CD28 is associated with and induces the immediate tyrosine phosphorylation and activation of the Tec family kinase ITK/EMT in the human Jurkat leukemic T-cell line.

T lymphocytes require two signals to be activated. The antigen-specific T-cell receptor can deliver the first signal, while ligation of the T-cell surface molecule CD28 by antibodies or its cognate ligands B7-1 (CD80) or B7-2 has been demonstrated to be sufficient for the delivery of the second signal. Signaling via CD28 and the T-cell receptor results (i) in their costimulation of T cells to produce numerous lymphokines including interleukin 2 and (ii) in the prevention of anergy induction. Little is known about the pathway by which CD28 mediates its signals except that protein-tyrosine phosphorylation is involved. We show here in human Jurkat cells that the Tec-family protein-tyrosine kinase ITK/EMT (p72ITK/EMT) is associated with CD28 and becomes tyrosine-phosphorylated and activated within seconds of CD28 ligation. This tyrosine phosphorylation of p72ITK/EMT is rapid (within 30 sec), occurs in the absence of LCK activation, and precedes tyrosine phosphorylation of the guanine nucleotide exchange factor VAV. Secondary crosslinking of CD28 is unnecessary for the induced tyrosine phosphorylation of p72ITK/EMT. Thus, tyrosine phosphorylation of p72ITK/EMT may represent one of the earliest events in CD28 signaling. This demonstrates that a member of the Tec family of protein tyrosine kinases, similar to members of the Src and Syk families, plays a role in the activation of T cells. Furthermore, the data demonstrate that p72ITK/EMT, and by analogy other members of the Tec family, responds to extracellularly generated signals.     

Identification, cloning, and characterization of a novel human T-cell- specific tyrosine kinase located at the hematopoietin complex on chromosome 5q

Signal transduction through the T-cell receptor and cytokine receptors on the surface of T lymphocytes occurs largely via tyrosine phosphorylation of intracellular substrates. Because neither the T-cell receptor nor cytokine receptors contain intrinsic kinase domains, signal transduction is thought to occur via association of these receptors with intracellular protein tyrosine kinases. Although several members of the SRC and SYK families of tyrosine kinases have been implicated in signal transduction in lymphocytes, it seems likely that additional tyrosine kinases involved in signal transduction remain to be identified. To identify unique T-cell tyrosine kinases, we used polymerase chain reaction-based cloning with degenerate oligonucleotides directed at highly conserved motifs of tyrosine kinase domains. We have cloned the complete cDNA for a unique human tyrosine kinase that is expressed mainly in T lymphocytes (EMT) and natural killer (NK) cells. The cDNA of EMT predicts an open reading frame of 1866 bp encoding a protein with a predicted size of 72 Kd, which is in keeping with its size on Western blotting. A single 6.2-kb EMT mRNA and 72-Kd protein were detected in T lymphocytes and NK-like cell lines, but were not detected in other cell lineages. EMT contains both SH2 and SH3 domains, as do many other intracellular kinases. EMT does not contain the N-terminal myristylation site or the negative regulatory tyrosine phosphorylation site in its carboxyterminus that are found in the SRC family of tyrosine kinases. EMT is related to the B-cell progenitor kinase (BPK), which has recently been implicated in X-linked hypogammaglobulinemia, to the TECI mammalian kinase, which has been implicated in liver neoplasia, to the more widely expressed TECII mammalian kinase, and to the Drosophila melanogaster Dsrc28 kinase. Sequence comparison suggests that EMT is likely the human homologue of a recently identified murine interleukin-2 (IL-2)-inducible T cell kinase (ITK). However, unlike ITK, EMT message and protein levels do not vary markedly on stimulation of human IL-2-responsive T cells with IL-2. Taken together, it seems that EMT is a member of a new family of intracellular kinases that includes BPK, TECI, and TECII. EMT was localized to chromosome 5q31-32, a region that contains the genes for several growth factors and receptors as well as early activation genes, particularly those involved in the hematopoietic system. Furthermore, the 5q31-32 region is implicated in the genesis of the 5q- syndrome associated with myelodysplasia and development of leukemia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Developmental regulation of a murine T-cell-specific tyrosine kinase gene, Tsk.

