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The Lck SH3 Domain Is Required for Activation of the Mitogen-activated Protein Kinase Pathway but Not the Initiation of T-cell Antigen Receptor Signaling

1999; Elsevier BV; Volume: 274; Issue: 8 Linguagem: Inglês

10.1074/jbc.274.8.5146

1083-351X

Michael F. Denny, Heather C. Kaufman, Andrew C. Chan, David B. Straus,

Immune Cell Function and Interaction

Initiation of T-cell antigen receptor (TCR) signaling is dependent upon the activity of protein tyrosine kinases. The Src family kinase Lck is required for the initial events in TCR signaling, such as the phosphorylation of the TCR complex and the activation of ZAP-70, but little is known of its role in downstream signaling. Expression of a mutated form of Lck lacking SH3 domain function (LckW97A) in the Lck-deficient T-cell line JCaM1 revealed a requirement for Lck beyond the initiation of TCR signaling. In cells expressing LckW97A, stimulation of the TCR failed to activate the mitogen-activated protein kinase (MAPK) pathway, despite normal TCR ζ chain phosphorylation, ZAP-70 recruitment, and ZAP-70 activation. Activation of extracellular signal-regulated kinase (ERK) and MAPK kinase (MEK), as well as the induction of CD69 expression, was greatly impaired in JCaM1/LckW97A cells. In contrast, the phosphorylation of phospholipase Cγ1 (PLCγ1) and corresponding elevations in intracellular calcium concentration ([Ca2+]i) were intact. Thus, cells expressing LckW97A exhibit a selective defect in the activation of the MAPK pathway. These results demonstrate that Lck has a role in the activation of signaling pathways beyond the initiation of TCR signaling and suggest that the MAPK pathway may be selectively controlled by regulating the function of Lck. Initiation of T-cell antigen receptor (TCR) signaling is dependent upon the activity of protein tyrosine kinases. The Src family kinase Lck is required for the initial events in TCR signaling, such as the phosphorylation of the TCR complex and the activation of ZAP-70, but little is known of its role in downstream signaling. Expression of a mutated form of Lck lacking SH3 domain function (LckW97A) in the Lck-deficient T-cell line JCaM1 revealed a requirement for Lck beyond the initiation of TCR signaling. In cells expressing LckW97A, stimulation of the TCR failed to activate the mitogen-activated protein kinase (MAPK) pathway, despite normal TCR ζ chain phosphorylation, ZAP-70 recruitment, and ZAP-70 activation. Activation of extracellular signal-regulated kinase (ERK) and MAPK kinase (MEK), as well as the induction of CD69 expression, was greatly impaired in JCaM1/LckW97A cells. In contrast, the phosphorylation of phospholipase Cγ1 (PLCγ1) and corresponding elevations in intracellular calcium concentration ([Ca2+]i) were intact. Thus, cells expressing LckW97A exhibit a selective defect in the activation of the MAPK pathway. These results demonstrate that Lck has a role in the activation of signaling pathways beyond the initiation of TCR signaling and suggest that the MAPK pathway may be selectively controlled by regulating the function of Lck. Activation of T-lymphocytes is initiated by engagement of the T-cell antigen receptor (TCR) 1The abbreviations TCRT-cell antigen receptorITAMsimmunoreceptor-based tyrosine activation motifsMAPKmitogen-activated protein kinaseMEKmitogen-activated protein kinase kinaseERKextracellular-signal regulated kinasePIP2phosphatidylinositolPI-3-Kphosphatidylinositol 3-kinaseSH2Src homology 2SH3Src homology 3PAGEpolyacrylamide gel electrophoresisFITCfluorescein isothiocyanatePHAphytohemagglutininPMAphorbol 12-myristate 13-acetateGSTglutathione S-transferase[Ca2+]iintracellular calcium concentrationPLCγ1phospholipase Cγ1 (1Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1981) Google Scholar, 2Cantrell D. Annu. Rev. Immunol. 1996; 14: 259-274Crossref PubMed Scopus (597) Google Scholar, 3Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). Signaling through the TCR is dependent upon the activity of protein-tyrosine kinases which induce the phosphorylation of a number of proteins, including the subunits of the TCR complex itself. Tyrosine phosphorylation of the TCR complex occurs within amino acid sequences known as immunoreceptor-based tyrosine activation motifs (ITAMs) and is required for receptor signaling function (4Letourneur F. Klausner R.D. Science. 1992; 255: 79-82Crossref PubMed Scopus (366) Google Scholar, 5Romeo C. Amiot M. Seed B. Cell. 1992; 68: 889-897Abstract Full Text PDF PubMed Scopus (277) Google Scholar, 6Irving B.A. Chan A.C. Weiss A. J. Exp. Med. 1993; 177: 1093-1103Crossref PubMed Scopus (260) Google Scholar). Activation of downstream signaling pathways, including the Ras/mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol (PIP2) pathway, and the phosphatidylinositol 3-kinase (PI-3-K) pathway, is dependent upon protein-tyrosine kinase function (7June C.H. Fletcher M.C. Ledbetter J.A. Schieven G.L. Siegel J.N. Phillips A.F. Samelson L.