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.     

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.     

itk, a T-cell-specific tyrosine kinase gene inducible by interleukin 2.

T lymphocytes are activated by interactions with antigens, lymphokines, and cell adhesion molecules. Tyrosine phosphorylation has been implicated as important in signaling through each of these pathways, but except for p56lck, a member of the Src family that associates with CD4 and CD8, the protein-tyrosine kinases involved have not been defined. We describe here a tyrosine kinase gene that we have designated itk (for IL-2-inducible T-cell kinase). The itk gene specifies a 72-kDa protein-tyrosine kinase that is related to members of the Src family but lacks two features characteristic of Src kinases: an N-terminal myristoylation consensus sequence and a regulatory tyrosine residue near the C terminus. Analysis of mouse tissues and cell lines indicates that itk is specifically expressed in the T-cell lineage, suggesting that the tyrosine kinase encoded by itk functions in a signal transduction pathway unique to T lymphocytes. On addition of IL-2 to responsive T cells, itk RNA increases in parallel with that of IL-2R alpha, implicating itk in T-cell activation.     

p21ras activation via hemopoietin receptors and c-kit requires tyrosine kinase activity but not tyrosine phosphorylation of p21ras GTPase-activating protein.

Products of the ras gene family, termed p21ras, are GTP-binding proteins that have been implicated in signal transduction via receptors encoding tyrosine kinase domains. Recent findings have defined a superfamily of hemopoietin receptors that includes receptors for a number of interleukins and colony-stimulating factors. The intracellular portions of these receptors show only restricted homologies, have no tyrosine kinase domain, and provide no clues to the mode of signal transduction. However, in most cases the factors stimulate tyrosine phosphorylation. We demonstrate here that ligand-induced activation of the interleukin (IL)-2, IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor receptors resulted in activation of p21ras in various hemopoietic cell lines. The only cytokine tested that binds to a hemopoietin receptor and that did not activate p21ras was IL-4. Activation of p21ras was also observed in response to Steel factor, which stimulates the endogenous tyrosine kinase activity of the c-kit receptor, as well as with phorbol esters, which activate protein kinase C. Experiments with protein kinase inhibitors implicated tyrosine kinase activity, but not protein kinase C activity, as the upstream signal in p21ras activation via these growth factor receptors. Attempts to demonstrate tyrosine phosphorylation of the p21ras GTPase-activating protein (GAP) were negative, suggesting that phosphorylation of GAP may not be the major mechanism for regulation of p21ras activity by tyrosine kinases.     

En bloc substitution of the Src homology region 2 domain activates the transforming potential of the c-Abl protein tyrosine kinase.

Src homology region 2 (SH2) domains are present in many proteins involved in signal transduction. In nonreceptor protein tyrosine kinases the SH2 domain has been implicated in regulation of tyrosine kinase activity and in mediating interactions involved in downstream signaling. Different SH2 domains exhibit distinct binding specificities for both phosphotyrosine- and phosphoserine/phosphothreonine-containing proteins. We show that different SH2 domains are not functionally equivalent within the context of the c-ABL1b protooncogene. c-ABL1b, altered by replacement of its SH2 domain with the N-terminal SH2 domain of Ras GTPase-activating protein, exhibited activated transforming capability, caused intracellular tyrosine phosphorylation of p62, and was relocalized from nucleus to cytoplasm. This en bloc substitution apparently uncouples two distinct functions of the SH2 domain so that c-ABL escapes normal regulatory control while it retains the capability to elicit signals that promote transformation. The SH2 domain of the ARG protein tyrosine kinase, which shares high amino acid-sequence homology with the SH2 domain of ABL, was less effective in activating the oncogenic potential of c-ABL. The effects that the N-terminal SH2 domain of Ras GTPase-activating protein has in the context of c-ABL resemble the effects of deleting the SH3 domain. Thus, the SH2 and SH3 domains may have coordinate roles as regulatory control elements within the context of c-ABL.     

