Polyubiquitination of Insulin-like Growth Factor I Receptor (IGF-IR) Activation Loop Promotes Antibody-induced Receptor Internalization and Down-regulation

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Poly-ubiquitination of the Insulin-like Growth Factor I receptor (IGF-

IR) Activation Loop Promotes Antibody-induced Receptor Internalization and Down-regulation

Yifan Mao1,4$, Yonglei Shang1,4, Victoria C. Pham2, James A. Ernst2, Jennie R. Lill2

Suzie J. Scales3* and Jiping Zha1*

1Department of Research Pathology, 2Department of Protein Chemistry, 3Department of Molecular Biology, Genentech, South San Francisco, CA

4, The first two authors contributed equally to this work. *Corresponding authors’ addresses: Dr. Jiping Zha, Department of Cancer Biology, Integrated Drug Discovery Service Business Unit, Crown Bioscience, Inc., 6 Beijing West Road, Taicang Economic Development Area, Jiangsu

Province, P.R. China 215400. Tel: +86 512-5387-9803. Fax: +86 512-5387-9801. E-mail: [email protected]

Dr. Suzie J. Scales, Department of Molecular Biology, Genentech, South San Francisco, CA 94080, USA. Fax: +1 650-467-8882. Email: [email protected]

Running Title: Mapping and characterization of IGF-IR ubiquitination sites Background: Insulin-like growth factor I receptor (IGF-IR) can be downregulated by antibodies and ligand, but its ubiquitination sites remain unknown Results: The IGF-IR ubiquitination sites were mapped to its activation loop, adjacent to the phosphorylation sites Conclusion: IGF-IR requires phosphorylation of its activation loop, followed by K48- and K29-linked ubiquitination for downregulation Significance: This is the first mapping of IGF-IR ubiquitination sites

Key Words IGF-IR Receptor tyrosine kinase Ubiquitination Protein degradation Signal Transduction + special keyword: Downregulation

http://www.jbc.org/cgi/doi/10.1074/jbc.M111.288514The latest version is at JBC Papers in Press. Published on October 12, 2011 as Manuscript M111.288514

Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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ABSTRACT Ubiquitination has been implicated in

negatively regulating insulin-like growth factor I receptor (IGF-IR) activity. Due to the relative stability of IGF-IR in the presence of ligand stimulation, IGF-IR ubiquitination sites have yet to be mapped and characterized, thus preventing a direct demonstration of how the receptor ubiquitination contributes to downstream molecular cascades. We took advantage of an anti-IGF-IR antibody (h10H5) that induces more efficient receptor down-regulation to show that IGF-IR is promptly and robustly ubiquitinated. The ubiquitination sites were mapped to the two lysine residues in the IGF-IR activation loop (K1138 and K1141), and consisted of poly-ubiquitin chains formed through both K48 and K29 linkages. Mutation of these ubiquitinated lysine residues resulted in decreased h10H5-induced IGF-IR internalization and down-regulation, as well as a reduced cellular response to h10H5 treatment. We have therefore demonstrated that IGF-IR ubiquitination contributes critically to the down-regulating and anti-proliferative activity of h10H5. This finding is physiologically relevant because IGF-I appears to mediate ubiquitination of the same major sites as h10H5 (albeit to a lesser extent), and ubiquitination is facilitated by pre-existing phosphorylation of the receptor in both cases. Furthermore, identification of a breast cancer cell line with a defect in IGF-IR ubiquitination suggests that this could be an important tumor resistance mechanism to evade down-regulation-mediated negative regulation of IGF-IR receptor activity in cancer. Key words: IGF-IR, phosphorylation, ubiquitination, internalization, degradation.

INTRODUCTION Ubiquitination plays an important role

in regulating the localization, stability, and activity of receptor tyrosine kinases (RTK) (1-3). The covalent attachment of ubiquitin (Ub) to a substrate can be in the form of mono-ubiquitination of single or multiple lysine residues (multi-ubiquitination), or poly-

ubiquitination connected through its own lysine residues. While mono-ubiquitination is implicated in RTK internalization and endocytosis (4), poly-ubiquitination can have distinct functions depending on the lysine linkage. Canonical K48-linked poly-ubiquitin is commonly assumed to play the role of targeting proteins for degradation by the 26S proteasome (5,6), whereas K63-linked poly-ubiquitin can function in non-degradation pathways as a scaffold to help assemble multi-subunit complexes involved in endocytosis, signal transduction and DNA repair (7-9). Much less is known about the functions of so-called “atypical ubiquitin” linked via other lysine residues, K6, 11, 27, 29 and 33 (7,10).

The type I insulin-like growth factor receptor (IGF-IR) is a RTK critical for cellular proliferation, survival and transformation (11-13). Upon IGF ligand binding, the intra-cellular kinase is activated through an autophosphorylation-induced conformational change within the activation loop, which functions as a pseudo-substrate, blocking the catalytic site in the un-stimulated state (14). However, the role of ubiquitination in IGF-IR signaling is less well characterized, partly due to the relative stability of IGF-IR upon ligand stimulation (15). Recent studies have identified specific systems and conditions under which IGF-IR exhibits IGF-I dependent ubiquitination and down-regulation (16-19). Similar to other RTKs (20,21), IGF-IR phosphorylation appears to be required for its ubiquitination upon ligand stimulation, since mutation of either the catalytic lysine residue (K1003R) or phosphate modifiable tyrosine residues (Y1131, 1135 and 1136) in the activation loop compromises ubiquitination (19).

Three E3 ligases, Nedd4, Mdm2 and c-Cbl, have been implicated in mediating IGF-IR ubiquitination (16,17,22). While exactly how c-Cbl interacts with IGF-IR remains unknown, Nedd4 and Mdm2 have been demonstrated to exhibit their effects through adaptor molecules Grb10 and β-arrestin, respectively (16,17). Since Nedd4, Mdm2 and c-Cbl are also E3 ligases for multiple other substrates, analyses of their effects on IGF-IR ubiquitination are often complicated by

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accompanying changes in these other substrates. Previous deletion analysis revealed that the carboxyl terminal domain of IGF-IR is required for receptor ubiquitination as well as for downstream MAPK pathway activation (19). However, whether this simply removes the ubiquitin modification sites, prevents binding of adaptor molecule(s)/E3 ligase(s) or causes a gross conformational change, remains to be determined. Identification of the exact ubiquitination sites on IGF-IR should permit dissection of the role of ubiquitination in IGF-IR signaling, endocytosis, and down-regulation.