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7722-7726Crossref PubMed Scopus (432) Google Scholar, 8Izquierdo M. Downward J. Graves J. Cantrell D. Mol. Cell. Biol. 1992; 12: 3305-3312Crossref PubMed Scopus (141) Google Scholar, 9Carrera A.C. Rodriguez-Borlado L. Martinez-Alonso C. Merida I. J. Biol. Chem. 1994; 269: 19435-19440Abstract Full Text PDF PubMed Google Scholar, 10von Willebrand M. Baier G. Couture C. Burn P. Mustelin T. Eur. J. Immunol. 1994; 24: 234-238Crossref PubMed Scopus (41) Google Scholar). However, the mechanisms by which the induction of tyrosine kinase activity initiates these diverse signaling processes is unknown. T-cell antigen receptor immunoreceptor-based tyrosine activation motifs mitogen-activated protein kinase mitogen-activated protein kinase kinase extracellular-signal regulated kinase phosphatidylinositol phosphatidylinositol 3-kinase Src homology 2 Src homology 3 polyacrylamide gel electrophoresis fluorescein isothiocyanate phytohemagglutinin phorbol 12-myristate 13-acetate glutathione S-transferase intracellular calcium concentration phospholipase Cγ1 Unlike receptor tyrosine kinases, the cytoplasmic portion of the TCR lacks intrinsic catalytic activity. Instead, the induction of tyrosine phosphorylation following engagement of the TCR requires the expression of non-receptor kinases. Both the Src family and the Syk/ZAP-70 family of tyrosine kinases are required for normal TCR signal transduction (11Karnitz L. Sutor S.L. Torigoe T. Reed J.C. Bell M.P. McKean D.J. Leibson P.J. Abraham R.T. Mol. Cell. Biol. 1992; 12: 4521-4530Crossref PubMed Scopus (187) Google Scholar, 12Straus D. 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A role for Lck has been identified in the initiation of TCR signaling; T-cells lacking functional Lck fail to initiate ITAM phosphorylation or induce ZAP-70 recruitment and activation (12Straus D. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (949) Google Scholar, 17van Oers N. Killeen N. Weiss A. J. Exp. Med. 1996; 183: 1053-1062Crossref PubMed Scopus (284) Google Scholar,19Iwashima M. Irving B.A. van Oers N. Chan A. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar). ZAP-70 has been implicated in downstream signaling events such as activation of the PIP2 and the MAPK pathways (18Williams B.L. Schreiber K.L. Zhang W. Wange R.L. Samelson L.E. Leibson P.J. Abraham R.T. Mol. Cell. Biol. 1998; 18: 1388-1399Crossref PubMed Scopus (228) Google Scholar, 20Kong G.H. Bu J.Y. Kurosaki T. Shaw A.S. Chan A.C. Immunity. 1995; 2: 485-492Abstract Full Text PDF PubMed Scopus (87) Google Scholar, 21Mege D. Di Bartolo V. Germain B. Tuosto L. Michel F. Acuto O. J. Biol. Chem. 1996; 271: 32644-32652Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 22Qian D. Mollenauer M. Weiss A. J. Exp. Med. 1996; 183: 611-620Crossref PubMed Scopus (93) Google Scholar). A current model for the initiation of TCR signaling proposes that Lck and ZAP-70 function sequentially (1Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1981) Google Scholar, 2Cantrell D. Annu. Rev. Immunol. 1996; 14: 259-274Crossref PubMed Scopus (597) Google Scholar, 3Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). Heterologous cell systems expressing ZAP-70 and the cytoplasmic domain of the TCR ζ chain displayed enhanced ITAM phosphorylation and ZAP-70 activation when Lck is co-transfected with ZAP-70 (19Iwashima M. Irving B.A. van Oers N. Chan A. Weiss A. Science. 1994; 263: 1136-1139Crossref PubMed Scopus (2) Google Scholar, 23Chan A.C. Dalton M. Johnson R. Kong G.H. Wang T. Thoma R. Kurosaki T. EMBO J. 1995; 14: 2499-2508Crossref PubMed Scopus (326) Google Scholar, 24Wange R.L. Guitian R. Isakov N. Watts J.D. Aebersold R. Samelson L.E. J. Biol. Chem. 1995; 270: 18730-18733Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), suggesting Lck mediates both the initial ITAM phosphorylation and the subsequent phosphorylation and activation of ZAP-70. However, this does not preclude a role for Lck in TCR signaling which is independent of the activation of ZAP-70 (25Wong J. Straus D. Chan A.C. Mol. Cel. Biol. 1998; 18: 2855-2866Crossref PubMed Scopus (25) Google Scholar). Like other Src family kinases, Lck contains a C-terminal kinase domain, a single Src homology 2 (SH2) and SH3 domain, and an N-terminal region that is distinct from other family members (26Bolen J.B. Rowley R.B. Spana C. Tsygankov A.Y. FASEB J. 1992; 6: 3403-3409Crossref PubMed Scopus (194) Google Scholar). SH3 domains are able to mediate protein interactions by binding certain proline-rich amino acid sequences (27Ren R. Mayer B.J. Cicchetti P. Baltimore D. Science. 