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.     

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.     

Sulfhydryl oxidation down-regulates T-cell signaling and inhibits tyrosine phosphorylation of phospholipase C gamma 1.

Early events in both T-cell receptor (CD3)- and CD4-induced signal transduction pathways include tyrosine phosphorylation of protein substrates, the generation of phosphatidylinositol-phosphate breakdown products, and the mobilization of intracellular Ca2+. Oxidative stress in T cells mediated by sulfhydryl-reactive nonpolar maleimides was shown previously to down-regulate both receptor-mediated Ca2+ mobilization and interleukin 2 production. Here we show that N-ethylmaleimide suppresses both CD3- and CD4-induced Ca2+ responses in human T cells correlating with a reduction in the level of phospholipase C gamma 1 (PLC gamma 1) tyrosine phosphorylation. The inhibition of tyrosine phosphorylation of PLC gamma 1 and additional protein substrates was observed at concentrations of N-ethylmaleimide above 20 microM, whereas lower concentrations of oxidant appeared to increase tyrosine kinase activity following cell stimulation. Sulfhydryl oxidation did not directly affect the catalytic activity of PLC gamma 1, since immunopurified enzyme from N-ethylmaleimide-treated T cells was fully active. Although N-ethylmaleimide treatment of T cells did not cause a direct effect on total pp56lck kinase activity measured in vitro, the interaction between CD4 and pp56lck was oxidation-sensitive in vivo. However, CD3-induced signaling was inhibited at N-ethylmaleimide concentrations lower than that required for CD4/pp56lck dissociation, suggesting that CD3-associated tyrosine kinase activity involves acutely sensitive regulatory thiols. In addition to chemically induced sulfhydryl oxidation, naturally regulated cellular redox states appear to dictate the potential for T-cell responsiveness, since degranulating human peripheral blood neutrophils inhibited CD3-induced Ca2+ mobilization in T lymphocytes. These data indicate that signal transduction in T cells involves the activation of PLC gamma 1 by tyrosine phosphorylation through an oxidation-sensitive intermediate between surface receptors and tyrosine kinases, perhaps including the interaction between CD4 and pp56lck.     

Delineation of a T-cell activation motif required for binding of protein tyrosine kinases containing tandem SH2 domains.

To define the T-cell receptor signal transduction motif, we have transfected human and murine T-cell lines with a chimeric receptor consisting of the extracellular and transmembrane domains of human CD8 alpha and the membrane-proximal portion of CD3 zeta containing at its C terminus either an 18-amino acid segment (NQLYNELNLGRREEYDVL) or alanine-scanning point mutant derivatives. Crosslinking of the extracellular domain of the chimera is sufficient to initiate Ca2+ flux, interleukin 2 production, and tyrosine phosphorylation of cellular proteins including the chimera. Subsequently, the chimera becomes associated with several tyrosine-phosphorylated proteins, among them the 70-kDa protein tyrosine kinase ZAP70. Mutational data identify the T-cell activation motif as Y(X)2L(X)7Y(X)2L and show that each of the four designated residues is necessary for the above activation events. Recombinant protein containing the two tandem SH2 domains derived from ZAP70 binds to a synthetic peptide corresponding to the above 18-amino acid motif but only when both tyrosines are phosphorylated; in contrast, little or no binding is observed to monophosphorylated or nonphosphorylated analogues. These results imply that after receptor crosslinking in T cells, and by inference also in B cells and mast cells, the motif is phosphorylated on both tyrosine residues, thereafter serving as a docking site for protein tyrosine kinases containing tandem SH2 domains.     

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.     

The Src homology 2 domain of the protein-tyrosine kinase p56lck mediates both intermolecular and intramolecular interactions.

