Requirement of multiple SH3 domains of Nck for ligand binding

Cell. Signal. Vol. 11, No. 4, pp. 253]262, 1999 Copyright Q 1999 Elsevier Science Inc.

ISSN 0898-6568r99 $ - see front matter PII S0898-6568Ž98.00054-0

Requirement of multiple SH3 domains of Nck for ligand binding ´ Goher,† Livius Wunderlich,† Agnes ´ Anna Farago,‡ ´ Julian Downward‡ and Laszlo ´ ´ Buday†U †DEPARTMENT OF MEDICAL CHEMISTRY, SEMMELWEIS UNIVERSITY MEDICAL SCHOOL, 9 PUSKIN STR., 1088 BUDAPEST, HUNGARY; AND ‡SIGNAL TRANSDUCTION LABORATORY, IMPERIAL CANCER RESEARCH FUND, LONDON, UK

ABSTRACT. The Nck adaptor protein comprises a single C-terminal SH2 domain and three SH3 domains. The domain structure of Nck suggests that Nck links tyrosine kinase substrates to proteins containing proline-rich motifs. Here we show that BcrrAbl tyrosine kinase, and three tyrosine phosphorylated proteins Ž115, 120 and 155 kDa. are co-immunoprecipitated with antibody against Nck from lysates of the human leukaemia cell line K562. By means of affinity purification with the Nck-binding phosphopeptide EPGPYŽP.AQPSV, we could also detect the association of endogenous Nck with the proto-oncogene product Cbl. An investigation of the nature of interactions revealed that BcrrAbl, Cbl, and the 155-kDa tyrosine phosphotyrosine bind exclusively to the SH3 domains of Nck. In addition, none of the single SH3 domains of Nck expressed as glutathione-S-transferase ŽGST. fusion proteins is able to interact with the proline-rich ligands. However, combined first and second SH3 domains have the capacity to bind BcrrAbl, Cbl and p155. Mutations of conserved tryptophan to lysine in either of the combined first and second SH3 domains completely abolish ligand binding. These data suggest that cooperation exists among the SH3 domains of Nck for a high-affinity binding of proteins containing proline-rich motifs. CELL SIGNAL 11;4:253]262, 1999. Q 1999 Elsevier Science Inc. KEY WORDS. Nck, BcrrAbl, Cbl, SH2rSH3 domains, K562 cells

INTRODUCTION The Nck protein belongs to the class of adaptor proteins containing SH2 and SH3 domains w1x. These proteins are thought to link signalling molecules to receptor tyrosine kinases. Whereas the SH2 domains recognise specific phosphotyrosine sequences w2x, the SH3 domains bind to proteins possessing proline-rich motifs w3, 4x. The bestcharacterised adaptor molecule is Grb2, which binds to the proline-rich C-terminal region of the guanine nucleotide exchange protein, Sos, recruiting it to the plasma membrane where it activates Ras w5, 6x. Recently, Nck, through its SH3 domains, was shown to be able to physically associate with a number of signalling molecules, including Sos w7, 8x, Cbl w9x, Pak1 w10, 11x, PRK2 w12x and the Wiskott]Aldrich syndrome protein ŽWASP. w13x. In addition, an unidentified seriner U

Author to whom all correspondence should be addressed. Fax: q36 1 2667480; e-mail: [email protected] Received 6 April 1998; accepted 6 July 1998.

threonine kinase was reported to associate with the second domain of Nck w14x. Pak1 and PRK2 preferentially also associate with the second SH3 domain w10]12x, whereas WASP binds to the third SH3 domain of Nck w13x. However, the physiological importance and the nature of these interaction are not clearly understood. BcrrAbl is a chimeric oncogene, implicated in the pathogenesis of Philadelphia chromosome positive human leukaemias w15, 16x. The bcr r abl oncogene is generated by a reciprocal translocation between the c-abl protooncogene on chromosome 9 and the breakpoint cluster region Ž bcr . on chromosome 22 w17x. Chimeric proteins encoded by the Abl oncogene exhibit deregulated tyrosine kinase activity w15x. It has been well established that Ras activation is an important component in BcrrAbl-mediated transformation. Autophosphorylation within the Bcr domain creates a direct physical link to Grb2rSos w18, 19x. Recently, in vitro studies have shown that Nck can bind Abl tyrosine kinase and Cbl in an SH3-dependent manner w9, 20x. However, Nck adaptor molecule contains three

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SH3 domains, and it is not clear whether proline-rich ligands may interact with one or more SH3 domains. In this report, we demonstrate that, in human leukaemic cell line K562, Nck binds to a number of phosphotyrosine proteins, including BcrrAbl, Cbl and an unidentified protein with a molecular mass of 155 kDa. To examine the nature of Nck complexes, a set of different glutathione-S-transferase ŽGST. fusion proteins of Nck were generated. We show here that in vitro Cbl, BcrrAbl and p155 bind only to combined SH3 domains but not to any of the individual SH3 domains of Nck.

