide analysis of pathogenic SH2 domain mutations. Proteins. 72, 779-792 58 Tzeng, S. R., Pai, M. T., Lung, F. D., Wu, C. W., Roller, P. P., Lei, B., Wei, C. J., Tu, S. C., Chen, S. H., Soong, W. J., and Cheng, J. W. (2000) Stability and peptide binding specificity of Btk SH2 domain: molecular basis for X-linked agammaglobulinemia. Protein Sci. 9, 2377-2385 59 Mattsson, P. T., Lappalainen, I., Backesjo, C. M., Brockmann, E., Lauren, S., Vihinen, M., and Smith, C. I. (2000) Six X-linked agammaglobulinemia-causing missense mutations in the Src homology 2 domain of Bruton's tyrosine kinase: phosphotyrosine-binding and circular dichroism analysis. J Immunol. 164, 4170-4177 60 Hashimoto, S., Tsukada, S., Matsushita, M., Miyawaki, T., Niida, Y., Yachie, A., Kobayashi, S., Iwata, T., Hayakawa, H., Matsuoka, H., Tsuge, I., Yamadori, T., Kunikata, T., Arai, S., Yoshizaki, K., Taniguchi, N., and Kishimoto, T. (1996) Identification of Bruton's tyrosine kinase (Btk) gene mutations and characterization of the derived proteins in 35 X-linked agammaglobulinemia families: a nationwide study of Btk deficiency in Japan. Blood. 88, 561-573 61 Saffran, D. C., Parolini, O., Fitch-Hilgenberg, M. E., Rawlings, D. J., Afar, D. E., Witte, O. N., and Conley, M. E. (1994) Brief report: a point mutation in the SH2 domain of Bruton's tyrosine kinase in atypical X-linked agammaglobulinemia. N Engl J Med. 330, 1488-1491 62 Bentires-Alj, M., Paez, J. G., David, F. S., Keilhack, H., Halmos, B., Naoki, K., Maris, J. M., Richardson, A., Bardelli, A., Sugarbaker, D. J., Richards, W. G., Du, J., Girard, L., Minna, J. D., Loh, M. L., Fisher, D. E., Velculescu, V. E., Vogelstein, B., Meyerson, M., Sellers, W. R., and Neel, B. G. (2004) Activating mutations of the noonan syndrome-associated SHP2/PTPN11 gene in human solid tumors and adult acute myelogenous leukemia. Cancer Res. 64, 8816-8820 63 Muller, P. J., Rigbolt, K. T., Paterok, D., Piehler, J., Vanselow, J., Lasonder, E., Andersen, J. S., Schaper, F., and Sobota, R. M. (2013) Protein tyrosine phosphatase SHP2/PTPN11 mistargeting as a consequence of SH2-domain point mutations associated with Noonan Syndrome and leukemia. J Proteomics. 84, 132-147
146
64 Chan, G., Kalaitzidis, D., and Neel, B. G. (2008) The tyrosine phosphatase Shp2 (PTPN11) in cancer. Cancer Metastasis Rev. 27, 179-192 65 Liu, X., and Qu, C. K. (2011) Protein Tyrosine Phosphatase SHP-2 (PTPN11) in Hematopoiesis and Leukemogenesis. J Signal Transduct. 2011, 195239 66 Xu, R. (2007) Shp2, a novel oncogenic tyrosine phosphatase and potential therapeutic target for human leukemia. Cell Res. 17, 295-297 67 Mohi, M. G., and Neel, B. G. (2007) The role of Shp2 (PTPN11) in cancer. Curr Opin Genet Dev. 17, 23-30 68 Sun, M., Hillmann, P., Hofmann, B. T., Hart, J. R., and Vogt, P. K. (2010) Cancer-derived mutations in the regulatory subunit p85alpha of phosphoinositide 3-kinase function through the catalytic subunit p110alpha. Proc Natl Acad Sci U S A. 107, 15547-15552 69 Jakovljevic, G., Kardum-Skelin, I., Rogosic, S., and Nakic, M. (2010) Juvenile myelomonocytic leukemia with PTPN11 mutation in a 23-month-old girl. Coll Antropol. 34, 251-254 70 Kratz, C. P., Niemeyer, C. M., Castleberry, R. P., Cetin, M., Bergstrasser, E., Emanuel, P. D., Hasle, H., Kardos, G., Klein, C., Kojima, S., Stary, J., Trebo, M., Zecca, M., Gelb, B. D., Tartaglia, M., and Loh, M. L. (2005) The mutational spectrum of PTPN11 in juvenile myelomonocytic leukemia and Noonan syndrome/myeloproliferative disease. Blood. 106, 2183-2185 71 Parsons, D. W., Jones, S., Zhang, X., Lin, J. C., Leary, R. J., Angenendt, P., Mankoo, P., Carter, H., Siu, I. M., Gallia, G. L., Olivi, A., McLendon, R., Rasheed, B. A., Keir, S., Nikolskaya, T., Nikolsky, Y., Busam, D. A., Tekleab, H., Diaz, L. A., Jr., Hartigan, J., Smith, D. R., Strausberg, R. L., Marie, S. K., Shinjo, S. M., Yan, H., Riggins, G. J., Bigner, D. D., Karchin, R., Papadopoulos, N., Parmigiani, G., Vogelstein, B., Velculescu, V. E., and Kinzler, K. W. (2008) An integrated genomic analysis of human glioblastoma multiforme. Science. 321, 1807-1812 72 Friedman, E., Gejman, P. V., Martin, G. A., and McCormick, F. (1993) Nonsense mutations in the C-terminal SH2 region of the GTPase activating protein (GAP) gene in human tumours. Nat Genet. 5, 242-247 73 Sumegi, J., Huang, D., Lanyi, A., Davis, J. D., Seemayer, T. A., Maeda, A., Klein, G., Seri, M., Wakiguchi, H., Purtilo, D. T., and Gross, T. G. (2000) Correlation of mutations of the SH2D1A gene and epstein-barr virus infection with clinical phenotype and outcome in X-linked lymphoproliferative disease. Blood. 96, 3118-3125 74 Pawson, T. (2004) Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell. 116, 191-203 75 Filippakopoulos, P., Muller, S., and Knapp, S. (2009) SH2 domains: modulators of nonreceptor tyrosine kinase activity. Curr Opin Struct Biol. 19, 643-649 76 Schlessinger, J., and Lemmon, M. A. (2003) SH2 and PTB domains in tyrosine kinase signaling. Sci STKE. 2003, RE12 77 Fischer, E. H. (1999) Cell signaling by protein tyrosine phosphorylation. Adv Enzyme Regul. 39, 359-369 78 Lowenstein, E. J., Daly, R. J., Batzer, A. G., Li, W., Margolis, B., Lammers, R., Ullrich, A., Skolnik, E. Y., Bar-Sagi, D., and Schlessinger, J. (1992) The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell. 70, 431-442
147
79 Okutani, T., Okabayashi, Y., Kido, Y., Sugimoto, Y., Sakaguchi, K., Matuoka, K., Takenawa, T., and Kasuga, M. (1994) Grb2/Ash binds directly to tyrosines 1068 and 1086 and indirectly to tyrosine 1148 of activated human epidermal growth factor receptors in intact cells. J Biol Chem. 269, 31310-31314 80 Parker, L. L., and Piwnica-Worms, H. (1992) Inactivation of the p34cdc2-cyclin B complex by the human WEE1 tyrosine kinase. Science. 257, 1955-1957 81 Rhind, N., and Russell, P. (2012) Signaling pathways that regulate cell division. Cold Spring Harb Perspect Biol. 4 82 Gertz, M. A., and Dingli, D. (2014) How we manage autologous stem cell transplantation for patients with multiple myeloma. Blood. 124, 882-890 83 Kyle, R. A., and Rajkumar, S. V. (2008) Multiple myeloma. Blood. 111, 2962-2972 84 Chesi, M., Nardini, E., Brents, L. A., Schrock, E., Ried, T., Kuehl, W. M., and Bergsagel, P. L. (1997) Frequent translocation t(4;14)(p16.3;q32.3) in multiple myeloma is associated with increased expression and activating mutations of fibroblast growth factor receptor 3. Nat Genet. 