A key event in signaling by many cell surface receptors is the activation of Src-like protein-tyrosine kinases and the assembly of protein complexes at the plasma membrane mediated by Src homology 2 and 3 (SH2 and SH3) domains. p56lck is a Src-related protein-tyrosine kinase which has SH2 and SH3 domains and is involved in T-cell signaling and oncogenic transformation. Here we demonstrate that purified recombinant SH2 and HSH3/SH2 domains of p56lck can mediate intermolecular interactions with a number of tyrosine-phosphorylated proteins present in lysates of NIH 3T3 cells transformed by a constitutively activated form of p56lck (p56lckF505). Two of the interacting tyrosine-phosphorylated proteins were identified as the p85 subunit of phosphatidylinositol 3-kinase and the GTPase-activating protein of p21ras. Using a synthetic phosphopeptide corresponding to the tyrosine-phosphorylated carboxyl terminus of p56lck (amino acids 494-509), purified recombinant Lck SH2 domain, and differentially phosphorylated forms of p56lck we provide evidence that the SH2 domain of p56lck can also mediate intramolecular interactions with the phosphorylated carboxyl terminus. Together these results suggest that the SH2 domain of p56lck has a dual function: (i) it can mediate intermolecular interactions with cellular proteins phosphorylated on tyrosine and thus might be involved in building up signaling complexes at the plasma membrane and (ii) it can bind to the tyrosine-phosphorylated carboxyl terminus of p56lck in an intramolecular fashion and thereby might be involved in the regulation of its intrinsic protein-tyrosine kinase activity. Phosphorylation/dephosphorylation of the regulatory tyrosine residue 505 might serve as a switch between these two functions.     

SLP-76 mediates and maintains activation of the Tec family kinase ITK via the T cell antigen receptor-induced association between SLP-76 and ITK

ITK (IL-2-inducible T cell kinase), a Tec family protein tyrosine kinase (PTK), is one of three PTKs required for T cell antigen receptor (TCR)-induced activation of phospholipase C-gamma1 (PLC-gamma1). Like Src and Abl family PTKs, ITK adopts an inactive, "closed" conformation, and its conversion to the active conformation is not well understood, nor have its direct substrates been identified. In a side-by-side comparison of ITK and ZAP-70 (zeta chain-associated protein kinase of 70 kDa), ITK efficiently phosphorylated Y(783) and Y(775) of PLC-gamma1, two phosphorylation sites that are critical for its activation, whereas ZAP-70 did not. SLP-76 (SH2-domain-containing leukocyte protein of 76 kDa), an adaptor required for TCR-induced activation of PLC-gamma1, was required for the phosphorylation of both PLC-gamma1 sites in intact cells. Furthermore, this event depended on the N-terminal tyrosines of SLP-76. Likewise, SLP-76, particularly its N-terminal tyrosines, was required for TCR-induced tyrosine phosphorylation and activation of ITK but was not required for the phosphorylation or activation of ZAP-70. Both ZAP-70 and ITK phosphorylated SLP-76 in vitro; thus, both PTKs are potential regulators of SLP-76, but only ITK is regulated by SLP-76. Upon TCR stimulation, a small fraction of ITK bound to SLP-76. This fraction, however, encompassed most of the catalytically active ITK. Catalytic activity was lost upon mild elution of ITK from the SLP-76-nucleated complex but was restored upon reconstitution of the complex. We propose that SLP-76 is required for ITK activation; furthermore, an ongoing physical interaction between SLP-76 and ITK is required to maintain ITK in an active conformation.     

Lymphocyte antigen receptor activation of a focal adhesion kinase-related tyrosine kinase substrate.

