FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2068675259> ?p ?o ?g. }

• W2068675259 endingPage "28246" @default.
• W2068675259 startingPage "28238" @default.
• W2068675259 abstract "The importance of the SH3 domain of Hck in kinase regulation, substrate phosphorylation, and ligand binding has been established. However, few in vivo ligands are known for the SH3 domain of Hck. In this study, we used mass spectrometry to identify ∼25 potential binding partners for the SH3 domain of Hck from the monocyte cell line U937. Two major interacting proteins were the actin binding proteins Wiskott-Aldrich syndrome protein (WASP) and WASP-interacting protein (WIP). We also focused on a novel interaction between Hck and ELMO1, an 84-kDa protein that was recently identified as the mammalian ortholog of the Caenorhabditis elegansgene, ced-12. In mammalian cells, ELMO1 interacts with Dock180 as a component of the CrkII/Dock180/Rac pathway responsible for phagocytosis and cell migration. Using purified proteins, we confirmed that WASP-interacting protein and ELMO1 interact directly with the SH3 domain of Hck. We also show that Hck and ELMO1 interact in intact cells and that ELMO1 is heavily tyrosine-phosphorylated in cells that co-express Hck, suggesting that it is a substrate of Hck. The binding of ELMO1 to Hck is specifically dependent on the interaction of a polyproline motif with the SH3 domain of Hck. Our results suggest that these proteins may be novel activators/effectors of Hck. The importance of the SH3 domain of Hck in kinase regulation, substrate phosphorylation, and ligand binding has been established. However, few in vivo ligands are known for the SH3 domain of Hck. In this study, we used mass spectrometry to identify ∼25 potential binding partners for the SH3 domain of Hck from the monocyte cell line U937. Two major interacting proteins were the actin binding proteins Wiskott-Aldrich syndrome protein (WASP) and WASP-interacting protein (WIP). We also focused on a novel interaction between Hck and ELMO1, an 84-kDa protein that was recently identified as the mammalian ortholog of the Caenorhabditis elegansgene, ced-12. In mammalian cells, ELMO1 interacts with Dock180 as a component of the CrkII/Dock180/Rac pathway responsible for phagocytosis and cell migration. Using purified proteins, we confirmed that WASP-interacting protein and ELMO1 interact directly with the SH3 domain of Hck. We also show that Hck and ELMO1 interact in intact cells and that ELMO1 is heavily tyrosine-phosphorylated in cells that co-express Hck, suggesting that it is a substrate of Hck. The binding of ELMO1 to Hck is specifically dependent on the interaction of a polyproline motif with the SH3 domain of Hck. Our results suggest that these proteins may be novel activators/effectors of Hck. high pressure liquid chromatography Chinese hamster ovary WASP-interacting protein Wiskott-Aldrich syndrome protein polyvinylidene difluoride glutathione S-transferase The members of the Src family of nonreceptor tyrosine kinases share a modular structure comprising unique SH3, SH2, and kinase catalytic domains (1Bjorge J.D. Jakymiw A. Fujita D.J. Oncogene. 2000; 19: 5620-5635Crossref PubMed Scopus (335) Google Scholar, 2Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Crossref PubMed Scopus (1080) Google Scholar, 3Schwartzberg P.L. Oncogene. 1998; 17: 1463-1468Crossref PubMed Scopus (132) Google Scholar, 4Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2149) Google Scholar). The enzymatic activity of Src family kinases is tightly regulated by intramolecular interactions. The autoinhibited state is maintained by two interactions, (i) an interaction between the SH2 domain and a phosphorylated C-terminal tyrosine (Tyr-527) and (ii) an interaction between the SH3 domain and a polyproline type II helix in the SH2 kinase linker sequence (residues Pro-244–Trp-254) (5Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1043) Google Scholar, 6Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1244) Google Scholar, 7Williams J.C. Weijland A. Gonfloni S. Thompson A. Courtneidge S.A. Superti-Furga G. Wierenga R.K. J. Mol. Biol. 1997; 274: 757-775Crossref PubMed Scopus (220) Google Scholar). Src kinases can be potently activated by exogenous ligands for the SH3 and SH2 domains (8Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (537) Google Scholar, 9Briggs S.D. Sharkey M. Stevenson M. Smithgall T.E. J. Biol. Chem. 1997; 272: 17899-17902Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 10Alexandropoulos K. Baltimore D. Genes Dev. 1996; 10: 1341-1355Crossref PubMed Scopus (221) Google Scholar, 11Liu X. Brodeur S.R. Gish G. Songyang Z. Cantley L.C. Laudano A.P. Pawson T. Oncogene. 