Coordinated Regulation of the Tyrosine Phosphorylation of Cbl by Fyn and Syk Tyrosine Kinases

• DOI: 10.1074/jbc.273.15.8867
• Publication Date: 2002-07-26T15:04:43Z
• Authors: Marcel Deckert; Chris Elly; Amnon Altman; Yun-Cai Liu
• Keywords: FYN; Tyrosine-protein kinase CSK

Cross-linking of the T cell antigen receptor (TCR)-CD3 complex induces rapid tyrosine phosphorylation and activation Src (Lck Fyn) Syk (Syk Zap-70) family protein kinases (PTKs) which, in turn, phosphorylate multiple intracellular substrates. Cbl is a prominent PTK substrate suggesting pivotal role for it early signal transduction events. However, regulation function cells by upstream PTKs remains poorly understood. In present study, we used genetic biochemical approaches to demonstrate that directly interacts with Fyn via its N-terminal C-terminal regions, respectively. Tyr-316 was required interaction as well maximal Cbl. both wild-type Y316F-mutated phosphorylated equally fragment vivo, existence an alternative, N terminus-independent mechanism Syk-induced This appears involve Fyn, since, addition association region Cbl, also associated enhanced These findings implicate adaptor facilitates between suggest coordinately regulate Binding antigenic peptides presented major histocompatibility molecules (TCR) 1The abbreviations are: TCR, receptor; GST, glutathione S-transferase; mAb, monoclonal antibody; PTK, kinase; Tyr(P), phosphotyrosine; PTB, phosphotyrosine binding; IP, immunoprecipitate; HA, hemagglutinin; SD, synthetic dropout. -CD3 increase activities two families nonreceptor PTKs,i.e. (Fyn Lck), (1Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1957) Google Scholar, 2Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Abstract (462) Scholar). Activation Lck and/or leads immunoreceptor tyrosine-based motifs domains CD3 ζ subunits, resulting subsequent recruitment Zap-70 Syk. The activated turn propagate signals phosphorylating proteins, eventually leading activation, lymphokine production, proliferation. many aspects underlying signaling cascades remain unclear. c-cbl (Casitas B-lineagelymphoma) protooncogene originally isolated cellular homologue v-cbl, part transforming gene Cas NS-1 murine leukemia retrovirus (3Langdon W.Y. Hartley J.W. Klinken S.P. Ruscetti S.K. Morse H.C. d. Proc. Natl. Acad. Sci. U. S. 1989; 86: 1168-1172Crossref (283) 4Blake T.J. Shapiro M. Langdon Oncogene. 1991; 6: 653-657PubMed 120-kDa product c-cbl, rapidly on residues response stimulation various surface receptors, including TCR (5Liu Y.-C. Altman Signalling. 1998; 10 (in press)Google 6Donovan J.A. Wange J. Biol. Chem. 269: 22921-22924Abstract 7Meisner H. Conway B.R. D. Czech M.P. Mol. 1995; 15: 3571-3578Crossref (212) Scholar) (reviewed Liu Scholar)), critical pathways. importance further emphasized observation associates several other molecules. Numerous studies have shown binds constitutively Grb2 (6Donovan 8Fukazawa T. Reedquist K.A. Trub Soltoff Panchamoorthy G. Druker B. Cantley L. Shoelson S.E. Band 270: 19141-19150Abstract (186) 9Odai Sasaki K. Iwamatsu Hanazono Y. Tanaka Mitani Yazaki Hirai 10800-10805Abstract (182) Tyrosine results inducible phosphatidylinositol 3-kinase (7Meisner 10Soltoff L.C. 271: 563-567Abstract (133) 11Hartley Meisner Corvera 18260-18263Abstract (90) 12Kim Kim Y.-T. Pillai 27504-27509Abstract (70) Scholar), CrkL (13de Jong R. ten Hoeve Heisterkamp N. Groffen 21468-21471Abstract (127) 14Ribon V. Hubbell Herrera Saltiel A.R. 16: 45-52Crossref (102) 15Sawasdikosol Ravichandran K.S. Lee K.K. Chang J.