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Fyn/Lck Src Homology 3 Domain-Binding Protein (T-Celil Actvation/Signal Transduction/Protein-Tyrws Kna/Src-Famfly Kinases) KRIS A

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Fyn/Lck Src Homology 3 Domain-Binding Protein (T-Celil Actvation/Signal Transduction/Protein-Tyrws Kna/Src-Famfly Kinases) KRIS A Proc. Nati. Acad. Sci. USA Vol. 91, pp. 4135-4139, May 1994 Immunology Rapid T-cell receptor-mediated tyrosine phosphorylation of p120, an Fyn/Lck Src homology 3 domain-binding protein (T-celil actvation/signal transduction/protein-tyrws kna/Src-famfly kinases) KRIS A. REEDQUIST*, TORU FUKAZAWA*, BRIAN DRUKERt, GOVINDASWAMY PANCHAMOORTHY*, STEVEN E. SHOELSONt, AND HAMID BAND*§ *Lymphocyte Biology Section, Department of Rheumatology and Immunology, and tResearch Division, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, SGMB 514, 250 Longwood Avenue, Boston, MA 02115; and tDivision of Hematology and Medical Oncology, Oregon Health Sciences University, Portland, OR 97201. Communicated by K. Frank Austen, December 30, 1993 ABSTRACT Tyrosine phosphorylation ofcellular proteins phosphotyrosyl (pY) peptide motifs and mediate assembly is the earliest identifiable event following T-cell antigen recep- of signaling complexes (15, 16). The SH3 domains bind to tor (TCR) stimulation and is essential for activating down- small proline-rich peptide motifs to mediate protein-protein stream signaling machiner. Two Src-family protein-tyrosine interactions (17, 18). A clear physiological role for SH3 kinas, the TCR-associated pS9f (Fyn) and the CD4/8- domains of the Sem-5/Grb-2 protein in linking receptor associated p561c (Lck), have emerged as the likely mediators of tyrosine kinases to the Ras pathway has been demonstrated early tyrosine phosphorylation in T cells. Here, we show direct (19). Recently, the SH3 domains of Src-family kinases have binding of a 120-kDa TCR-induced phosphotyrosyl polypep- been shown to bind to the p85 subunit of phosphatidylino- tide, p120, to glutathione S-transferase fusion proteins of the sitol (PI) 3-kinase (3-K) (20, 21), paxillin (a focal adhesion Src homology 3 (SH3) domains of Fyn, Lck, and p60 (Src) protein) (22), actin-associated protein p110 (23), and micro- but not other proteins. While binding of p120 to Fyn SH2 tubule-associated GTPase dynamin (24). Thus, SH3 do- domain was phosphotyrosine-dependent as expected, its bind- mains may couple tyrosine kinases to multiple signaling ing to the SH3 domain was independent of tyrosine phosphor- pathways. Here we provide evidence for a role of SH3 ylation, as shown by lack ofcompetition with a phosphotyrosyl in competitor peptide. In contrast, an SH3-specific proline-rich domains recruiting a cellular substrate to TCR-associated peptide completely abolished p120 binding to SH3. p120 was tyrosine kinases. tyrosine-phosphorylated within 10 sec following stimulation of Jurkat cells with anti-CD3 monoclonal antibody, with mmal MATERIALS AND METHODS phosphorylation at 30 sec. Importantly, p120 was found asso- ciated with Fyn and Lck proteins in unstimulated Jurkat cells Peptides. The pY peptide including Tyr324 of the hamster and served as an in vitro substrate for these kinases. These medium-sized tumor antigen (EPQpYEEIPIYL, or pYEEI; results provide evidence for a role of the SH3 domains of Fyn bold type indicates the SH2-binding motif) (16) and its un- and Lck in the recruitment of early tyrosine-phosphorylation phosphorylated analog (YEEI) were synthesized and HPLC substrates to the TCR-associated tyrosine kinses. purified (25). The p85 SH3-N proline-rich peptide (PI 3-kinase p85a aa 83-101, ISPPTPKPRPPRPLPVAPG) (26) was syn- Engagement of the T-cell antigen receptor (TCR) induces a thesized with an Applied Biosystems peptide synthesizer. rapid tyrosine phosphorylation of cellular proteins as an Generation of Glutathione S-Transferase (GST) Fusion Pro- obligatory step in T-cell activation (1-3). While the TCR teins. Various domains of human Lck (27) (from R. Perlmut- components lack intrinsic tyrosine kinase domains, TCR {/'q ter, University ofWashington, Seattle) or Fyn (28) (American and CD3 y and e chains physically complex with the unique Type Culture Collection) were amplified by PCR and cloned N-terminal domain of the Src-family protein-tyrosine kinase as BamHI-EcoRI fragments into pGEX2T.K vector (from p59fyn (Fyn) (4, 5). A role for Fyn in TCR-induced tyrosine Bill Kaelin and Erik Flemington, Dana-Farber Cancer Insti- phosphorylation was suggested by enhanced antigen-induced tute, Boston) in-frame with GST (29, 30) (Fig. 1A). DNA or anti-TCR monoclonal antibody (mAb)-induced tyrosine sequences of all constructs were confirmed. The non-SH3 phosphorylation in a T-cell hybridoma overexpressing an C-terminal extension of Fyn SH3 fusion protein carried a activated Fyn and by increased or decreased TCR signaling, proline instead of serine (aa 143). The primer/vector se- respectively, in thymocytes of mice expressing activated or quences added glycine and serine to the N terminus and dominant-negative Fyn transgenes (6, 7). Defective re- EFIVTD to the C terminus of fusion proteins. Murine c-Src, sponses of thymocytes in fyn-/- knockout mice further neuronal Src [Src(+)], Abl type IV (aa 84-138) and v-Crk (aa underscore the critical role of Fyn in TCR signaling (8, 9). 