1993; 8: 1119-1126PubMed Google Scholar). These ligands disrupt the autoinhibitory interactions, promote autophosphorylation at Tyr-416 within the activation loop, and stimulate tyrosine kinase activity. Binding of Src family kinases to cellular proteins can regulate kinase activity by at least three mechanisms, (i) release of autoinhibitory interactions as described above, (ii) relocalization of Src kinases to sites of cellular action, and (iii) tethering the kinases to potential substrates (1Bjorge J.D. Jakymiw A. Fujita D.J. Oncogene. 2000; 19: 5620-5635Crossref PubMed Scopus (335) Google Scholar, 2Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Crossref PubMed Scopus (1080) Google Scholar, 3Schwartzberg P.L. Oncogene. 1998; 17: 1463-1468Crossref PubMed Scopus (132) Google Scholar). These mechanisms often operate in combination, as is seen when Src kinases are targeted to their substrates by SH3/SH2 domain interactions. Many cases have been described in which Src kinases are recruited to their substrates via SH3 domain interactions. For example, c-Src is known to associate with at least eight substrates via direct binding of its SH3 domain to ligand binding motifs in the substrates (2Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Crossref PubMed Scopus (1080) Google Scholar); well studied cases include Cas (12Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), FAK (13Thomas J.W. Ellis B. Boerner R.J. Knight W.B. White G.C., II Schaller M.D. J. Biol. Chem. 1998; 273: 577-583Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), and AFAP-110 (14Guappone A.C. Flynn D.C. Mol. Cell Biochem. 1997; 175: 243-252Crossref PubMed Scopus (47) Google Scholar). The polyproline motifs in such substrates strongly activate Src kinases by SH3 displacement and concomitantly tether the substrate to the kinase, facilitating phosphorylation (13Thomas J.W. Ellis B. Boerner R.J. Knight W.B. White G.C., II Schaller M.D. J. Biol. Chem. 1998; 273: 577-583Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 15Pellicena P. Miller W.T. J. Biol. Chem. 2001; 276: 28190-28196Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 16Porter M. Schindler T. Kuriyan J. Miller W.T. J. Biol. Chem. 2000; 275: 2721-2726Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The dual role of the SH3 domain in substrate targeting is illustrated in experiments with synthetic peptides, where phosphorylation of substrates is greatly enhanced by the incorporation of an SH3 domain ligand (17Scott M.P. Miller W.T. Biochemistry. 2000; 39: 14531-14537Crossref PubMed Scopus (43) Google Scholar). These observations suggest that good substrates for particular Src kinases might be discovered as SH3 domain ligands. Hematopoietic cell kinase (Hck) is a Src family tyrosine kinase that is expressed predominantly in granulocytic and monocytic cells (18Quintrell N. Lebo R. Varmus H. Bishop J.M. Pettenati M.J., Le Beau M.M. Diaz M.O. Rowley J.D. Mol. Cell. Biol. 1987; 7: 2267-2275Crossref PubMed Scopus (202) Google Scholar, 19Ziegler S.F. Marth J.D. Lewis D.B. Perlmutter R.M. Mol. Cell. Biol. 1987; 7: 2276-2285Crossref PubMed Scopus (178) Google Scholar). In granulocytes, Hck is found in a secretory granule-enriched fraction and in a granule-free membrane fraction (20Welch H. Maridonneau-Parini I. J. Biol. Chem. 1997; 272: 102-109Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The different subcellular localizations of Hck are consistent with its proposed functional roles in phagocytosis (21Kedzierska K. Vardaxis N.J. Jaworowski A. Crowe S.M. J. Leukocyte Biol. 2001; 70: 322-328PubMed Google Scholar, 22Suzuki T. Kono H. Hirose N. Okada M. Yamamoto T. Yamamoto K. Honda Z. J. Immunol. 