16, 260-264 85 Trudel, S., Ely, S., Farooqi, Y., Affer, M., Robbiani, D. F., Chesi, M., and Bergsagel, P. L. (2004) Inhibition of fibroblast growth factor receptor 3 induces differentiation and apoptosis in t(4;14) myeloma. Blood. 103, 3521-3528 86 Ronchetti, D., Greco, A., Compasso, S., Colombo, G., Dell'Era, P., Otsuki, T., Lombardi, L., and Neri, A. (2001) Deregulated FGFR3 mutants in multiple myeloma cell lines with t(4;14): comparative analysis of Y373C, K650E and the novel G384D mutations. Oncogene. 20, 3553-3562 87 Bharti, A. C., Shishodia, S., Reuben, J. M., Weber, D., Alexanian, R., Raj-Vadhan, S., Estrov, Z., Talpaz, M., and Aggarwal, B. B. (2004) Nuclear factor-kappaB and STAT3 are constitutively active in CD138+ cells derived from multiple myeloma patients, and suppression of these transcription factors leads to apoptosis. Blood. 103, 3175-3184 88 Hilbert, D. M., Kopf, M., Mock, B. A., Kohler, G., and Rudikoff, S. (1995) Interleukin 6 is essential for in vivo development of B lineage neoplasms. J Exp Med. 182, 243-248 89 Chng, W. J., Gualberto, A., and Fonseca, R. (2006) IGF-1R is overexpressed in poor-prognostic subtypes of multiple myeloma. Leukemia. 20, 174-176 90 Bataille, R., Robillard, N., Avet-Loiseau, H., Harousseau, J. L., and Moreau, P. (2005) CD221 (IGF-1R) is aberrantly expressed in multiple myeloma, in relation to disease severity. Haematologica. 90, 706-707 91 Standal, T., Borset, M., Lenhoff, S., Wisloff, F., Stordal, B., Sundan, A., Waage, A., and Seidel, C. (2002) Serum insulinlike growth factor is not elevated in patients with multiple myeloma but is still a prognostic factor. Blood. 100, 3925-3929 92 Liang, S. B., Yang, X. Z., Trieu, Y., Li, Z., Zive, J., Leung-Hagesteijn, C., Wei, E., Zozulya, S., Coss, C. C., Dalton, J. T., Fantus, I. G., and Trudel, S. (2011) Molecular target characterization and antimyeloma activity of the novel, insulin-like growth factor 1 receptor inhibitor, GTx-134. Clin Cancer Res. 17, 4693-4704 93 Chim, C. S., Fung, T. K., Cheung, W. C., Liang, R., and Kwong, Y. L. (2004) SOCS1 and SHP1 hypermethylation in multiple myeloma: implications for epigenetic activation of the Jak/STAT pathway. Blood. 103, 4630-4635
148
94 Somani, A. K., Bignon, J. S., Mills, G. B., Siminovitch, K. A., and Branch, D. R. (1997) Src kinase activity is regulated by the SHP-1 protein-tyrosine phosphatase. J Biol Chem. 272, 21113-21119 95 Krautwald, S., Buscher, D., Kummer, V., Buder, S., and Baccarini, M. (1996) Involvement of the protein tyrosine phosphatase SHP-1 in Ras-mediated activation of the mitogen-activated protein kinase pathway. Mol Cell Biol. 16, 5955-5963 96 Johnson, K. J., Peck, A. R., Liu, C., Tran, T. H., Utama, F. E., Sjolund, A. B., Schaber, J. D., Witkiewicz, A. K., and Rui, H. (2010) PTP1B suppresses prolactin activation of Stat5 in breast cancer cells. Am J Pathol. 177, 2971-2983 97 Han, Y., Amin, H. M., Franko, B., Frantz, C., Shi, X., and Lai, R. (2006) Loss of SHP1 enhances JAK3/STAT3 signaling and decreases proteosome degradation of JAK3 and NPM-ALK in ALK+ anaplastic large-cell lymphoma. Blood. 108, 2796-2803 98 Wu, C., Sun, M., Liu, L., and Zhou, G. W. (2003) The function of the protein tyrosine phosphatase SHP-1 in cancer. Gene. 306, 1-12 99 Wu, C., Guan, Q., Wang, Y., Zhao, Z. J., and Zhou, G. W. (2003) SHP-1 suppresses cancer cell growth by promoting degradation of JAK kinases. J Cell Biochem. 90, 1026-1037 100 Hayslip, J., and Montero, A. (2006) Tumor suppressor gene methylation in follicular lymphoma: a comprehensive review. Mol Cancer. 5, 44 101 Saha, S., Bardelli, A., Buckhaults, P., Velculescu, V. E., Rago, C., St Croix, B., Romans, K. E., Choti, M. A., Lengauer, C., Kinzler, K. W., and Vogelstein, B. (2001) A phosphatase associated with metastasis of colorectal cancer. Science. 294, 1343-1346 102 Xing, X., Lian, S., Hu, Y., Li, Z., Zhang, L., Wen, X., Du, H., Jia, Y., Zheng, Z., Meng, L., Shou, C., and Ji, J. (2013) Phosphatase of regenerating liver-3 (PRL-3) is associated with metastasis and poor prognosis in gastric carcinoma. J Transl Med. 11, 309 103 Al-Aidaroos, A. Q., and Zeng, Q. (2010) PRL-3 phosphatase and cancer metastasis. J Cell Biochem. 111, 1087-1098 104 Deschler, B., and Lubbert, M. (2006) Acute myeloid leukemia: epidemiology and etiology. Cancer. 107, 2099-2107 105 Hasserjian, R. P. (2013) Acute myeloid leukemia: advances in diagnosis and classification. Int J Lab Hematol. 35, 358-366 106 Schiffer, C. A. (2003) Hematopoietic growth factors and the future of therapeutic research on acute myeloid leukemia. N Engl J Med. 349, 727-729 107 Fathi, A. T., and Chabner, B. A. (2011) FLT3 inhibition as therapy in acute myeloid leukemia: a record of trials and tribulations. Oncologist. 16, 1162-1174 108 Daver, N., and Cortes, J. (2012) Molecular targeted therapy in acute myeloid leukemia. Hematology. 17 Suppl 1, S59-62 109 Stirewalt, D. L., and Meshinchi, S. (2010) Receptor tyrosine kinase alterations in AML - biology and therapy. Cancer Treat Res. 145, 85-108 110 Lee, H., Kim, M., Lim, J., Kim, Y., Han, K., Cho, B. S., and Kim, H. J. (2013) Acute myeloid leukemia associated with FGFR1 abnormalities. Int J Hematol. 97, 808-812 111 Kentsis, A., Reed, C., Rice, K. L., Sanda, T., Rodig, S. J., Tholouli, E., Christie, A., Valk, P. J., Delwel, R., Ngo, V., Kutok, J. L., Dahlberg, S. E., Moreau, L. A., Byers, R. J., Christensen, J. G., Vande Woude, G., Licht, J. D., Kung, A. L., Staudt, L. M., and
149
Look, A. T. (2012) Autocrine activation of the MET receptor tyrosine kinase in acute myeloid leukemia. Nat Med. 18, 1118-1122 112 Lee-Sherick, A. B., Eisenman, K. M., Sather, S., McGranahan, A., Armistead, P. M., McGary, C. S., Hunsucker, S. A., Schlegel, J., Martinson, H., Cannon, C., Keating, A. K., Earp, H. S., Liang, X., Deryckere, D., and Graham, D. K. (2013) Aberrant Mer receptor tyrosine kinase expression contributes to leukemogenesis in acute myeloid leukemia. Oncogene 113 Davies, H., Bignell, G. R., Cox, C., Stephens, P., Edkins, S., Clegg, S., Teague, J., Woffendin, H., Garnett, M. J., Bottomley, W., Davis, N., Dicks, E., Ewing, R., Floyd, Y., Gray, K., Hall, S., Hawes, R., Hughes, J., Kosmidou, V., Menzies, A., Mould, C., Parker, A., Stevens, C., Watt, S., Hooper, S., Wilson, R., Jayatilake, H., Gusterson, B. A., Cooper, C., Shipley, J., Hargrave, D., Pritchard-Jones, K., Maitland, N., Chenevix-Trench, G., Riggins, G. J., Bigner, D. D., Palmieri, G., Cossu, A., Flanagan, A., Nicholson, A., Ho, J. W., Leung, S. Y., Yuen, S. T., Weber, B. L., Seigler, H. F., Darrow, T. L., Paterson, H., Marais, R., Marshall, C. J., Wooster, R., Stratton, M. R., and Futreal, P. A. (2002) Mutations of the BRAF gene in human cancer. Nature. 