In contrast to the minimal to modest effect of IGF-I on IGF-IR stability, several antagonist anti-IGF-IR antibodies induce efficient receptor endocytosis and degradation (15,23-30), a mechanism likely contributing to their inhibitory activities. We took advantage of one such antibody, h10H5, to investigate its mechanism of IGF-IR internalization and down-regulation. h10H5 inhibits IGF-IR-mediated signaling by a dual mechanism, mediated both by blocking IGF-I/IGF-II binding and by inducing receptor down-regulation without any detectable agonist activity (30). Here we identify the lysines ubiquitinated upon h10H5 treatment and demonstrate that prior phosphorylation and ubiquitination are required for efficient IGF-IR internalization, down-regulation and inhibition of proliferation by this antibody. The discovery of a human cancer cell line with a defect in IGF-IR ubiquitination and down-regulation, but normal signaling capacity, lends further support to the importance of this negative regulatory mechanism for IGF-IR function.

EXPERIMENTAL PROCEDURES Reagents and cell lines:

Mouse anti-human IGF-IR antibodies h10H5 and 10F5 (Genentech) were generated as previously described (30). Commercially available antibodies were purchased as follows: Anti-Mdm2 (Calbiochem); anti-Nedd4 and anti-phosphotyrosine 4G10 (Upstate Biotechnology); anti-HA tag clone 12CA5 (Berkeley Antibody company); anti-

Cbl (BD Transduction Laboratories); anti-Cbl-b (Santa Cruz Biotechnology); anti-beta actin (Sigma); anti-IGF-IR beta and anti-ubiquitin P4D1 (Cell Signaling Technology). A549, SK-N-AS, HCC1419 and MHH-ES-1 cells were purchased from ATCC and cultured according to their recommendations. IGF-IR null murine embryonic fibroblasts (R- MEF cells; (31)) were kindly provided by Dr. Renato Baserga (Thomas Jefferson University, Philadelphia, PA). Since serum contains IGF-I, all experiments examining the effect of IGF-I (R&D Systems, catalog number 291-G1) stimulation were performed in serum-free media following 5 hours serum starvation. Unless otherwise indicated, h10H5 treatment was performed in complete (10% FBS) media. Mass spectrometric analysis

Immunoprecipitated samples were reduced (with 10 mM DTT) in SDS sample buffer, alkylated with 20 mM iodoacetamide and separated by 4-20% Tris-Glycine SDS-PAGE. After Coomassie-blue staining and destaining, gel bands were excised, and digested overnight at 37°C with 0.1 µg of trypsin (Promega). The reactions were quenched, concentrated and subjected to mass spectrometric analysis. Samples were injected via an auto-sampler onto an Agilent 1100 micro-flow HPLC system (Agilent, CA). Peptides were eluted directly into a nanospray ionization source and were analyzed using an LTQ-FT hybrid mass spectrometer (Thermo Fisher, San Jose, CA). Data was collected in data dependent mode with the parent ion being analyzed in the FTMS and the top 5 most abundant ions being selected for fragmentation and analysis in the LTQ. Resultant data was analyzed either using the search algorithm Mascot (Matrix Sciences, London, UK) or by de novo interpretation. siRNA knockdowns

The following siRNAs were transfected into A549 or SK-N-AS cells for 72 hrs using Dharmafect-1 according to the manufacturer’s protocol:- CCACAAAUCUGAUAGUAUUTT and AAUACUAUCAGAUUUGUGGTT for

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Mdm2, GGAGAGACCAUAUACAUUUTT and AAAUGUAUAUGGUCUCUCCTT for Nedd4, GACACUUUCCGGAUUACUATT and UAGUAAUCCGGAAAGUGUCTT for Cbl, and GGACAGACGGAAUCUCACATT and UGUGAGAUUCCGUCUGUCCTT for Cbl-b. Non-targeting control siRNAs were ordered from Dharmacon. Expression constructs and retroviral transduction

Mutations in the kinase domain of IGF-IR were generated by replacing the lysine residues with arginines (KR mutants) using the QuikChangeTM Multi Site-Directed Mutagenesis Kit (Stratagene) according to manufacturer’s protocol. The mutants were 2KR (K1138R, K1141R), 5KR (2KR + K1155, 1203, 1224) and 13KR (5KR + K968, 989, 993, 1025, 1058, 1081, 1100, 1120), K1003R and 3YF (Y1131F, Y1135F, Y1136F). In addition, an HA-tag was introduced at the carboxy terminus of WT and mutant IGF-IR. R-, A549 or MHH-ES-1 cells stably expressing WT or mutant IGF-IR were generated by retroviral transduction as described (32). Bulk puromycin-resistant cells with comparable IGF-IR levels were used for biochemical and/or functional analysis. Immunoprecipitation and Western blot analysis

Cells were lysed with RIPA buffer with protease and phosphatase inhibitor cocktails (Sigma), and IGF-IR was immunoprecipitated from lysates using anti-IGF-IR antibody 10F5 (which does not compete with h10H5) as before (30), or alternatively with anti-HA for IGF-IRβ-HA, where stated. For analyzing IGF-IR ubiquitination, cell lysis was carried out in dissociation buffer (120 mM sodium chloride, 50 mM HEPES pH 7.2, 1% Triton-X100, 0.5% deoxycholate, 25 µM MG-132 and 10 mM N-ethylmaleimide). To ensure that IGF-IR-associating proteins were separated from the receptor, cell lysates were dissolved in 1% (v/v) SDS and heated at 90˚C for 5 minutes, followed by 10-fold dilution with dissociation buffer prior to immunoprecipitation as described above. Cell lysates or

immunoprecipitates were separated by SDS-PAGE, transferred onto nitrocellulose membranes (Invitrogen), and probed with the indicated antibodies. Immunofluorescence

R- MEFs stably transfected with WT or mutant IGF-IR were incubated in 8-well glass slides (LabTekII) for 7 or 60 minutes with 5 µg/ml h10H5 and 25 µg/ml Alexa488-transferrin (Molecular Probes) in cell media containing lysosomal protease inhibitors (5 µM pepstatin A, 10 µg/ml leupeptin (Roche)), washed 5x on ice, fixed in 3% paraformaldhehyde. Following saponin permeabilization, samples were and processed for immunofluorescence as previously described (30), using Cy3-anti human (Jackson Immunoresearch, catalog number 709-166-149) to detect internalized h10H5 and rat anti-mouse LAMP1 (1D4B, BD Pharmingen) followed by Cy5-anti rat (Jackson Immunoresearch, catalog number 712-175-153) to label lysosomes. Slides were viewed by deconvolution microscopy using a DeltaVision® RT System (Applied Precision), using a 60x Olympus Uplano Apo objective. Images were captured with a Photometrics CH350 CCD camera powered by SoftWorx (version 3.4.4) software and assembled in Adobe Photoshop CS2. Quantitation of h10H5 uptake