2000; 165: 473-482Crossref PubMed Scopus (82) Google Scholar, 23Majeed M. Caveggion E. Lowell C.A. Berton G. J. Leukocyte Biol. 2001; 70: 801-811PubMed Google Scholar, 24N′Diaye E.N. Darzacq X. Astarie-Dequeker C. Daffe M. Calafat J. Maridonneau-Parini I. J. Immunol. 1998; 161: 4983-4991PubMed Google Scholar) and in receptor-mediated signaling (25Daeron M. Annu. Rev. Immunol. 1997; 15: 203-234Crossref PubMed Scopus (1035) Google Scholar, 26Ghazizadeh S. Bolen J.B. Fleit H.B. J. Biol. Chem. 1994; 269: 8878-8884Abstract Full Text PDF PubMed Google Scholar, 27Mocsai A. Jakus Z. Vantus T. Berton G. Lowell C.A. Ligeti E. J. Immunol. 2000; 164: 4321-4331Crossref PubMed Scopus (254) Google Scholar, 28Chiaradonna F. Fontana L. Iavarone C. Carriero M.V. Scholz G. Barone M.V. Stoppelli M.P. EMBO J. 1999; 18: 3013-3023Crossref PubMed Scopus (58) Google Scholar). In monocytes, FcγRII clustering leads to an array of biological responses including phagocytosis, cell killing, secretion of inflammatory mediators, and activation (25Daeron M. Annu. Rev. Immunol. 1997; 15: 203-234Crossref PubMed Scopus (1035) Google Scholar). Clustering of FcγRII promotes activation of Hck and subsequent phosphorylation of the receptor by direct association of Hck and FcγRII (26Ghazizadeh S. Bolen J.B. Fleit H.B. J. Biol. Chem. 1994; 269: 8878-8884Abstract Full Text PDF PubMed Google Scholar). Association of Hck with an acidic region of the interleukin 6 receptor β chain, gp130, leads to Hck activation and cell proliferation (29Schaeffer M. Schneiderbauer M. Weidler S. Tavares R. Warmuth M. de Vos G. Hallek M. Mol. Cell. Biol. 2001; 21: 8068-8081Crossref PubMed Scopus (52) Google Scholar). There are several indications that Hck plays an important role in integrin-mediated signal transduction. Neutrophils isolated from Hck/Fgr double knockout mice are deficient in integrin-mediated respiratory burst and granule secretion (30Suen P.W. Ilic D. Caveggion E. Berton G. Damsky C.H. Lowell C.A. J. Cell Sci. 1999; 112: 4067-4078PubMed Google Scholar, 31Lowell C.A. Fumagalli L. Berton G. J. Cell Biol. 1996; 133: 895-910Crossref PubMed Scopus (323) Google Scholar, 32Mocsai A. Ligeti E. Lowell C.A. Berton G. J. Immunol. 1999; 162: 1120-1126PubMed Google Scholar). Integrin signaling in polymorphonuclear leukocytes also activates Hck (33Piccardoni P. Sideri R. Manarini S. Piccoli A. Martelli N. de Gaetano G. Cerletti C. Evangelista V. Blood. 2001; 98: 108-116Crossref PubMed Scopus (80) Google Scholar) and leads to increased association of Hck and with the cytoskeleton (34Nair K.S. Zingde S.M. Cell. Immunol. 2001; 208: 96-106Crossref PubMed Scopus (50) Google Scholar). Another role for Hck in actin rearrangement is suggested by the fact that Hck is activated during E-selectin-mediated induction of monocyte chemotaxis (35Kumar P. Hosaka S. Koch A.E. J. Biol. Chem. 2001; 276: 21039-21045Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Hck has been implicated in a wide variety of signaling pathways in hematopoietic cells. However, relatively few substrates or effectors of Hck have been identified. One in vivo substrate for Hck, the multidomain signaling protein Cbl, has been shown to interact with the SH3 domain of Hck. As described above, we would expect that many of the best cellular substrates for Hck would contain binding motifs for the SH3 domain of the enzyme. The focus of this study was to identify and characterize novel SH3 domain ligands for Hck. Two proteins that we identified, Wiskott-Aldrich syndrome protein (WASP) and WASP-interacting Protein (WIP), are known regulators of the actin cytoskeleton (36Zigmond S.H. J. Cell Biol. 2000; 150: 117-120Crossref PubMed Google Scholar, 37Ramesh N. Anton I.M. Martinez-Quiles N. Geha R.S. Trends Cell Biol. 1999; 9: 15-19Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). A third Hck-interacting protein, ELMO1, has recently been described as a component of signaling pathways that regulate phagocytosis and cell migration (38Gumienny T.L. Brugnera E. Tosello-Trampont A.C. Kinchen J.M. Haney L.B. Nishiwaki K. Walk S.F. Nemergut M.E. Macara I.G. Francis R. Schedl T. Qin Y. Van Aelst L. Hengartner M.O. Ravichandran K.S. Cell. 2001; 107: 