Pseudopodial and -arrestin-interacting proteomes from migrating breast cancer cells upon PAR2 activation. Nikolaos Parisis 1,2,3 , Gergana Metodieva 1 , Metodi V. Metodiev 1, * 1 Department of Biological Sciences/Proteomics Unit, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, United Kingdom; 2 Institute of Molecular Genetics (IGMM), CNRS, UMR5535, University of Montpellier I and II, 34293 Montpellier, France; (current address) 3 Laboratory of Functional Proteomics, INRA, 34060 Montpellier, France (current address) *Author for correspondence ([email protected]) Running title: Pseudopodial and -arrestin proteomes Key words: PAR-2; pseudopodium; beta-arrestin; cell migration; proteomics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
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Pseudopodial and -arrestin-interacting proteomes from migrating breast cancer cells upon PAR2 activation.
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Pseudopodial and -arrestin-interacting proteomes from migrating breast cancer cells upon PAR2 activation.Nikolaos Parisis1,2,3, Gergana Metodieva1, Metodi V. Metodiev1, *
1Department of Biological Sciences/Proteomics Unit, University of Essex, Wivenhoe
Park, Colchester, Essex CO4 3SQ, United Kingdom;2Institute of Molecular Genetics (IGMM), CNRS, UMR5535, University of Montpellier I
and II, 34293 Montpellier, France; (current address)3Laboratory of Functional Proteomics, INRA, 34060 Montpellier, France (current
In addition, several isoforms of proteins that are known to interact with -arrestins, such
as phosphatases and ribosomal proteins, were also found here. We also excluded proteins
that were found in other proteomic screens as -arrestin-interactors but in our strategy
were considered as background due to their identification in the negative controls (SI
table 11). Also note that in this study, we did not over-express the receptor, reducing the
possibility of unspecific binding due to exogenous protein expression.
Importantly, we identify new interactions, such as TRIM29, glutaredoxin, periplakin and
others, and these interactions might play a role in PAR-2 signalling in cancer. The finding
of alpha-actinin as well as several other actin isoforms in our screens suggests that actin
cytoskeleton and -arrestins interact to manage receptor endocytosis [49] and leading
edge formation [50]. Similarly, their interaction with S100A7 increases the role of -
arrestins in metastatic pathways [51]. It is also shown here that PAR-2 regulates the
activity of kinases such as cyclin-dependent kinase (cdk) 13, N-acetyl-D-glucosamine
kinase and microtubule-associated serine/threonine-protein kinase 2, in a -arrestin-
dependent manner. More specifically, cdk13 and N-acetyl-D-glucosamine kinase are
pulled down with both -arrestins in uninduced conditions but in induced conditions this
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association is lost. The opposite seems to be the case for microtubule-associated
serine/threonine-protein kinase 2 (Tables 2, 3 and SI Table 9). Another two proteins we
showed here by mass spectrometry and independent co-IPs that are genuine -arrestin-
interacting proteins downstream of PAR-2 are nucleophosmin B23 (NPM1) and heat
shock protein 70 (HSP70) (Fig. 5). This is specifically interesting in the case of NPM1, as
it is a multifunctional protein that is over-expressed in cancer cells, compared to normal
cells, and it is considered a putative therapeutic target for hematological diseases [52-54].
Another protein found in our proteomic screen, the eukaryotic translation initiation factor
3 (eIF3), has been recently found as a -arrestin-2 interactor by another study [55]. This
interaction, which is mediated by epidermal growth factor (EGF) stimulation, promotes
binding of eIF3a to SHC and Raf-1 and subsequent suppression of the ERK pathway
[55]. Whether all these interactions take place in other types of cancer or are also
regulated by other GPCRs is a question that requires further study.
Furthermore, we compared the overlap between the -arrestin-interactors and the PD-
enriched protein screens in order to identify other proteins of special interest (Table 4).