MATERIALS AND METHODS Cell Culture and Preparation of Cell Lysates The human myelogenous leukaemia cell line K562 was obtained from the American Type Culture Collection ŽATCC. and maintained in RPMI 1640 medium containing penicillin Ž100 unitsrmL., streptomycin Ž50 mgrmL. and 10% foetal calf serum. Cells were grown to a density of 10 6rmL, centrifuged, and washed twice in serum-free RPMI 1640. Cells were then harvested by centrifugation at 15,000 = g for 20 sec and lysed in ice-cold 50 mM HEPES buffer, pH 7.4 Ž5 = 10 6 cell equivalents per millilitre. containing 1% Triton X-100, 100 mM NaCl, 20 mM NaF, 1 mM EGTA, 0.1 mM Na 3VO4 , 1 mM p-nitrophenyl phosphate, 10 mM benzamidine, 1 mM phenylmethylsulphonyl fluoride, 25 mgrmL each of leupeptin, trypsin inhibitor and aprotinin. Lysates were clarified by centrifugation at 15,000 = g for 15 min at 48C.

Antibodies, GST Fusion Proteins and Peptides Monoclonal anti-phosphotyrosine antibody Žclone 4G10. was obtained from Upstate Biotechnology, Inc. Polyclonal anti-Cbl, polyclonal anti-Pak1 and monoclonal anti-Abl antibodies were purchased from Santa Cruz Biotechnology. Monoclonal anti-FAK and monoclonal antip130 CA S antibodies were purchased from Transduction Laboratories. Polyclonal anti-Nck antibody was generated against the full-length GST-Nck fusion protein. pGEX-2T expression vector containing the full-length encoding sequence of Nck ŽGST-Nck. was donated by M. Waterfield. Fusion proteins of various domains of Nck with GST were generated. The DNA fragments encoding the first Žamino-terminal. SH3 domain ŽGST-SH3-1, amino acids 1]102., the second SH3 domain ŽGST-SH3-2, amino acids 91]182., the third SH3 domain ŽGST-SH3-3, amino acids 182]260. and the SH2 domain of Nck ŽGST-NckSH2, amino acids 259]377. were amplified by polymerase chain reaction and sub-cloned into the pGEX-2TK vector

FIGURE 1. Association of Nck with tyrosine phosphoproteins and Bcr / Abl in vivo. Proteins from lysates of K562 cells (10 7 cells per sample) were immunoprecipitated with control (1) or with anti-Nck (2) antibodies. After SDS-PAGE and transfer to nitrocellulose, samples were analysed by anti-phosphotyrosine (4G10) (A) and anti-Abl (B) antibodies. Whole-cell lysates of K562 cells immunoblotted with anti-Abl (B, lane 3) antibody are also shown. The positions of molecular mass markers are indicated at the left (in kilodaltons).

ŽPharmacia.. Combined SH3 domain constructs of Nck encoded the first plus second ŽGST-SH3-12., the second plus third ŽGST-SH3-23. and all SH3 domains ŽGSTSH3-123. ŽFig. 4.. Point mutations within the first ŽW38K. or the second ŽW143K. SH3 domains of the GST-NckSH3-12 construct were generated by a USE mutagenesis kit ŽPharmacia.. In this way, the first tryptophan of the tryptophan doublet of the SH3 domains was changed to lysine w21x. In all cases, the constructs were verified by DNA sequencing. GST fusion proteins were purified by binding to glutathione agarose ŽSigma. and gave essen-

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basis of the Y221 phosphorylation site of CrkII. For protein precipitation with peptide beads, 5 mg of different phosphopeptides or their unphosphorylated forms covalently bound to Affi-gel 10 ŽBio-Rad. were added to cell lysates.