27-41Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). Hck has been implicated in these pathways as well, suggesting that ELMO1 may represent an important downstream substrate/effector of Hck. GST and GST-SH3(Hck) were expressed in Escherichia coli NB42 cells. Cells were lysed in a French pressure cell in buffer containing 50 mm HEPES, pH 7.4, 100 mm NaCl, 100 mm EDTA, 1% Triton X-100, 10% glycerol, and protease inhibitors (5 mg/liter of aprotinin, 5 mg/liter leupeptin, 0.1 mm phenylmethylsulfonyl fluoride). Cell lysates were centrifuged, and the supernatants were added to glutathione-agarose (Molecular Probes). After a 1-h incubation at 4 °C the beads were washed 5 times with buffer containing 50 mm HEPES, pH 7.4, and 100 mm EDTA. Glutathione-agarose with immobilized GST or GST-SH3 was used directly in ligand binding experiments. The concentration of GST or GST-SH3 on the beads was determined by treating 50 μl of beads with 20 mm glutathione in 50 mm Tris, pH 8. The total amount of protein eluted was determined by the Bradford method (Bio-Rad) and divided by 50 μl to calculate the total concentration on the beads. Control beads were added to dilute the protein concentration to 1 mg/ml. U937 cells were maintained in RPMI 1640 medium supplemented with 5% fetal bovine serum. Before the pull-down experiment, 800 ml of U937 cells at a density of 106 cells/ml were treated with 10 ng/ml phorbol 12-myristate 13-acetate. After 48 h, cells were harvested and then washed 2× with phosphate-buffered saline. Cells were lysed for 30 min with rocking in 4 ml of lysis buffer containing 1% Triton X-100, 10 mm Tris, pH 7.5, 150 mm NaCl, 5 mm EDTA, 2 mm sodium orthovanadate, and protease inhibitors (5 mg/liter aprotinin, 5 mg/liter leupeptin, 0.1 mm phenylmethylsulfonyl fluoride). After centrifugation, a 1-ml portion of this lysate (containing 5 mg of protein) was added to 50 μl of glutathione-agarose containing 50 μg of GST-SH3. The remaining portion (3 ml, containing 15 mg protein) was added to 50 μl of glutathione-agarose containing 50 μg of GST. Beads and lysate were rocked 2 h at 4 °C and then washed 5 times with 10 ml of lysis buffer. Bound proteins were eluted in 50 μl of gel loading buffer by boiling. Proteins that eluted from the GST-SH3(Hck) affinity column were separated by one-dimensional SDS-PAGE and visualized with colloidal Coomassie stain. Protein bands specific to the Hck-SH3 pull-downs were excised and digested with trypsin as previously described (39Joyal J.L. Annan R.S., Ho, Y.D. Huddleston M.E. Carr S.A. Hart M.J. Sacks D.B. J. Biol. Chem. 1997; 272: 15419-15425Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). According to the intensity of the Coomassie band, 1/20 to1/3 of each unfractionated tryptic digest was analyzed by on-line liquid chromatography-electrospray tandem mass spectrometry using a micro-column (75 μm × 10 cm, 3-μm particles) reverse phase HPLC1 interfaced to an LCQ Deca ion trap mass spectrometer. Electrospray tandem mass spectrometry-based sequencing was performed on line in a data-dependent manner (40McCormack A.L. Schieltz D.M. Goode B. Yang S. Barnes G. Drubin D. Yates III, J.R. Anal. Chem. 1997; 69: 767-776Crossref PubMed Scopus (445) Google Scholar) as peptides eluted from the HPLC. Uninterpreted spectra were searched for protein matches against a non-redundant protein data base using the program Mascot (41Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6732) Google Scholar). Similar experiments were carried out to measure binding between immobilized GST-SH3(Hck) and purified proteins or proteins in Chinese hamster ovary (CHO) cell lysates, with the exception that binding reactions contained 10 μl of glutathione-agarose beads. In some experiments, various concentrations of polyproline-containing peptides were added together with the immobilized SH3 domain. C-terminally phosphorylated wild type Hck, WIP, and ELMO1 were produced inSpodoptera frugiperda (Sf9) cells using baculovirus expression vectors. Hck was expressed and purified as described previously (16Porter M. Schindler T. Kuriyan J. Miller W.T. J. Biol. Chem. 2000; 275: 2721-2726Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The cDNA for WIP was the kind gift