Identification of a Novel Proline-Arginine Motif Involved in CIN85-dependent Clustering of Cbl and Down-regulation of Epidermal Growth Factor Receptors

CIN85 is a multidomain adaptor protein implicated in Cbl-mediated down-regulation of receptor tyrosine kinases. CIN85 binding to Cbl is increased after growth factor stimulation and is critical for targeting receptor tyrosine kinases to clathrin-mediated endocytosis. Here we report the identification of a novel polyproline-arginine motif (PXXXPR), specifically recognized by the SH3 domains of CIN85 and its homologue CMS/CD2AP. This motif was indispensable for CIN85 binding to Cbl/Cbl-b, to other CIN85 SH3 domains' effectors, and for mediating an intramolecular interaction between the SH3-A domain and the proline-rich region of CIN85. Individual SH3 domains of CIN85 bound to PXXXPR peptides of Cbl/Cbl-b with micromolar affinities, whereas an extended structure of two or three SH3 domains bound with higher stoichiometry and increased affinity to the same peptides. This enabled full size CIN85 to simultaneously interact with multiple Cbl molecules, promoting their clustering in mammalian cells. The ability of CIN85 to cluster Cbl was important for ligand-induced stabilization of CIN85·Cbl·epidermal growth factor receptor complexes, as well as for epidermal growth factor receptor degradation in the lysosome. Thus, specific interactions of CIN85 SH3 domains with the PXXXPR motif in Cbl play multiple roles in down-regulation of receptor tyrosine kinases. CIN85 is a multidomain adaptor protein implicated in Cbl-mediated down-regulation of receptor tyrosine kinases. CIN85 binding to Cbl is increased after growth factor stimulation and is critical for targeting receptor tyrosine kinases to clathrin-mediated endocytosis. Here we report the identification of a novel polyproline-arginine motif (PXXXPR), specifically recognized by the SH3 domains of CIN85 and its homologue CMS/CD2AP. This motif was indispensable for CIN85 binding to Cbl/Cbl-b, to other CIN85 SH3 domains' effectors, and for mediating an intramolecular interaction between the SH3-A domain and the proline-rich region of CIN85. Individual SH3 domains of CIN85 bound to PXXXPR peptides of Cbl/Cbl-b with micromolar affinities, whereas an extended structure of two or three SH3 domains bound with higher stoichiometry and increased affinity to the same peptides. This enabled full size CIN85 to simultaneously interact with multiple Cbl molecules, promoting their clustering in mammalian cells. The ability of CIN85 to cluster Cbl was important for ligand-induced stabilization of CIN85·Cbl·epidermal growth factor receptor complexes, as well as for epidermal growth factor receptor degradation in the lysosome. Thus, specific interactions of CIN85 SH3 domains with the PXXXPR motif in Cbl play multiple roles in down-regulation of receptor tyrosine kinases. Growth factor binding to receptor tyrosine kinases (RTKs) 1The abbreviations used are: RTK, receptor tyrosine kinase; CIN85, Cbl interacting protein of 85 kDa; CD2AP, CD2-associated protein; CMS, Cas ligand with multiple Src homology 3 domains; EGF(R), epidermal growth factor (receptor); GST, glutathione S-transferase; SH3, Src homology 3; HA, hemagglutinin; GFP, green fluorescent protein; HEK, human embryonic kidney; CHO, Chinese hamster ovary; ITC, isothermal calorimetry.1The abbreviations used are: RTK, receptor tyrosine kinase; CIN85, Cbl interacting protein of 85 kDa; CD2AP, CD2-associated protein; CMS, Cas ligand with multiple Src homology 3 domains; EGF(R), epidermal growth factor (receptor); GST, glutathione S-transferase; SH3, Src homology 3; HA, hemagglutinin; GFP, green fluorescent protein; HEK, human embryonic kidney; CHO, Chinese hamster ovary; ITC, isothermal calorimetry. promotes receptor autophosphorylation, association of intracellular signaling proteins with receptors, and phosphorylation of multiple substrates (1Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4569) Google Scholar). Subsequent changes in receptor substrates, including modulations of enzymatic activities and