417, 949-954 114 Testa, U., and Pelosi, E. (2013) The Impact of FLT3 Mutations on the Development of Acute Myeloid Leukemias. Leuk Res Treatment. 2013, 275760 115 Al-Mawali, A., Gillis, D., and Lewis, I. (2013) Characteristics and Prognosis of Adult Acute Myeloid Leukemia with Internal Tandem Duplication in the FLT3 Gene. Oman Med J. 28, 432-440 116 Meshinchi, S., and Appelbaum, F. R. (2009) Structural and functional alterations of FLT3 in acute myeloid leukemia. Clin Cancer Res. 15, 4263-4269 117 Thiede, C., Steudel, C., Mohr, B., Schaich, M., Schakel, U., Platzbecker, U., Wermke, M., Bornhauser, M., Ritter, M., Neubauer, A., Ehninger, G., and Illmer, T. (2002) Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood. 99, 4326-4335 118 Ray, R. J., Paige, C. J., Furlonger, C., Lyman, S. D., and Rottapel, R. (1996) Flt3 ligand supports the differentiation of early B cell progenitors in the presence of interleukin-11 and interleukin-7. Eur J Immunol. 26, 1504-1510 119 Piacibello, W., Fubini, L., Sanavio, F., Brizzi, M. F., Severino, A., Garetto, L., Stacchini, A., Pegoraro, L., and Aglietta, M. (1995) Effects of human FLT3 ligand on myeloid leukemia cell growth: heterogeneity in response and synergy with other hematopoietic growth factors. Blood. 86, 4105-4114 120 Mackarehtschian, K., Hardin, J. D., Moore, K. A., Boast, S., Goff, S. P., and Lemischka, I. R. (1995) Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors. Immunity. 3, 147-161 121 Kiyoi, H., Naoe, T., Nakano, Y., Yokota, S., Minami, S., Miyawaki, S., Asou, N., Kuriyama, K., Jinnai, I., Shimazaki, C., Akiyama, H., Saito, K., Oh, H., Motoji, T., Omoto, E., Saito, H., Ohno, R., and Ueda, R. (1999) Prognostic implication of FLT3 and N-RAS gene mutations in acute myeloid leukemia. Blood. 93, 3074-3080 122 Kottaridis, P. D., Gale, R. E., Frew, M. E., Harrison, G., Langabeer, S. E., Belton, A. A., Walker, H., Wheatley, K., Bowen, D. T., Burnett, A. K., Goldstone, A. H., and Linch, D. C. (2001) The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk
150
group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood. 98, 1752-1759 123 Kottaridis, P. D., Gale, R. E., Langabeer, S. E., Frew, M. E., Bowen, D. T., and Linch, D. C. (2002) Studies of FLT3 mutations in paired presentation and relapse samples from patients with acute myeloid leukemia: implications for the role of FLT3 mutations in leukemogenesis, minimal residual disease detection, and possible therapy with FLT3 inhibitors. Blood. 100, 2393-2398 124 Nabinger, S. C., Li, X. J., Ramdas, B., He, Y., Zhang, X., Zeng, L., Richine, B., Bowling, J. D., Fukuda, S., Goenka, S., Liu, Z., Feng, G. S., Yu, M., Sandusky, G. E., Boswell, H. S., Zhang, Z. Y., Kapur, R., and Chan, R. J. (2013) The protein tyrosine phosphatase, Shp2, positively contributes to FLT3-ITD-induced hematopoietic progenitor hyperproliferation and malignant disease in vivo. Leukemia. 27, 398-408 125 Zanke, B., Squire, J., Griesser, H., Henry, M., Suzuki, H., Patterson, B., Minden, M., and Mak, T. W. (1994) A hematopoietic protein tyrosine phosphatase (HePTP) gene that is amplified and overexpressed in myeloid malignancies maps to chromosome 1q32.1. Leukemia. 8, 236-244 126 Aebersold, R., and Mann, M. (2003) Mass spectrometry-based proteomics. Nature. 422, 198-207 127 Fenn JB, M. M., Meng CK, Wong SF, Whitehouse CM. (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science. 246, 64-71 128 Perkins, D. N., Pappin, D. J., Creasy, D. M., and Cottrell, J. S. (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis. 20, 3551-3567 129 Eng, J. K., McCormack, A. L., and Yates, J. R. (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom. 5, 976-989 130 Craig, R., and Beavis, R. C. (2004) TANDEM: matching proteins with tandem mass spectra. Bioinformatics. 20, 1466-1467 131 Chen, G., Pramanik, BN. (2008) LC-MS for protein characterization: current capabilities and future trends. Expert review of proteomics. 5, 435-444 132 Wysocki, V. H., Resing, K. A., Zhang, Q., and Cheng, G. (2005) Mass spectrometry of peptides and proteins. Methods. 35, 211-222 133 Lee, M. S., and Kerns, E. H. (1999) LC/MS applications in drug development. Mass Spectrom Rev. 18, 187-279 134 Mann, M. (2006) Functional and quantitative proteomics using SILAC. Nat Rev Mol Cell Biol. 7, 952-958 135 Kirkpatrick, D. S., Gerber, S. A., and Gygi, S. P. (2005) The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications. Methods. 35, 265-273 136 Smith, J. R., Olivier, M., and Greene, A. S. (2007) Relative quantification of peptide phosphorylation in a complex mixture using 18O labeling. Physiol Genomics. 31, 357-363 137 Wang, M., You, J., Bemis, K. G., Tegeler, T. J., and Brown, D. P. (2008) Label-free mass spectrometry-based protein quantification technologies in proteomic analysis. Brief Funct Genomic Proteomic. 7, 329-339
151
138 Asara, J. M., Christofk, H. R., Freimark, L. M., and Cantley, L. C. (2008) A label-free quantification method by MS/MS TIC compared to SILAC and spectral counting in a proteomics screen. Proteomics. 8, 994-999 139 Jin, L. L., Tong, J., Prakash, A., Peterman, S. M., St-Germain, J. R., Taylor, P., Trudel, S., and Moran, M. F. (2010) Measurement of protein phosphorylation stoichiometry by selected reaction monitoring mass spectrometry. J Proteome Res. 9, 2752-2761 140 Wang, G., Wu, W. W., Zeng, W., Chou, C. L., and Shen, R. F. (2006) Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: Reproducibility, linearity, and application with complex proteomes. J Proteome Res. 5, 1214-1223 141 Cutillas, P. R., and Vanhaesebroeck, B. (2007) Quantitative profile of five murine core proteomes using label-free functional proteomics. Mol Cell Proteomics. 6, 1560-1573 142 Cox, J., and Mann, M. (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 26, 1367-1372 143 Lange, V., Picotti, P., Domon, B., and Aebersold, R. (2008) Selected reaction monitoring for quantitative proteomics: a tutorial. Mol Syst Biol. 4, 222 144 Wolf-Yadlin, A., Hautaniemi, S., Lauffenburger, D. A., and White, F. M. (2007) Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks. Proc Natl Acad Sci U S A. 104, 5860-5865 145 Breitkopf, S. B., and Asara, J. M. (2012) Determining in vivo phosphorylation sites using mass spectrometry. Curr Protoc Mol Biol. Chapter 18, Unit18 19 11-27 146 Delom, F., and Chevet, E. (2006) Phosphoprotein analysis: from proteins to proteomes. Proteome Sci. 4, 15 147 Rush, J., Moritz, A., Lee, K. A., Guo, A., Goss, V. L., Spek, E. J., Zhang, H., Zha, X. M., Polakiewicz, R. D., and Comb, M. J. (2005) Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotechnol. 