1993; 259: 1157-1161Crossref PubMed Scopus (1044) Google Scholar, 28Musacchio A. Saraste M. Wilmanns M. Nat. Struct. Biol. 1994; 1: 546-551Crossref PubMed Scopus (287) Google Scholar), and a number of proteins have been reported to bind the Lck SH3 domain including c-Cbl, PI-3-K, Ras-GAP, HS1, and CD2 (29Reedquist K. Fukazawa T. Druker B. Panchamoorthy G. Shoelson S. Band H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4135-4139Crossref PubMed Scopus (77) Google Scholar, 30Prasad K.V.S. Kapeller R. Janssen O. Repke H. Duke-Cohan J.S. Cantley L.C. Rudd C.E. Mol. Cell. Biol. 1993; 13: 7708-7717Crossref PubMed Scopus (141) Google Scholar, 31Briggs S.D. Bryant S.S. Jove R. Sanderson S.D. Smithgall T.E. J. Biol. Chem. 1995; 270: 14718-14724Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 32Takemoto Y. Furuta M. Li X.-K. Strong-Sparks W.J. Hashimoto Y. EMBO J. 1995; 14: 3403-3414Crossref PubMed Scopus (78) Google Scholar, 33Bell G.M. Fargnoli J. Bolen J.B. Kish L. Imboden J.B. J. Exp. Med. 1996; 183: 169-178Crossref PubMed Scopus (80) Google Scholar). The SH3 domain of Lck has also been proposed to stabilize the formation of Lck homodimers which may potentiate TCR signaling following co-ligation of the receptor and CD4 (34Eck M.J. Atwell S.K. Shoelson S.E. Harrison S.C. Nature. 1994; 368: 764-769Crossref PubMed Scopus (244) Google Scholar, 35Lee-Fruman K.K. Collins T.L. Burakoff S.J. J. Biol. Chem. 1996; 271: 25003-25010Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Previous work indicated that deletion of the Lck SH3 domain interfered with the ability of an oncogenic form of Lck to enhance interleukin-2 production, supporting a role for the Lck SH3 domain in regulating T-cell activation (36Caron L. Abraham N. Pawson T. Veillette A. Mol. Cell. Biol. 1992; 12: 2720-2729Crossref PubMed Scopus (81) Google Scholar). In the present study, we have examined the involvement of the Lck SH3 domain in TCR signaling processes by expressing an altered form of Lck which contained an inactive SH3 domain in the Lck-deficient T-cell line JCaM1 (12Straus D. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (949) Google Scholar). TCR signaling pathways displayed a differential sensitivity to loss of Lck SH3 domain function. The induction of tyrosine phosphorylation and activation of the PIP2 pathway was independent of the Lck SH3 domain, whereas activation of the MAPK pathway was strictly SH3 domain-dependent. The inability of this altered form of Lck to activate the MAPK pathway despite ZAP-70 activation suggests that Lck participates directly in the stimulation of downstream signaling pathways following TCR ligation. Derivatives of the Lck-deficient Jurkat cell line JCaM1 were maintained at 37 °C and 5% CO2 in RPMI 1640 supplemented with 10% fetal bovine serum, glutamine, penicillin, and streptomycin. The LckW97A mutant cDNA was generated from wild type human Lck cDNA using base pair mismatched primers and polymerase chain reaction amplification and was confirmed by sequencing. The Lck cDNA was subcloned into pBP1, a plasmid derived from pUCH-13 (37Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4315) Google Scholar) which contains a cytomegalovirus promoter sequence regulated by tetracycline operators, and a gene conferring resistance to the antibiotic hygromycin. Clones that express wild type Lck or LckW97A were generated by electroporation of a G418-resistant JCaM1 derivative, which expresses a VP16-tetracycline repressor fusion protein (37Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4315) Google Scholar), and were isolated by plating at limiting dilution in the presence of hygromycin and G418. Clones that expressed TCR and wild type Lck, or LckW97A, at levels equivalent to the parental Jurkat cell line were maintained for further analysis. TCR expression was measured by staining cells with a mouse antibody recognizing CD3 (Leu-4), followed by a FITC-conjugated goat anti-mouse secondary antibody, and analyzed by fluorescence flow cytometry. To assess the induction of CD69, cells expressing wild type Lck or LckW97A, or Lck-deficient control cells, were incubated with media alone, PHA (0.3 μg/ml), or PMA (50 ng/ml) for 16–20 h at 37 °C. Cells were stained with a mouse anti-CD69 antibody (PharMingen) and a FITC-conjugated goat anti-mouse secondary antibody then evaluated by flow cytometry. Cell suspensions (40 × 106 per ml in phosphate-buffered saline) were stimulated for 2 min at 37 °C with the anti-Jurkat TCR monoclonal antibody C305 (38Weiss A. Stobo J.D. J. Exp. Med. 1984; 160: 1284-1299Crossref PubMed Scopus (419) Google