modifications of proteins and lipids, lead to the assembly of signaling networks that ultimately control cellular responses including cell proliferation, migration, or differentiation (2Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3456) Google Scholar). Activated RTKs are also rapidly relocalized from the cell surface into the endosomal compartment, from where they can be recycled back to the plasma membrane or alternatively sorted to the lysosome for degradation. The processes of receptor internalization and endocytosis are regulated via a network of protein-protein and protein-lipid interactions, as well as protein post-translational modifications, such as phosphorylation or ubiquitination (3Dikic I. Giordano S. Curr. Opin. Cell Biol. 2003; 15: 128-135Crossref PubMed Scopus (281) Google Scholar, 4Waterman H. Yarden Y. FEBS Lett. 2001; 490: 142-152Crossref PubMed Scopus (271) Google Scholar). Several RTKs, including epidermal growth factor (EGF) receptors, are ubiquitinated and down-regulated upon interactions with the Cbl family of ubiquitin ligases (5Joazeiro C.A. Hunter T. Science. 2000; 289: 2061-2062Crossref PubMed Scopus (39) Google Scholar, 6Thien C.B. Walker F. Langdon W.Y. Mol. Cell. 2001; 7: 355-365Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Cbl binding to EGF receptors occurs at the plasma membrane (7Stang E. Johannessen L.E. Knardal S.L. Madshus I.H. J. Biol. Chem. 2000; 275: 13940-13947Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 8de Melker A.A. van der Horst G. Calafat J. Jansen H. Borst J. J. Cell Sci. 2001; 114: 2167-2178Crossref PubMed Google Scholar), and following receptor internalization they remain associated throughout the endocytic compartment (8de Melker A.A. van der Horst G. Calafat J. Jansen H. Borst J. J. Cell Sci. 2001; 114: 2167-2178Crossref PubMed Google Scholar, 9Longva K.E. Blystad F.D. Stang E. Larsen A.M. Johannessen L.E. Madshus I.H. J. Cell Biol. 2002; 156: 843-854Crossref PubMed Scopus (318) Google Scholar). Recent data (10Haglund K. Sigismund S. Polo S. Szymkiewicz I. Di Fiore P.P. Dikic I. Nat. Cell Biol. 2003; 5: 461-466Crossref PubMed Scopus (651) Google Scholar) have indicated that attachment of a single ubiquitin, rather than polyubiquitin chains, to EGF and platelet-derived growth factor receptors is sufficient for receptor endocytosis and that multiple monoubiquitination events ensure proper receptor sorting and subsequent degradation in the lysosome. Cbl can also promote receptor endocytosis via a pathway that is functionally separable from its ubiquitin ligase activity and is dependent on Cbl interactions with the adaptor protein CIN85 (11Petrelli A. Gilestro G.F. Lanzardo S. Comoglio P.M. Migone N. Giordano S. Nature. 2002; 416: 187-190Crossref PubMed Scopus (371) Google Scholar, 12Soubeyran P. Kowanetz K. Szymkiewicz I. Langdon W.Y. Dikic I. Nature. 2002; 416: 183-187Crossref PubMed Scopus (484) Google Scholar). CIN85, also known as Ruk and SETA, is a broadly expressed protein containing three SH3 domains, a proline-rich region, and a coiled-coil domain (13Bogler O. Furnari F.B. Kindler-Roehrborn A. Sykes V.W. Yung R. Huang H.J. Cavenee W.K. Neuro-oncol. 2000; 2: 6-15Crossref PubMed Google Scholar, 14Gout I. Middleton G. Adu J. Ninkina N.N. Drobot L.B. Filonenko V. Matsuka G. Davies A.M. Waterfield M. Buchman V.L. EMBO J. 2000; 19: 4015-4025Crossref PubMed Scopus (121) Google Scholar, 15Take H. Watanabe S. Takeda K. Yu Z.X. Iwata N. Kajigaya S. Biochem. Biophys. Res. Commun. 2000; 268: 321-328Crossref PubMed Scopus (136) Google Scholar). The multidomain structure of CIN85 and its homologue CMS/CD2AP enables them to associate with various proteins including Cbl, Grb2, p85 subunit of phosphatidylinositol 3-kinase, CD2 receptors, SETA-binding protein 1 (SB-1), SLP-65/BLNK, Alg2-interacting protein 1 (AIP1), and p130Cas (16Dikic I. FEBS Lett. 2002; 529: 110-115Crossref PubMed Scopus (163) Google Scholar). These interactions promote formation of CIN85-linked protein networks that are implicated in the control of RTK signaling, actin reorganization, T cell functions, kidney architecture, and apoptotic signals (16Dikic I. FEBS Lett. 2002; 529: 110-115Crossref PubMed Scopus (163) Google Scholar). CIN85 binds to Cbl via its SH3 domains, and their association is enhanced by growth factor-induced tyrosine phosphorylation of Cbl (15Take H. Watanabe S. Takeda K. Yu Z.X. Iwata N. Kajigaya S. Biochem. Biophys. Res. Commun. 