23, 94-101 148 Gorla, L., Cantu, M., Micciche, F., Patelli, C., Mondellini, P., Pierotti, M. A., and Bongarzone, I. (2006) RET oncoproteins induce tyrosine phosphorylation changes of proteins involved in RNA metabolism. Cell Signal. 18, 2272-2282 149 Salomon AR, F. S., Brill LM, Brinker A, Phung QT, Ericson C, Sauer K, Brock A, Horn DM, Schultz PG, Peters EC. (2003) Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry. Proceedings of the National Academy of Sciences of the United States of America. 100, 443-448 150 St-Germain, J. R., Taylor, P., Tong, J., Jin, L. L., Nikolic, A., Stewart, II, Ewing, R. M., Dharsee, M., Li, Z., Trudel, S., and Moran, M. F. (2009) Multiple myeloma phosphotyrosine proteomic profile associated with FGFR3 expression, ligand activation, and drug inhibition. Proc Natl Acad Sci U S A. 106, 20127-20132 151 Karisch, R., Fernandez, M., Taylor, P., Virtanen, C., St-Germain, J. R., Jin, L. L., Harris, I. S., Mori, J., Mak, T. W., Senis, Y. A., Ostman, A., Moran, M. F., and Neel, B. G. (2011) Global proteomic assessment of the classical protein-tyrosine phosphatome and "redoxome". Cell. 146, 826-840
152
152 Persson, C., Kappert, K., Engstrom, U., Ostman, A., and Sjoblom, T. (2005) An antibody-based method for monitoring in vivo oxidation of protein tyrosine phosphatases. Methods. 35, 37-43 153 Abdi, H., and Williams, L. J. (2013) Partial least squares methods: partial least squares correlation and partial least square regression. Methods Mol Biol. 930, 549-579 154 Ergon, R. (2014) Principal component regression (PCR) and partial least squares regression (PLSR). In Mathematical and Statistical Methods in Food Science and Technology (Granato, D. and Ares, D., eds.), John Wiley & Sons, Ltd, Chichester, United Kingdom 155 Jernberg-Wiklund, H., and Nilsson, K. (2000) Multiple myeloma cell lines. In Human Cell Culture (Palsson, B. Ø. and Masters, J., eds.). pp. 81-155, Dordrecht, Netherlands 156 Hadari, Y., and Schlessinger, J. (2009) FGFR3-targeted mAb therapy for bladder cancer and multiple myeloma. J Clin Invest. 119, 1077-1079 157 Grand, E. K., Chase, A. J., Heath, C., Rahemtulla, A., and Cross, N. C. (2004) Targeting FGFR3 in multiple myeloma: inhibition of t(4;14)-positive cells by SU5402 and PD173074. Leukemia. 18, 962-966 158 Nilsson, K., Georgii-Hemming, P., Spets, H., and Jernberg-Wiklund, H. (1999) The control of proliferation, survival and apoptosis in human multiple myeloma cells in vitro. Curr Top Microbiol Immunol. 246, 325-332; discussion 333 159 Georgii-Hemming, P., Wiklund, H. J., Ljunggren, O., and Nilsson, K. (1996) Insulin-like growth factor I is a growth and survival factor in human multiple myeloma cell lines. Blood. 88, 2250-2258 160 Steinbrunn, T., Stuhmer, T., Gattenlohner, S., Rosenwald, A., Mottok, A., Unzicker, C., Einsele, H., Chatterjee, M., and Bargou, R. C. (2011) Mutated RAS and constitutively activated Akt delineate distinct oncogenic pathways, which independently contribute to multiple myeloma cell survival. Blood. 117, 1998-2004 161 Zollinger, A., Stuhmer, T., Chatterjee, M., Gattenlohner, S., Haralambieva, E., Muller-Hermelink, H. K., Andrulis, M., Greiner, A., Wesemeier, C., Rath, J. C., Einsele, H., and Bargou, R. C. (2008) Combined functional and molecular analysis of tumor cell signaling defines 2 distinct myeloma subgroups: Akt-dependent and Akt-independent multiple myeloma. Blood. 112, 3403-3411 162 Lentzsch, S., Chatterjee, M., Gries, M., Bommert, K., Gollasch, H., Dorken, B., and Bargou, R. C. (2004) PI3-K/AKT/FKHR and MAPK signaling cascades are redundantly stimulated by a variety of cytokines and contribute independently to proliferation and survival of multiple myeloma cells. Leukemia. 18, 1883-1890 163 Ikediobi, O. N., Davies, H., Bignell, G., Edkins, S., Stevens, C., O'Meara, S., Santarius, T., Avis, T., Barthorpe, S., Brackenbury, L., Buck, G., Butler, A., Clements, J., Cole, J., Dicks, E., Forbes, S., Gray, K., Halliday, K., Harrison, R., Hills, K., Hinton, J., Hunter, C., Jenkinson, A., Jones, D., Kosmidou, V., Lugg, R., Menzies, A., Mironenko, T., Parker, A., Perry, J., Raine, K., Richardson, D., Shepherd, R., Small, A., Smith, R., Solomon, H., Stephens, P., Teague, J., Tofts, C., Varian, J., Webb, T., West, S., Widaa, S., Yates, A., Reinhold, W., Weinstein, J. N., Stratton, M. R., Futreal, P. A., and Wooster, R. (2006) Mutation analysis of 24 known cancer genes in the NCI-60 cell line set. Mol Cancer Ther. 5, 2606-2612
153
164 Voutsina, A., Tzardi, M., Kalikaki, A., Zafeiriou, Z., Papadimitraki, E., Papadakis, M., Mavroudis, D., and Georgoulias, V. (2012) Combined analysis of KRAS and PIK3CA mutations, MET and PTEN expression in primary tumors and corresponding metastases in colorectal cancer. Mod Pathol. 26, 302-313 165 Teoh, G., Urashima, M., Ogata, A., Chauhan, D., DeCaprio, J. A., Treon, S. P., Schlossman, R. L., and Anderson, K. C. (1997) MDM2 protein overexpression promotes proliferation and survival of multiple myeloma cells. Blood. 90, 1982-1992 166 Mao, X., Cao, B., Wood, T. E., Hurren, R., Tong, J., Wang, X., Wang, W., Li, J., Jin, Y., Sun, W., Spagnuolo, P. A., MacLean, N., Moran, M. F., Datti, A., Wrana, J., Batey, R. A., and Schimmer, A. D. (2011) A small-molecule inhibitor of D-cyclin transactivation displays preclinical efficacy in myeloma and leukemia via phosphoinositide 3-kinase pathway. Blood. 117, 1986-1997 167 Shou, Y., Martelli, M. L., Gabrea, A., Qi, Y., Brents, L. A., Roschke, A., Dewald, G., Kirsch, I. R., Bergsagel, P. L., and Kuehl, W. M. (2000) Diverse karyotypic abnormalities of the c-myc locus associated with c-myc dysregulation and tumor progression in multiple myeloma. Proc Natl Acad Sci U S A. 97, 228-233 168 Schwarz, F., and Aebi, M. (2011) Mechanisms and principles of N-linked protein glycosylation. Curr Opin Struct Biol. 21, 576-582 169 Malumbres, M., and Barbacid, M. (2009) Cell cycle, CDKs and cancer: a changing paradigm. Nat Rev Cancer. 