of Dr. Narayanaswamy Ramesh (Harvard Medical School). The WIP-coding sequence was amplified by PCR and subcloned into the baculovirus expression vector pFastBac-Htb (Invitrogen). The cDNA for ELMO1 was obtained as clone DKFZp434B0819 from the German Cancer Research Center. We amplified the 2184-base pair DNA sequence by PCR and ligated it as anEcoRI/KpnI fragment into the baculovirus expression vector pFastBac-Htb. WIP and ELMO1 were expressed in Sf9 cells using the Invitrogen Bac-to-Bac system. Cells expressing His-tagged WIP and ELMO1 were lysed in a French pressure cell in buffer A (20 mm Tris, pH 8.5, 10% glycerol, 5 mm β-mercaptoethanol) containing protease inhibitors (5 mg/liter of aprotinin, 5 mg/liter leupeptin, 0.1 mm phenylmethylsulfonyl fluoride, and 1 mmEDTA). Cell lysate was diluted to 200 ml with column loading buffer (20 mm Tris, pH 8.5, 5% glycerol, 5 mmβ-mercaptoethanol, 1 m NaCl, and 20 mmimidazole) and centrifuged. Lysate was passed over a 5-ml nickel nitrilotriacetic acid Superflow column (Qiagen). WIP and ELMO1 were eluted with 100 mm imidazole. The concentration of ELMO1 and WIP were determined using the Bradford method (Bio-Rad). The substrate peptide for the coupled assay (AEEEIYGEFEAKKKKG (42Songyang Z. Carraway III, K.L. Eck M.J. Harrison S.C. Feldman R.A. Mohammadi M. Schlessinger J. Hubbard S.R. Smith D.P. Eng C. Lorenzo M.J. Poner B.A.J. Mayer B.J. Cantley L.C. Nature. 1995; 373: 536-539Crossref PubMed Scopus (844) Google Scholar)) was prepared by solid phase synthesis on an Applied Biosystems automated 431A Peptide Synthesizer. It was purified by reverse phase high pressure liquid chromatography and characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The polyproline-containing peptide (DFPLGPPPPLPPRATPSR (43Boonyaratanakornkit V. Scott M.P. Ribon V. Sherman L. Anderson S.M. Maller J.L. Miller W.T. Edwards D.P. Mol. Cell. 2001; 8: 269-280Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar)) was the generous gift of Dean Edwards (University of Colorado). Kinase assays were performed by a coupled spectrophotometric assay (44Barker S.C. Kassel D.B. Weigl D. Huang X. Luther M.A. Knight W.B. Biochemistry. 1995; 34: 14843-14851Crossref PubMed Scopus (158) Google Scholar). In this assay, the production of ADP is coupled to the oxidation of NADH measured as a reduction in absorbance at 340 nm. All experiments were carried out at 30 °C. Reactions were performed in buffer containing 100 mm Tris, pH 7.5, 10 mm MgCl2, 1 mmphosphoenolpyruvate, 0.28 mm NADH, 89 units/ml pyruvate kinase, and 124 units/ml lactate dehydrogenase. The assays contained 400 μm peptide substrate and 10 nm Hck. The ELMO1 cDNA was subcloned into two expression vectors to generate epitope-tagged versions of ELMO1 for mammalian expression, 1) pEF1/V5-HisA (Invitrogen) for a C-terminal V5 tag and 2) pCM45 (gift of Steve Lang and Pat Hearing, SUNY Stony Brook) for an N-terminal M45 tag. Full-length human Hck cDNA was subcloned into plasmid pCDNA6 (Invitrogen). Two 150-mm tissue culture plates were transfected for each condition tested. Transfections were carried out with 3.5 μl of TransIt (Panvera)/μg of DNA according to the manufacturer's protocol. After 40–48 h, cells from the two plates were combined and washed 2× in phosphate-buffered saline. Cells were lysed in lysis buffer (1% Triton X-100, 10 mm Tris, pH 7.5, 150 mm NaCl, 5 mm EDTA, 2 mmvanadate, and protease inhibitors) and clarified by centrifugation, and the protein concentration was determined. An equal amount (between 1 and 5 mg) of total cell protein was diluted to 1 ml for each reaction and precleared with 50 μl of protein A- or G-Sepharose beads for 1 h at 4 °C with rocking. After pre-clearing, 2 μg of appropriate antibody (or 15 μl of serum containing M45 antibody) or control antibody was added to lysate and incubated overnight at 4 °C with rocking. Antibodies used were mouse anti-M45 (P. Hearing, SUNY Stony Brook), mouse anti-Hck (Transduction Laboratories), rabbit anti-Hck (Santa Cruz), rabbit anti-WIP (a gift of Dr. Narayanaswamy Ramesh, Harvard Medical School), rabbit anti-WASP (Santa Cruz), and mouse anti-V5 (Invitrogen). Antibody-protein complexes were collected with 50 μl of