These proteins are very likely to spatiotemporally translocate to the pseudopodium or
retained away from it in a -arrestin-dependent manner. For example, copine, T-plastin,
coatomer subunit beta and LDH-A were found associated with -arrestin-2 in untreated
conditions but when PAR-2 is activated this interaction is lost and the proteins are found
in pseudopodia. Alpha-actinin, AHNAK and elongation factor 1-alpha 1 are likely to be
recruited to the pseudopodia by -arrestins when PAR-2 is activated. Very importantly,
RNA helicase DDX5 has also been found in this overlap. DDX5 is a nuclear DEAD-box
containing protein with roles in development, miRNA pathway, in regulation of
transcription and RNA processing and ribosome biogenesis. It is mainly nuclear but it
was reported to translocate to the cytoplasm in order to recruit beta-catenin back to the
nucleus and control transcription [56]. In our screens, it was found as -arrestin-2 partner
only when cells were treated with FCS. Therefore, as the pseudopodia in our study were
formed upon PAR-2 activation, this shows that DDX5 is enriched at the PD when not
sequestered by -arrestin-2. As an additional validation of our screens, AP-2 complex
subunits alpha-1 and beta were found in both screens.
Conclusion
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In conclusion, this study presents the proteins and the signalling pathways enriched in the
pseudopodia of migrating breast cancer cells by using pseudopodia purification and mass
spectrometry-based proteomics. Furthermore, by using immunoprecipitation and mass
spectrometry, several PAR-2-regulated -arrestin-1 and -arrestin-2 interactors were
identified and revealed novel functions. This study therefore expanded the possible
functions of -arrestins, especially during breast cancer cell migration, and provides
directions for future research.
Acknowledgment
We acknowledge all members of the Metodiev laboratory for support and useful
discussions. We are grateful to Robert Lefkowitz, for kindly providing the -arrestin-
expressing vectors used in the study, Yoanne Mousseau for technical help and critical
advice in setting up the agarose migration assay. NP is grateful to Daniel Fisher for his
continuous support. We also acknowledge Daniel Fisher, Liliana Krasinska and James
Hutchins for proofreading and for critical recommendations while writing the first
version of the manuscript.
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Figure legends.Figure 1.
Schematic representation of the experimental strategies performed in this study.
Figure 2.
Endogenous expression of PAR-2 in MCF7 cells and ERK activity in untreated, FCS
(10%) or PAR-2 (50 M) treated conditions. (A) Detection of PAR-2 mRNA in MCF7
cells by reverse transcription PCR. (B) Levels of ERK activity upon treatment with P2AP
(50 M) or FCS. Cells were serum-starved and then treated or not with the indicated
agents for the indicated times and pERK was detected by anti-phospho p44/p42 MAPK
(Cell Signaling) in clear lysates by western blotting. Tubulin was used as loading control
Figure 3.
Effect of gradient concentrations of P2AP on chemotaxis and on ERK activity in MCF-7
cells. (A) Under agarose migration assay: Three wells were generated essentially as
described in [28]. cells were seeded in the middle well while 50 M P2AP or RPMI only
were added in the side wells. The chemoattractant diffused through the agarose creating a
gradient to which cells responded by migrating towards the side with the chemoattractant.
The image is representative of 3 individual experiments. Bar: 100 m; (B) The pools of
proteins from cell bodies (CB) or pseudopodia (PD) separated by the pseudopodia
purification assay were analysed by 10% SDS-PAGE. Arrow indicates the histone
proteins. Lines indicate the sliced gel pieces from which proteins were digested and
analysed by mass spectrometry. (C) Immunoblots of PARP-1 for validation of PD
purification (upper panel) and pERK (lower panel) in PD and CB. (D) Levels of
pERK1/2, pERK1 and pERK2 in PD and CB.
Figure 4.
Comparison of the protein identifications from the 2 different proteomic workflows used
to identify PD and CB enriched proteins (see text for details). In parallel, proteins are
categorized based on the PD/CB ratio, with the PD enriched proteins to the right-hand
side and CB-enriched to the left-hand side. The most enriched signalling pathways
represented by these proteins in each fraction are listed; the complete PANTHER
pathway analysis is shown in SI figure 2.
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Figure 5.
Validation of nucleophosmin (A) and heat shock protein 70 (HSP70; B) as -arrestin
interacting proteins by co-immunoprecipitation (co-IP). Control experiments were co-IPs
with -FLAG beads from untransfected cells or with unreactive protein A/G beads from
FLAG-tagged -arrestin-transfected cells.
SI Figure 1.
PANTHER pathway analysis of proteins enriched in the pseudopodia (left-hand panel) or
cell bodies (right-hand panel) [34]. Numbers in square brackets indicate the percentage of
the number of proteins corresponding to this pathway.