Immunoprecipitation and Western Blotting Immunoprecipitation and Western blotting were performed as described previously w5, 22x. Briefly, cell lysates were pre-cleared with protein A-agarose, and then proteins were immunoprecipitated with 10 mg of polyclonal anti-Nck antibody. Immunoprecipitating antibodies were cross-linked to protein A-agarose. Washing was performed with ice-cold 50 mM HEPES buffer, pH 7.5, containing 250 mM NaCl, 0.2% Triton-X100 and 0.1 mM Na 3VO4 . Bound proteins were separated by SDS polyacrylamide gel electrophoresis Žin 7.5% gel., transferred to nitrocellulose membrane and immunoblotted with the indicated antibodies. Blots were developed by the enhanced chemiluminescence system ŽAmersham.. For precipitation with GST fusion proteins, 5-mg aliquots of different Nck fusion proteins were used as non-covalently bound adducts to glutathione]agarose beads.

RESULTS Nck Associates with Bcr r Abl, Cbl, p120 and p155 In Vivo Recently, Nck was shown to bind to Abl tyrosine kinase in a SH3 domain-dependent manner w20x. To determine whether Nck associates with BcrrAbl or any other tyrosine phosphorylated proteins in K562 cells, proteins were immunoprecipitated from cell lysates with anti-Nck antibody raised against the full-length GST-Nck molecule, and then immunoprecipitates were probed with anti-phosphotyrosine antibody. Fig. 1A demonstrates that Nck associates with four phosphotyrosine proteins with molecular

tially single bands on Coomassie blue-stained SDS polyacrylamide gels. The Y1068 phosphopeptide PVPEYŽP.INQS derived from the autophosphorylated epidermal growth factor ŽEGF. receptor w22, 23x and the Y751 phosphopeptide corresponding to sequence residues 746]756 of the human platelet-derived growth factor ŽPDGF. receptor were synthesised. The Nck-binding peptide EPGPYŽP.AQPSV and its unphosphorylated form were also synthesised on the

FIGURE 2. Nck / Cbl complex is precipitated with specific phosphopeptide. (A) Lysates from K562 cells (10 7 cells per sample) were subjected to affinity purification with the following peptides immobilised on Affi-gel 10: EPGPYAQPSV (1), EPGPY(P)AQPSV (2), ADTTQY(P)-VPMLE (3) and PVPEY(P)INQS (4). Bound proteins were then resolved by SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-Nck antibody. (B) Proteins from lysates of K562 cells were precipitated with unphosphorylated (1) and phosphorylated (2) forms of peptide EPGPYAQPSV. After SDS-PAGE and transfer to nitrocellulose, samples were analysed by anti-Cbl antibody. Whole-cell lysate of K562 cells immunoblotted with anti-Cbl antibody is also shown (3). The positions of molecular mass markers are indicated at the left (in kilodaltons).