Protein-tyrosine kinases have been implicated in signal transduction in T lymphocytes after stimulation of many cell-surface molecules, including the T-cell antigen receptor, CD4, CD8, CD2, CD5, and CD28. Yet the identities of many of these tyrosine kinases remain unknown. We have isolated a murine tyrosine kinase gene, called Tsk for T-cell-specific kinase, that appears to be exclusively expressed in T lymphocytes. The Tsk cDNA clone encodes a polypeptide of 70 kDa, which is similar in sequence to both the src and abl families of tyrosine kinases. Sequence comparisons also indicate that Tsk contains one src-homology region 2 domain and one src-homology 3 domain but lacks the negative regulatory tyrosine (src Tyr-527) common to src-family kinases. In addition, Tsk expression is developmentally regulated. Steady-state Tsk mRNA levels are 5- to 10-fold higher in thymocytes than in peripheral T cells and increase in the thymus during mouse development from neonate to adult. Furthermore, Tsk is expressed in day 14 fetal thymus, suggesting a role for Tsk in early T-lymphocyte differentiation.     

Binding of Bruton''s tyrosine kinase to Fyn, Lyn, or Hck through a Src homology 3 domain-mediated interaction.

Bruton's tyrosine kinase (Btk) is a recently described B-cell-specific tyrosine kinase. Mutations in this gene lead to human X chromosome-linked agammaglobulinemia and murine X-linked immunodeficiency. Although genetic evidence strongly suggests that Btk plays a crucial role in B-lymphocyte differentiation and activation, its precise mechanism of action remains unknown, primarily because the proteins that it interacts with have not yet been identified. Here, we show that Btk interacts with Src homology 3 domains of Fyn, Lyn, and Hck, protein-tyrosine kinases that get activated upon stimulation of B- and T-cell receptors. These interactions are mediated by two 10-aa motifs in Btk. An analogous site with the same specificity is also present in Itk, the T-cell-specific homologue of Btk. Our data extend the range of interactions mediated by Src homology 3 domains and provide an indication of a link between Btk and established signaling pathways in B lymphocytes.     

7,12-Dimethylbenz[a]anthracene activates protein-tyrosine kinases Fyn and Lck in the HPB-ALL human T-cell line and increases tyrosine phosphorylation of phospholipase C-gamma 1, formation of inositol 1,4,5-trisphosphate, and mobilization of intracellular calcium.

Previous studies have shown that the immunosuppressive and carcinogenic polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) impairs Ca(2+)-dependent transmembrane signaling in human and murine lymphocytes. The purpose of the present studies was to analyze potential mechanisms of immunosuppression by DMBA and to examine effects on Ca2+ homeostasis and antigen-receptor signaling in human T cells. DMBA produced a rapid and sustained increase in Ca2+ levels in HPB-ALL cells by release of cytoplasmic Ca2+. DMBA also inhibited anti-CD3/CD4 mobilization of Ca2+ in HPB-ALL cells, with half-maximal inhibition occurring at approximately 4 hr. Thus, the kinetics for initial Ca2+ mobilization and inhibition of the anti-CD3/CD4 response differed. The rapid rise in intracellular Ca2+ induced by DMBA alone was accompanied by a rapid but transient increase in inositol 1,4,5-trisphosphate and tyrosine phosphorylation of phospholipase C-gamma 1. The pattern of tyrosine phosphorylation induced by DMBA in HPB-ALL cells was remarkably similar to that induced by anti-CD3/CD4 activation. Thus, DMBA-induced phosphorylation may mimic antigen-receptor activation in T cells, which may lead to alterations in antigen responsiveness. The mechanism of DMBA-induced tyrosine phosphorylation of phospholipase C-gamma 1 may have been due to an increase in protein-tyrosine kinase activity, since it was found that DMBA produced a > 2-fold increase in the activity of the T-cell receptor-associated Src-family kinases Fyn and Lck. The kinetics of activation of protein-tyrosine kinases demonstrated that Fyn activity was increased within 10 min of exposure to DMBA, whereas maximal Lck activation required 30 min. Thus, it is likely that the Fyn kinase or other protein-tyrosine kinases may be responsible for the early tyrosine phosphorylation of phospholipase C-gamma 1, which results in inositol 1,4,5-trisphosphate release and mobilization of intracellular Ca2+.     