R- MEFs stably transfected with WT or mutant IGF-IR were plated in 6-well plates to reach ~80% confluency 2 days later. Cells were incubated with 3 µg/ml Alexa488-labeled h10H5 (3.3 dye/Ab, labeled according to Molecular Probes’ instructions) on ice in complete (10% FBS, 2mM glutamine, 10 mM HEPES-containing) carbonate-independent medium (Invitrogen) for 1 hour, washed 3x, then chased in growth media at 37°C for 0, 20 or 60 minutes. Cells were then chilled, detached with 5 mM EDTA in PBS and either fixed immediately in 2% paraformaldehyde (unquenched) or after quenching for 1 hour on ice with 25 µg/ml anti-Alexa488 (Molecular Probes). Fluorescence intensities were measured by flow cytometry using a FACSCalibur (BD Biosciences), and the

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amount of internalization for each construct was calculated as the fluorescence of the quenched sample as a percentage of the unquenched sample at each timepoint, after correcting for incomplete quenching and any antibody losses during the chase, as previously described (33). Each sample was performed in duplicate and the results are the average and standard deviation of 3 independent experiments. Statistical significance compared to WT normalized to 100% was determined using the student’s t-test with 2 degrees of freedom. Proliferation Assay

MHH-ES-1 cells stably expressing WT or mutant IGF-IR were seeded onto 96-well plates at 8,000 cells/well, and incubated overnight with 10% serum-containing media containing 4 µg/ml h10H5 or isotype control antibody (gp120) for 1, 2 or 3 days, then cell viability was determined using the CellTiter-Glo Luminescence kit (Promega) according to the manufacturer’s instructions.

RESULTS h10H5 Induces IGF-IR Ubiquitination within the activation loop.

We previously showed that, unlike IGF-I ligand, the anti-IGF-IR antibody h10H5 induces rapid (within one hour) down-regulation of IGF-IR in SK-N-AS cells, via both lysosomal and proteasomal pathways (30). Here we further investigate the molecular mechanisms of this antibody-mediated downregulation. Since ubiquitination of IGF-IR has recently been implicated in IGF-I and anti-IGF-IR antibody-mediated receptor down-regulation (16,17,19,34), we tested whether a similar mechanism was also employed after engagement with the antibody h10H5. Serum-starved SK-N-AS cells were thus treated with 100 ng/ml (13nM) IGF-I or 4 µg/ml (28 nM) h10H5, after which IGF-IR was immuno-precipitated using the non-competing monoclonal antibody 10F5 (30) and subsequently analyzed by Western blotting using an anti-ubiquitin antibody. IGF-I treatment only slightly increased IGF-IR ubiquitination above basal levels (Fig. 1A);

however, h10H5 induced robust, albeit transient, IGF-IR ubiquitination, which peaked at 5-10 min of treatment and waned by 30 min, preceding any significant down-regulation at the receptor level.

This strong h10H5-mediated ubiquitination prompted us to search for modification sites on IGF-IR. IGF-IR was therefore immunoprecipitated from SK-N-AS cells that had been treated with h10H5 for 5 min to maximize receptor ubiquitination and analyzed by high resolution tandem mass spectrometry. Ubiquitinated tryptic peptides are characterized by the addition of two glycine residues (MW of 114 Da) for each lysine modification site (35). HPLC-MS/MS analysis revealed a doubly ubiquitinated IGF-IR tryptic peptide (Fig. 1B), which was manually validated and found to match with the precursor mass within 0.7 ppm (parts per million) mass accuracy. This peptide was only identified in the h10H5-treated sample and was absent in the control antibody-treated cells. The identified tryptic peptide contained two lysine residues, K1138 and K1141, which are located in the activation loop of IGF-IR kinase domain, both residues interestingly being juxtaposed to the phosphorylatable tyrosine residues Y1131, 1135 and 1136. Based on the crystal structures of inactive and active IGF-IR kinase domains (14,36), K1138 and K1141 become more surface-exposed upon phosphorylation-induced conformational changes in the activation loop (Supplemental Fig. 1), likely rendering these sites more accessible to ubiquitination. Since various ubiquitin linkages are associated with distinct functions, we performed similar HPLC-MS/MS analyses to determine the nature of the IGF-IR ubiquitination. This revealed poly-ubiquitination via both canonical K48 and non-canonical K29 poly-ubiquitin chain linkages in qualitatively similar amounts (Supplemental Fig. 2), thus potentially marking IGF-IR for degradation by different pathways. K29 is not a commonly observed poly-ubiquitin chain linkage and in a global cell lysate analysis typically contributes towards a minimal amount of the total linkages (37) and so this was an unexpected finding.

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IGF-IR Ubiquitination is Required for Efficient Receptor Down-regulation.

To confirm whether ubiquitination is the key triggering event for h10H5-mediated downstream processes, we took a loss-of-function approach to disrupt this regulation. Three E3 ligases, Nedd4, Mdm2 and c-Cbl, have been reported to participate in ligand-mediated IGF-IR ubiquitination (16,17,22). Since A549 human lung cancer cells have been shown to undergo IGF-IR ubiquitination upon IGF-I treatment and are more readily transfected than SK-N-AS cells (18), we transfected them with siRNAs to the above E3 ligases. SiRNAs against Nedd4, Mdm2, or c-Cbl/Cbl-b, but not the non-targeting control siRNA, caused significant depletion of their respective proteins, but did not alter the kinetics of h10H5-mediated IGF-IR down-regulation (Figure 2), suggesting redundancy among these and/or use of other E3 ligases.

Identification of the ubiquitination sites allowed us to take an alternative approach to investigate the role of IGF-IR ubiquitination by mutating them to prevent ubiquitination. We thus generated lysine to arginine mutations in IGF-IR: 2KR has both the identified lysines mutated (K1138/1141R); and 5KR (2KR + K1155/1203/1224R) has the three additional neighboring lysine residues mutated to prevent the utilization of cryptic ubiquitination sites when the major ones are mutated (Figure 3A). WT, 2KR and 5KR IGF-IR expression vectors were retrovirally transduced into IGF-IR null (R-) MEFs (31), chosen to avoid any potential interference from endogenous IGF-IR signaling. Similar levels of IGF-IR proteins were produced from the puromycin-resistant cell pools for all three versions of IGF-IR (Fig. 3B, bottom panel), so we compared their signaling capacity and their response to h10H5 treatment. Serum-starved R- MEF lines were stimulated with IGF-I, then IGF-IRs were immunoprecipitated and examined for tyrosine phosphorylation by Western blotting. 2KR IGF-IR exhibited comparable (or only slightly decreased) phosphorylation to the WT receptor, whereas 5KR showed more decreased levels (Fig. 3B). Phosphorylation of AKT and MAPK, two

