SRChing for the substrates of Src.

By the mid 1980's, it was clear that the transforming activity of oncogenic Src was linked to the activity of its tyrosine kinase domain and attention turned to identifying substrates, the putative next level of control in the pathway to transformation. Among the first to recognize the potential of phosphotyrosine-specific antibodies, Parsons and colleagues launched a risky shotgun-based approach that led ultimately to the cDNA cloning and functional characterization of many of today's best-known Src substrates (for example, p85-Cortactin, p110-AFAP1, p130Cas, p125FAK and p120-catenin). Two decades and over 6000 citations later, the original goals of the project may be seen as secondary to the enormous impact of these protein substrates in many areas of biology. At the request of the editors, this review is not restricted to the current status of the substrates, but reflects also on the anatomy of the project itself and some of the challenges and decisions encountered along the way.

Nuclear localization of the tyrosine kinase Itk and interaction of its SH3 domain with karyopherin alpha (Rch1alpha).

We report a physical and functional association between the Tec-family tyrosine kinase Itk (Emt/Tsk) and the nuclear import chaperone karyopherin alpha (Rch1alpha) in human T cells. The Itk-SH3 domain and the Rch1alpha proline-rich (PR) motif were crucial for the Itk/Rch1alpha constitutive interaction as demonstrated by directed mutagenesis of the Rch1alpha PR motif (proline 242 to alanine, P242A). TCR-CD3 stimulation of Jurkat T cells resulted in increased Itk/Rch1alpha complex formation, recruitment of karyopherin beta to the protein complex and Rch1alpha tyrosine phosphorylation. Analysis of in vitro kinase reactions with a panel of recombinant glutathione-S-transferase (GST) fusion tyrosine kinases (Itk, Lck, ZAP-70 and Jak3) revealed that only GST-Itk efficiently phosphorylated a recombinant GST-Rch1alpha fusion. We observed constitutive nuclear localization of Itk that was up-regulated following either TCR-CD3 stimulation or over-expression of wild-type Rch1alpha in T cells. Further, nuclear localization of Itk and TCR-CD3-mediated IL-2 production were significantly down-regulated following expression of the Rch1alpha-P242A mutant, implicating a role for Rch1alpha in the nuclear translocation of Itk.

Focal adhesion kinase regulates beta1 integrin-dependent T cell migration through an HEF1 effector pathway.

Although beta1 integrin-dependent T cell migration is required for immune function, little is known of the signaling pathways regulating this migration. We now show that the cytoplasmic tyrosine kinase, focal adhesion kinase (FAK) plays an essential role in the beta1 integrin-stimulated migration of T cells through regulation of the unique Crk-associated substrate (Cas) family docking protein, human enhancer of filamentation 1 (HEF1) and effects on "outside-in" beta1 integrin signaling. Overexpression of wild-type FAK promoted beta1 integrin-dependent Jurkat T cell migration, whereas FAK mutated in either its autophosphorylation site or proline rich region 1 (PR1)/HEF1 SH3 domain-binding site had a dominant negative effect on migration. In contrast, neither wild-type nor mutant FAK affected Jurkat cell adhesion to fibronectin, a beta1 integrin ligand. The migration of FAK-overexpressing cells directly correlated with the beta1 integrin-inducible tyrosine phosphorylation of endogenous plus wild-type exogenous FAK, and not with phosphorylation of the FAK-related kinase, Pyk2. FAK was also found to regulate both HEF1-promoted migration, and HEF1 tyrosine phosphorylation in beta1 integrin-stimulated cells, in a manner dependent upon the FAK autophosphorylation and PR1 sites, and HEF1 SH3 domain. Together, our results indicate that beta1 integrin-stimulated T cell migration requires a linear beta1 integrin-FAK-HEF1 effector pathway.

Engagement of the T lymphocyte antigen receptor regulates association of son-of-sevenless homologues with the SH3 domain of phospholipase Cgamma1.

One mechanism for transducing signals downstream of lymphocyte receptor activation involves the stable association between signaling proteins. To identify protein ligands of the signal activator phospholipase Cgamma1 (PLCgamma1), we screened T cell cDNA libraries with the PLCgamma1-SH3 domain by the yeast two-hybrid assay. We observed association between the PLCgamma1-SH3 domain and the human Ras guanine nucleotide exchange factor son-of-sevenless-2 (hSos2) through a proline-rich domain interaction. Stable and abundant hSos2 / PLCgamma1 and hSos1 / PLCgamma1 complexes were observed in unstimulated T cells. The interaction between these enzymes was augmented following engagement of the T cell antigen receptor (TCR / CD3). The kinetics of protein complex enhancement correlated with TCR / CD3-induced tyrosine phosphorylation of PLCgamma1; however, those PLCgamma1 molecules in complex with hSos2 were non-phosphorylated after TCR / CD3 stimulation, in contrast to the phosphorylated PLCgamma1 associated with the linker for activation of T cells, LAT. The Grb2 adapter protein was detected in complex with hSos / PLCgamma1, suggesting a regulatory role for Grb2. SH3 domains from both Grb2 and PLCgamma1, but not RasGAP, bound directly to hSos homologues. The SH2 domain from Grb2 formed an association with the hSos / PLCgamma1 complex, which was enhanced following TCR / CD3 ligation. Together, the data suggest a mechanism for the son-of-sevenless and PLCgamma1 signal transducing enzymes in recruitment to protein complexes with potentially differential signaling consequences in T lymphocytes.

Regulated association between the tyrosine kinase Emt/Itk/Tsk and phospholipase-C gamma 1 in human T lymphocytes.