Ctk: a protein-tyrosine kinase related to Csk that defines an enzyme family.

We used the polymerase chain reaction with degenerate oligonucleotide primers to search for Csk-related kinases. A cDNA coding for a Csk-like protein-tyrosine kinase was cloned from mouse brain and was designated ctk, for csk-type protein-tyrosine kinase. The 1.9-kb ctk mRNA was found to be expressed predominantly in brain and capable of encoding a 52-kDa protein-tyrosine kinase. The amino acid sequence of Ctk was found to possess 53% identity with mouse Csk, shared all the predicted structural features of Csk, and was capable of phosphorylating the carboxyl-terminal conserved tyrosine of Src family members. Our results thereby indicate that ctk represents a gene that defines a family of structurally and functionally related Csk-like protein-tyrosine kinases.     

p56Lck and p59Fyn regulate CD28 binding to phosphatidylinositol 3-kinase, growth factor receptor-bound protein GRB-2, and T cell-specific protein-tyrosine kinase ITK: implications for T-cell costimulation.

T-cell activation requires cooperative signals generated by the T-cell antigen receptor zeta-chain complex (TCR zeta-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, zeta-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.     

T-lymphocyte interleukin 2-dependent tyrosine protein kinase signal transduction involves the activation of p56lck.

Addition of interleukin 2 (IL-2) to IL-2-dependent T cells results in tyrosine protein kinase signal transduction events even though the IL-2 receptor alpha and beta chains lack intrinsic enzymatic activity. Here we report that addition of IL-2 to IL-2-dependent human T cells transiently stimulates the specific activity of p56lck, a member of the src family of nonreceptor tyrosine protein kinases expressed at high levels in T lymphocytes. The ability of IL-2 to induce p56lck activation was found to be independent of the capacity of p56lck to associate with either CD4 or CD8. Following IL-2 treatment, p56lck was found to undergo serine/threonine phosphorylation modifications that resulted in altered mobility of the lck gene product on polyacrylamide gels. These observations raise the possibility that p56lck participates in IL-2-mediated signal transduction events in T cells.     

Src-induced activation of inducible T cell kinase (ITK) requires phosphatidylinositol 3-kinase activity and the Pleckstrin homology domain of inducible T cell kinase

The Tec family of tyrosine kinases are involved in signals emanating from cytokine receptors, antigen receptors, and other lymphoid cell surface receptors. One family member, ITK (inducible T cell kinase), is involved in T cell activation and can be activated by the T cell receptor and the CD28 cell surface receptor. This stimulation of tyrosine phosphorylation and activation of ITK can be mimicked by the Src family kinase Lck. We have explored the mechanism of this requirement for Src family kinases in the activation of ITK. We found that coexpression of ITK and Src results in increased membrane association, tyrosine phosphorylation and activation of ITK, which could be blocked by inhibitors of the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase) as well as overexpression of the p85 subunit of PI 3-kinase. Removal of the Pleckstrin homology domain (PH) of ITK resulted in a kinase that could no longer be induced to localize to the membrane or be activated by Src. The PH of ITK was also able to bind inositol phosphates phosphorylated at the D3 position. Membrane targeting of ITK without the PH recovered its ability to be activated by Src. These results suggest that ITK can be activated by a combination of Src and PI 3-kinase.     

CD5 acts as a tyrosine kinase substrate within a receptor complex comprising T-cell receptor zeta chain/CD3 and protein-tyrosine kinases p56lck and p59fyn.