Scholar). Cells were lysed in 1% (v/v) Nonidet P-40 solution containing (in mm) 10 Tris (pH 7.8), 150 NaCl, 1 phenylmethylsulfonyl fluoride, 0.4 sodium orthovanadate, 10 NaF, as well as 1 μg/ml leupeptin. Particulate matter was removed by centrifugation at 12,500 × g for 10 min at 4 °C, and lysates were precleared with fixed Staphylococcus aureus(Pansorbin, Calbiochem). Lck, ZAP-70, and PLCγ1 were immunoprecipitated using rabbit antisera (Upstate Biotechnology), and TCR ζ chain was immunoprecipitated using the 6B10.2 monoclonal antibody (Zymed Laboratories Inc.). Immunoprecipitates were collected on protein A-Sepharose, washed twice each in Nonidet P-40 lysis buffer and lysis buffer containing 0.5 m NaCl, analyzed by SDS-PAGE, and transferred to polyvinylidene difluoride membranes. Monoclonal antibodies were used to detect Lck (1F6; A. Burkhardt and J. Bolen), phosphotyrosine (4G10; Upstate Biotechnology, Inc.), ZAP-70 (2F3.2; Upstate Biotechnology, Inc.), PLCγ1 (mixed monoclonal, Upstate Biotechnology, Inc.), and TCR ζ chain (6B10.2;Zymed Laboratories Inc.). The activated forms of ERK1 and ERK2 or MEK1 and MEK2 were detected using rabbit antibodies which recognize phospho-ERK or phospho-MEK (New England Biolabs). Proteins were detected using horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG secondary antibody and enhanced chemiluminescence. Lck immunoprecipitates were washed twice in Nonidet P-40 lysis buffer followed by two washes in 20 mm Tris (pH 7.4), 0.5m LiCl, 0.4 mm sodium vanadate, and a single wash in distilled H2O. The immobilized Lck was resuspended at 37 °C in Lck kinase buffer containing (in mm) 20 Tris (pH 7.4), 10 MnCl2, 0.01 ATP, as well as 1 μg of GST-ζ fusion protein and 10 μCi of [γ-32P]ATP. Aliquots were withdrawn and transferred to microcentrifuge tubes containing ice-cold Nonidet P-40 lysis buffer, 2 mm EDTA, 10 mm ATP, and glutathione-agarose; after 30 min at 4 °C the samples were washed twice with Nonidet P-40 lysis buffer and resuspended in SDS sample buffer. ZAP-70 immunoprecipitates were prepared from unstimulated and TCR-stimulated cells using Nonidet P-40 lysis buffer containing 2 mm EDTA, washed twice in the same buffer, then twice 10 mm Tris (pH 7.4), 0.5 m LiCl, and once with ZAP-70 kinase buffer containing (in mm) 10 Tris (pH 7.4), 10 MnCl2, 10 MgCl2. The ZAP-70 kinase assay was conducted at room temperature for 10 min by resuspending in ZAP-70 kinase buffer with 20 μm ATP, 1 μg of GST-Band III fusion protein, and 15 μCi of [γ-32P]ATP. The reactions were terminated by addition of 2× SDS sample buffer. Following SDS-PAGE and staining with Coomassie Blue, the gels were dried to filter paper and exposed to x-ray-sensitive film (X-OMat, Eastman Kodak Co.). The regions corresponding to phospho-GST-ζ and phospho-Lck or phospho-GST-Band III were excised, and the incorporation of 32P was measured by counting Cerenkov radiation. Cells were loaded with the fluorescent calcium indicator indo-1, washed extensively in Hepes-buffered saline (HeBS, pH 7.4) solution containing (in mm) 25 Hepes, 125 NaCl, 5 KCl, 1 CaCl2, 0.5 MgCl2, 1 Na2HPO4, 0.1% (w/v) bovine serum albumin and 0.1% (w/v) d-glucose, and kept on ice. Prior to use, the aliquots were warmed to 37 °C for 10 min and then placed in a spectrofluorometer equipped with a thermally jacketed cuvette holder maintained at 37 °C. The fluorescence intensity was monitored continuously at the emission wavelength of 400 nm following excitation at 334 nm. Fluorescence intensity values were corrected for cell autofluorescence and then converted to [Ca2+]iusing a K d value of indo-1 for Ca2+ of 250 nm (39Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). To examine the role of Lck in TCR signaling, we have utilized the Lck-deficient JCaM1 T-cell line, derived from the Jurkat cell line (12Straus D. Weiss A. Cell. 1992; 70: 585-593Abstract Full Text PDF PubMed Scopus (949) Google Scholar,40Goldsmith M.A. Weiss A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6879-6883Crossref PubMed Scopus (123) Google Scholar). The advantage of using JCaM1 is that TCR signaling can be studied in clones expressing an altered form of Lck independent of the contributions of endogenous wild type Lck (41Straus D.B. Chan A.C. Patai B. Weiss A. J. Biol. Chem. 1996; 271: 9976-9981Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). As such, the importance of the SH3 domain of Lck in TCR signaling was examined by transfecting JCaM1 cells with an Lck cDNA that encoded a protein containing an inactive SH3 domain. A conserved tryptophan in SH3 domains stabilizes their association with proline-rich target peptides (28Musacchio A. Saraste M. Wilmanns M. Nat. Struct. Biol. 1994; 1: 546-551Crossref PubMed Scopus (287) Google