SI Figure 2
Gene Ontology (GO) terms of PD- and CD-enriched proteins in pie charts generated by
PANTHER Classification System (http://www.pantherdb.org/).
SI Figure 3
Ingenuity Pathway Analysis of proteins that co-immunoprecipitate with -arrestin-1 (dark
blue bars) or -arrestin-2 (light blue bars).
SI Figure 4 A-I.
Gene Ontology (GO) terms Molecular Function and Biological Process of -arrestin-
interacting proteins, upon treatment with FCS, P2AP or not treated, in pie charts
generated by PANTHER Classification System (http://www.pantherdb.org/).
References
[1] Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, et al. Cell migration: integrating signals from front to back. Science. 2003;302:1704-9.[2] Devreotes P, Janetopoulos C. Eukaryotic chemotaxis: distinctions between directional sensing and polarization. Journal of Biological Chemistry. 2003;278:20445-8.[3] Chodniewicz D, Klemke RL. Guiding cell migration through directed extension and stabilization of pseudopodia. Experimental Cell Research. 2004;301:31-7.[4] Wang Y, Ding SJ, Wang W, Jacobs JM, Qian WJ, Moore RJ, et al. Profiling signaling polarity in chemotactic cells. Proceedings of the National Academy of Sciences of the United States of America. 2007;104:8328-33.
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536537538539540541542543544
[5] Wang Y, Kelber JA, Tran Cao HS, Cantin GT, Lin R, Wang W, et al. Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression [corrected]. Proc Natl Acad Sci U S A. 2010;107:10920-5.[6] Nystedt S, Emilsson K, Larsson AK, Strombeck B, Sundelin J. Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor 2. European Journal of Biochemistry. 1995;232:84-9.[7] Dery O, Corvera CU, Steinhoff M, Bunnett NW. Proteinase-activated receptors: novel mechanisms of signaling by serine proteases. American Journal of Physiology. 1998;274:C1429-52.[8] Ramachandran R, Noorbakhsh F, Defea K, Hollenberg MD. Targeting proteinase-activated receptors: therapeutic potential and challenges. Nat Rev Drug Discov. 2012;11:69-86.[9] D'Andrea MR, Derian CK, Santulli RJ, Andrade-Gordon P. Differential expression of protease-activated receptors-1 and -2 in stromal fibroblasts of normal, benign, and malignant human tissues. American Journal of Pathology. 2001;158:2031-41.[10] Ge L, Shenoy SK, Lefkowitz RJ, DeFea K. Constitutive protease-activated receptor-2-mediated migration of MDA MB-231 breast cancer cells requires both beta-arrestin-1 and -2. Journal of Biological Chemistry. 2004;279:55419-24.[11] Ikeda O, Egami H, Ishiko T, Ishikawa S, Kamohara H, Hidaka H, et al. Signal of proteinase-activated receptor-2 contributes to highly malignant potential of human pancreatic cancer by up-regulation of interleukin-8 release. International Journal of Oncology. 2006;28:939-46.[12] Shi X, Gangadharan B, Brass LF, Ruf W, Mueller BM. Protease-activated receptors (PAR1 and PAR2) contribute to tumor cell motility and metastasis. Mol Cancer Res. 2004;2:395-402.[13] Hjortoe GM, Petersen LC, Albrektsen T, Sorensen BB, Norby PL, Mandal SK, et al. Tissue factor-factor VIIa-specific up-regulation of IL-8 expression in MDA-MB-231 cells is mediated by PAR-2 and results in increased cell migration. Blood. 2004;103:3029-37.[14] Kamath L, Meydani A, Foss F, Kuliopulos A. Signaling from protease-activated receptor-1 inhibits migration and invasion of breast cancer cells. Cancer Research. 2001;61:5933-40.[15] Morris DR, Ding Y, Ricks TK, Gullapalli A, Wolfe BL, Trejo J. Protease-activated receptor-2 is essential for factor VIIa and Xa-induced signaling, migration, and invasion of breast cancer cells. Cancer Research. 2006;66:307-14.[16] Krupnick JG, Benovic JL. The role of receptor kinases and arrestins in G protein-coupled receptor regulation. Annual Review of Pharmacology and Toxicology. 1998;38:289-319.[17] Ossovskaya VS, Bunnett NW. Protease-activated receptors: contribution to physiology and disease. Physiological Reviews. 2004;84:579-621.[18] DeFea KA, Zalevsky J, Thoma MS, Dery O, Mullins RD, Bunnett NW. beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2. Journal of Cell Biology. 