T-cell antigens including CD2, CD4, CD6, CD8, and CD28 serve as coreceptors with the T-cell receptor (TCR)/CD3 complex in control of T-cell growth. The molecular basis by which these antigens fulfill this role has remained a major issue. An initial clue to this question came with our finding that the sensitivity of in vitro kinase labeling (specifically using protein-tyrosine kinase p56lck) allowed detection of a physical association between CD4-p56lck and the TCR/CD3 complexes. Another T-cell antigen, CD5, is structurally related to the macrophage scavenger receptor family and, as such, can directly stimulate and/or potentiate T-cell proliferation. In this study, we reveal that in Brij 96-based cell lysates, anti-CD5 antibodies coprecipitated TCR zeta chain (TCR zeta)/CD3 subunits as well as the protein-tyrosine kinases p56lck and p59fyn. Conversely, anti-CD3 antibody coprecipitated CD5, p56lck, and p59fyn. Indeed, anti-CD5 and anti-CD3 gel patterns were virtually identical, except for a difference in relative intensity of polypeptides. Anti-CD4 coprecipitated p56lck, p32, and CD3/TCR zeta subunits but precipitated less CD5, suggesting the existence of CD4-TCR zeta/CD3 complexes distinct from the CD5-TCR zeta/CD3 complexes. Consistent with the formation of a multimeric CD5-TCR zeta/CD3 complex, anti-CD5 crosslinking induced tyrosine phosphorylation of numerous T-cell substrates, similar to those phosphorylated by TCR zeta/CD3 ligation. Significantly, as for TCR zeta, CD5 was found to act as a tyrosine kinase substrate induced by TCR/CD3 ligation. The kinetics of phosphorylation of CD5 (t1/2 = 20 sec) was among the earliest of activation events, more rapid than seen for TCR zeta (t1/2 = 1 min). CD5 represents a likely TCR/CD3-associated substrate for protein-tyrosine kinases (p56lck or p59fyn) and an alternative signaling pathway within a multimeric TCR complex.     

The murine c-fgr gene product associated with Ly6C and p70 integral membrane protein is expressed in cells of a monocyte/macrophage lineage.

The c-fgr gene is a member of the Src family of protooncogene tyrosine kinases. A monoclonal antibody (2H2) that recognizes the specific region of the N-terminal domain of the murine c-fgr gene product (Fgr) has been established. With an immune complex kinase assay in a monocytic leukemia cell line, 2H2 monoclonal antibody was shown to precipitate a 59-kDa protein that corresponds in molecular mass to murine Fgr. Fgr was expressed highly in lymph nodes, slightly in spleen and peripheral blood leukocytes, and barely in the thymus and was not detected in bone marrow. In the presence of a mild detergent, Fgr was coimmunoprecipitated with a 70-kDa protein (p70) or with p70 plus several other molecules that were expressed on the cell-surface membrane of macrophage tumor cell lines PU5-1.8 and J774.1, respectively. By contrast, Fgr was not coimmunoprecipitated with a low-affinity receptor for the Fc portion of IgG that is associated with human Fgr. The molecule was also coimmunoprecipitated with the Ly6C molecule from a macrophage cell line (J774.1) that showed protein-tyrosine kinase activity. Peptide mapping revealed that this kinase activity was derived from Fgr. The similarity of relationship between this intramembrane p70 and/or Ly6C and cytoplasmic Fgr to relationships previously reported between T-cell antigen receptor complex, including CD4 and CD8 coreceptors, and Lck or Fyn in T cells and between surface IgM and Lyn or Blk in B cells, suggests that the Fgr and p70 or Ly6C are, indeed, associated with each other and in the murine system may be responsible for recognition of extracellular substances (either cellular or noncellular) and for signal transduction in cells of monocyte/macrophage lineage.     