2000; 268: 321-328Crossref PubMed Scopus (136) Google Scholar, 17Szymkiewicz I. Kowanetz K. Soubeyran P. Dinarina A. Lipkowitz S. Dikic I. J. Biol. Chem. 2002; 277: 39666-39672Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), whereas the proline-rich region of CIN85 constitutively interacts with endophilins (11Petrelli A. Gilestro G.F. Lanzardo S. Comoglio P.M. Migone N. Giordano S. Nature. 2002; 416: 187-190Crossref PubMed Scopus (371) Google Scholar, 12Soubeyran P. Kowanetz K. Szymkiewicz I. Langdon W.Y. Dikic I. Nature. 2002; 416: 183-187Crossref PubMed Scopus (484) Google Scholar), regulatory components of clathrin-coated pits (18Brodin L. Low P. Shupliakov O. Curr. Opin. Neurobiol. 2000; 10: 312-320Crossref PubMed Scopus (190) Google Scholar). Based on these features, CIN85 can rapidly recruit endophilins to complexes with activated receptors, thus controlling receptor internalization (11Petrelli A. Gilestro G.F. Lanzardo S. Comoglio P.M. Migone N. Giordano S. Nature. 2002; 416: 187-190Crossref PubMed Scopus (371) Google Scholar, 12Soubeyran P. Kowanetz K. Szymkiewicz I. Langdon W.Y. Dikic I. Nature. 2002; 416: 183-187Crossref PubMed Scopus (484) Google Scholar). Importantly, an analogous mechanism has been shown recently (19Lynch D.K. Winata S. Lyons R.J. Hughes W.E. Lehrbach G.M. Wasinger V. Corthals G. Cordwell S. Daly R.J. J. Biol. Chem. 2003; 278: 21805-21813Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) for CMS/CD2AP. Interestingly, the CMS/CD2AP proline-rich region also interacts with cortactin in a ligand-dependent manner, recruiting it to the CD2AP· Cbl·EGFR complex. These data open an intriguing possibility that CIN85/CD2AP adaptor proteins link receptor endocytosis with cytoskeletal rearrangements (19Lynch D.K. Winata S. Lyons R.J. Hughes W.E. Lehrbach G.M. Wasinger V. Corthals G. Cordwell S. Daly R.J. J. Biol. Chem. 2003; 278: 21805-21813Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). CIN85 associates with two members of the Cbl family, Cbl and Cbl-b, but not Cbl-3, and this interaction depends on the presence of a minimal binding domain in their distal carboxyl-terminal tails (17Szymkiewicz I. Kowanetz K. Soubeyran P. Dinarina A. Lipkowitz S. Dikic I. J. Biol. Chem. 2002; 277: 39666-39672Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Surprisingly, binding of SH3 domains of CIN85 or CMS to Cbl/Cbl-b is not mediated via a PXXP motif (17Szymkiewicz I. Kowanetz K. Soubeyran P. Dinarina A. Lipkowitz S. Dikic I. J. Biol. Chem. 2002; 277: 39666-39672Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 20Kirsch K.H. Georgescu M.M. Shishido T. Langdon W.Y. Birge R.B. Hanafusa H. J. Biol. Chem. 2001; 276: 4957-4963Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), known to be critical for ligand recognition by the majority of SH3 domains (21Mayer B.J. J. Cell Sci. 2001; 114: 1253-1263Crossref PubMed Google Scholar). Proline-rich sequences are among the most abundant motifs found in signaling proteins (22Kay B.K. Williamson M.P. Sudol M. Faseb J. 2000; 14: 231-241Crossref PubMed Scopus (1031) Google Scholar). Numerous protein interaction modules including the SH3 domain, the WW domain, the EVH1 domain, and the GYF domain have been shown to bind to distinct proline-based sequences found in various signaling proteins (21Mayer B.J. J. Cell Sci. 2001; 114: 1253-1263Crossref PubMed Google Scholar, 22Kay B.K. Williamson M.P. Sudol M. Faseb J. 2000; 14: 231-241Crossref PubMed Scopus (1031) Google Scholar, 23Cesareni G. Panni S. Nardelli G. Castagnoli L. FEBS Lett. 2002; 513: 38-44Crossref PubMed Scopus (121) Google Scholar, 24Macias M.J. Wiesner S. Sudol M. 