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weights of 115, 120, 155 and 210 kDa. Although the in vivo interaction of Nck with the 115, 120 and 210 kDa phosphoproteins seemed to be strong, the presence of the 155 kDa protein in the Nck immunoprecipitate was hardly detectable, suggesting a weak association between the two molecules. To identify the Nck-associated proteins, Nck immunoprecipitates were tested with various candidate antibodies. Immunoblot with monoclonal anti-Abl antibody shows that the 210 kDa tyrosine phosphoprotein is identical with BcrrAbl ŽFig. 1B.. To identify any of the two tyrosine phosphoproteins at ; 120 kDa, Nck immunoprecipitates were probed with anti-Cbl, anti-FAK and anti-p130 CA S antibodies; however, none of the aforementioned antibodies recognised the proteins. By using GST fusion proteins of Nck, we found that a 120-kDa protein associates predominantly with the Nck SH2 domain Ždata not shown.. Characterisation and identification of this phosphoprotein requires further investigations. The physical interaction of Nck with Cbl was recently reported w9x. Furthermore, the 120-kDa Cbl is one of the prominent phosphotyrosine proteins in K562 cells w24x. In contrast, we failed to show the presence of Cbl in the Nck immunoprecipitates. Therefore, a different approach was used to demonstrate the in vivo interaction of Nck with Cbl. We have recently used an immobilised tyrosine phosphopeptide derived from the Y1068 autophosphorylation site of the EGF receptor to precipitate complexes of Grb2rSos and Grb2rCbl w22, 23x. To investigate other phosphopeptiderSH2 domain interactions, a phosphopeptide wEPGPYŽP.AQPSVx derived from the Y221 phosphorylation site of CrkII was synthesised w25x. This phosphopeptide was immobilised on agarose-beads ŽY221-P beads. and used for affinity purification of proteins. It was found that the Y221-P beads precipitated one major protein Ž47 kDa. from lysates of Jurkat, PC12, Rat 1 and K562 cells based on Coomassie blue-stained SDS-PAGE gels ŽL. Buday and J. Downward, unpublished observations.. We identified the 47-kDa protein as a Nck adaptor molecule. Fig. 2A shows that immobilised EPGPYŽP.AQPSV phosphopeptide specifically precipitates Nck from K562 cell lysates, whereas its unphosphorylated form ŽY221 beads. does not bind Nck. In addition, a phosphopeptide ADTTQYŽP.VPMLE derived from the Y751 autophosphorylation site of the PDGF receptor, which is a known in vivo binding site for the Nck SH2 domain w26x, also precipitates the 47-kDa Nck ŽFig. 2A.. Other tyrosine phosphorylated peptides}for example, the PVPEYŽP.INQS Žfrom the EGF receptor. }did not bind to the SH2 domain of Nck ŽFig. 2A.. The phosphorylation of Crk Y221 was reported to create a binding site for the Crk SH2 domain and is thus implicated in the regulation of the Crk molecule w25, 27x. Interestingly, the binding preference of the SH2 domains of Crk and Nck are very similar, both having high affinity for pTyr-D-X-Pro phosphorylation motifs w2, 28x. However, the exact consensus phosphorylation site for the SH2 domain of Nck is not known, because its binding sites on the PDGF receptor ŽYVPM., on the rat IRS-I ŽYDTG. or on a synthetic phosphopeptide ŽYDEP.

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FIGURE 3. In vitro binding of Cbl to Nck. Lysates from K562 cells (10 7 cells per sample) were subjected to affinity purification with the indicated fusion proteins immobilised on agarose beads. Bound proteins were then resolved by SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-phosphotyrosine (A) or with anti-Cbl antibodies (B). Immunoblot of whole-cell lysates of K562 cells with anti-phosphotyrosine (A) and with anti-Cbl antibodies are also shown. Numbered arrows indicate tyrosine phosphoproteins in the precipitate of Nck in the following order: [1 ]2] ª pp52]54; [3 ]4] ª pp58]60, [5] ª p75, [6] ª p110, [7] ª p120, [8] ª p155, [9] ª p180, [10] ª p210. The positions of molecular mass markers are indicated at the left (in kilodaltons).

Interactions of Nck with BcrrAbl and Cbl

obtained by using degenerate phosphopeptide libraries show very different sequences w2, 26, 29x. To demonstrate the interaction of endogenous Nck with Cbl, the immobilised Y221 tyrosine phosphopeptide was used for affinity chromatography, because this peptide precipitates Nck predominantly, whereas the Y751 phosphopeptide binds several other SH2 domain-containing proteins from cell lysates, including, for example, the phosphatidylinositol 3-kinase Ždata not shown.. Fig. 2B demonstrates that Cbl Ž120 kDa. is present in the precipitates of the Y221 phosphopeptide, whereas the unphosphorylated peptide is not able to interact with the NckrCbl complex. It is noteworthy that, although the Y221 phosphopeptide binds to the SH2 domains of Crk proteins, Cbl could not interact with the SH3 domains of Crk proteins w22x.

Nck Interacts with Bcr r Abl, Cbl and p155 via its SH3 Domains To investigate the nature of interactions between Nck and the associated phosphotyrosine proteins, glutathione-Stransferase fusion proteins of full-length Nck and its SH3