C-terminal Src kinase (CSK) modulates insulin-like growth factor-I signaling through Src in 3T3-L1 differentiation

IGF-I stimulates both proliferation and differentiation of adipocyte-precursor cells, preadipocytes in vivo and in vitro. We have previously shown that IGF-I stimulates proliferation of 3T3-L1 preadipocytes through activation of MAPK and MAPK activation by IGF-I is mediated through the Src family of nonreceptor tyrosine kinases. In addition, we have shown that when 3T3-L1 cells reach growth arrest and are stimulated to differentiate, IGF-I can no longer activate the MAPK pathway. We hypothesized that the loss of IGF-I signaling to MAPK in differentiating 3T3-L1 cells is due to loss of IGF-I activation of Src family kinases. We measured c-Src kinase activity in cell lysates from proliferating, growth-arrested and differentiating 3T3-L1 cells. Src activity increased 2- to 4-fold in IGF-I-stimulated proliferating cells; however, IGF-I had a marginal affect on Src activity in growth-arrested cells and inhibited Src activity localized at the membrane in differentiating cells. C-terminal Src kinase (CSK), a ubiquitously expressed nonreceptor tyrosine kinase, negatively regulates the Src family kinases by phosphorylation of the Src C-terminal tyrosine. IGF-I decreased phosphorylation of the Src C-terminal tyrosine in proliferating cells and increased phosphorylation of this site in differentiating cells. IGF-I stimulated CSK kinase activity 2-fold in differentiating 3T3-L1 cells. An association between CSK and c-Src was detected by immunoprecipitation following IGF-I stimulation of differentiating but not proliferating 3T3-L1 cells. These results suggest that the loss of IGF-I downstream mitogenic signaling in differentiating 3T3-L1 cells is due to a change in IGF-I activation of c-Src and CSK may mediate the inactivation of c-Src by IGF-I in 3T3-L1 adipogenesis.     

Structure of the murine Jak2 protein-tyrosine kinase and its role in interleukin 3 signal transduction.

Interleukin 3 (IL-3) regulates the proliferation and differentiation of hematopoietic cells. Although the IL-3 receptor chains lack kinase catalytic domains, IL-3 induces tyrosine phosphorylation of cellular proteins. To investigate the potential role of the JAK family of protein-tyrosine kinases in IL-3 signal transduction, we have obtained full-length cDNA clones for murine Jak1 and Jak2 protein-tyrosine kinases and prepared antiserum against the predicted proteins. Using antisera against Jak2, we demonstrate that IL-3 stimulation results in the rapid and specific tyrosine phosphorylation of Jak2 and activates its in vitro kinase activity.     

Association of the fyn protein-tyrosine kinase with the T-cell antigen receptor.

Activation of the T-cell antigen receptor (TCR) results in tyrosine phosphorylation of the TCR zeta chain and other intracellular substrates. Two other T-cell integral membrane proteins, CD4 and CD8, are associated with the protein-tyrosine kinase (PTK), lck. Despite evidence that activation of this enzyme results in TCR-zeta chain phosphorylation, it has not been shown that the TCR activates lck. We have sought evidence that the TCR is associated with a PTK. In this study we use digitonin to solubilize a murine T-cell hybridoma and demonstrate that antibodies binding extracellular but not intracellular domains of the TCR specifically coprecipitate only the fyn PTK and not lck or yes, two other kinases found in these cells. The association of the fyn PTK with the TCR might enable the T cell to independently regulate two PTKs through surface receptors.     

Dysregulation of signaling pathways in CD45-deficient NK cells leads to differentially regulated cytotoxicity and cytokine production

CD45, a protein tyrosine phosphatase that regulates Src family kinases, is important for regulating T cell and B cell receptor signaling; however, little is known about how CD45 regulates immunoreceptor tyrosine-based activation motif (ITAM)-dependent natural killer (NK) cell receptor signaling and the resulting effector functions. NK cells from CD45-deficient mice are relatively competent for ITAM receptor-induced cell-mediated cytotoxicity, yet completely deficient for cytokine secretion after stimulation with ligands to or antibodies against NK1.1, CD16, Ly49H, Ly49D, and NKG2D. This deficiency in cytokine/chemokine production occurs at the level of mRNA expression. After receptor engagement, extracellular signal-regulated kinase and c-Jun N-terminal kinase activation was markedly perturbed, whereas p38 activation was not substantially affected. The pattern and amounts of basal tyrosine phosphorylation were altered in freshly isolated NK cells and were surprisingly and markedly increased in IL-2-expanded NK cells from CD45-/- mice. These findings indicate that CD45-dependent regulation of ITAM-dependent signaling pathways is essential for NK cell-mediated cytokine production but not cytolytic activity.     