9, 153-166 170 Bjorge, J. D., Pang, A., and Fujita, D. J. (2000) Identification of protein-tyrosine phosphatase 1B as the major tyrosine phosphatase activity capable of dephosphorylating and activating c-Src in several human breast cancer cell lines. J Biol Chem. 275, 41439-41446 171 Valverde, A. M., and Gonzalez-Rodriguez, A. (2011) IRS2 and PTP1B: Two opposite modulators of hepatic insulin signalling. Arch Physiol Biochem. 117, 105-115 172 Chatterjee, M., Stuhmer, T., Herrmann, P., Bommert, K., Dorken, B., and Bargou, R. C. (2004) Combined disruption of both the MEK/ERK and the IL-6R/STAT3 pathways is required to induce apoptosis of multiple myeloma cells in the presence of bone marrow stromal cells. Blood. 104, 3712-3721 173 Zhu, S., Bjorge, J. D., and Fujita, D. J. (2007) PTP1B contributes to the oncogenic properties of colon cancer cells through Src activation. Cancer Res. 67, 10129-10137 174 Zhang, S. Q., Yang, W., Kontaridis, M. I., Bivona, T. G., Wen, G., Araki, T., Luo, J., Thompson, J. A., Schraven, B. L., Philips, M. R., and Neel, B. G. (2004) Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment. Mol Cell. 13, 341-355 175 Rikova, K., Guo, A., Zeng, Q., Possemato, A., Yu, J., Haack, H., Nardone, J., Lee, K., Reeves, C., Li, Y., Hu, Y., Tan, Z., Stokes, M., Sullivan, L., Mitchell, J., Wetzel, R., Macneill, J., Ren, J. M., Yuan, J., Bakalarski, C. E., Villen, J., Kornhauser, J. M., Smith, B., Li, D., Zhou, X., Gygi, S. P., Gu, T. L., Polakiewicz, R. D., Rush, J., and Comb, M. J. (2007) Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell. 131, 1190-1203 176 Harsha, H. C., Jimeno, A., Molina, H., Mihalas, A. B., Goggins, M. G., Hruban, R. H., Schulick, R. D., Kamath, U., Maitra, A., Hidalgo, M., and Pandey, A. (2008) Activated epidermal growth factor receptor as a novel target in pancreatic cancer therapy. J Proteome Res. 7, 4651-4658
154
177 Yu, L. R., Issaq, H. J., and Veenstra, T. D. (2007) Phosphoproteomics for the discovery of kinases as cancer biomarkers and drug targets. Proteomics Clin Appl. 1, 1042-1057 178 Pan, C., Olsen, J. V., Daub, H., and Mann, M. (2009) Global effects of kinase inhibitors on signaling networks revealed by quantitative phosphoproteomics. Mol Cell Proteomics. 8, 2796-2808 179 Bose, R., Molina, H., Patterson, A. S., Bitok, J. K., Periaswamy, B., Bader, J. S., Pandey, A., and Cole, P. A. (2006) Phosphoproteomic analysis of Her2/neu signaling and inhibition. Proc Natl Acad Sci U S A. 103, 9773-9778 180 Godl, K., Wissing, J., Kurtenbach, A., Habenberger, P., Blencke, S., Gutbrod, H., Salassidis, K., Stein-Gerlach, M., Missio, A., Cotten, M., and Daub, H. (2003) An efficient proteomics method to identify the cellular targets of protein kinase inhibitors. Proc Natl Acad Sci U S A. 100, 15434-15439 181 Guha, U., Chaerkady, R., Marimuthu, A., Patterson, A. S., Kashyap, M. K., Harsha, H. C., Sato, M., Bader, J. S., Lash, A. E., Minna, J. D., Pandey, A., and Varmus, H. E. (2008) Comparisons of tyrosine phosphorylated proteins in cells expressing lung cancer-specific alleles of EGFR and KRAS. Proc Natl Acad Sci U S A. 105, 14112-14117 182 Huang, P. H., Mukasa, A., Bonavia, R., Flynn, R. A., Brewer, Z. E., Cavenee, W. K., Furnari, F. B., and White, F. M. (2007) Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma. Proc Natl Acad Sci U S A. 104, 12867-12872 183 Chesi, M., Brents, L. A., Ely, S. A., Bais, C., Robbiani, D. F., Mesri, E. A., Kuehl, W. M., and Bergsagel, P. L. (2001) Activated fibroblast growth factor receptor 3 is an oncogene that contributes to tumor progression in multiple myeloma. Blood. 97, 729-736 184 McLean, G. W., Carragher, N. O., Avizienyte, E., Evans, J., Brunton, V. G., and Frame, M. C. (2005) The role of focal-adhesion kinase in cancer - a new therapeutic opportunity. Nat Rev Cancer. 5, 505-515 185 Wang, S. Y., Hao, H. L., Deng, K., Li, Y., Cheng, Z. Y., Lv, C., Liu, Z. M., Yang, J., and Pan, L. (2012) Expression levels of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and focal adhesion kinase in patients with multiple myeloma and their relationship to clinical stage and extramedullary infiltration. Leuk Lymphoma. 53, 1162-1168 186 Irby, R. B., Mao, W., Coppola, D., Kang, J., Loubeau, J. M., Trudeau, W., Karl, R., Fujita, D. J., Jove, R., and Yeatman, T. J. (1999) Activating SRC mutation in a subset of advanced human colon cancers. Nat Genet. 21, 187-190 187 Gertz, M. A. (2008) New targets and treatments in multiple myeloma: Src family kinases as central regulators of disease progression. Leuk Lymphoma. 49, 2240-2245 188 Neri, P., Ren, L., Azab, A. K., Brentnall, M., Gratton, K., Klimowicz, A. C., Lin, C., Duggan, P., Tassone, P., Mansoor, A., Stewart, D. A., Boise, L. H., Ghobrial, I. M., and Bahlis, N. J. (2011) Integrin beta7-mediated regulation of multiple myeloma cell adhesion, migration, and invasion. Blood. 117, 6202-6213 189 An, X., Tiwari, A. K., Sun, Y., Ding, P. R., Ashby, C. R., Jr., and Chen, Z. S. (2010) BCR-ABL tyrosine kinase inhibitors in the treatment of Philadelphia chromosome positive chronic myeloid leukemia: a review. Leuk Res. 34, 1255-1268 190 Breitkopf, S. B., Yuan, M., Pihan, G. A., and Asara, J. M. (2012) Detection of a rare BCR-ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using
155
immunoprecipitation (IP)-tandem mass spectrometry (MS/MS). Proc Natl Acad Sci U S A. 109, 16190-16195 191 Shaw, L. M. (2011) The insulin receptor substrate (IRS) proteins: at the intersection of metabolism and cancer. Cell Cycle. 10, 1750-1756 192 Grossmann, K. S., Rosario, M., Birchmeier, C., and Birchmeier, W. The tyrosine phosphatase Shp2 in development and cancer. Adv Cancer Res. 106, 53-89 193 Matozaki, T., Murata, Y., Saito, Y., Okazawa, H., and Ohnishi, H. (2009) Protein tyrosine phosphatase SHP-2: a proto-oncogene product that promotes Ras activation. Cancer Sci. 100, 1786-1793 194 Luo, Y., Liang, F., and Zhang, Z. Y. (2009) PRL1 promotes cell migration and invasion by increasing MMP2 and MMP9 expression through Src and ERK1/2 pathways. Biochemistry. 48, 1838-1846 195 Zhang, S., and Zhang, Z. Y. (2007) PTP1B as a drug target: recent developments in PTP1B inhibitor discovery. Drug Discov Today. 12, 373-381 196 Chauhan, D., Pandey, P., Hideshima, T., Treon, S., Raje, N., Davies, F. E., Shima, Y., Tai, Y. T., Rosen, S., Avraham, S., Kharbanda, S., and Anderson, K. C. (2000) SHP2 mediates the protective effect of interleukin-6 against dexamethasone-induced apoptosis in multiple myeloma cells. J Biol Chem. 275, 27845-27850 197 Agazie, Y. M., Movilla, N., Ischenko, I., and Hayman, M. J. (2003) The phosphotyrosine phosphatase SHP2 is a critical mediator of transformation induced by the oncogenic fibroblast growth factor receptor 3. Oncogene. 