-H. Burakoff S.J. 2893-2896Abstract (74) 16Reedquist Fukazawa B.J. 8435-8442Abstract (165) 17Buday Khwaja Sipeki Farago Downward 6159-6163Abstract (135) 18Andoniou C.E. Thien C.B. 12: 1981-1989PubMed 19Smit van der Horst Borst 8564-8569Abstract (111) 20Ingham R.J. Krebs D.L. Barbazuk S.M. Turck C.W. Matsuda Gold M.R. 32306-32314Abstract (68) Vav (21Marengere Mirtsos C. Kozieradzki I. Veillette Mak Penninger Immunol. 1997; 159: 70-76PubMed We recently demonstrated 14-3-3 proteins (22Liu Elly Yoshida Bonnefoy-Berard 14591-14595Abstract (100) which depends serine (23Liu Lipkowitz 272: 9979-9985Abstract (125) Reconstitution COS-1 showed phosphorylates Lck- Fyn-dependent manner (24Fournel Davidson Weil Exp. Med. 183: 301-306Crossref (123) has been greater intrinsic enzymatic activity than terms (25Latour Chow L.M.L. 22782-22790Abstract (132) alone can cause coexpression Overexpression mast enhances FcεRI-mediated (26Ota Beitz L.O. Scharenberg A.M. Donovan Kinet J.P. 184: 1713-1723Crossref (86) notion complemented observations physically associate these PTKs. Both kinases, such 24Fournel 27Panchamoorthy Miyake 3187-3194Abstract (176) 28Tsygankov A.Y. Mahajan Fincke J.E. Bolen J.B. 23137-27130Abstract (72) or Lyn (29Tezuka Umemori Fusaki Yagi Takata Kurosaki Yamamoto 675-680Crossref (93) 30Lupher Jr., M.L. 24063-24068Abstract (166) reported activation-dependent, mediated (Tyr(P))-binding (PTB) domain (30Lupher A point mutation (G306E) this c-Cbl, corresponds loss-of-function Sli-1, Caenorhabditis elegans (31Yoon C.H. Jongeward G.D. Sternberg P.W. Science. 1102-1105Crossref (282) disrupts Zap-70, indicating portion interaction. implicated constitutive interactions Syk/Zap-70 strongly involved Syk/Zap-70- pathways are stimulated ligation. As first step toward understanding biological undertaken study using approaches. terminus whereas C terminus. define residue Syk, Tyr-316, direct with, of, addition, our act phsophorylation coordinated Polyclonal rabbit anti-Cbl (c-15) anti-Fyn antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Tyr(P) antibody (mAb) 4G10 anti-glutathioneS-transferase (GST) polyclonal Upstate (Lake Placid, NY). Anti-hemagglutinin (HA) mAb (12CA5) Boehringer Mannheim. An anti-CD3ε (OKT3) anti-HIV-derived tag epitope (H902) (32Deckert Tartare-Deckert Couture Mustelin 591-604Abstract (244) mAbs purified ascites fluid A-Sepharose affinity chromatography. Horseradish peroxidase-conjugated F(ab′)2 fragments donkey anti-rabbit IgG sheep anti-mouse Amersham Corp. pLex pACT2 vectors (CLONTECH) yeast two-hybrid system. cDNAs corresponding full-length v-Cbl (Cbl 1–365), (Cbl-C; amino acid 450–906) fused in-frame Gal4 encoded vector. containing mutants SykK395R, Y518F/Y519F, 1–298, 1–212, 213–628, Y341F, Y345F, vector Vav, described previously Additional mutations Y316F Y500F made site-directed mutagenesis based pLex-Syk encoding subcloned into mammalian vector, pEFneo (33Liu Kawagishi Mikayama Inagaki Takeuchi Ohashi 1993; 90: 8957-8961Crossref (43) tagged HA epitope. H902-tagged pME18S cDNA provided Kawakami. derived fashion. HA-tagged 70Z, v-Cbl, Cbl-C, successive deletion 70Z (34Liu 168-173Abstract (60) Cbl-C constructed polymerase chain reaction downstream H902 system essentially same Briefly, strain L40 cotransformed appropriate cotransformants screened plates dropout (SD) medium lacking Leu Trp. Colonies then picked up grown SD Leu, Trp, His examine protein-protein interactions. transformants tested β-galactosidase filter assay. Simian virus 40 (TAg)-transfected human leukemic Jurkat (Jurkat-TAg) RPMI 1640 (Life Technologies, Inc.) supplemented 10% fetal bovine serum antibiotics. Cells resuspended (2 × 107/ml) 0.5 ml medium, equilibrated at 37 °C 5 min, OKT3 (4 μg/ml) min. Stimulation terminated