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domains were used. Lysates of K562 cells were mixed with GST fusion proteins immobilised on glutathione]agarose beads, and then protein precipitates were probed with anti-phosphotyrosine antibody. As shown in Fig. 3A, fulllength Nck precipitates a number of tyrosine phosphoproteins with molecular weights of 52]54, 58]60, 75, 110, 120, 155, 180 and 210 kDa. However, when the SH3 domains of Nck ŽSH3]123. were used for protein precipitation, four major tyrosine phosphoproteins were detected with the following molecular masses: 120, 155, 180 and 210 kDa ŽFig. 3A.. This finding suggests that, although these proteins contain tyrosine phosphorylated residues, their interactions with Nck are mediated via SH3 domains. To demonstrate that the 120-kDa tyrosine phosphoprotein found to be associated with the Nck SH3 domains is Cbl, GST-Nck protein precipitates were probed with antiCbl antibody. The immunoblot shows that the full-length Nck binds Cbl ŽFig. 3B.. Furthermore, although Cbl is a prominent tyrosine phosphoprotein in the lysates of K562 cells w24x, the SH2 domain of Nck is virtually unable to associate with the phosphorylated Cbl ŽFig. 3B.. In agreement with a previous report w9x, our results confirm that the interaction of Nck with Cbl is mainly mediated through SH3 domains Žsee also Fig. 6B..

FIGURE 4. GST fusion constructs containing various domains of Nck. Schematic representation of GST fusion proteins. The name of each construct is denoted at the right. The GST-Nck fusion protein represents the entire Nck molecule.

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Combined SH3 Domains of Nck are Necessary for Ligand Binding It has been well characterised that PAK1 and PRK2 kinases bind to the second SH3 domain of Nck w10]12x, whereas WASP associates with the third SH3 domain w13x. To further characterise the SH3-mediated interactions of Nck with Cbl, BcrrAbl and p155, various GST fusion proteins were generated with distinct domains of Nck ŽFig. 4.. To test whether our GST fusion protein constructs containing single SH3 domains work properly, protein precipitates from lysates of K562 cells were probed with anti-Pak1, anti-Sos and anti-dynamin antibodies, respectively. Fig. 5 shows that Pak1 binds to the second SH3 domain of Nck. In agreement with previous studies w10x, binding of Pak1 to full-length Nck was stronger than to the second SH3 domain alone ŽFig. 5.. Immunoblot analysis with the use of anti-Sos and anti-dynamin antibodies showed that both Sos and dynamin bind to the third Nck SH3 domain w30x. In subsequent experiments, individual SH3 domains ŽGST-SH3-1, GST-SH3-2 and GST-SH3-3. were used to precipitate proteins from the lysate of K562 cells. However, testing protein precipitates with anti-phosphotyrosine antibody revealed that none of the known tyrosine phosphoproteins binds to single SH3 domains ŽFig. 6A.. The fact that none of the individual SH3 domains of Nck has the ability to bind proline-rich proteins with high affinity prompted us to test whether combined SH3 domains would associate with ligands. As demonstrated in Fig. 6A, combined first and second SH3 domains ŽGST-

FIGURE 6A,B.

FIGURE 5. Pak1 binds to the second SH3 domain of Nck. Lysates prepared from K562 cells (10 7 cells per sample) were precipitated with GST fusion protein constructs containing individual Nck SH3 domains and the full-length protein. Bound proteins were resolved by SDS-PAGE (in 10% gel) followed by immunoblotting with anti-Pak1 antibody. The positions of molecular mass markers are indicated at the left (in kilodaltons).

SH3-12. bind 120- and 155-kDa tyrosine phosphoproteins. In addition, the 210-kDa phosphoprotein that corresponds to BcrrAbl shows a lower-affinity binding to GST-SH3-12. Interestingly, when the three SH3 domains ŽGST-SH3-123. or the full-length Nck were used for protein precipitations, all ligands exhibit a high-affinity interaction ŽFig. 6A.. Moreover, it appears that, in vitro, the 180-kDa tyrosine phosphoprotein is able to associate only with the three SH3 domains as a minimal structure required for its binding. These results suggest that, in K562 cells, the proline-rich phosphoprotein ligands need combined SH3 domains of Nck for interactions. To determine which regions of the Nck protein are required for its association with Cbl, single SH3 domains were used separately for protein precipitations from lysates of K562 cells. Probing SH3-associated proteins with antiCbl antibody demonstrates that none of the GST fusion

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FIGURE 6C. FIGURE 6. Combined SH3 domains of Nck are neccessary forligand binding. Proteins from lysates of K562 cells (10 7 cells per sample) were precipitated with the indicated GST fusion proteins immobilised on glutathione]agarose beads. After SDS-PAGE and transfer to nitrocellulose, samples were analysed by anti-phosphotyrosine (A), anti-Cbl (B) or anti-Abl (C) antibodies. Whole-cell lysate of K562 cells immunoblotted with anti-Abl antibody also is shown (C). The positions of molecular mass markers are indicated at left (in kilodaltons).