Activation of human peripheral blood T lymphocytes by pharmacological induction of protein-tyrosine phosphorylation.

Protein-tyrosine kinase and protein-tyrosine phosphatase (PTPase) activities are essential for T-cell antigen receptor-mediated signaling. To assess the functional consequences of alteration of the levels of tyrosine phosphorylation in normal human T cells, the effects of vanadate and hydrogen peroxide were studied. In combination, these agents induced tyrosine phosphorylation of cellular substrates, elevated cytosolic free calcium, and induced interleukin 2 receptor (IL-2R) alpha chain expression but not IL-2 secretion. However, anti-CD28 antibody in combination with vanadate and hydrogen peroxide induced IL-2 secretion, consistent with the requirement for a costimulatory signal in the induction of this gene. The effects of vanadate and hydrogen peroxide were enhanced in the absence of the T-cell PTPase, CD45. Thus, acute pharmacologic manipulation of the level of tyrosine phosphorylation in normal T cells correlates with partial, but not full, activation of these cells; in concert with a costimulatory signal provided by perturbation of the CD28 molecule, the complete program of activation is initiated. These agents should prove useful in dissecting signaling pathways involved in the regulation of genes critical to the immune response.     

Disruption of the CD4-p56lck complex is required for rapid internalization of CD4.

CD4 is a cell surface glycoprotein expressed by a subset of T lymphocytes and functions to enhance T-cell activation. CD4 is noncovalently associated via the cytoplasmic domain with the protein-tyrosine kinase p56lck, a member of the src protein-tyrosine kinase family. Upon activation of protein kinase C by phorbol ester, CD4 is phosphorylated on cytoplasmic serine residues and internalized from the cell surface, and disruption of the CD4-p56lck complex occurs. The exact relationship between these events is likely to be functionally significant, as cytoplasmic-domain serine phosphorylation and internalization have been shown to regulate the function of receptors that possess intrinsic protein-tyrosine kinase activity. Here we demonstrate that p56lck slows the rate of phorbol 12-myristate 13-acetate-induced internalization of CD4 in a manner that depends on a physical association between p56lck and CD4. This decreased rate is due at least in part to a requirement for disruption of the CD4-p56lck complex prior to internalization of CD4. Furthermore, disruption of the CD4-p56lck complex appears to depend on the integrity of the cytoplasmic-domain serine at position 408, probably due to a requirement for phosphorylation.     

Correlation between Src family member regulation by the protein-tyrosine-phosphatase CD45 and transmembrane signaling through the T-cell receptor.

Stimulation of tyrosine phosphorylation is an early and important event in antigen-induced T-cell activation. T-cell clones deficient in expression of CD45, a transmembrane protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), are impaired in their ability to respond to either antigen or T-cell receptor cross-linking. Analysis of the CD45-deficient CD8+ T-cell clone L3M-93 demonstrates that the Src family members p56lck and p59fyn show increased immunoreactivity with anti-phosphotyrosine antibody and exhibit decreased kinase activity. The site of increased tyrosine phosphorylation in Src family members was identified by comparison of cyanogen bromide peptide maps. Phosphorylation of the C-terminal phosphopeptide, containing the negative regulatory site of tyrosine phosphorylation, from the CD45-deficient cells was increased 8-fold for p56lck and 2-fold for p59fyn. These data suggest that CD45 dephosphorylates the negative regulatory site of multiple Src family members in the cytotoxic T-lymphocyte clone L3 and show a correlation between the ability to respond efficiently to antigen and the dephosphorylation of Src family members by CD45.