adding 2× Nonidet P-40 lysis buffer (2% P-40, mm Tris-HCl, pH 7.5, 300 mmNaCl, EDTA, sodium pyrophosphate, NaF, 4 Na3VO4, 20 μg/ml each aprotinin leupeptin). lysed min insoluble materials removed centrifugation 15,000 g min). cultured Iscove's modified Dulbecco's heat-inactivated 5% CO2. For expression Jurkat-TAg cells, transfected amount plasmids (usually μg total) electroporation baculovirus GST fusion pAc-GST (Pharmingen) partner, encodes 6× Recombinant carrying constructs generated cotransfection DNA insect line, Sf9, Baculogold transfection kit (Pharmingen), amplified, infection Sf9 cells. GST-Cbl Ni2+-nitrilotriacetic columns. Forin vitro binding assays, lysates incubated 2 h °C, followed μl glutathione-Sepharose beads (35Bonnefoy B.N. von W.M. Sung Ishizaka 92: 10142-10146Crossref After 1 mixtures washed extensively 1× analysis. procedure Far-Western blotting GST-Fyn SH2 purchased Biotechnology. Lysates (1 107 cells) mixed h, A/G Plus-Sepharose Biotechnology) additional °C. Immunoprecipitates (IPs) four times buffer, boiled 30 Laemmli's buffer. Samples subjected SDS-polyacrylamide gel electrophoresis, gel, analysis electrotransferred onto polyvinylidene difluoride membranes (Millipore). Membranes immunoblotted indicated primary μg/ml), horseradish secondary antibodies. visualized chemiluminescence detection (ECL, Corp.). When necessary, stripped incubation 62.5 6.7, 100 mm2-mercaptoethanol, 2% SDS 70 constant agitation, washed, reprobed indicated. It increased upon (28Tsygankov To Fig. A, transformed (and Fyn), but not exhibited growth Trp His, negative control, no observed empty Next, wished more precisely dissect Fyn-binding Two proximal (amino 450–730) distal (residues 731–906) portions B, similar fragment, selection medium. result indicates encompassing 450–730 contains site(s) Fyn. performedin assays proteins. Cell unstimulated anti-CD3 (OKT3)-stimulated alone, GST-Cbl-C, GST-v-Cbl. precipitates analyzed antibody. bound activation-dependent (top panel). membrane anti-Grb2 antibody, showing (bottom panel), earlier Fyn-Cbl vivo cotransfecting plasmid plus panel deletions B (70Z) coimmunoprecipitated (lanes 6). 1–450 1–655 did coimmunoprecipitate 3). mutant acids 1–698 weakly (lane 4), 1–730 displayed even stronger versus 5). Equivalent levels all samples revealed reprobing (Fig. middle Similarly, expressed equivalent B,bottom map interaction, prepared series Cbl-C. above. 538–906 552–906 degree C, top panel; lanes 3 lane 1). Deletion 63 (615–906 mutant) 146 (698–906 markedly reduced 5, respectively). immunoprecipitated different groups immunoblotting C,middle bottom panels, indicate 552–614 binding. found interact rat basophilic (27Panchamoorthy grew His. No any three constructs. positive interacted region. expanded Cbl-binding kinase-inactive (K395R), kinase-deficient tandem 1–298), Y518F/Y519F regulatory autophosphorylation sites mutated, 1–212 lacks kinase second domain. 213–628 catalytically active, still v-Cbl. linker/kinase Of tyrosine-mutated only B). note, Y341F Y345F mutations, mediate Y500F, had discernible effect specificity finding disrupt under conditions C). establish physiological relevance Cbl-Syk explored assay transiently anti-CD3-stimulated GST-v-Cbl, probed anti-HA GST-v-Cbl 6 8). weak consistent GST-Cbl-C OKT3-stimulated 10), Y316 11 12). detected 13 14). suggests independent domain, mediates (directly indirectly) analyze cotransfected IPs anti-Tyr(P) Cbl-associated phosphoprotein comigrating authentic immunoprecipitates 2). weaker parallel immunoblotting, confirm previous contributes observedin suggests, again, alternative Syk-Cbl N- regions respectively, suggested d