proteins can bind Cbl ŽFig. 6B.. When the same experiments were performed with combined SH3 domains, the minimal structure necessary for Cbl binding was found to be the combined first and second SH3 domains ŽFig. 6B.. To determine the required regions of Nck for binding BcrrAbl, different GST proteins were used again for protein precipitation, and then the immunoblot was tested with anti-Abl antibody. Fig. 6C shows that BcrrAbl binds to those fusion proteins that contain all SH3 domains ŽGST-SH3-123 or GST-Nck.. This experiment is in agreement with previous results ŽFig. 6A., where the 210-kDa tyrosine phosphoprotein interacted mostly with the GST-SH3-123 and GST-Nck fusion proteins. Interestingly, in the precipitate of the full-length Nck, a protein with an approximate molecular weight of 180 appeared and cross-reacted with the anti-Abl antibody. The characterisation of this 180-kDa protein requires further investigations.

Mutations of Conserved Tryptophans to Lysines in GST-SH3-12 Abolishes Ligand Binding To confirm our hypothesis that, for high-affinity binding of ligands, Nck needs at least two SH3 domains acting together, point mutation were introduced in the structure of GST-SH3-12. The first absolutely conserved tryptophan of the characteristic tryptophan doublet found in the SH3 domains of Nck was changed to lysine w21x. We introduced this mutation into the first SH3 domain ŽGSTSH3-12, W38K. or into the second SH3 domain ŽGSTSH3-12, W143K. of the combined fusion protein construct. Mutant fusion proteins covalently bound to Affi-gel

10 were used for protein precipitation from lysates of K562 cells. Analysis of protein precipitates with anti-Cbl antibody shows that fusion protein constructs containing point mutations in either the first ŽGST-SH3-1U 2. or the second ŽGST-SH3-12U . SH3 domain of Nck are not able to bind Cbl from cell lysates ŽFig. 7A.. Moreover, reprobing the immunoblot with anti-phosphotyrosine antibody demonstrates that associations of phosphoproteins Žp120 Cbl , p155 and p210 BcrrAbl . with the mutant SH3 fusion proteins were also strongly inhibited ŽFig. 7B.. These results suggest that at least two intact SH3 domains of Nck are necessary for binding of the aforementioned phosphoproteins.

DISCUSSION In this study, we demonstrate that Nck adaptor protein associates with different phosphotyrosine proteins in human leukaemia cell line K562. One of the prominent tyrosine phosphoproteins in the Nck immunoprecipitate is identical with BcrrAbl tyrosine kinase. Recent observations have shown that in vitro Nck can bind to Abl in an SH3 domain-dependent manner w20x. The SH3 domain binding sites were mapped on the C-terminal part of Abl w20x. In Philadelphia chromosome positive cells, the Cterminal region of Abl remains intact in the BcrrAbl fusion protein; therefore, Nck can bind to it. Our data confirm that association of Nck with the chimeric BcrrAbl protein occurs in vivo and is mediated through SH3 domains. Another tyrosine phosphoprotein with a molecular weight of 120 kDa was also found in the immunoprecipi-

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FIGURE 7. Mutations of conserved tryptophans to lysines in GST-SH3-12 abolish ligand binding. Lysates prepared from K562 cells (10 7 cells per sample) were precipitated with GST, GST-SH3-12, GST-SH3-12 or GST-SH3-12 fusion proteins immobilised on glutathione]agarose beads. GST-SH3-12 contains the W38 ª K mutation in the first SH3 domain of Nck, whereas GST-SH3-12 is mutated (W143ª K) in the second SH3 domain. The bound proteins were resolved by SDS-PAGE followed by immunoblotting with anti-Cbl (A) or anti-phosphotyrosine (B) antibodies. (B) Arrows indicate the 120-, 155-, and 210-kDa tyrosine phosphoproteins bound to the GST-SH3-12 fusion protein construct. The positions of molecular mass markers are indicated at the left (in kilodaltons).