major downstream pathways, was similar among WT, 2KR and 5KR IGF-IRs, (data not shown), although IGF-IR phosphorylation may not be a rate limiting step for these signaling events. Therefore, the conservative changes in 2KR and 5KR have a mild effect on IGF-IR kinase activity, which was somewhat unexpected. We next extended our analysis to the antibody-mediated IGF-IR ubiquitination by treating the (non-starved) cells with h10H5 and probing the IGF-IR immunoprecipitates with anti-ubiquitin. While WT IGF-IR exhibited the expected rapid increase ubiquitination within 5-30 min of h10H5 treatment, there was significantly less ubiquitination of 2KR and even less of 5KR over the same time course (Fig. 3C), supporting the identification of K1138 and K1141 in the activation loop as the primary modification sites. However, residual ubiquitination was still present in both mutants, indicating additional minor modification sites and/or that other lysine residues can partially substitute for mutated K1138/1141. In accordance with the reduced ubiquitination in 2KR and 5KR mutants, their kinetics of h10H5-induced receptor down-regulation were also delayed (Fig 3D). WT IGF-IR was depleted by 80% within 4 hours of treatment, whereas 2KR IGF-IR was more stable, with only ~50% degraded in 4 hours and ~70% in 8 hours. 5KR IGF-IR was more stable still, with essentially no detectable degradation during the 8-hour time course. Taken together, these data suggest that IGF-IR ubiquitination induced by h10H5 is required for efficient receptor down-regulation. Since anti-IGF-IR antibodies represent a therapeutic intervention rather than normal receptor physiology, we next examined whether the same ubiquitination sites are also utilized during normal ligand-dependent stimulation by IGF-I. To this end, we transduced A549 cells with HA-tagged WT, 2KR or 5KR IGF-IR to enable immunoprecipitation of exogenous IGF-IR with anti-HA tag antibodies. HA-tagged WT and mutant IGF-IRs exhibited comparable expression (Fig. 4A, 0 minute lanes, lower panel) and similar levels of IGF-I induced receptor tyrosine phosphorylation (Fig. 4A

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upper panel). The lack of changes in phosphorylation compared to the mild decreases in R- cells (Fig. 3B) may be attributable to the existence of endogenous IGF-IR in A549 cells, which can heterodimerize with the transfected proteins and obscure any small changes in phosphorylation. Nonetheless, compared to h10H5, IGF-I stimulation resulted in much slower kinetics of receptor down-regulation (Fig. 4B), as previously seen for endogenous IGF-IR in MCF7 cells (30). WT IGF-IR did not show any detectable down-regulation until 42 hr, by which time it was depleted by 81%, compared to only 36% and 25% for 2KR and 5KR, respectively, suggesting that these mutations also reduce ligand-dependent IGF-IR turnover. We also examined their ubiquitination and found it was defective in both mutants, although the differences compared to WT were smaller because IGF-I-mediated ubiquitination levels were so low even in WT that they were only just detectable at the 30-minute timepoint (Fig 1C). Thus, our data suggest that K1138 and K1141 are utilized as the key modification sites in IGF-I-induced as well as h10H5-induced receptor ubiquitination. Phosphorylation Facilitates h10H5-induced Ubiquitination.

Phosphorylation of IGF-IR appears to be required for IGF-I mediated receptor ubiquitination (19). To determine whether a similar relationship also exits for h10H5, we generated R- cells expressing two mutants defective in IGF-IR phosphorylation: an inactive kinase with its catalytic lysine replaced with arginine (K1003R) and a mutant IGF-IR with its activation loop locked in the inactive conformation (3YF: Y1131F/Y1135F/Y1136F; Figure 5A). In addition, we substituted all 13 lysine residues in the kinase domain except K1003 with arginine to control for background levels of ubiquitination (13KR). All mutants were expressed at similar levels (Fig. 5B, bottom panel), and as expected, neither K1003R nor 3YF exhibited any IGF-I dependent receptor phosphorylation (Fig. 5B, top panel). Surprisingly, the 13KR ubiquitination mutant

was as defective in phosphorylation as K1003R and 3YF, despite retaining the catalytic lysine at K1003. It is unclear whether this is due to a conformational change induced by so many mutations or whether ubiquitination affects the stability of the phosphorylation groups. The kinetics of h10H5-mediated IGF-IR ubiquitination revealed a moderately decreased in ubiquitination in 3YF and a strong decrease in K1003R, although to a lesser extent than the positive control ubiquitination mutant 13KR. Thus, IGF-IR phosphorylation is important for efficient receptor ubiquitination, although is not absolutely required. The time-courses of degradation were also compared: correlating with the extent of the receptor ubiquitination, WT IGF-IR was depleted 28%, 72% and 76% after 1, 4 and 8 hours continuous h10H5 treatment, respectively, whereas K1003R and 3YF were only decreased 29% and 51% at 8 hours, respectively. 13KR exhibited minimal changes in receptor levels over the entire time course, in agreement with its lack of ubiquitination. Taken together, these data demonstrate that IGF-IR phosphorylation, ubiquitination, and down-regulation are a series of inter-dependent events, with the robustness of the latter events depending on the former ones. IGF-IR Ubiquitination Facilitates Efficient Receptor Endocytosis.

Our studies have clearly demonstrated a link between h10H5-induced IGF-IR ubiquitination and down-regulation. However, there are several intermediate steps, such as receptor internalization and trafficking, before IGF-IR becomes accessible to the proteasomal and lysosomal degradation machineries (30,34). We therefore examined internalization of h10H5 by all five ubiquitination-defective IGF-IR mutants in R- cells by immunofluorescence. Consistent with previous studies in MCF7 cells (30), WT IGF-IR was rapidly internalized by h10H5, as demonstrated by numerous h10H5-positive cytoplasmic puncta after 7 minutes, most of which colocalized with the clathrin-mediated endocytic cargo transferrin (Supplemental Figure 3). 2KR and 5KR mutants produced

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far fewer h10H5-positive vesicles, although their similar co-localization with transferrin implies they also internalize via a clathrin-dependent route, while no vesicles were detected at all for the other three mutants (13KR, K1003R and 3YF) at this early time-point, suggesting autophosphorylation may be more important than ubiquitination for initiation of IGF-IR endocytosis. After 60 minutes uptake, the majority of the h10H5 signal was internal in WT IGF-IR cells (Fig. 6A), mostly co-localizing with the late endosomal and lysosomal marker LAMP1 (Supplemental Figure 4). By contrast, plasma membrane signal was still readily visible in all the mutants, indicating less uptake, particularly for 13KR, which remained almost exclusively at the plasma membrane (Fig. 6A), comparable to cells retained on ice (data not shown). 2KR, 5KR, K1003R and 3YF mutants all exhibited decreases in numbers of cytoplasmic vesicles and extent of lysosomal colocalization compared to WT (Fig. 6A and Supplemental Figure 4).