Scholar); mutation of this tryptophan residue abolishes ligand binding (42Erpel T. Superti-Furga G. Courtneidge S.A. EMBO J. 1995; 14: 963-975Crossref PubMed Scopus (143) Google Scholar, 43Panchamoorthy G. Fukazawa T. Stolz L. Payne G. Reedquist K. Shoelson S. Songyang Z. Cantley L. Walsh C. Band H. Mol. Cell. Biol. 1994; 14: 6372-6385Crossref PubMed Scopus (62) Google Scholar). In this study, the SH3 domain of Lck was inactivated by mutating this essential tryptophan, located at position 97, to alanine using site-directed mutagenesis (LckW97A). In preliminary experiments, we observed that the SH3 domain of LckW97A, when expressed as a fusion protein with glutathione S-transferase (GST), was unable to bind the known substrate c-Cbl (not shown). Thus, the LckW97A mutation represents a loss of function mutation for the Lck SH3 domain. Stable transfection of JCaM1 cells generated several independent clones expressing similar levels of TCR, and levels of wild type Lck or LckW97A, equivalent to those in the parental Jurkat cell line (Fig.1). Lck kinase activity is essential for the initiation of TCR signaling, and it is possible that disruption of the SH3 domain of Lck affects its catalytic activity. To determine if the SH3 domain of Lck influenced its catalytic activity in vitro, we incubated immunoprecipitates of LckW97A or wild type Lck with [γ-32P]ATP and an exogenous substrate consisting of the cytosolic domain of the ζ chain of the TCR fused to GST (Fig.2 A). Both wild type Lck and LckW97A displayed autophosphorylation and GST-ζ phosphorylation, indicating that the SH3 domain of Lck was not required for kinase activity. In fact, LckW97A displayed enhanced catalytic activity relative to wild type Lck, consistent with an autoinhibitory role for the Lck SH3 domain (Fig. 2 B). Thus, any disturbances in TCR signaling in cells expressing LckW97A cannot be attributed to a general loss of catalytic activity. Although LckW97A was active in vitro, mutation of the SH3 domain may disrupt TCR-induced substrate phosphorylation within the cell, possibly by altering Lck distribution or by interfering with the binding of potential substrates. Therefore, we examined the levels of cellular tyrosine phosphoproteins in unstimulated and TCR-stimulated JCaM1 transfectants expressing wild type Lck, or LckW97A, by immunoblotting lysates with an anti-phosphotyrosine antibody (Fig.3 A). The profiles of tyrosine phosphoproteins in unstimulated and TCR-stimulated cells expressing LckW97A closely resembled those of cells expressing wild type Lck, whereas TCR stimulation of plasmid vector-transfected JCaM1 cells failed to induce tyrosine phosphorylation (not shown). Although cells expressing LckW97A displayed a slight, but reproducible, elevation in the basal level of tyrosine phosphorylation, it is apparent that mutation of SH3 domain of Lck did not substantially alter the tyrosine phosphorylation of cellular phosphoproteins prior to or following TCR stimulation. As such, LckW97A was capable of inducing tyrosine phosphorylation upon TCR stimulation. Although the induction of tyrosine phosphorylation appeared to be intact, we examined in greater detail the effects of mutation of the Lck SH3 domain on the initiation of TCR signaling. Specifically, we focused on the induction of ITAM and ZAP-70 phosphorylation in cells expressing LckW97A since these events are mediated directly by Lck. Immunoprecipitates of TCR ζ chain were immunoblotted for phosphotyrosine to evaluate ITAM phosphorylation and associated ZAP-70 phosphorylation (Fig. 3 B). TCR stimulation of cells expressing either wild type Lck or LckW97A induced the phosphorylation of TCR ζ chain and association of tyrosine-phosphorylated ZAP-70 (Fig. 3 B), but vector-transfected control cells failed to induce detectable levels of TCR ζ chain or ZAP-70 phosphorylation (not shown). To confirm that TCR stimulation of cells expressing LckW97A elicited ZAP-70 activation, we examined ZAP-70 phosphorylation in the JCaM1 transfectants and evaluated ZAP-70 kinase activityin vitro. Anti-phosphotyrosine blotting of ZAP-70 immunoprecipitates revealed that TCR stimulation induced similar levels of ZAP-70 phosphorylation in cells expressing either LckW97A or wild type Lck but not vector-transfected cells (Fig.4 A). The induction of ZAP-70 phosphorylation in TCR-stimulated cells corresponded to an elevation in ZAP-70 kinase activity in vitro, demonstrating that the SH3 domain of Lck is not required for the activation of ZAP-70 (Fig.4 B). Although a slight reduction in TCR-induced ZAP-70 activation was observed in JCaM1 cells expressing LckW97A, this difference was not statistically significant (p > 0.05). Thus, the previously identified roles for Lck in the initiation of TCR