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Two prolines in the PXXP motif occupy the hydrophobic pockets, whereas the third binding pocket associates with a positively charged residue flanking the PXXP motif (21Mayer B.J. J. Cell Sci. 2001; 114: 1253-1263Crossref PubMed Google Scholar). In addition, several reports have indicated that unconventional proline-based sequences, which are distinct from PXXP motifs, can functionally interact with various SH3 domains (27Kami K. Takeya R. Sumimoto H. Kohda D. EMBO J. 2002; 21: 4268-4276Crossref PubMed Scopus (143) Google Scholar, 28Mongiovi A.M. Romano P.R. Panni S. Mendoza M. Wong W.T. Musacchio A. Cesareni G. Di Fiore P.P. EMBO J. 1999; 18: 5300-5309Crossref PubMed Scopus (154) Google Scholar, 29Lewitzky M. Kardinal C. Gehring N.H. Schmidt E.K. Konkol B. Eulitz M. Birchmeier W. Schaeper U. Feller S.M. Oncogene. 2001; 20: 1052-1062Crossref PubMed Scopus (87) Google Scholar, 30Kato M. Miyazawa K. Kitamura N. J. Biol. Chem. 2000; 275: 37481-37487Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Here we identified a novel atypical polyproline motif present in the distal tail of Cbl/Cbl-b, which serves as a high affinity binding site for the SH3 domains of CIN85/CMS adaptor molecules. We also show that CIN85, via its three SH3 domains, clusters Cbl molecules, which is a critical step for oligomerization of CIN85·Cbl·EGFR complexes leading to internalization and efficient degradation of activated EGF receptors. CIN85 can thus function as an RTK scaffold molecule, like G protein-coupled receptor specific β-arrestins, controlling receptor endocytosis and degradation. Products, Antibodies, and Expression Vectors—EGF was purchased from Intergen; antibodies recognizing Cbl-b (C-20), phosphotyrosine (PY99), autophosphorylated EGF receptor (phosphotyrosine 1173, anti-pEGFR), or extracellular signal-regulated kinase 2 (C14) were from Santa Cruz Biotechnology, Inc., mouse anti-HA (12CA5) was from Roche Applied Science, mouse anti-FLAG M2 and M5 antibodies were from Sigma, and rabbit anti-GFP antibodies were from Molecular Probes. Anti-Cbl (RF), anti-CIN85 (CT), and anti-EGFR receptor (RK2) antibodies were used as described previously (17Szymkiewicz I. Kowanetz K. Soubeyran P. Dinarina A. Lipkowitz S. Dikic I. J. Biol. Chem. 2002; 277: 39666-39672Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 31Haglund K. Shimokawa N. Szymkiewicz I. Dikic I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12191-12196Crossref PubMed Scopus (125) Google Scholar). Constructs of Cbl, Cbl-b, FLAG-CMS, FLAG-CIN85, FLAG-CIN85–3SH3, FLAG-CIN85-PCc, GST fusion proteins encoding the SH3 domains of CIN85, and FLAG-tagged ubiquitin were described previously (12Soubeyran P. Kowanetz K. Szymkiewicz I. Langdon W.Y. Dikic I. Nature. 2002; 416: 183-187Crossref PubMed Scopus (484) Google Scholar, 17Szymkiewicz I. Kowanetz K. Soubeyran P. Dinarina A. Lipkowitz S. Dikic I. J. Biol. Chem. 2002; 277: 39666-39672Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 31Haglund K. Shimokawa N. Szymkiewicz I. Dikic I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12191-12196Crossref PubMed Scopus (125) Google Scholar). FLAG-CIN85-ΔA construct encodes amino acids 78 to 661 of CIN85, and FLAG-CIN85-ΔAB encodes amino acids 211 to 661. Expression vectors encoding for Ruk SH3-A domain in pGEX vector was provided by Vladimir Buchman, HA-SLP-65 in pSGN vector was provided by Juergen Wienands, and pEGFP-Cbl was provided by Nancy Lill. Site-directed Mutagenesis—All mutant constructs were generated by PCR using QuikChange (Stratagene). The following tryptophan residues in pcDNA-FLAG-CIN85 were mutated to alanine: SH3-A*-W36A, SH3-B*-W135A, SH3-C*-W306A, SH3-AB*-W36A, W135A, SH3-BC*-W135A, W306A, and SH3-ABC*-W36A, W135A, W306A. The arginine residues were mutated to alanines in the following proteins: Cbl-R829A, Cbl-b-R911A, SLP-65-R248A,R313A, CIN85-R404A. Cbl-b-P906A, -P908A, and -P910A constructs have respective proline residues mutated to alanines. The constructs were verified by sequencing. The sequences of the oligonucleotides used are available upon request. Cell Culture and Transfections—HEK293T and CHO-EGFR cells were used as described previously (12Soubeyran P. Kowanetz K. Szymkiewicz I. Langdon W.Y. Dikic I. Nature. 