These results were confirmed quantitatively by flow cytometry using Alexa-488 conjugated h10H5 pre-bound to cells on ice. After chasing at 37oC for 20 or 60 minutes, any remaining surface 488-h10H5 was quenched with anti-Alexa488 antibodies to determine the percentage internalized. After 20 minutes, internalization was significantly defective compared to WT in all the mutants: 13KR (internalization decreased to 12% of WT levels), 2KR (44%), 5KR (53%), 3YF (29%) and K1003R (35%). Prolonged incubation for 60 minutes did not further enhance the internalization of the 13KR, 2KR or 5KR mutants, while a modest increase was observed for the phosphorylation-defective mutants, albeit still less than WT: 66% of WT levels for K1003R and 49% for 3YF. Taken together, these data confirm that h10H5-induced IGF-IR ubiquitination is important for efficient receptor internalization and subsequent delivery to lysosomes, with the two identified lysines (2KR) playing a major, but not the only, role.

Ubiquitination-dependent IGF-IR Down-regulation Contributes to the Inhibitory Activity of h10H5.

Two independent mechanisms of action have been described for several anti-IGF-IR antibodies; firstly blocking IGF ligand binding to IGF-IR, thereby inhibiting ligand-dependent downstream signaling, and secondly actively inducing IGF-IR down-regulation (15,23,26-28,30). However, it remains to be determined how these two mechanisms contribute to the full activity of the antibodies. The ubiquitination mutants provide us with an opportunity to analyze these two distinct roles separately for h10H5. We first identified a cell line that was responsive to h10H5 inhibition (presumably possessing the downstream signaling machinery), but expressed IGF-IR at low enough levels not to interfere with transfected IGF-IR constructs: MHH-ES1 cells, a human Ewing’s sarcoma cell line. WT, 2KR and 5KR IGF-IR expression vectors were transduced into MHH-ES1 cells and growth was measured using an in vitro proliferation assay. MHH-ES1 cells expressing WT and mutant IGF-IRs were comparable in their growth rates in the presence of a control antibody (Fig. 7A and B). However, h10H5 treatment resulted in 37% growth inhibition in the cells expressing WT IGF-IR at 72 hr, but only 23% and 16% inhibition in the 2KR and 5KR expressing cells, respectively (Fig. 7A and B), both mutants being statistically significantly different to WT. As expected, mutant IGF-IRs, especially 5KR, in the MHH-ES1 cells were relatively stable despite h10H5 treatment (Fig. 7C), suggesting that the low extent of growth inhibition in these mutants is primarily due to the ligand-blocking activity of h10H5. The extra inhibition (37% - 16% = 21% i.e. ~half) observed in the cells carrying WT IGF-IR likely results from combined down-regulation of the receptor in addition to ligand blocking, thus h10H5 likely utilizes both mechanisms to inhibit tumor growth (30). Deficiency of h10H5-induced Ubiquitination in Cancer Cells.

Our data suggest that IGF-IR ubiquitination appears to negatively regulate

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receptor-mediated signaling by promoting receptor down-regulation. We therefore examined IGF-IR ubiquitination in a panel of cancer cell lines to examine whether any of the cell types had developed mechanisms of evading such regulation. One cell line, human breast cancer HCC1419 cells, exhibited increased receptor phosphorylation upon IGF-I treatment (Fig. 8A), showing that they are ligand-responsive, in line with their lack of IGF-IR mutations as determined by sequencing (data not shown). However, unlike SK-N-AS cells, they failed to show any IGF-IR poly-ubiquitination upon h10H5 treatment (Fig. 8B). Furthermore, HCC1419 cells were specifically defective in IGF-IR internalization, but were competent at internalizing transferrin (Fig. 8C) and basic FGF (fibroblast growth factor; data not shown). Correlating with these ubiquitination and internalization defects, IGF-IR was stable throughout the entire 8-hour time course of h10H5 treatment (Fig. 8D). Thus, HCC1419 cells do not downregulate IGF-IR upon antibody treatement due to a defect in receptor ubiquitination.

DISCUSSION We have mapped h10H5-mediated

IGF-IR ubiquitination sites to K1138 and K1141 in the activation loop of the kinase domain adjacent to phosphorylation sites Y1134 and Y1135, and provided clear evidence through mutagenesis that IGF-IR ubiquitination is a key triggering event leading to efficient receptor internalization and degradation in multiple cell lines, thereby contributing to the anti-proliferative activity of this antibody. Substitution of the two major ubiquitinated lysines with arginines (2KR) decreased h10H5-induced ubiquitination, endocytosis and down-regulation, suggesting IGF-IR ubiquitination at these sites is important for endocytic down-regulation. The same mutations also decreased IGF-I-dependent receptor down-regulation, suggesting that K1138 and K1141 are also crucial for ligand-induced IGF-IR ubiquitination. The similarity between IGF-I and h10H5-induced IGF-IR ubiquitination extends beyond the conserved ubiquitination

sites, with both processes having similar wax and wane kinetics and possibly depending on IGF-IR phosphorylation, since IGF-IR phosphorylation mutants also fail to exhibit significant IGF-I or antibody-induced ubiquitination. These data help delineate an interdependent molecular cascade, which starts with IGF-I/II-induced IGF-IR autophosphorylation followed sequentially by h10H5-induced receptor ubiquitination, endocytosis and down-regulation, with IGF-IR ubiquitination as a critical trigger for the latter events. While IGF-IR phosphorylation appears to facilitate ubiquitination, it is not absolutely required, since the 3YF mutant (which blocks phosphorylation of the activation loop) and the K1003R mutatant (which is catalytically inactive) did exhibit low levels of ubiquitination. It is unclear, however, whether ubiquitination reciprocally regulates IGF-IR phosphorylation, given that in R- cells, the 5KR and 13KR (and to a lesser extent the 2KR) ubiquitination mutants also showed defects in phosphorylation. In WT IGF-IR, the K1138 residue forms a salt bridge to phosphorylated Y1136, stabilizing the active conformation of the kinase domain (14). Ubiquitination of K1138 would disrupt this salt bridge, with correspondingly reduced kinase activity and hence autophosphorylation. Mutation of K1138 to R would not necessarily prevent salt bridge formation, but could change its conformation somewhat. However, we cannot exclude the possibility that other kinases may mediate IGF-IR phosphorylation at other sites, although current models posit that IGF-IR is only transphosphorylated by itself (14,44).