protein A- or G-Sepharose beads for 1 h at 4 °C with rocking. The beads were washed 5 times in lysis buffer and boiled in 40 μl of gel loading buffer. After separation by SDS-PAGE, proteins were transferred to PVDF membranes. Incubation with primary antibody was carried out according to the manufacturer's protocol. After washing, the appropriate horseradish peroxidase-conjugated secondary antibody was added, and proteins were detected using the enhanced chemiluminescent detection kit (Amersham Biosciences). Hck is expressed predominantly in myeloid cells such as monocytes (18Quintrell N. Lebo R. Varmus H. Bishop J.M. Pettenati M.J., Le Beau M.M. Diaz M.O. Rowley J.D. Mol. Cell. Biol. 1987; 7: 2267-2275Crossref PubMed Scopus (202) Google Scholar). Furthermore, differentiation of monocytes to macrophages is associated with increased Hck expression and activity (18Quintrell N. Lebo R. Varmus H. Bishop J.M. Pettenati M.J., Le Beau M.M. Diaz M.O. Rowley J.D. Mol. Cell. Biol. 1987; 7: 2267-2275Crossref PubMed Scopus (202) Google Scholar). We reasoned that the more differentiated phenotype would also be accompanied by increases in specific binding complexes between Hck and other signaling proteins. Therefore, we treated U937 monocytes with phorbol 12-myristate 13-acetate for 48 h to induce the cells to differentiate to a more macrophage-like phenotype (45Schwende H. Fitzke E. Ambs P. Dieter P. J. Leukocyte Biol. 1996; 59: 555-561Crossref PubMed Scopus (437) Google Scholar). Clarified lysate from these cells was added to glutathione-agarose beads containing immobilized GST-SH3(Hck). As a control, 3-fold more U937 lysate was added to beads containing GST alone. After extensive washing, bound proteins were eluted by boiling in Laemmli buffer and then subjected to SDS-PAGE and visualized with colloidal Coomassie stain (Fig. 1). All visible bands were excised from the GST-SH3(Hck) lane. Bands in the control lane having similar molecular weight and intensity as bands in the GST-SH3(Hck) lane were also excised. The proteins in the bands were reduced, alkylated, and digested overnight with trypsin. The tryptic digests were analyzed by on-line liquid chromatography-electrospray tandem mass spectrometry, and the uninterpreted data was searched against the non-redundant protein data base using the program Mascot (41Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6732) Google Scholar). Proteins identified in the GST-SH3(Hck) lane but not the control lane are presented in TableI. The table also briefly summarizes what is known about the biological function of the interacting proteins and lists whether they have previously been shown to bind to any Src family kinase.Table IU937 proteins that interact with Hck SH3 domainSH3 ligandsNumber of peptides sequencedKnown to bind Src family kinasesBiological rolesDynamin II18No (related protein dynamin is Src substrate (63Ahn S. Maudsley S. Luttrell L.M. Lefkowitz R.J. Daaka Y. J. Biol. Chem. 1999; 274: 1185-1188Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar))Proline rich GTPase that mediates clathrin-dependent endocytosis (64Henley J.R. Krueger E.W. Oswald B.J. McNiven M.A. J. Cell Biol. 1998; 141: 85-99Crossref PubMed Scopus (619) Google Scholar)SIP-14516NoSH2 domain-containing member of the inositol polyphosphate 5-phosphatase family (65Kavanaugh W.M. Pot D.A. Chin S.M. Deuter-Reinhard M. Jefferson A.B. Norris F.A. Masiarz F.R. Cousens L.S. Majerus P.W. Williams L.T. Curr. Biol. 1996; 6: 438-445Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar)Cbl13Substrate for Hck (46Howlett C.J. Bisson S.A. Resek M.E. Tigley A.W. Robbins S.M. Biochem. Biophys. Res. Commun. 1999; 257: 129-138Crossref PubMed Scopus (26) Google Scholar), Src (66Yokouchi M. Kondo T. Sanjay A. Houghton A. Yoshimura A. Komiya S. Zhang H. Baron R. J. Biol. Chem. 2001; 276: 35185-35193Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), and Fyn (67Andoniou C.E. Lill N.L. Thien C.B. Lupher M.L., Jr. Ota S. Bowtell D.D. Scaife R.M. Langdon W.Y. Band H. Mol. Cell. Biol. 2000; 20: 851-867Crossref PubMed Scopus (99) Google Scholar)Many functions (68Tsygankov A.Y. Teckchandani A.M. Feshchenko E.A. Swaminathan G. Oncogene. 