2000;148:1267-81.[19] Ge L, Ly Y, Hollenberg M, DeFea K. A beta-arrestin-dependent scaffold is associated with prolonged MAPK activation in pseudopodia during protease-activated receptor-2-induced chemotaxis. Journal of Biological Chemistry. 2003;278:34418-26.[20] Alldridge L, Metodieva G, Greenwood C, Al-Janabi K, Thwaites L, Sauven P, et al. Proteome profiling of breast tumors by gel electrophoresis and nanoscale electrospray ionization mass spectrometry. J Proteome Res. 2008;7:1458-69.[21] Greenwood C, Metodieva G, Al-Janabi K, Lausen B, Alldridge L, Leng L, et al. Stat1 and CD74 overexpression is co-dependent and linked to increased invasion and lymph node metastasis in triple-negative breast cancer. J Proteomics. in print.[22] Laporte SA, Oakley RH, Zhang J, Holt JA, Ferguson SS, Caron MG, et al. The beta2-adrenergic receptor/betaarrestin complex recruits the clathrin adaptor AP-2 during endocytosis.
Proceedings of the National Academy of Sciences of the United States of America. 1999;96:3712-7.[23] Luttrell LM, Ferguson SS, Daaka Y, Miller WE, Maudsley S, Della Rocca GJ, et al. Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes. Science. 1999;283:655-61.[24] Blackhart BD, Emilsson K, Nguyen D, Teng W, Martelli AJ, Nystedt S, et al. Ligand cross-reactivity within the protease-activated receptor family. Journal of Biological Chemistry. 1996;271:16466-71.[25] Compton SJ, Cairns JA, Palmer KJ, Al-Ani B, Hollenberg MD, Walls AF. A polymorphic protease-activated receptor 2 (PAR2) displaying reduced sensitivity to trypsin and differential responses to PAR agonists. Journal of Biological Chemistry. 2000;275:39207-12.[26] Inoue H, Nojima H, Okayama H. High efficiency transformation of Escherichia coli with plasmids. Gene. 1990;96:23-8.[27] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-5.[28] Mousseau Y, Leclers D, Faucher-Durand K, Cook-Moreau J, Lia-Baldini AS, Rigaud M, et al. Improved agarose gel assay for quantification of growth factor-induced cell motility. Biotechniques. 2007;43:509-16.[29] Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc. 2006;1:2856-60.[30] Rauch A, Bellew M, Eng J, Fitzgibbon M, Holzman T, Hussey P, et al. Computational Proteomics Analysis System (CPAS): an extensible, open-source analytic system for evaluating and publishing proteomic data and high throughput biological experiments. J Proteome Res. 2006;5:112-21.[31] Keller A, Eng J, Zhang N, Li XJ, Aebersold R. A uniform proteomics MS/MS analysis platform utilizing open XML file formats. Mol Syst Biol. 2005;1:2005 0017.[32] Nesvizhskii AI, Keller A, Kolker E, Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem. 2003;75:4646-58.[33] Liu H, Sadygov RG, Yates JR, 3rd. A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Analytical Chemistry. 2004;76:4193-201.[34] Thomas PD, Kejariwal A, Campbell MJ, Mi H, Diemer K, Guo N, et al. PANTHER: a browsable database of gene products organized by biological function, using curated protein family and subfamily classification. Nucleic Acids Res. 2003;31:334-41.[35] Calvano SE, Xiao W, Richards DR, Felciano RM, Baker HV, Cho RJ, et al. A network-based analysis of systemic inflammation in humans. Nature. 2005;437:1032-7.[36] Brahmbhatt AA, Klemke RL. ERK and RhoA differentially regulate pseudopodia growth and retraction during chemotaxis. Journal of Biological Chemistry. 2003;278:13016-25.[37] Thompson EW, Paik S, Brunner N, Sommers CL, Zugmaier G, Clarke R, et al. Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines. Journal of Cellular Physiology. 1992;150:534-44.[38] Eden E, Hammel J, Rouhani FN, Brantly ML, Barker AF, Buist AS, et al. Asthma features in severe alpha1-antitrypsin deficiency: experience of the National Heart, Lung, and Blood Institute Registry. Chest. 2003;123:765-71.[39] Beckner ME, Chen X, An J, Day BW, Pollack IF. Proteomic characterization of harvested pseudopodia with differential gel electrophoresis and specific antibodies. Lab Invest. 2005;85:316-27.[40] Thingholm TE, Jensen ON, Larsen MR. Analytical strategies for phosphoproteomics. Proteomics. 2009;9:1451-68.