One of the earliest responses of T and B lymphocytes to stimulation through their antigen receptors is the activation of protein tyrosine kinases and the tyrosine phosphorylation of multiple cellular substrates. Here we describe a tyrosine kinase substrate, fakB, a putative homologue of the focal adhesion kinase pp125FAK. Tyrosine phosphorylation of fakB was rapidly augmented in human T and B cells following antigen receptor cross-linking with antibody, while pp125FAK was nonresponsive. Costimulation of the T-cell antigen receptor (TCR/CD3) with either the CD2 or CD4 costimulatory receptors induced synergistic fakB tyrosine phosphorylation in normal human T cells. Engagement of TCR/CD3 induced the stable association of fakB with ZAP-70, the TCR/CD3 sigma-chain-associated tyrosine kinase involved in antigen receptor-induced T-cell activation. In addition, preformed complexes of fakB and ZAP-70 were observed in T-cell leukemia lines. Phosphorylation of fakB on serine, threonine, and tyrosine residues was observed both in vivo and in vitro, where a functional increase of in vitro kinase activity was observed following TCR/CD3 stimulation. fakB is thus a focal adhesion kinase-related tyrosine kinase substrate that is differentially regulated from that of pp125FAK and likely plays a role in antigen-induced lymphocyte signaling.     

Extracellular ligation-dependent CD45RB enzymatic activity negatively regulates lipid raft signal transduction

CD45 is the most prominent membrane protein on lymphocytes. The function and regulation of this protein tyrosine phosphatase remain largely obscure, mainly because of the lack of a known ligand, and it still remains unknown whether such tyrosine phosphatases are subject to extracellular control at all. We report that an anti-CD45RB antibody (Ab) that prevents rejection and induces tolerance activates CD45RB tyrosine phosphatase enzymatic activity in T lymphocytes, allowing us to directly monitor the effects of increased CD45RB activity on signal transduction. Using both kinase substrate peptide arrays as well as conventional biochemistry, we also provide evidence of the various kinases involved in bringing about the inhibitory effect of this Ab on CD3-induced T-cell receptor signaling. Furthermore, we report that activated CD45RB translocates to lipid rafts and interferes with lipid raft localization and activation state of CD45 substrate Lck. Thus, these findings indeed prove that CD45 is subject to extracellular control and also define a novel mechanism by which receptor tyrosine phosphatases control lymphocyte biology and provide further insight into the intracellular signaling pathways effected by anti-CD45RB monoclonal Ab treatment.     

Evidence for different interleukin 1 receptors in murine B- and T-cell lines.

Previous studies have shown that binding of interleukin 1 (IL-1) to its receptor and intracellular processing of the IL-1/IL-1 receptor complex appear to be different in B- and T-lymphocyte cell lines. In this study we used a B-lymphoid cell line, 70Z/3, and T-lymphoid cell line, EL-4 6.1 C10, to explore further the differences that exist between IL-1 receptors on cells of B and T lineage. We show that a monoclonal antibody against the IL-1 receptor on EL-4 cells does not bind to the IL-1 receptor on 70Z/3 cells. This finding suggests that there are structural differences in the extracellular domains of the IL-1 receptors on the two cell lines. Furthermore, affinity crosslinking showed that the molecular mass of the IL-1 receptor on EL-4 is 87 kDa, whereas that of 70Z/3 is significantly lower (66 kDa). Activation of phospholipid/Ca2+-dependent protein kinase, protein kinase C, by phorbol 12-myristate 13-acetate (PMA) greatly reduced the number of IL-1 binding sites on 70Z/3. But, in sharp contrast, PMA had no effect on surface IL-1 receptor expression on EL-4 cells despite having an equally potent effect in activating protein kinase C. The different effects of protein kinase C suggest that the cytoplasmic domains of the IL-1 receptors in 70Z/3 and EL-4 may also be different. Lastly, a probe containing the entire coding region of the murine T-cell IL-1 receptor hybridized under high stringency conditions with mRNA from EL-4 cells but not with mRNA from 70Z/3 cells. Taken together, the observations made in this study suggest that major structural differences exist between the IL-1 receptors on B and T lymphocytes.     