2001; 20: 6382-6402Crossref PubMed Scopus (106) Google Scholar) including negative regulation of Src kinases by targeting them to ubiquitin ligase (66Yokouchi M. Kondo T. Sanjay A. Houghton A. Yoshimura A. Komiya S. Zhang H. Baron R. J. Biol. Chem. 2001; 276: 35185-35193Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 67Andoniou C.E. Lill N.L. Thien C.B. Lupher M.L., Jr. Ota S. Bowtell D.D. Scaife R.M. Langdon W.Y. Band H. Mol. Cell. Biol. 2000; 20: 851-867Crossref PubMed Scopus (99) Google Scholar)Sos9Binds Src, Fyn, and Lck (69Park C. Choi Y. Yun Y. Mol. Cell. 1998; 8: 518-523Google Scholar)Ras GEF (70Nimnual A.S. Yatsula B.A. Bar-Sagi D. Science. 1998; 279: 560-563Crossref PubMed Scopus (388) Google Scholar)c-Cbl-interacting protein (CIN85)8NoAdaptor protein that associates with Cbl as well as BLNK, Crk-I, Crk-II, p130(Cas), p85-phosphatidylinositol 3-kinase Grb2, and Sos1 (71Watanabe S. Take H. Takeda K., Yu, Z.X. Iwata N. Kajigaya S. Biochem. Biophys. Res. Commun. 2000; 278: 167-174Crossref PubMed Scopus (69) Google Scholar)Actin6NoForms actin filaments that are part of the cytoskeleton of eukarotic cells (72Bear J.E. Krause M. Gertler F.B. Curr. Opin. Cell Biol. 2001; 13: 158-166Crossref PubMed Scopus (91) Google Scholar)WASP6Binds Fyn (49Banin S. Gout I. Brickell P. Mol. Biol. Rep. 1999; 26: 173-177Crossref PubMed Scopus (14) Google Scholar), Lyn (54Guinamard R. Aspenstrom P. Fougereau M. Chavrier P. Guillemot J.C. FEBS Lett. 1998; 434: 431-436Crossref PubMed Scopus (94) Google Scholar), and Fgr (50Finan P.M. Soames C.J. Wilson L. Nelson D.L. Stewart D.M. Truong O. Hsuan J.J. Kellie S. J. Biol. Chem. 1996; 271: 26291-26295Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar)Binding of WASP to the Arp2/3 complex at a cell membrane is the initial stimulation of actin nucleation (36Zigmond S.H. J. Cell Biol. 2000; 150: 117-120Crossref PubMed Google Scholar, 37Ramesh N. Anton I.M. Martinez-Quiles N. Geha R.S. Trends Cell Biol. 1999; 9: 15-19Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar)ASAP1/DEF-16Substrate for Src (73Brown M.T. Andrade J. Radhakrishna H. Donaldson J.G. Cooper J.A. Randazzo P.A. Mol. Cell. Biol. 1998; 18: 7038-7051Crossref PubMed Scopus (190) Google Scholar)Phospholipid-dependent arf GTPase-activating protein, which is phosphorylated by Src (73Brown M.T. Andrade J. Radhakrishna H. Donaldson J.G. Cooper J.A. Randazzo P.A. Mol. Cell. Biol. 1998; 18: 7038-7051Crossref PubMed Scopus (190) Google Scholar) and promotes adipogenesis in fibroblastic cell lines (74King F.J., Hu, E. Harris D.F. Sarraf P. Spiegelman B.M. Roberts T.M. Mol. Cell. Biol. 1999; 19: 2330-2337Crossref PubMed Scopus (36) Google Scholar)Heterogeneous nuclear ribonucleoprotein K5Binds Src, Fyn, and Lyn (75Weng Z. Thomas S.M. Rickles R.J. Taylor J.A. Brauer A.W. Seidel-Dugan C. Michael W.M. Dreyfuss G. Brugge J.S. Mol. Cell. Biol. 1994; 14: 4509-4521Crossref PubMed Scopus (206) Google Scholar)Nuclear RNA-binding protein that functions in transcription and translational silencing (76Krecic A.M. Swanson M.S. Curr. Opin. Cell Biol. 1999; 11: 363-371Crossref PubMed Scopus (709) Google Scholar)WIP5NoRegulates N-WASP-mediated actin polymerization (53Vetterkind S. Miki H. Takenawa T. Klawitz I. Scheidtmann K.H. Preuss U. J. Biol. Chem. 2001; 277: 87-95Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 77Martinez-Quiles N. Rohatgi R. Anton I.M. Medina M. Saville S.P. Miki H. Yamaguchi H. Takenawa T. Hartwig J.H. Geha R.S. Ramesh N. Nat. Cell Biol. 2001; 3: 484-491Crossref PubMed Scopus (225) Google Scholar)RNB64NoMember of Ena VASP protein family implicated in control of actin filament assembly and cell motility (78Ohta S. Mineta T. Kimoto M. Tabuchi K. Biochem. Biophys. Res. Commun. 1997; 237: 307-312Crossref PubMed Scopus (8) Google Scholar)α-Tubulin3No (but Src associates via other proteins with microtubules) (1Bjorge J.D. Jakymiw A. Fujita D.J. Oncogene. 2000; 19: 5620-5635Crossref PubMed Scopus (335) Google Scholar)Forms α-β tubulin heterodimers that form into microtubule polymers (79Dutcher S.K. Curr. Opin. Cell Biol. 2001; 13: 49-54Crossref PubMed Scopus (171) Google S" @default.
• W2068675259 created "2016-06-24" @default.
• W2068675259 creator A5022609500 @default.