22, 6909-6918 198 Shu, S. T., Sugimoto, Y., Liu, S., Chang, H. L., Ye, W., Wang, L. S., Huang, Y. W., Yan, P., and Lin, Y. C. (2010) Function and regulatory mechanisms of the candidate tumor suppressor receptor protein tyrosine phosphatase gamma (PTPRG) in breast cancer cells. Anticancer Res. 30, 1937-1946 199 Yuan, T., Wang, Y., Zhao, Z. J., and Gu, H. (2010) Protein-tyrosine phosphatase PTPN9 negatively regulates ErbB2 and epidermal growth factor receptor signaling in breast cancer cells. J Biol Chem. 285, 14861-14870 200 Dhillon, A. S., Hagan, S., Rath, O., and Kolch, W. (2007) MAP kinase signalling pathways in cancer. Oncogene. 26, 3279-3290 201 Hideshima, T., Akiyama, M., Hayashi, T., Richardson, P., Schlossman, R., Chauhan, D., and Anderson, K. C. (2003) Targeting p38 MAPK inhibits multiple myeloma cell growth in the bone marrow milieu. Blood. 101, 703-705 202 Chang-Yew Leow, C., Gerondakis, S., and Spencer, A. (2013) MEK inhibitors as a chemotherapeutic intervention in multiple myeloma. Blood Cancer J. 3, e105 203 Zheng, H., Hu, P., Quinn, D. F., and Wang, Y. K. (2005) Phosphotyrosine proteomic study of interferon alpha signaling pathway using a combination of immunoprecipitation and immobilized metal affinity chromatography. Mol Cell Proteomics. 4, 721-730 204 Velazquez, L., Fellous, M., Stark, G. R., and Pellegrini, S. (1992) A protein tyrosine kinase in the interferon alpha/beta signaling pathway. Cell. 70, 313-322 205 Minegishi, Y., Saito, M., Morio, T., Watanabe, K., Agematsu, K., Tsuchiya, S., Takada, H., Hara, T., Kawamura, N., Ariga, T., Kaneko, H., Kondo, N., Tsuge, I., Yachie, A., Sakiyama, Y., Iwata, T., Bessho, F., Ohishi, T., Joh, K., Imai, K., Kogawa, K., Shinohara, M., Fujieda, M., Wakiguchi, H., Pasic, S., Abinun, M., Ochs, H. D., Renner, E. D., Jansson, A., Belohradsky, B. H., Metin, A., Shimizu, N., Mizutani, S., Miyawaki, T.,
156
Nonoyama, S., and Karasuyama, H. (2006) Human tyrosine kinase 2 deficiency reveals its requisite roles in multiple cytokine signals involved in innate and acquired immunity. Immunity. 25, 745-755 206 Karaghiosoff, M., Neubauer, H., Lassnig, C., Kovarik, P., Schindler, H., Pircher, H., McCoy, B., Bogdan, C., Decker, T., Brem, G., Pfeffer, K., and Muller, M. (2000) Partial impairment of cytokine responses in Tyk2-deficient mice. Immunity. 13, 549-560 207 Zhang, Q., Sturgill, J. L., Kmieciak, M., Szczepanek, K., Derecka, M., Koebel, C., Graham, L. J., Dai, Y., Chen, S., Grant, S., Cichy, J., Shimoda, K., Gamero, A., Manjili, M., Bear, H., Conrad, D., and Larner, A. C. (2011) The role of Tyk2 in regulation of breast cancer growth. J Interferon Cytokine Res. 31, 671-677 208 Lemaire, M., Deleu, S., De Bruyne, E., Van Valckenborgh, E., Menu, E., and Vanderkerken, K. (2011) The microenvironment and molecular biology of the multiple myeloma tumor. Adv Cancer Res. 110, 19-42 209 Keller, A., Nesvizhskii, A. I., Kolker, E., and Aebersold, R. (2002) Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem. 74, 5383-5392 210 Nesvizhskii, A. I., Keller, A., Kolker, E., and Aebersold, R. (2003) A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem. 75, 4646-4658 211 Thomsen, M. C., and Nielsen, M. (2012) Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion. Nucleic Acids Res. 40, W281-287 212 Eisen, M. B., Spellman, P. T., Brown, P. O., and Botstein, D. (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U S A. 95, 14863-14868 213 Lainey, E., Thepot, S., Bouteloup, C., Sebert, M., Ades, L., Tailler, M., Gardin, C., de Botton, S., Baruchel, A., Fenaux, P., Kroemer, G., and Boehrer, S. (2011) Tyrosine kinase inhibitors for the treatment of acute myeloid leukemia: delineation of anti-leukemic mechanisms of action. Biochem Pharmacol. 82, 1457-1466 214 Kornblau, S. M., Tibes, R., Qiu, Y. H., Chen, W., Kantarjian, H. M., Andreeff, M., Coombes, K. R., and Mills, G. B. (2009) Functional proteomic profiling of AML predicts response and survival. Blood. 113, 154-164 215 Irish, J. M., Hovland, R., Krutzik, P. O., Perez, O. D., Bruserud, O., Gjertsen, B. T., and Nolan, G. P. (2004) Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell. 118, 217-228 216 Walters, D. K., Goss, V. L., Stoffregen, E. P., Gu, T. L., Lee, K., Nardone, J., McGreevey, L., Heinrich, M. C., Deininger, M. W., Polakiewicz, R., and Druker, B. J. (2006) Phosphoproteomic analysis of AML cell lines identifies leukemic oncogenes. Leuk Res. 30, 1097-1104 217 Kabir, N. N., Ronnstrand, L., and Kazi, J. U. (2013) Deregulation of protein phosphatase expression in acute myeloid leukemia. Med Oncol. 30, 517 218 Cairoli, R., Beghini, A., Grillo, G., Nadali, G., Elice, F., Ripamonti, C. B., Colapietro, P., Nichelatti, M., Pezzetti, L., Lunghi, M., Cuneo, A., Viola, A., Ferrara, F., Lazzarino, M., Rodeghiero, F., Pizzolo, G., Larizza, L., and Morra, E. (2006) Prognostic impact of c-KIT mutations in core binding factor leukemias: an Italian retrospective study. Blood. 107, 3463-3468
157
219 Hahn, C. K., Berchuck, J. E., Ross, K. N., Kakoza, R. M., Clauser, K., Schinzel, A. C., Ross, L., Galinsky, I., Davis, T. N., Silver, S. J., Root, D. E., Stone, R. M., DeAngelo, D. J., Carroll, M., Hahn, W. C., Carr, S. A., Golub, T. R., Kung, A. L., and Stegmaier, K. (2009) Proteomic and genetic approaches identify Syk as an AML target. Cancer Cell. 16, 281-294 220 Jain, N., Curran, E., Iyengar, N. M., Diaz-Flores, E., Kunnavakkam, R., Popplewell, L., Kirschbaum, M. H., Karrison, T., Erba, H. P., Green, M., Poire, X., Koval, G., Shannon, K., Reddy, P. L., Joseph, L., Atallah, E. L., Dy, P., Thomas, S. P., Smith, S. E., Doyle, L. A., Stadler, W. M., Larson, R. A., Stock, W., and Odenike, O. (2014) Phase II study of the oral MEK inhibitor selumetinib in advanced acute myelogenous leukemia: a University of Chicago phase II consortium trial. Clin Cancer Res. 20, 490-498 221 Arora, D., Kothe, S., van den Eijnden, M., Hooft van Huijsduijnen, R., Heidel, F., Fischer, T., Scholl, S., Tolle, B., Bohmer, S. A., Lennartsson, J., Isken, F., Muller-Tidow, C., and Bohmer, F. D. (2012) Expression of protein-tyrosine phosphatases in Acute Myeloid Leukemia cells: FLT3 ITD sustains high levels of DUSP6 expression. Cell Commun Signal. 