signaling, namely ITAM phosphorylation and the subsequent phosphorylation and activation of ZAP-70, can occur independently of Lck SH3 domain function. Since mutation of the SH3 domain did not alter the initial TCR signaling events that are known to require Lck, we investigated the possibility that the SH3 domain of Lck participates in the activation of downstream signaling events. Specifically, we examined the ability of this altered form of Lck to activate the PIP2 and the MAPK signaling pathways following TCR stimulation. Activation of the PIP2 pathway upon TCR stimulation was assessed by measuring elevations in [Ca2+]i and the induction of PLCγ1 phosphorylation. Elevations in [Ca2+]ielicited by TCR stimulation were examined using cells loaded with the Ca2+-sensitive fluorescent dye indo-1. We analyzed a range of anti-TCR antibody concentrations since subtle disturbances in the activation of the PIP2 pathway would be more apparent at submaximal levels of TCR stimulation. A deficit in the activation of the PIP2 pathway would be manifested as a shift in the concentration-response curve of TCR-stimulated elevations in [Ca2+]i. However, TCR stimulation of cells expressing wild type Lck or LckW97A induced similar elevations in [Ca2+]i at every concentration of anti-TCR antibody tested (Fig. 5 A). Similarly, the kinetics of these TCR-stimulated elevations in [Ca2+]i were identical in cells expressing wild type Lck or LckW97A (Fig. 5 B). The time-to-onset, peak elevation, decline phase, and plateau were all unaltered by inactivation of the Lck SH3 domain. We confirmed that the Lck SH3 domain was not required for the activation of the PIP2pathway by measuring PLCγ1 phosphorylation in cells expressing LckW97A. Anti-phosphotyrosine immunoblots of PLCγ1 immunoprecipitates from cells expressing LckW97A showed that PLCγ1 tyrosine phosphorylation was induced upon TCR stimulation (Fig. 5 C). Although lower levels of inducible PLCγ1 phosphorylation were observed in JCaM1/LckW97A cells, it did not alter the ability of these cells to elicit elevations in [Ca2+]i following TCR stimulation. Thus, the activation of the PIP2 pathway following TCR stimulation is not dependent upon the SH3 domain of Lck. We also examined the activation of the MAPK pathway following TCR stimulation of cells expressing LckW97A. In contrast to the initiation of TCR signaling and the activation of the PIP2 pathway, the activation of the MAPK pathway was strictly dependent upon the SH3 domain of Lck. MAPK pathway activity was assessed initially by immunoblotting cell lysates with antibodies that specifically recognize the activated forms of the MAP kinases ERK1 and ERK2 and the MAPK kinases MEK1 and MEK2. TCR stimulation of cells expressing LckW97A failed to induce ERK or MEK phosphorylation, whereas stimulation of cells expressing wild type Lck elicited substantial ERK and MEK phosphorylation (Fig. 6 A). A kinetic analysis demonstrated that extending the period of TCR stimulation as much as 20 min did not enhance ERK phosphorylation in JCaM1 cells expressing LckW97A (not shown). TCR-independent activation of the MAPK pathway using PMA resulted in phosphorylation of both ERK and MEK irrespective of Lck expression (Fig. 6 A). The selective disruption of the MAPK pathway in cells expressing LckW97A demonstrates that an additional requirement for Lck exists in the activation of downstream signaling events, which is distinct from its previously identified role in mediating the initiation of TCR signaling. Engagement of the TCR initiates intracellular signaling processes that culminate in characteristic changes in gene expression, including expression of the cell-surface marker CD69 (44Testi R. Phillips J.H. Lanier L.L. J. Immunol. 1989; 142: 1854-1860PubMed Google Scholar). In light of the pronounced deficits in MAPK pathway activation, we suspected that TCR stimulation of JCaM1 cells expressing LckW97A would be incapable of inducing CD69 expression (45D'Ambrosio D. Cantrell D. Frati L. Santoni A. Testi R. Eur. J. Immunol. 