• W2068675259 creator A5030517432 @default.
• W2068675259 creator A5032711040 @default.
• W2068675259 creator A5050315454 @default.
• W2068675259 creator A5064563580 @default.
• W2068675259 date "2002-08-01" @default.
• W2068675259 modified "2023-10-14" @default.
• W2068675259 title "Identification of Novel SH3 Domain Ligands for the Src Family Kinase Hck" @default.
• W2068675259 cites W1496101665 @default.
• W2068675259 cites W1509485250 @default.
• W2068675259 cites W1510401881 @default.
• W2068675259 cites W1530340727 @default.
• W2068675259 cites W1574164310 @default.
• W2068675259 cites W1786780592 @default.
• W2068675259 cites W1926532709 @default.
• W2068675259 cites W1959893769 @default.
• W2068675259 cites W1965383843 @default.
• W2068675259 cites W1966680042 @default.
• W2068675259 cites W1968422877 @default.
• W2068675259 cites W1971997730 @default.
• W2068675259 cites W1981593008 @default.
• W2068675259 cites W1988965362 @default.
• W2068675259 cites W1991683855 @default.
• W2068675259 cites W1993742172 @default.
• W2068675259 cites W1997372415 @default.
• W2068675259 cites W1998734127 @default.
• W2068675259 cites W1999857129 @default.
• W2068675259 cites W2002473468 @default.
• W2068675259 cites W2004460632 @default.
• W2068675259 cites W2007765297 @default.
• W2068675259 cites W2008351137 @default.
• W2068675259 cites W2013691294 @default.
• W2068675259 cites W2014211428 @default.
• W2068675259 cites W2015827627 @default.
• W2068675259 cites W2016207825 @default.
• W2068675259 cites W2022495919 @default.
• W2068675259 cites W2025138357 @default.
• W2068675259 cites W2025459204 @default.
• W2068675259 cites W2025925786 @default.
• W2068675259 cites W2026446377 @default.
• W2068675259 cites W2026907934 @default.
• W2068675259 cites W2027266132 @default.
• W2068675259 cites W2033250227 @default.
• W2068675259 cites W2034429144 @default.
• W2068675259 cites W2034716886 @default.
• W2068675259 cites W2043761739 @default.
• W2068675259 cites W2049360086 @default.
• W2068675259 cites W2052092007 @default.
• W2068675259 cites W2052934075 @default.
• W2068675259 cites W2058778918 @default.
• W2068675259 cites W2059161447 @default.
• W2068675259 cites W2062823148 @default.
• W2068675259 cites W2063760096 @default.
• W2068675259 cites W2065289702 @default.
• W2068675259 cites W2067948561 @default.
• W2068675259 cites W2068456278 @default.
• W2068675259 cites W2071481601 @default.
• W2068675259 cites W2072273093 @default.
• W2068675259 cites W2074590221 @default.
• W2068675259 cites W2078802143 @default.
• W2068675259 cites W2080489609 @default.
• W2068675259 cites W2081607618 @default.
• W2068675259 cites W2089291239 @default.
• W2068675259 cites W2095393268 @default.
• W2068675259 cites W2096204372 @default.
• W2068675259 cites W2101257013 @default.
• W2068675259 cites W2102487598 @default.
• W2068675259 cites W2105420135 @default.
• W2068675259 cites W2108131452 @default.
• W2068675259 cites W2109126307 @default.
• W2068675259 cites W2113249117 @default.
• W2068675259 cites W2119691249 @default.
• W2068675259 cites W2121634203 @default.
• W2068675259 cites W2121645313 @default.
• W2068675259 cites W2124504624 @default.
• W2068675259 cites W2127943666 @default.
• W2068675259 cites W2128640800 @default.
• W2068675259 cites W2137298291 @default.
• W2068675259 cites W2150486782 @default.
• W2068675259 cites W2153689647 @default.
• W2068675259 cites W2153844283 @default.
• W2068675259 cites W2154608911 @default.
• W2068675259 cites W2154901790 @default.
• W2068675259 cites W2159018836 @default.
• W2068675259 cites W2162795346 @default.
• W2068675259 cites W2170170334 @default.
• W2068675259 cites W2170449608 @default.
• W2068675259 cites W2188590912 @default.
• W2068675259 cites W224148732 @default.
• W2068675259 cites W2318731751 @default.
• W2068675259 cites W4241533536 @default.
• W2068675259 cites W4312243655 @default.
• W2068675259 cites W4312652157 @default.
• W2068675259 doi "https://doi.org/10.1074/jbc.m202783200" @default.
• W2068675259 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12029088" @default.
• W2068675259 hasPublicationYear "2002" @default.

• next