10, 19 222 Scott, L. M., Lawrence, H. R., Sebti, S. M., Lawrence, N. J., and Wu, J. (2010) Targeting protein tyrosine phosphatases for anticancer drug discovery. Curr Pharm Des. 16, 1843-1862 223 Vogelstein, B., and Kinzler, K. W. (2004) Cancer genes and the pathways they control. Nat Med. 10, 789-799 224 Valk, P. J., Verhaak, R. G., Beijen, M. A., Erpelinck, C. A., Barjesteh van Waalwijk van Doorn-Khosrovani, S., Boer, J. M., Beverloo, H. B., Moorhouse, M. J., van der Spek, P. J., Lowenberg, B., and Delwel, R. (2004) Prognostically useful gene-expression profiles in acute myeloid leukemia. N Engl J Med. 350, 1617-1628 225 Bullinger, L., Dohner, K., Bair, E., Frohling, S., Schlenk, R. F., Tibshirani, R., Dohner, H., and Pollack, J. R. (2004) Use of gene-expression profiling to identify prognostic subclasses in adult acute myeloid leukemia. N Engl J Med. 350, 1605-1616 226 Chin, L., Andersen, J. N., and Futreal, P. A. (2011) Cancer genomics: from discovery science to personalized medicine. Nat Med. 17, 297-303 227 Varambally, S., Yu, J., Laxman, B., Rhodes, D. R., Mehra, R., Tomlins, S. A., Shah, R. B., Chandran, U., Monzon, F. A., Becich, M. J., Wei, J. T., Pienta, K. J., Ghosh, D., Rubin, M. A., and Chinnaiyan, A. M. (2005) Integrative genomic and proteomic analysis of prostate cancer reveals signatures of metastatic progression. Cancer Cell. 8, 393-406 228 Tian, Q., Stepaniants, S. B., Mao, M., Weng, L., Feetham, M. C., Doyle, M. J., Yi, E. C., Dai, H., Thorsson, V., Eng, J., Goodlett, D., Berger, J. P., Gunter, B., Linseley, P. S., Stoughton, R. B., Aebersold, R., Collins, S. J., Hanlon, W. A., and Hood, L. E. (2004) Integrated genomic and proteomic analyses of gene expression in Mammalian cells. Mol Cell Proteomics. 3, 960-969 229 Gygi, S. P., Rochon, Y., Franza, B. R., and Aebersold, R. (1999) Correlation between protein and mRNA abundance in yeast. Mol Cell Biol. 19, 1720-1730 230 Lu, P., Vogel, C., Wang, R., Yao, X., and Marcotte, E. M. (2007) Absolute protein expression profiling estimates the relative contributions of transcriptional and translational regulation. Nat Biotechnol. 25, 117-124
158
231 Deng, X., Kohanfars, M., Hsu, H. M., Souda, P., Capri, J., Whitelegge, J. P., and Chang, H. R. (2014) Combined phosphoproteomics and bioinformatics strategy in deciphering drug resistant related pathways in triple negative breast cancer. Int J Proteomics. 2014, 390781 232 Ali, N. A., Wu, J., Hochgrafe, F., Chan, H., Nair, R., Ye, S., Zhang, L., Lyons, R. J., Pinese, M., Lee, H. C., Armstrong, N., Ormandy, C. J., Clark, S. J., Swarbrick, A., and Daly, R. J. (2014) Profiling the tyrosine phosphoproteome of different mouse mammary tumour models reveals distinct, model-specific signalling networks and conserved oncogenic pathways. Breast Cancer Res. 16, 437 233 Yoshida, T., Zhang, G., Smith, M. A., Lopez, A. S., Bai, Y., Li, J., Fang, B., Koomen, J., Rawal, B., Fisher, K. J., Chen, A. Y., Kitano, M., Morita, Y., Yamaguchi, H., Shibata, K., Okabe, T., Okamoto, I., Nakagawa, K., and Haura, E. B. (2014) Tyrosine phosphoproteomics identifies both codrivers and cotargeting strategies for T790M-related EGFR-TKI resistance in non-small cell lung cancer. Clin Cancer Res. 20, 4059-4074 234 Casado, P., Alcolea, M. P., Iorio, F., Rodriguez-Prados, J. C., Vanhaesebroeck, B., Saez-Rodriguez, J., Joel, S., and Cutillas, P. R. (2013) Phosphoproteomics data classify hematological cancer cell lines according to tumor type and sensitivity to kinase inhibitors. Genome Biol. 14, R37 235 Lim, Y. P. (2005) Mining the tumor phosphoproteome for cancer markers. Clin Cancer Res. 11, 3163-3169 236 Beghini, A., Ripamonti, C. B., Peterlongo, P., Roversi, G., Cairoli, R., Morra, E., and Larizza, L. (2000) RNA hyperediting and alternative splicing of hematopoietic cell phosphatase (PTPN6) gene in acute myeloid leukemia. Hum Mol Genet. 9, 2297-2304 237 Sun, T., Aceto, N., Meerbrey, K. L., Kessler, J. D., Zhou, C., Migliaccio, I., Nguyen, D. X., Pavlova, N. N., Botero, M., Huang, J., Bernardi, R. J., Schmitt, E., Hu, G., Li, M. Z., Dephoure, N., Gygi, S. P., Rao, M., Creighton, C. J., Hilsenbeck, S. G., Shaw, C. A., Muzny, D., Gibbs, R. A., Wheeler, D. A., Osborne, C. K., Schiff, R., Bentires-Alj, M., Elledge, S. J., and Westbrook, T. F. (2011) Activation of multiple proto-oncogenic tyrosine kinases in breast cancer via loss of the PTPN12 phosphatase. Cell. 144, 703-718 238 Porcu, M., Kleppe, M., Gianfelici, V., Geerdens, E., De Keersmaecker, K., Tartaglia, M., Foa, R., Soulier, J., Cauwelier, B., Uyttebroeck, A., Macintyre, E., Vandenberghe, P., Asnafi, V., and Cools, J. (2012) Mutation of the receptor tyrosine phosphatase PTPRC (CD45) in T-cell acute lymphoblastic leukemia. Blood. 119, 4476-4479 239 Bentires-Alj, M., and Neel, B. G. (2007) Protein-tyrosine phosphatase 1B is required for HER2/Neu-induced breast cancer. Cancer Res. 67, 2420-2424 240 Julien, S. G., Dube, N., Read, M., Penney, J., Paquet, M., Han, Y., Kennedy, B. P., Muller, W. J., and Tremblay, M. L. (2007) Protein tyrosine phosphatase 1B deficiency or inhibition delays ErbB2-induced mammary tumorigenesis and protects from lung metastasis. Nat Genet. 39, 338-346 241 Harder, K. W., Moller, N. P., Peacock, J. W., and Jirik, F. R. (1998) Protein-tyrosine phosphatase alpha regulates Src family kinases and alters cell-substratum adhesion. J Biol Chem. 273, 31890-31900 242 Gil-Henn, H., Patsialou, A., Wang, Y., Warren, M. S., Condeelis, J. S., and Koleske, A. J. (2013) Arg/Abl2 promotes invasion and attenuates proliferation of breast cancer in vivo. Oncogene. 32, 2622-2630
159