The identification of ubiquitination sites K1138 and K1141 in the intracellular activation loop helps explain our previous finding that the stability of the intracellular domain of IGF-IR was increased by proteasomal inhibition (30). Although we did not map IGF-dependent IGF-IR ubiquitination sites, the 2KR and 5KR mutations that inhibited h10H5 ubiquitination and down-regulation also delayed the kinetics of ligand-induced down-regulation, suggesting that the primary K1138 and K1141 modification sites are shared by h10H5 and IGF-I. This is

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perhaps not surprising, given that both the antibody and ligand require receptor phosphorylation for efficient ubiquitination: IGF-I induces phosphorylation, while h10H5 ubiquitination is facilitated prior phosphorylation of the receptor (likely via IGF-I/II in the FBS, since this antibody blocks IGF binding). Such priming of ubiquitination by prior phosphorylation has been well documented for cytosolic proteins such as SCF E3 ligase substrates (reviewed by (38)), but is poorly documented for cell surface receptors. In the case of IGF-IR, priming may be explained by structural modeling, which suggests that phosphorylation-induced structural changes in the activation loop could render K1138 and K1141 more accessible to ubiquitination. Alternatively, since the phosphorylation mutants (3YF and K1003R) also show delayed internalization (especially at early timepoints), it is possible that phosphorylation facilitates endocytic trafficking of IGF-IR to a site harboring the E3 ligases that mediate ubiquitination. Despite sharing the same ubiquitination sites, the efficiency of ubiquitination is far lower for IGF-I than h10H5, likely explaining the much less efficient ligand-mediated down-regulation.

Other than the extent of ubiquitination and down-regulation, there are also notable differences between ligand and antibody-induced IGF-IR ubiquitination with respect to the E3 ligases employed. Three E3 ligases, Nedd4, Mdm2 and Cbl, have been implicated in ligand-mediated ubiquitination (16,17,22). Nedd4-dependent ubiquitination was shown, by use of antibodies preferentially recognizing poly-ubiquitin chains over single ubiquitin molecules, to induce multi-ubiquitination of IGF-IR (39). In contrast, Mdm2 and c-Cbl conjugate poly-ubiquitin chains onto IGF-IR, with distinct K63 and K48 linkages in an in vitro reconstitution system, respectively (22). The engagement of Mdm2 or c-Cbl correlates with low (5 ng/ml) and high (50-100 ng/ml) IGF-I levels, respectively. Therefore, ligand-mediated IGF-IR ubiquitination is subject to a complex regulation that is influenced by cellular context and growth conditions.

By contrast anti-IGF-IR antibodies, including h10H5, can induce IGF-IR down-regulation more robustly and consistently than ligand in a variety of cells, employing both lysosomal and proteasomal degradation pathways (30). Depletion of Nedd4, Mdm2 or c-Cbl/Cbl-b individually by siRNA treatment did not affect the kinetics of h10H5-induced down-regulation, suggesting simultaneous engagement of at least two of the known E3 ligases or the involvement of uncharacterized E3 ligase(s) in this process. Nedd4 and Mdm2 may be the most likely candidates because they have been associated with clathrin-dependent endocytosis, the route by which h10H5 likely internalizes in our cell lines (Supplemental Figure 3 and (30)), while c-Cbl has been associated with caveolin-dependent endocytosis of IGF-IR (22). h10H5 treatment resulted not only in K48-linked poly-ubiquitination, marking the receptor for proteasomal degradation similar to c-Cbl, but also in K29-linked poly-ubiquitination. This adds IGF-IR to a short list of proteins known to use K29 linkages, including NEMO and p65 (ubiquitinated by TRAF7) in the NF kappaB pathway (9,40), and Deltex (by AIP4/Itch) in the Notch pathway (41), all of which are thus targeted for lysosomal degradation, as we now observe for IGF-IR. However, not all known cases of K29-mediated poly-ubiquitination result in lysosomal degradation, since on the AMPK-related kinases NUAK1 and MARK4, ubiquitination via K29 (and K33) linkages appears to inhibit their phosphorylation and activation by the kinase LKB1 instead (42), thus the known functions of K29-linked ubiquitin are not yet fully understood. As discussed above, we cannot exclude a role for ubiquitination in regulating IGF-IR phosphorylation, although our data clearly show lysosomal trafficking and IGF-IR degradation is impaired if ubiquitination is defective. Ubiquitination of receptor tyrosine kinases is frequently associated with their internalization. However, mutation of the major ubiquitination sites in EGFR only resulted in increased receptor stability, while failing to decrease EGFR internalization (43).

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In our study, 2KR and 5KR IGF-IR mutants exhibited decreased internalization as well as increased stability, suggesting that IGF-IR internalization is a prerequisite for intracellular lysosomal and proteasomal degradation. The difference between the two systems could be due to their different ubiquitin modifications. Over 50% EGF receptors have K63-linked poly-ubiquitin in response to EGF stimulation, whereas IGF-IR exhibits exclusively K48 and K29 linked poly-ubiquitin upon h10H5 treatment (although it unclear if IGF-I ligand does the same). Thus, the nature and extent of poly-ubiquitination seems to dictate the functional outcome (although again the triggers are different).

In conclusion, we have delineated the order of a series of h10H5-triggered molecular events, starting with pre-existing IGF-IR phosphorylation followed by sequential receptor ubiquitination, internalization and down-regulation. Furthermore we identified a cell line (HCC1419) that is resistant to ubiquitination and down-regulation, despite having no ubiquitination site or other IGF-IR mutations. It therefore appears possible for cancer cells to develop a defect in IGF-IR ubiquitination that prevents the negative

regulation of receptor activity, further supporting IGF-IR ubiquitination as an upstream event required for receptor internalization and down-regulation. While it remains to be determined how this defect in ubiquitination occurs and how common this is among tumor cells, we speculate that resistance to ubiquitination might be an important mechanism to maximize IGF-dependent signaling for tumor growth or to become resistant to anti-IGF-IR antibody therapeutics. Furthermore, monitoring receptor ubiquitination could possibly be a means of identifying anti-IGF-IR non-responders in the clinic. Anti-IGF-IR antibodies have provided successful examples of how to inhibit RTK activity by increasing receptor ubiquitination and down-regulation. Promoting the turnover of key signaling molecules required for cancer growth and survival could be applied as a general therapeutic strategy. In addition to enhancing recruitment of E3 ligases, inhibiting deubiquitinating enzymes offers an alternative approach to regulate protein stability. Combination of both strategies may further augment the inhibition of the signaling pathways critical for tumorigenesis.