1994; 24: 616-620Crossref PubMed Scopus (141) Google Scholar). Treatment of cells expressing LckW97A with phytohemagglutinin (PHA) resulted in little or no induction of CD69 expression, whereas PHA treatment of cells expressing wild type Lck elicited a 10-fold elevation in median fluorescence intensity (Fig.6 B). TCR-independent stimulation with PMA elicited similar levels of CD69 expression in all cell lines, regardless of Lck expression. Thus, the SH3 domain of Lck is required for TCR-dependent CD69 expression. The inability of LckW97A to activate the MAPK pathway or induce CD69 expression demonstrates a critical role for the SH3 domain of Lck in TCR signaling and T-cell activation. We have identified a crucial role for the SH3 domain of Lck in activation of the MAPK pathway following TCR stimulation. The Lck-deficient cell line JCaM1 was transfected with an altered form of Lck in which a conserved tryptophan located within the SH3 domain was mutated to alanine, thereby abolishing Lck SH3 domain function. Substantial biochemical and genetic evidence has demonstrated that this conserved tryptophan is essential for ligand binding (28Musacchio A. Saraste M. Wilmanns M. Nat. Struct. Biol. 1994; 1: 546-551Crossref PubMed Scopus (287) Google Scholar, 42Erpel T. Superti-Furga G. Courtneidge S.A. EMBO J. 1995; 14: 963-975Crossref PubMed Scopus (143) Google Scholar, 43Panchamoorthy G. Fukazawa T. Stolz L. Payne G. Reedquist K. Shoelson S. Songyang Z. Cantley L. Walsh C. Band H. Mol. Cell. Biol. 1994; 14: 6372-6385Crossref PubMed Scopus (62) Google Scholar, 46Feng S. Chen J.K. Yu H. Simon J.A. Schreiber S.L. Science. 1994; 266: 1241-1246Crossref PubMed Scopus (755) Google Scholar); thus, it is very unlikely that LckW97A has any residual SH3 domain function. JCaM1 clones expressing LckW97A failed to induce CD69 expression and MAPK activation upon TCR stimulation, revealing a defect in TCR signaling. Since LckW97A possessed kinase activity in vitro, the block in T-cell activation in cells expressing LckW97A could not be attributed to loss of catalytic function. Furthermore, cells expressing LckW97A had only minor alterations in the tyrosine phosphorylation of cellular proteins and exhibited inducible TCR ζ chain phosphorylation and ZAP-70 activation upon TCR stimulation. Thus, the Lck SH3 domain is not required for the initiation of TCR signaling but is essential for events that are independent of, or subsequent to, the recruitment and activation of ZAP-70. Additional analysis of downstream signaling pathways demonstrated that cells expressing LckW97A were capable of activating the PIP2 pathway following TCR stimulation; both PLCγ1 phosphorylation and subsequent elevations in [Ca2+]i were independent of Lck SH3 domain function. However, unlike the PIP2 pathway, TCR stimulation of cells expressing LckW97A failed to induce activation of ERK or MEK, indicating that there was a selective loss of this TCR signaling pathway. Thus, the Lck SH3 domain is required specifically for the activation of the MAPK pathway but not the initiation of TCR signaling or the activation of the PIP2 pathway. A current model of TCR signal transduction proposes that Lck and ZAP-70 function sequentially to initiate signaling (1Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1981) Google Scholar, 2Cantrell D. Annu. Rev. Immunol. 1996; 14: 259-274Crossref PubMed Scopus (597) Google Scholar, 3Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). Lck phosphorylates the TCR which allows ZAP-70 recruitment, followed by ZAP-70 phosphorylation and activation, which is also mediated by Lck. The subsequent stimulation of the PIP2 and MAPK pathways is dependent upon the activation of ZAP-70. In the simplest model, the sole role of Lck in TCR signaling would be to recruit and activate ZAP-70. However, our data demonstrate that the involvement of Lck in TCR signaling is more complex and establish that a selective requirement for Lck exists in the activation of downstream signaling pathways. TCR stimulation of JCaM1/LckW97A cells failed to activate the MAPK pathway, despite activation of ZAP-70 and the PIP2pathway. Recent studies with a constitutively active form of ZAP-70 support the notion that Lck is required for downstream signaling (25Wong J. Straus D. Chan A.C. Mol. Cel. Biol. 1998; 18: 2855-2866Crossref PubMed Scopus (25) Google Scholar). Constitutively active ZAP-70 is able to elicit interleukin-2 gene expression independently of TCR stimulation but only when co-expressed with Lck. Taken together, these studies indicate an additional role for Lck in TCR signaling beyond the recruitment and activation of ZAP-70. Several models could explain the requirement for both activated ZAP-70 and Lck in the stimulation of the MAPK pathway. Lck and activated ZAP-70 may provide separate signals, both of which are needed for activation of the downstream pathway. Alternatively, ZAP-70 and Lck may act together on a common substrate that is essential for the stimulation of the MAPK pathway. The Lck SH3 domain has been shown to bind several signaling molecules implicated in the regulation of the MAPK pathway. The binding of these target proteins by the Lck SH3 domain may modify their activity as a result of conformational changes, subsequent tyrosine phosphorylation, or recruitment to the membrane. PI-3-K binds to the Lck SH3 domain (30Prasad K.V.S. Kapeller R. Janssen O. Repke H. Duke-Cohan J.S. Cantley L.C. Rudd C.E. Mol. Cell. Biol. 1993; 13: 7708-7717Crossref PubMed Scopus (141) Google Scholar) and is capable of activating the MAPK pathway in some cell types (47Hu Q. Klippel A. Muslin A.J. Fantl W.J. Williams L.T. Science. 