2002; 416: 183-187Crossref PubMed Scopus (484) Google Scholar, 17Szymkiewicz I. Kowanetz K. Soubeyran P. Dinarina A. Lipkowitz S. Dikic I. J. Biol. Chem. 2002; 277: 39666-39672Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 31Haglund K. Shimokawa N. Szymkiewicz I. Dikic I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12191-12196Crossref PubMed Scopus (125) Google Scholar). Cells were transfected with LipofectAMINE reagent (Invitrogen) following the manufacturer's instructions. 30 h after transfection the cells were starved for an additional 12 h and stimulated with 100 ng/ml EGF for indicated times. Cells were lysed in ice-cold 1% Triton X-100 lysis buffer (pH 7.4; 50 mm Hepes, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 10% glycerol) containing a mixture of protease and phosphatase inhibitors. The lysates were cleared by centrifugation at 13000 rpm for 20 min at 4 °C. Peptide Synthesis and Peptide Binding Assays—All peptides were synthesized following the Fmoc (N-(9-fluorenyl)methoxycarbonyl) strategy using Fmoc amide resin resulting in carboxyl-terminally amidated peptides. Peptides were purified by reverse phase high pressure liquid chromatography using a C18 column. Matrix-assisted laser desorption ionization time-of-flight-mass spectrometry analysis confirmed the correct molecular weight for each peptide. Each peptide had a single amino-terminal cysteine residue that was used for coupling to a SulfoLink resin (following the product instruction; Pierce). In peptide binding assays, 5 nmol of each SulfoLink-immobilized peptide was incubated with cell lysates for 2 h, and after washing three times in the lysis buffer the protein complex was resolved by SDS-PAGE. In peptide competition experiments, the lysates were incubated with increasing amounts of peptides 1 h before and during immunoprecipitation. Isothermal Calorimetry (ITC) Measurements—ITC experiments were conducted on a VP ITC (Microcal Inc., Northampton, MA) as described previously (32Renzoni D.A. Pugh D.J. Siligardi G. Das P. Morton C.J. Rossi C. Waterfield M.D. Campbell I.D. Ladbury J.E. Biochemistry. 1996; 35: 15646-15653Crossref PubMed Scopus (82) Google Scholar, 33O'Brien R. DeDecker B. Fleming K.G. Sigler P.B. Ladbury J.E. J. Mol. Biol. 1998; 279: 117-125Crossref PubMed Scopus (105) Google Scholar). Experiments were performed at 25 °C in phosphate-buffered saline at pH 7.4. The data were analyzed using the ORIGIN software supplied with the calorimeter. In a typical titration ∼1.5 ml of 25 μm SH3 domain was placed in the calorimeter cell, and 250 μm peptide was injected in 25 × 10-μl injections. The reported data for the stoichiometry and KD are based on at least two repetitions of the titrations. The actual measurement data for these experiments are available upon request. Ligand Internalization Assays—Ligand internalization assays were performed as described previously (12Soubeyran P. Kowanetz K. Szymkiewicz I. Langdon W.Y. Dikic I. Nature. 2002; 416: 183-187Crossref PubMed Scopus (484) Google Scholar). Briefly, cell monolayers of transfected CHO cells were incubated for 1 h at 4 °C with 125I-labeled EGF, washed twice with binding buffers, and then incubated at 37 °C for the indicated time intervals. Cells were transferred on ice and washed with either cold phosphate-buffered saline or mild acidic buffer to remove surface-bound radiolabeled EGF. The remaining radioactivity in cells was quantified by a γ-counter following cell lysis. Each point was measured in quadruplicate and expressed as a percentage (average ± S.D.) of internalized versus total cell-associated radioactive EGF. The amounts of transfected proteins were monitored by Western blotting (data not shown). Biochemical Assays—Lysates with adjusted protein concentration (Bradford assay; Bio-Rad) were incubated with antibody for 2 h at 4 °C. Immune complexes were precipitated following 1-h incubation with protein A-agarose beads. After washing in cold lysis buffer, the complexes were resuspended in Laemmli