[41] Kubota Y, Ohkura K, Tamai KK, Nagata K, Nishiwaki K. MIG-17/ADAMTS controls cell migration by recruiting nidogen to the basement membrane in C. elegans. Proceedings of the National Academy of Sciences of the United States of America. 2008;105:20804-9.[42] Baranowsky A, Mokkapati S, Bechtel M, Krugel J, Miosge N, Wickenhauser C, et al. Impaired wound healing in mice lacking the basement membrane protein nidogen 1. Matrix Biology. 2008;29:15-21.[43] Kuk C, Gunawardana CG, Soosaipillai A, Kobayashi H, Li L, Zheng Y, et al. Nidogen-2: a new serum biomarker for ovarian cancer. Clinical Biochemistry. 2010;43:355-61.[44] Vishnubhotla R, Sun S, Huq J, Bulic M, Ramesh A, Guzman G, et al. ROCK-II mediates colon cancer invasion via regulation of MMP-2 and MMP-13 at the site of invadopodia as revealed by multiphoton imaging. Lab Invest. 2007;87:1149-58.[45] Xiao K, McClatchy DB, Shukla AK, Zhao Y, Chen M, Shenoy SK, et al. Functional specialization of beta-arrestin interactions revealed by proteomic analysis. Proc Natl Acad Sci U S A. 2007;104:12011-6.[46] Lefkowitz RJ, Rajagopal K, Whalen EJ. New roles for beta-arrestins in cell signaling: not just for seven-transmembrane receptors. Mol Cell. 2006;24:643-52.[47] Lefkowitz RJ, Shenoy SK. Transduction of receptor signals by beta-arrestins. Science. 2005;308:512-7.[48] Min J, Defea K. beta-arrestin-dependent actin reorganization: bringing the right players together at the leading edge. Mol Pharmacol.80:760-8.[49] Lamaze C, Fujimoto LM, Yin HL, Schmid SL. The actin cytoskeleton is required for receptor-mediated endocytosis in mammalian cells. J Biol Chem. 1997;272:20332-5.[50] Min J, Defea K. beta-arrestin-dependent actin reorganization: bringing the right players together at the leading edge. Mol Pharmacol. 2011;80:760-8.[51] Nasser MW, Qamri Z, Deol YS, Ravi J, Powell CA, Trikha P, et al. S100A7 enhances mammary tumorigenesis through upregulation of inflammatory pathways. Cancer Res.72:604-15.[52] Colombo E, Alcalay M, Pelicci PG. Nucleophosmin and its complex network: a possible therapeutic target in hematological diseases. Oncogene. 2011;30:2595-609.[53] Okuwaki M. The structure and functions of NPM1/Nucleophsmin/B23, a multifunctional nucleolar acidic protein. J Biochem. 2008;143:441-8.[54] Yung BY. Oncogenic role of nucleophosmin/B23. Chang Gung Med J. 2007;30:285-93.[55] Xu TR, Lu RF, Romano D, Pitt A, Houslay MD, Milligan G, et al. Eukaryotic translation initiation factor 3, subunit a, regulates the extracellular signal-regulated kinase pathway. Mol Cell Biol.32:88-95.[56] Yang L, Lin C, Liu ZR. P68 RNA helicase mediates PDGF-induced epithelial mesenchymal transition by displacing Axin from beta-catenin. Cell. 2006;127:139-55.[57] Ulazzi L, Sabbioni S, Miotto E, Veronese A, Angusti A, Gafa R, et al. Nidogen 1 and 2 gene promoters are abberantly methylated in human gastointestinal cancer. Mol Cancer. 2007;6:17