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FOOTNOTES Conflict of interest statement: All authors conducted this work while employees of Genentech. Acknowledgement: We thank Dr. Renato Baserga for generously providing IGF-IR null MEFs. $ Yifan Mao’s current address: Mendel Biotechnology, Inc., 3935 Point Eden Way, Hayward, CA 94545-3720. Abbreviations: IGF-IR, insulin-like growth factor I receptor; IGF-I, insulin-like growth factor I; RTK, receptor tyrosine kinase; Ub, ubiquitin; R- cells, IGF-IR knockout cells; LAMP1, lysosomal-associated membrane protein.

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FIGURE LEGENDS

Figure 1. h10H5 Induces IGF-IR Ubiquitination within the activation loop. (A) SK-N-AS cells were serum starved overnight, followed by stimulation with either 100 ng/ml IGF-I or 4 µg/ml h10H5 for the indicated times (in minutes). Cell lysates were immunoprecipitated with 10F5 anti-IGF-IR and Western blotted with anti-ubiquitin P4D1 (upper panel) and anti-IGF-IRβ subunit (lower panel, ~ 100 kDa) antibodies. (B) Tandem mass spectrum of the [M+2H]2+ ion (m/z 626.8779) of the doubly ubiquitinated peptide K*GGK*GLLPVR from IGF-IR. The spectrum was found during data dependent analysis of the total immunoprecipitate of IGF-IR on the LTQ-FT hybrid mass spectrometer. The m/z matches within -.06 Da and the b ion series (in blue, right to left above the sequence) and y ion series (in green, left to right below the sequence) show complete sequence coverage of this peptide. Figure 2. siRNA-mediated Mdm2, Nedd4, or Cbl and Cbl-b depletion does not affect 10H5-induced IGF-IR degradation. Non-targeting control (NTC) siRNA (A), Nedd4 (B), Mdm2 (C), or a mixture of Cbl and Cbl-b (D) siRNAs were transfected into A549 cells. After 72 hrs, the cells were treated with 2 µg/ml h10H5, and harvested at the indicated time points. Lysates were separated by SDS-PAGE and analyzed by Western blotting (without prior immunoprecipitation) with anti-IGF-IRβ antibodies to examine down-regulation of endogenous IGF-IR (A-D); and anti-Nedd4 (~100 kDa) (B), anti-Mdm2 (~ 90 kDa) (C), and anti-Cbl and anti-Cbl-b (~ 120 kDa) (D) antibodies to verify gene knockdown. β-actin was used as a loading control. Figure 3. 2KR and 5KR IGF-IRs exhibit decreased h10H5-induced ubiquitination and receptor down-regulation. (A) Schematic of the IGF-IR kinase domain (aa 944-1264 of human IGF-IR in gray, with the activation loop (AL, aa 1122-1143) in white) drawn to scale, showing the positions of the lysine to arginine substitutions in mutants 2KR (above) and 5KR (below). (B) 2KR and 5KR IGF-IRs are competent for signaling. R- MEFs stably expressing WT, 2KR, 5KR IGF-IR or empty vector were serum-starved for 5 hr and stimulated with 100 ng/ml IGF-I for the indicated time points. Lysates were immunoprecipitated with anti-IGF-IR antibody 10F5, and Western blotted with anti-phosphotyrosine antibody 4G10 (upper panel) or IGF-IRβ subunit (lower panel, loading control). (C) 2KR and 5KR IGF-IRs exhibit decreased h10H5-mediated IGF-IR ubiquitination. R- MEFs stably expressing WT or mutant IGF-IR were treated with 4 µg/ml 10H5 (in 10% FBS media) for the indicated time points. IGF-IR was immunoprecipitated with 10F5 and analyzed by Western blotting with anti ubiquitin antibody P4D1 and IGF-IRβ subunit (loading control). (D) 2KR and 5KR IGF-IRs are more resistant to h10H5-induced IGF-IR down-regulation. Cells were treated as in (C) except for longer timepoints and the kinetics of IGF-IR down-regulation was analyzed by direct Western blotting with anti-IGF-IRβ versus β-actin loading control. Figure 4. 2KR and 5KR mutations affect IGF-I-induced IGF-IR down-regulation and ubiquitination. (A) HA-tagged WT, 2KR and 5KR IGF-IRs are comparable in their signaling capacity. A549 cells stably expressing WT, 2KR or 5KR mutant IGF-IRβ-HA were serum-starved for 5 hr and stimulated with 100 ng/ml IGF-I for 10 or 30 minutes. Transfected IGF-IR was immunoprecipitated with anti-HA antibodies and analyzed by Western blotting with anti-phosphotyrosine antibody 4G10. (B) 2KR and 5KR IGF-IRs are more resistant to IGF-I-mediated IGF-IR down-regulation. A549 cells stably expressing HA-tagged WT or mutant IGF-IR were treated with 4 µg/ml 10H5 for 3, 24 or 42 hours. The kinetics of receptor down-regulation were analyzed by direct Western blotting with an anti-HA antibody. (C) 2KR and 5KR IGF-IRs exhibit decreased IGF-I-mediated IGF-IR ubiquitination. As in (A) except