sample buffer (Bio-Rad), boiled, and resolved by SDS-PAGE. For GST binding assays, GST fusion proteins were adsorbed on glutathione-Sepharose beads, incubated with the lysates for 2 h at 4 °C, washed in the lysis buffer, and proceeded for Western blotting as above. In vitro translation was performed with the TNT transcription/translation system (Promega), according to the manufacturer's instructions. EGF receptor degradation assays were performed as described previously (31Haglund K. Shimokawa N. Szymkiewicz I. Dikic I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12191-12196Crossref PubMed Scopus (125) Google Scholar). Briefly, HEK293T were transfected with EGFR, Cbl, FLAG-ubiquitin, and CIN85 or indicated CIN85 mutants, serum-starved, and stimulated with EGF (100 ng/ml) for indicated times. The cell lysates were subjected to immunoblotting with anti-EGFR (RK2) and anti-extracellular signal-regulated kinase 2 antibodies. The levels of EGFR were quantified for each time point from three independent experiments by using the NIH Image program (1.62) and expressed as the percentage of remaining EGFR for every time point as compared with unstimulated cells in the same experiment. Identification of the Consensus Binding Motif for the SH3 Domains of CIN85/CMS—To identify the binding site responsible for selective recognition of Cbl proteins by CIN85/CMS molecules, we synthesized all proline-rich peptides found in the distal tail of Cbl-b (Fig. 1A) and tested their binding to CIN85 (Fig. 1B). Peptide 893 bound strongly to CIN85, peptide 968 bound much less, and the other Cbl-b-derived polyproline peptides failed to interact with CIN85 (Fig. 1B). This is consistent with the fact that peptide 893 is located in the minimal binding domain of Cbl-b (residues 891–927), which is critical for interactions with CIN85 (17Szymkiewicz I. Kowanetz K. Soubeyran P. Dinarina A. Lipkowitz S. Dikic I. J. Biol. Chem. 2002; 277: 39666-39672Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Furthermore, addition of increasing amounts of peptide 893, but not peptide 768, to the cell lysates, efficiently competed against co-precipitation between Cbl-b and CIN85 (Fig. 1C). The amino acid sequence of peptide 893 of Cbl-b (PARPPKPRPR) is similar to peptide 814 of Cbl (PERPPKPFPR) (Fig. 1A). Consistently, peptide 814 of Cbl also bound very potently to CIN85 in peptide binding assays and competed effectively against Cbl/CIN85 binding in co-precipitation assays (Fig. 1, B and D). Close inspection of these sequences revealed a double PXXP motif followed by a charged arginine residue. However, a similar double PXXP motif found in peptide 768 of Cbl-b (PLPPARP) was not involved in binding to CIN85 (Fig. 1, A and B). To identify amino acids critical for CIN85 recognition, we synthesized peptides with proline and arginine residues in the PXXPPXPXPR motif substituted to alanines (Fig. 2A). The peptide containing mutations of three prolines (3A peptide) involved in the formation of double PXXP motifs (A XX APX A XPR), bound as strongly as wild type 814 peptide, whereas mutation of two additional prolines in 5A peptide (A XX AA X A X AR) completely blocked binding to CIN85 (Fig. 2B, left panel). More detailed analysis indicated that mutation of proline 906 in Cbl-b peptide (PXXPA XPXPR) significantly reduced its ability to associate with CIN85, and the effect was even more prominent when proline 906 was mutated together with proline 910 in Cbl-b sequence (PXXPA XPX AR) but not when mutated together with proline 905 (PXX AA X-PXPR) (Fig. 2B, right panel). On the other hand, single mutation of arginine residue 829 in Cbl or arginine 911 in Cbl-b peptides (PXPPPXPXPA) completely abrogated their ability to bind to CIN85 or CMS (Fig. 2, B and C), indicating that the minimal recognition sequence for the SH3 domains of CIN85 may contain the PXXXPR motif. However, additional peptides containing the PXXXPR core sequence were found in Cbl-b, such as peptide 968 (PpvsPR) and peptide 500 (PpvpPR) (Fig. 2A). Peptide 968 bound much less efficiently to CIN85 as compared with peptide 