Cloning and characterization of a lymphoid-specific, inducible human protein tyrosine phosphatase, Lyp

Protein tyrosine phosphatases act in conjunction with protein kinases to regulate the tyrosine phosphorylation events that control cell activation and differentiation. We have isolated a previously undescribed human phosphatase, Lyp, that encodes an intracellular 105-kD protein containing a single tyrosine phosphatase catalytic domain. The noncatalytic domain contains four proline-rich potential SH3 domain binding sites and an NXXY motif that, if phosphorylated, may be recognized by phosphotyrosine binding (PTB) domains. Comparison of the Lyp amino acid sequence with other known proteins shows 70% identity with the murine phosphatase PEP. The human Lyp gene was localized to chromosome 1p13 by fluorescence in situ hybridization analysis. We also identified an alternative spliced form of Lyp RNA, Lyp2. This isoform encodes a smaller 85-kD protein with an alternative C-terminus. The lyp phosphatases are predominantly expressed in lymphoid tissues and cells, with Lyp1 being highly expressed in thymocytes and both mature B and T cells. Increased Lyp1 expression can be induced by activation of resting peripheral T lymphocytes with phytohemagglutinin or anti-CD3. Lyp1 was found to be constitutively associated with the proto-oncogene c-Cbl in thymocytes and T cells. Overexpression of lyp1 reduces Cbl tyrosine phosphorylation, suggesting that it may be a substrate of the phosphatase. Thus, Lyp may play a role in regulating the function of Cbl and its associated protein kinases.     

Permissive roles of hematopoietin and cytokine tyrosine kinase receptors in early T-cell development

Although several cytokines have been demonstrated to be critical regulators of development of multiple blood cell lineages, it remains disputed to what degree they act through instructive or permissive mechanisms. Signaling through the FMS-like tyrosine kinase 3 (FLT3) receptor and the hematopoietin IL-7 receptor alpha (IL-7Ralpha) has been demonstrated to be of critical importance for sustained thymopoiesis. Signaling triggered by IL-7 and thymic stromal lymphopoietin (TSLP) is dependent on IL-7Ralpha, and both ligands have been implicated in T-cell development. However, we demonstrate that, whereas thymopoiesis is abolished in adult mice doubly deficient in IL-7 and FLT3 ligand (FLT3L), TSLP does not play a key role in IL-7-independent or FLT3L-independent T lymphopoiesis. Furthermore, whereas previous studies implicated that the role of other cytokine tyrosine kinase receptors in T lymphopoiesis might not involve permissive actions, we demonstrate that ectopic expression of BCL2 is sufficient not only to partially correct the T-cell phenotype of Flt3l(-/-) mice but also to rescue the virtually complete loss of all discernable stages of early T lymphopoiesis in Flt3l(-/-)Il7r(-/-) mice. These findings implicate a permissive role of cytokine receptors of the hematopoietin and tyrosine kinase families in early T lymphopoiesis.     

The cbl oncogene: a novel substrate of protein tyrosine kinases

The cbl oncogene was first identified as part of a transforming retrovirus which arose in a mouse pre-B cell lymphoma. Its protein product, p120^cbl, is cytoplasmic and has several distinctive domains including a highly basic region, a RING finger motif and a large proline-rich domain. A mutation to cbl in the 70Z/3 pre-B cell lymphoma produces an oncogenic protein which exhibits a marked enhancement of tyrosine phosphorylation. Parallel studies have demonstrated that P120^cbl is a substrate of protein tyrosine kinases activated by engagement of the T cell antigen receptor and that cbl is phosphorylated by oncogenic forms of the Abl tyrosine kinase. These studies also demonstrated a constitutive association between cbl and the SH3 domains of the Grb2 and Nek adaptor proteins in a range of haemopoietic cell lines. More recently it has been found that cbl is rapidly phosphorylated following stimulation of the EGF receptor, Fey receptor, c-Kit receptor and CSF-1 receptor. A genetic analysis in Caenorhabditis elegans has identified a cbl homologue, called sli-1, that negatively regulates the LET-23 tyrosine kinase receptor. These characteristics indicate a central role for cbl in the regulation of intracellular signals that are mediated by growth factors and antigenic stimuli which activate protein tyrosine kinases.