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immunoprecipitates were Western blotted with the anti-ubiquitin antibody P4D1. Total IGF-IR (A and C) or β-actin (B) were used as loading controls. Note the ubiquitination induced by IGF-I is much less extensive than that induced by h10H5 in Figure 3. The asterisk denotes a non-specific ubiquitin band immunoprecipitated by the anti-HA antibody (present also in the empty vector control cells). Figure 5. IGF-IR phosphorylation is required for efficient receptor ubiquitination. (A) To-scale schematic of the IGF-IR kinase domain (gray, with the activation loop (AL) in white) showing the positions of the K1003R, 3YF (three tyrosine to phenylalanine) and 13KR (all 13 lysines in the kinase domain to arginine (except K1003)) mutants. (B) K1003R, 3YF and 13KR IGF-IRs are defective in IGF-I mediated signaling. R- MEFs with WT or mutant IGF-IR or empty vector were serum-starved for 5 hours and stimulated with 100 ng/ml IGF-I for 10 or 30 minutes. IGF-IR was immunoprecipitated with 10F5 and analyzed by Western blotting with anti-phosphotyrosine antibody 4G10. (C) K1003R, 3YF and 13KR IGF-IRs exhibit decreased h10H5-mediated receptor ubiquitination. R- MEFs stably expressing WT or mutant IGF-IR were treated with 4 µg/ml 10H5 in complete (10% FBS, which contains IGF-I) media for 5, 10 or 30 minutes, then IGF-IR was immunoprecipitated with 10F5 and Western blotted with anti-ubiquitin antibody P4D1. (D) As in (C) except h10H5 treatment was for 1, 4 or 8 hours and the lysates Western blotted with anti-IGF-IRβ subunit antibody without prior immunoprecipitation to analyze the kinetics of receptor down-regulation. Total IGF-IR (B and C) and β-actin (D) were used loading controls. Figure 6. Immunofluorescence and quantitative flow cytometry analysis of 10H5 uptake. (A) R- MEFs transfected with WT or all the mutant IGF-IRs (or vector alone, which gave no specific signal, not shown) were incubated for 60 minutes with h10H5 in the presence of lysosomal protease inhibitors to inhibit lysosomal degradation, fixed, permeabilized and the total antibody detected with Cy3-anti human. Scale bar = 30µm. See Supplemental Figure 4 for overlays with lysosomes. (B) R- MEFs transfected with WT or mutant IGF-IR were incubated on ice for 1 hour with Alexa488-labeled h10H5, washed, chased for 20 min or 60 min in the presence of lysosomal protease inhibitors, then any remaining surface antibody was quenched with anti-Alexa488 antibodies. Following flow cytometry measurements, the percentage internalized at each time point (after correcting for any losses or incomplete quenching) was calculated from three independent duplicate experiments, each normalized to WT as 100%, with mean and standard deviations shown. *, p = 0.05; **, p = 0.01; ***, p = 0.001 vs WT (by Student’s t-test). Figure 7. Cells expressing 2KR or 5KR IGF-IR are more resistant to h10H5-mediated growth inhibition. (A) Timecourse of the effect of h10H5 treatment on viability of MHH-ES-1 cells stably expressing HA-tagged WT, 2KR or 5KR mutant IGF-IR. Cells were treated with 10 µg/ml h10H5 or isotype control anti-gp120 antibody (Ctrl Ab) and cell growth was monitored by the CellTiter-Glo luminescence viability assay at 1, 2 and 3 days, with means and standard deviations of triplicates shown. (B) 3-day Cell Titer-Glo timepoint from (A) normalized to 100% for the gp120 control antibody. Statistical analysis using the Student’s t-test was performed by comparing mutant versus WT IGF-IR groups treated with h10H5. (C) MHH-ES-1 cells expressing WT, 2KR and 5KR IGF-IR-HA were treated with 2 µg/ml 10H5 for the indicated time points. The kinetics of receptor down-regulation were analyzed by direct Western blotting with anti-HA versus β-actin loading control. Figure 8. HCC1419 breast cancer cells exhibit defective h10H5-mediated IGF-IR ubiquitination, internalization, and down-regulation. (A) HCC1419 cells were serum-starved overnight and stimulated with 100 ng/ml IGF-I for 0, 10 or 30 minutes. Endogenous IGF-IR was

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immunoprecipitated with 10F5 and analyzed with anti-phosphotyrosine antibody 4G10. Total IGF-IRβ subunit was used a loading control. (B) HCC1419 and SK-N-AS cells were treated with h10H5 for the time points indicated, IGF-IR was immunoprecipitated with 10F5 and analyzed with anti-ubiquitin antibody P4D1. (C) HCC1419 cells were incubated on ice for 30 min with Alexa555-h10H5 (top) or Alexa555-transferrin (bottom), washed and chased at 37ºC for 0.5, 1 or 5 hours, then fixed and imaged. (D) HCC1419 cells were treated with h10H5 for the indicated time points, and IGF-IRβ subunit levels were analyzed by direct Western blotting compared to β-actin loading control.

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All ubiquitin-modified lysines identified are located in the activation loop of the IGF-IR kinase domain.The kinase domain (aa 944-1264 of human IGF-IR (ref. 36) is represented as a ribbon diagram shown in gray. The unphosphorylated activation loop from Gly1122 to Leu1143 is shown in red, while the phosphorylated activation loop is shown in orange (PDB code IK3A (refs. 14,44)). Ubiquitin-modified lysines in the unphosphorylated (unP) activation loop are shown in green, while those in the phosphorylated (P) form are inpurple and unmodified lysines are in blue. The ATP analog AMP-PCP is shown in yellow (upper left). The right panel is rotated 90° leftwards with respect to the left panel.

Free lysinesUb-lysines (unP)

Unphosphorylated activation loopPhosphorylated activation loop

K1138

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Mao et al., Supplemental Figure 1

AMao et al., Supplemental Figure 3

Distribution of h10H5 in IGF-IR WT and mutant R- cells after 7 minutes incubation(A) R- MEF cells stably transfected with wild-type or mutant IGF-IR were incubated with 5 µg/ml h10H5 and 25 µg/ml Alexa488-transferrin on ice for 1h to allow binding. Cells were then shifted to 37°C for 7 minutes to promote internalization, then washed, fixed, permeabilized and stained with Cy3-anti-human to detect h10H5 (shown in (A)). h10H5-positive vesicles are evident in WT and to a lesser extent in the 2KR and 5KR mutants, but not in the 3YF, K1003R or 13KR mutants at this early timepoint. (B) The internalizing IGF-IR constructs from (A) are shown (top row) with the Alexa488-transferrin signal (a clathrin-dependent endocytic cargo, middle row). Yellowish staining in the overlay (bottom)indicates overlap between h10H5 (red) and transferrin (green), which were similar in all cases. Insets show 2x magnification of the boxed areas (left, h10H5; middle, transferrin; right, merge), with white and yellow arrows indicating colocalization and no colocalization of h10H5 with transferrin, respectively.

WT 3YF K1003R

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rinh1

0H5

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Mao et al., Supplemental Figure 4WT 3YF K1003R

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Colocalization of h10H5 with lysosomes in IGF-IR transfected R- MEFs after 60 minutes internalization.R- MEF cells stably transfected with wild-type or mutant IGF-IR were incubated for 60 minutes with 5 µg/ml h10H5 in the presence of lysosomal protease inhibitors to minimize degradation. Cells were then washed, fixed, permeabilized and stained with Cy3-anti-human (red channel) to detect h10H5. The same h10H5 images as in Figure 6A are shown in red overlaid with rat anti-mouse LAMP1 (1D4B monoclonal) staining, detected with Cy5-anti-rat (shown in the green channel for optimal contrast). Yellowish staining indicates overlap between h10H5 and lysosomal LAMP1antibodies and is more prominent in the WT IGF-IR cells than in the mutants. Scale bar is 30 µm.

Scales and Jiping ZhaYifan Mao, Yonglei Shang, Victoria C. Pham, James A. Ernst, Jennie R. Lill, Suzie J.loop promotes antibody-induced receptor internalization and down-regulation

Poly-ubiquitination of the insulin-like growth factor I receptor (IGF-IR) activation

published online October 12, 2011J. Biol. Chem. 

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