372-428) SH3 domains encoded in pGEX2T were provided p56ock (Lck) interacts with the cytoplasmic tails of the CD4 by Bruce Mayer (Children's Hospital, Boston), and human and CD8 coreceptors and participates in CD4/8-dependent Lck SH3SH2 in pGEX3b by J. Shin (Dana-Farber Cancer enhancement ofantigen stimulation (10-12). In addition, Lck Institute). plays a critical role in early T-cell development (3). Fusion proteins were affinity purified on glutathione- The Src-family kinases contain the noncatalytic Src ho- Sepharose beads (Pharmacia) from the Triton X-100-soluble mology 3 and 2 (SH3 and SH2) domains that are required for fraction of isopropyl (-D-thiogalactopyranoside-induced negative regulation of kinase activity and are essential for Escherichia coli (strain DH5a or NB42) (29, 30), quantitated mediating T-cell activation as shown with Lck transfectants by Bradford assay (Bio-Rad; bovine '>'-globulin standard), (13, 14). The SH2 domains of signaling proteins bind to Abbreviations: TCR, T-cell antigen receptor; SHn, Src homology n; The publication costs ofthis article were defrayed in part by page charge GST, glutathione S-transferase; mAb, monoclonal antibody; PI, phos- payment. This article must therefore be hereby marked "advertisement" phatidylinositol. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 4135 Downloaded by guest on October 2, 2021 4136 Immunology: Reedquist et al. Proc. Natl. Acad. Sci. USA 91 (1994) A NT SH3 SH2 Kinase Tween 20 (Bio-Rad) containing anti-pY mAb 4G10 (31) at 1 N I~ _... ___ _.h-73A--1, i pCg/ml followed by TBS with 1251-labeled protein A (Amer- sham) at 10 ng/ml (0.3 ,Ci/ml; 1 uCi = 37 kBq), with six A.A. washes in between. Autoradiograms were developed with NTSH3SH2 --- Fyn 1-252 Kodak XAR-5 film at -80WC. In Vio KInase Reaction. Unlabeled Jurkat cells were lysed NTSH3 ==. Fyn :1-148 in lysis buffer with 1% (vol/vol) Brij 96 (Fluka) instead of SH3SH2 ---- Fyn 84-252 Triton X-100. Lysates from 5 x 107 cells were immunopre- FGST j- cipitated with normal rabbit serum, anti-integrin ,B7-antiserum Lck 66-225 (aa 736-755, from Chris Parker, Harvard Medical School) .SH2 [ - -- 3 Fyn : 143-252 anti-Lck (aa 22-51; Upstate Biotechnology, Lake Placid, NY), or anti-Fyn antiserum (aa 35-51, from Chris Rudd, Dana- Lck: 118233 Farber Cancer Institute) (32), and immunocomplexes were bound to protein A-Sepharose (Pharmacia). Immunocom- SH3 LS Fyn 84-148 plexes or products of fusion-protein binding reactions were Lck: 64-126 washed once in kinase buffer without ATP and then incubated Src: 88-141 in 100 id of kinase buffer (20 mM Hepes, pH 7.3/100 mM Src(+) 88-147 NaCl/5 mM MgCl2/5 mM MnCl2) in the absence or presence of 0.5 mM unlabeled ATP. After 20 min at 25°C, beads were B Fyn Lck washed three times with lysis buffer and anti-pY blotting was I performed. 0N = I MI Uc U' t| F tNC4 C-)X IVU'II CN cn C u U' U3 M: m -r ' :I I : 2~(DEnU)0I (n u' cn Mr(x 10 ) RESULTS Fyn and Lck SH3 Fusion Proteins Bind to a TCR-Induced p120- p 4 Tyrosine-Phosphorylated Protein of 120 kDa. Purified GST - 97 fusion proteins (Fig. 1A) noncovalently immobilized on glu- tathione-Sepharose beads were incubated with Triton X-100 *_ 3_ S_ - 69 lysates of Jurkat T cells stimulated with anti-CD3 mAb for 2 min, and the proteins adsorbed by the fusion proteins were analyzed by anti-pY immunoblotting. The Fyn SH2 fusion protein bound to essentially all induced pY proteins (Fig. 1B, - 46 compare lanes 1 and 3). Surprisingly, the binding of several phosphoproteins was increased when either the SH3 domain (lane 5) or the N-terminal and SH3 domains (lane 6) were included as part of the fusion protein. Increased phospho- protein binding to SH3SH2 and NTSH3SH2 fusion proteins 2 3 4 r 8 9 is partly due to enhanced affinity of the SH2 domain for pY FIG. 1. (A) Schematic representation and nomenclature of GST peptide motifs (G.P., T.F., L. Stolz, G. Payne, K.A.R., fusion proteins in relation to full-length Src kinases (top). NT, S.E.S., Z. Songyang, L. Cantley, C. Walsh, and H.B., N-terminal unique domain. Amino acid residues (A.A.) are indi- unpublished work). To test the additional possibility that the cated. (B) Binding ofFyn and Lck GST fusion proteins to pY proteins SH3 domain might directly bind to pY proteins, binding in anti-CD3-stimulated Jurkat cell lysates. Triton X-100 lysates of 5 reactions were performed with GST-SH3 fusion protein. x 107 Jurkat cells stimulatedfor 2 min with anti-CD3e mAb SPV-T3b Indeed, a prominent 120-kDa pY polypeptide (p120) was were incubated with 50 pg of indicated fusion proteins, and bound observed to bind to Fyn SH3 (lane 4).
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