893, and this low affinity binding was also dependent on the intact arginine 979, whereas peptide 500 did not bind to CIN85 (Fig. 2B, right panel). Peptide 968 may represent the low affinity binding site, associating only in in vitro binding assays, as in the context of the full size Cbl-b molecule, introduction of a stop codon at a residue 927, which preserves only the motif corresponding to the peptide 893 but not 968 of Cbl-b, did not affect the binding to CIN85 (17Szymkiewicz I. Kowanetz K. Soubeyran P. Dinarina A. Lipkowitz S. Dikic I. J. Biol. Chem. 2002; 277: 39666-39672Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar).Fig. 2Identification of the consensus binding site for the SH3 domains of CIN85. Different mutants of peptide 814 from Cbl and peptide 893, 968, or 500 from Cbl-b are listed (A) and were used for pull down assays with lysates of HEK293T cells transfected with CIN85 (B) or CMS (C). After recovery of the bound material, detection was performed by immunoblotting with anti-CIN85 or anti-FLAG (CMS) antibodies. D, HEK-293T cells were transiently transfected with CIN85 or CMS together with Cbl or Cbl-b or their mutants Cbl-R829A or Cbl-b-R911A. Lysates were subjected to immunoprecipitation (IP) with anti-Cbl or anti-Cbl-b antibodies and immunoblotting (IB) with anti-FLAG, anti-CIN85, or anti-Cbl/Cbl-b antibodies. TCL, total cell lysate. E, HEK293T cells were transiently transfected with CIN85 and HA-Cbl-b or indicated Cbl-b proline mutants. Lysates were subjected to immunoprecipitation (IP) with anti-HA antibodies and immunoblotting (IB) with anti-CIN85 or anti-HA antibodies. F, sequences from several CIN85-binding proteins. The consensus PXPXPR motifs are emphasized in bold, and amino acid positions of arginines in each motif in SLP-65 sequence are indicated. G, HEK293T cells were transiently transfected with CIN85 and HA-SLP-65 or HA-SLP-65 arginine mutants (R248A, R313A, and R248,313A). Lysates were subjected to immunoprecipitation (IP) with anti-HA or anti-CIN85 antibodies and immunoblotting (IB) with indicated antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The difference between peptides 893 or 814 and these additional PXXXPR peptides was based on spacing between proline residues, PXPXPR found in peptide 893 as compared with PPXXPR or PPXPPR found in peptides 968 and 500, respectively. Thus, it seems that variations in X residues in PXXXPR motif might, in addition to the structural availability of the motif, define the strength of interactions with the SH3 domains of CIN85/CMS. Because peptide analysis indicated that the arginine in the PXXXPR motif is essential for binding to CIN85 and CMS, we further tested whether the same residue is necessary for interactions between CIN85/CMS and Cbl/Cbl-b in the context of full size molecules. Mutation of arginine 829 in Cbl or arginine 911 in Cbl-b abolished their co-precipitation with CIN85 or CMS (Fig. 2D). Mutations of proline residues within the high affinity PXXXPR motif of Cbl-b led to similar results as our peptide pull-down assays, with the first proline residue (Pro-906) being the most important for the association (Fig. 2E). Substituting the last proline (Pro-910) to alanine significantly affected the binding to CIN85, whereas the middle (Pro-908) could be exchanged for alanine without any effect on the interaction (Fig. 2E). Interestingly, several proteins shown previously to interact with the SH3 domains of CIN85 and CMS, including the CD2 receptor and AIP1, contained the conserved PXXXPR motif, whereas the SETA-binding protein 1 (SB1) and B cell linker adaptor protein SLP-65 had two conserved binding sites (Fig. 2F) (16Dikic I. FEBS Lett. 2002; 529: 110-115Crossref PubMed Scopus (163) Google Scholar). Furthermore, mutation of individual arginine residues in the corresponding motifs in SLP-65, as well as in SB1, reduced their association with CIN85, whereas mutation of both arginines completely blocked co-precipitation with CIN85 (Fig. 2G) (data not shown). Taken together, our data demonstrate that the PXXXPR motif found in numerous signaling proteins is