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tates of Nck. This protein binds to the SH2 domain of Nck and seems to be different from the p120 Cbl Ždata not shown.. In this study, we have investigated the interactions of Nck with proline-rich ligands. Recently, Andoniou et al. w23x reported that Cbl is the prominent tyrosine phosphoprotein in cells that express activated forms of the abl oncogene. Cbl binds to Nck in an SH3 domain-dependent manner w9x; therefore, we studied the association of Nck with Cbl in K562 cells. Although Cbl was not present in the Nck immunoprecipitates, by means of affinity purification with a Nck-binding phosphopeptide, we could detect that Cbl associates with the endogenous Nck. The EPGPYŽP.AQPSV peptide totally depletes Nck from lysates of K562 cells Ždata not shown.; therefore, it is likely that the efficiency of the Nck precipitation with the phosphopeptide is significantly higher than that of immunoprecipitation with an anti-Nck antibody. Investigating the nature of interactions of Nck with the phosphoproteins, we demonstrate that none of the individual SH3 domains is able to mediate the association. Therefore, we generated combined GST fusion proteins containing either the first and second or the second and third SH3 domains of Nck. Three phosphotyrosine proteins, including p120, p155 and p210, were found to bind to the first and second combined SH3 domains with low affinity. However, when the three SH3 domains or the full-length Nck were used, all three tyrosine phosphoproteins showed a higher-affinity binding to Nck. These results suggest that cooperation exists among the SH3 domains of Nck for binding proline-rich ligands. To confirm our hypothesis, we have changed conserved tryptophans to lysine in the first or the second SH3 domains, respectively. We found that introduction of a point mutation in either of the two SH3 domains of the combined fusion protein construct ŽGST-SH3-12. markedly reduced the capacity of Nck to bind Cbl or the tyrosine phosphoproteins. Recently, Rivero-Leczano et al. w13x reported that WASP binds to the third, carboxy-terminal SH3 domain of Nck. However, when they used a fusion protein containing the three SH3 domains together, WASP binding in vitro was much higher than when only the carboxy-terminal SH3 domain was present. Similar data were reported by Bokoch et al. w10x, who investigated the interaction of PAK1 with Nck. PAK1 was seen to bind to the second SH3 domain; but, when combined second and third SH3 domains of Nck were used, binding of PAK1 was always greater than it was to the second SH3 domain alone. We recently reported that, although the Sos exchange factor binds to the third domain of Nck, other SH3 domains also may contribute to the high-affinity binding of Sos w30x. These findings strongly suggest that many of the Nck ligands bind to more than one SH3 domain of Nck, and this cooperative interaction is necessary for a tight complex formation.

Interactions of Nck with BcrrAbl and Cbl

Proteins containing SH3-binding sites usually contain more than one proline-rich stretch in their sequence. It has been well documented that Grb2, the best-characterised adapter protein, forms complexes with a number of proline-rich effector proteins including Sos w5, 6x, dynamin w31x, Cbl w22, 32x and Slp-76 w33x. These proteins can bind preferentially to either the C- or the N-terminal SH3 domains of Grb2. Sos, for example, prefers the N-terminal SH3 domain w6, 34x. However, it has been suggested that a stable association between Grb2 and Sos requires interactions of Sos with both of the SH3 domains of Grb2 w3, 4x. Nck is unique among the adapter proteins in that it contains three SH3 domains. Why does Nck need three SH3 domains? One of the possible explanations is that the individual SH3 domains may bind to at least three effector proteins. This would mean that, in theory, three SH3 ligands could bind to one Nck molecule at the same time. In contrast, we present data here that many ligands are not able to associate with single SH3 domains. The second possibility, which we favour, is that cooperation exists among the SH3 domains of Nck for ligand binding. Therefore, a given Nck molecule may not bind three effector proteins at the same time. It is well known that effector proteins generally have more than one proline-rich motif in their structure. Abl, for example, contains at least three proline-rich motifs that were found close to the nuclear localisation signal of the protein w20x. The sequence of Cbl also contains more than four potential SH3-binding sites w35x. Therefore, it is possible that at least two proline-rich motifs and two SH3 domains are involved in the complex formation. This hypothesis is confirmed by our experiment in which point mutations in either of the SH3 domains of GST-Nck-3-12 abolish the capacity of Cbl and BcrrAbl to bind to the fusion protein. Nevertheless, further experiments will be performed to evaluate the exact mechanism by which SH3 ligands bind to Nck.

261 4. 5. 6.

7. 8. 9. 10. 11. 12.

13. 14. 15. 16. 17. 18.

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20. 21. 22. 23. 24.

We thank M. Waterfield for Nck cDNA. This work was supported by Association for International Cancer Research Grant No. 96-106 and in part by research Grant No. MKM 0070 r 1997.

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