1995; 268: 100-102Crossref PubMed Scopus (520) Google Scholar). However, it is unlikely that the loss of MAPK activation in JCaM1/LckW97A cells is due to alterations in the function of PI-3-K since we did not observe any alteration in PI-3-K localization or activity following TCR stimulation of JCaM1 cells expressing either wild type Lck or LckW97A (not shown). The proto-oncogene product c-Cbl also associates with the SH3 domain of Lck (29Reedquist K. Fukazawa T. Druker B. Panchamoorthy G. Shoelson S. Band H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4135-4139Crossref PubMed Scopus (77) Google Scholar). c-Cbl acts as a negative regulator of MAPK activation (48Yoon C.H. Lee J. Jongeward G.D. Sternberg P.W. Science. 1995; 269: 1102-1105Crossref PubMed Scopus (282) Google Scholar), although this appears to be the result of a block in the activation of Syk/ZAP-70 making it unlikely that c-Cbl would selectively regulate the MAPK pathway (49Ota Y. Samelson L.E. Science. 1997; 276: 418-420Crossref PubMed Scopus (226) Google Scholar). Lck has also been shown to interact directly with the kinases comprising the MAPK signaling pathway, including Raf-1, MEK, and MAPK (50Ettehadieh E. Sanghera J.S. Pelech S.L. Hess-Bienz D. Watts J. Shastri N. Aebersold R. Science. 1992; 255: 853-855Crossref PubMed Scopus (96) Google Scholar, 51Thompson P.A. Ledbetter J.A. Rapp U.R. Bolen J.B. Cell Growth Differ. 1994; 2: 609-617Google Scholar, 52Popik W. Pitha P. Mol. Cell. Biol. 1996; 16: 6532-6541Crossref PubMed Google Scholar, 53August A. Dupont B. J. Biol. Chem. 1996; 271: 10054-10059Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) as well as the inhibitor of Ras activity, Ras-GAP (31Briggs S.D. Bryant S.S. Jove R. Sanderson S.D. Smithgall T.E. J. Biol. Chem. 1995; 270: 14718-14724Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Future studies will be required to understand the specific function of the Lck SH3 domain in the activation of the MAPK pathway following stimulation of the TCR. The SH3 domains of Src family kinases have been proposed to be autoinhibitory. X-ray crystallographic studies examining the structure of Src and Hck revealed that the SH3 domain mediates an intramolecular interaction with an atypical binding site located in the region linking the SH2 and kinase domains (54Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1050) Google Scholar, 55Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1260) Google Scholar). Inactivating the SH3 domain of Src induces an 8–10-fold elevation in its kinase activity (42Erpel T. Superti-Furga G. Courtneidge S.A. EMBO J. 1995; 14: 963-975Crossref PubMed Scopus (143) Google Scholar). However, in the present study, mutation of the SH3 domain of Lck caused a comparatively modest 2-fold elevation in its kinase activity. Moreover, deleting the SH3 domain of Lyn reduces its kinase activity (56Abrams C.S. Zhao W. J. Biol. Chem. 1995; 270: 333-339Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). These findings suggest that despite their significant structural similarity, the SH3 domains of Src family kinases may not be functionally identical, although it is possible that other regulatory mechanisms may compensate for loss of SH3 domain function in Lck and Lyn. In summary, we have identified an additional role for Lck in TCR signaling which is distinct from its ability to mediate ITAM phosphorylation and ZAP-70 activation. Our results demonstrate that TCR-induced activation of the MAPK pathway, but not the PIP2 pathway, requires the SH3 domain of Lck. The selective requirement for Lck SH3 domain function in the MAPK pathway suggests that the activation of specific downstream signaling events could be controlled by regulating the function of Lck. Such a mechanism may be involved in thymocyte development where activation of the MAPK pathway is required for positive selection but not negative selection (57Alberola-Ila J. Forbush K.A. Seger R. Krebs E.G. Perlmutter R.M. Nature. 1995; 373: 620-623Crossref PubMed Scopus (370) Google Scholar, 58Swan K.A. Alberola-Ila J. Gross J.A. Appleby M.W. Forbush K.A. Thomas J.F. Perlmutter R.M. EMBO J. 1995; 14: 276-285Crossref PubMed Scopus (272) Google Scholar). Future studies will address the molecular basis of the role of the Lck SH3 domain in activation of the MAPK pathway. We thank Anne Burkhardt, Joe Bolen, Nicolai van Oers, and Arthur Weiss for providing reagents; Jean Maguire and Angus MacNicol for critical review of the manuscript; and Barbara Patai and Judith Austin for technical assistance.