243 Greer, P. A., Kanda, S., and Smithgall, T. E. (2012) The contrasting oncogenic and tumor suppressor roles of FES. Front Biosci (Schol Ed). 4, 489-501 244 Dos Santos, C., McDonald, T., Ho, Y. W., Liu, H., Lin, A., Forman, S. J., Kuo, Y. H., and Bhatia, R. (2013) The Src and c-Kit kinase inhibitor dasatinib enhances p53-mediated targeting of human acute myeloid leukemia stem cells by chemotherapeutic agents. Blood. 122, 1900-1913 245 Okamoto, M., Hayakawa, F., Miyata, Y., Watamoto, K., Emi, N., Abe, A., Kiyoi, H., Towatari, M., and Naoe, T. (2007) Lyn is an important component of the signal transduction pathway specific to FLT3/ITD and can be a therapeutic target in the treatment of AML with FLT3/ITD. Leukemia. 21, 403-410 246 Paschka, P., Marcucci, G., Ruppert, A. S., Mrozek, K., Chen, H., Kittles, R. A., Vukosavljevic, T., Perrotti, D., Vardiman, J. W., Carroll, A. J., Kolitz, J. E., Larson, R. A., and Bloomfield, C. D. (2006) Adverse prognostic significance of KIT mutations in adult acute myeloid leukemia with inv(16) and t(8;21): a Cancer and Leukemia Group B Study. J Clin Oncol. 24, 3904-3911 247 Nakao, M., Yokota, S., Iwai, T., Kaneko, H., Horiike, S., Kashima, K., Sonoda, Y., Fujimoto, T., and Misawa, S. (1996) Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia. 10, 1911-1918 248 Voisset, E., Lopez, S., Chaix, A., Georges, C., Hanssens, K., Prebet, T., Dubreuil, P., and De Sepulveda, P. (2010) FES kinases are required for oncogenic FLT3 signaling. Leukemia. 24, 721-728 249 Puissant, A., Fenouille, N., Alexe, G., Pikman, Y., Bassil, C. F., Mehta, S., Du, J., Kazi, J. U., Luciano, F., Ronnstrand, L., Kung, A. L., Aster, J. C., Galinsky, I., Stone, R. M., DeAngelo, D. J., Hemann, M. T., and Stegmaier, K. (2014) SYK is a critical regulator of FLT3 in acute myeloid leukemia. Cancer Cell. 25, 226-242 250 Carnevale, J., Ross, L., Puissant, A., Banerji, V., Stone, R. M., DeAngelo, D. J., Ross, K. N., and Stegmaier, K. (2013) SYK regulates mTOR signaling in AML. Leukemia. 27, 2118-2128 251 Ozawa, Y., Williams, A. H., Estes, M. L., Matsushita, N., Boschelli, F., Jove, R., and List, A. F. (2008) Src family kinases promote AML cell survival through activation of signal transducers and activators of transcription (STAT). Leuk Res. 32, 893-903 252 Willman, C. L., Stewart, C. C., Longacre, T. L., Head, D. R., Habbersett, R., Ziegler, S. F., and Perlmutter, R. M. (1991) Expression of the c-fgr and hck protein-tyrosine kinases in acute myeloid leukemic blasts is associated with early commitment and differentiation events in the monocytic and granulocytic lineages. Blood. 77, 726-734 253 Iijima, Y., Ito, T., Oikawa, T., Eguchi, M., Eguchi-Ishimae, M., Kamada, N., Kishi, K., Asano, S., Sakaki, Y., and Sato, Y. (2000) A new ETV6/TEL partner gene, ARG (ABL-related gene or ABL2), identified in an AML-M3 cell line with a t(1;12)(q25;p13) translocation. Blood. 95, 2126-2131 254 Vadlamudi, R. K., Sahin, A. A., Adam, L., Wang, R. A., and Kumar, R. (2003) Heregulin and HER2 signaling selectively activates c-Src phosphorylation at tyrosine 215. FEBS Lett. 543, 76-80 255 Goss, V. L., Lee, K. A., Moritz, A., Nardone, J., Spek, E. J., MacNeill, J., Rush, J., Comb, M. J., and Polakiewicz, R. D. (2006) A common phosphotyrosine signature for the Bcr-Abl kinase. Blood. 107, 4888-4897
160
256 Crooks, G. E., Hon, G., Chandonia, J. M., and Brenner, S. E. (2004) WebLogo: a sequence logo generator. Genome Res. 14, 1188-1190 257 Chou, M. F., and Schwartz, D. (2011) Biological sequence motif discovery using motif-x. Curr Protoc Bioinformatics. Chapter 13, Unit 13 15-24 258 Koncz, G., Pecht, I., Gergely, J., and Sarmay, G. (1999) Fcgamma receptor-mediated inhibition of human B cell activation: the role of SHP-2 phosphatase. Eur J Immunol. 29, 1980-1989 259 Whiting, R. J., Payne, C. J., Satiaputra, J., Kucera, N., Qiu, T. W., Irtegun, S., Gunn, N. J., Slavova-Azmanova, N. S., Mulhern, T. D., and Ingley, E. (2012) Targeting Lyn tyrosine kinase through protein fusions encompassing motifs of Cbp (Csk-binding protein) and the SOCS box of SOCS1. Biochem J. 442, 611-620 260 Ladbury, J. E., Lemmon, M. A., Zhou, M., Green, J., Botfield, M. C., and Schlessinger, J. (1995) Measurement of the binding of tyrosyl phosphopeptides to SH2 domains: a reappraisal. Proc Natl Acad Sci U S A. 92, 3199-3203 261 Safran, M., Dalah, I., Alexander, J., Rosen, N., Iny Stein, T., Shmoish, M., Nativ, N., Bahir, I., Doniger, T., Krug, H., Sirota-Madi, A., Olender, T., Golan, Y., Stelzer, G., Harel, A., and Lancet, D. (2010) GeneCards Version 3: the human gene integrator. Database (Oxford). 2010, baq020 262 Remmert, M., Biegert, A., Hauser, A., and Soding, J. (2011) HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment. Nat Methods. 9, 173-175 263 Hornbeck, P. V., Kornhauser, J. M., Tkachev, S., Zhang, B., Skrzypek, E., Murray, B., Latham, V., and Sullivan, M. (2012) PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse. Nucleic Acids Res. 40, D261-270 264 Reimand, J., Arak, T., and Vilo, J. (2011) g:Profiler--a web server for functional interpretation of gene lists (2011 update). Nucleic Acids Res. 39, W307-315 265 Soula, M., Rothhut, B., Camoin, L., Guillaume, J. L., Strosberg, D., Vorherr, T., Burn, P., Meggio, F., Fischer, S., and Fagard, R. (1993) Anti-CD3 and phorbol ester induce distinct phosphorylated sites in the SH2 domain of p56lck. J Biol Chem. 268, 27420-27427 266 Zhao, M., Sun, J., and Zhao, Z. (2013) TSGene: a web resource for tumor suppressor genes. Nucleic Acids Res. 41, D970-976 267 Spurling, C. C., Godman, C. A., Noonan, E. J., Rasmussen, T. P., Rosenberg, D. W., and Giardina, C. (2008) HDAC3 overexpression and colon cancer cell proliferation and differentiation. Mol Carcinog. 47, 137-147 268 Summers, A. R., Fischer, M. A., Stengel, K. R., Zhao, Y., Kaiser, J. F., Wells, C. E., Hunt, A., Bhaskara, S., Luzwick, J. W., Sampathi, S., Chen, X., Thompson, M. A., Cortez, D., and Hiebert, S. W. (2013) HDAC3 is essential for DNA replication in hematopoietic progenitor cells. J Clin Invest. 123, 3112-3123 269 Amsellem, V., Kryszke, M. H., Hervy, M., Subra, F., Athman, R., Leh, H., Brachet-Ducos, C., and Auclair, C. (2005) The actin cytoskeleton-associated protein zyxin acts as a tumor suppressor in Ewing tumor cells. Exp Cell Res. 304, 443-456 270 Firlej, V., Mathieu, J. R., Gilbert, C., Lemonnier, L., Nakhle, J., Gallou-Kabani, C., Guarmit, B., Morin, A., Prevarskaya, N., Delongchamps, N. B., and Cabon, F. (2011)
161
Thrombospondin-1 triggers cell migration and development of advanced prostate tumors. Cancer Res. 71, 7649-7658 271 Barrett, C. W., Ning, W., Chen, X., Smith, J. J., Washington, M. K., Hill, K. E., Coburn, L. A., Peek, R. M., Chaturvedi, R., Wilson, K. T., Burk, R. F., and Williams, C. S. (2012) Tumor suppressor function of the plasma glutathione peroxidase gpx3 in colitis-associated carcinoma. Cancer Res. 73, 1245-1255 272 Chen, C. I., Bergsagel, P. L., Paul, H., Xu, W., Lau, A., Dave, N., Kukreti, V., Wei, E., Leung-Hagesteijn, C., Li, Z. H., Brandwein, J., Pantoja, M., Johnston, J., Gibson, S., Hernandez, T., Spaner, D., and Trudel, S. (2011) Single-agent lenalidomide in the treatment of previously untreated chronic lymphocytic leukemia. J Clin Oncol. 29, 1175-1181 273 Smith, J., Figeys, D. (2008) Recent developments in mass spectrometry-based quantitative phosphoproteomics. Biochemistry and cell biology 86, 137-148