The Emt/Itk/Tsk tyrosine kinase is involved in intracellular signaling events induced by several lymphocyte surface receptors. Modulation of TCR/CD3-induced phospholipase-C gamma 1 (PLC gamma 1) activity by the tyrosine kinase Emt/Itk/Tsk has been demonstrated based on studies of Itk-deficient murine T lymphocytes. Here we report a TCR/CD3-regulated association between Emt and PLC gamma 1 in both normal and leukemic T cells. In addition, this association was enhanced following independent ligation of the CD2, CD4, or CD28 costimulatory molecules, but not of CD5 or CD6 surface receptors, correlating to the induced tyrosine phosphorylation of Emt. Before Ab-induced T cell activation, we found that the Emt-SH3 domain was crucial for the constitutive Emt/PLC gamma 1 association; however, upon TCR/CD3 engagement, the Emt-SH2 domain was more efficient in mediating the enhanced Emt/PLC gamma 1 interaction. Furthermore, the PLC gamma 1-SH3 domain, but not the two PLC gamma 1-SH2 domains, contributed to formation of the protein complex. Thus, we describe a regulated interaction between Emt and PLC gamma 1, and based on our studies with individual Emt and PLC gamma 1 SH2/SH3 domains, we propose a mechanism for this association.

CD5 negatively regulates the T-cell antigen receptor signal transduction pathway: involvement of SH2-containing phosphotyrosine phosphatase SHP-1.

The negative regulation of T- or B-cell antigen receptor signaling by CD5 was proposed based on studies of thymocytes and peritoneal B-1a cells from CD5-deficient mice. Here, we show that CD5 is constitutively associated with phosphotyrosine phosphatase activity in Jurkat T cells. CD5 was found associated with the Src homology 2 (SH2) domain containing hematopoietic phosphotyrosine phosphatase SHP-1 in both Jurkat cells and normal phytohemagglutinin-expanded T lymphoblasts. This interaction was increased upon T-cell receptor (TCR)-CD3 cell stimulation. CD5 co-cross-linking with the TCR-CD3 complex down-regulated the TCR-CD3-increased Ca2+ mobilization in Jurkat cells. In addition, stimulation of Jurkat cells or normal phytohemagglutinin-expanded T lymphoblasts through TCR-CD3 induced rapid tyrosine phosphorylation of several protein substrates, which was substantially diminished after CD5 cross-linking. The CD5-regulated substrates included CD3zeta, ZAP-70, Syk, and phospholipase Cgammal but not the Src family tyrosine kinase p56(lck). By mutation of all four CD5 intracellular tyrosine residues to phenylalanine, we found the membrane-proximal tyrosine at position 378, which is located in an immunoreceptor tyrosine-based inhibitory (ITIM)-like motif, crucial for SHP-1 association. The F378 point mutation ablated both SHP-1 binding and the down-regulating activity of CD5 during TCR-CD3 stimulation. These results suggest a critical role of the CD5 ITIM-like motif, which by binding to SHP-1 mediates the down-regulatory activity of this receptor.

Integrin signalling defects in T-lymphocytes in systemic lupus erythematosus.

OBJECTIVE: To establish the relationship between T cell responses to integrin coreceptor stimulation and B cell hyperreactivity as measured by pathologic autoantibody production. METHODS: Peripheral blood mononuclear cells from 42 patients with SLE according to the American Rheumatism Association criteria were examined for their ability to adhere to plate-immobilised fibronectin. Co-stimulation assays were performed on the same cells using anti-CD3 antibody alone or co-immobilised with an anti-beta1-integrin antibody. Proliferative responses were measured by 3[H]thymidine pulsing on day 3 and activation was determined using a commercial protein kinase C assay, the protocol being established by our group in association with Promega. Beta-integrin expression was established by FACS analysis. RESULTS: An impaired PKC response to integrin-mediated activation was found in T-lymphocytes from 6/21 (29%) SLE patients, which correlated significantly with an absence of anti-dsDNA antibody in patient sera, irrespective of prednisolone treatment. Integrin co-stimulation of TcR/CD3-induced proliferation and T cell adhesion to fibronectin were also impaired among 5/21 (24%) and 6/15 (40%) patients studied, respectively. CONCLUSION: We hypothesise that the integrity of beta1-integrin signalling pathways may influence pathological antibody production in SLE by affecting T-lymphocyte activation and interactions between T- and B-lymphocytes.

Filamin binds to the cytoplasmic domain of the beta1-integrin. Identification of amino acids responsible for this interaction.

Integrins play an important role in regulating cell adhesion, motility, and activation. In an effort to identify intracellular proteins expressed by activated T cells that interact with the cytoplasmic domain of beta1-integrin (CD29), we used the beta1-integrin cytoplasmic domain as bait in the yeast two-hybrid system. Here we report that the cytoplasmic domain of beta1-integrin specifically interacts with the cytoskeletal protein filamin. This interaction required all but the most carboxyl-terminal three residues of the cytoplasmic domain of beta1, and the carboxyl-terminal 477 residues of filamin containing the terminal 4. 5 approximately 96-residue tandem repeats of filamin. To verify this interaction in vivo, we showed that filamin specifically coprecipitated with beta1 in mammalian cells. We also showed that recombinant filamin chimeric proteins were able to bind to the beta1 cytoplasmic domain in vitro. We observed that a subset of single point mutations in the cytoplasmic domain of beta1, which had been previously reported to impair its function, disrupt the interaction between beta1 and filamin. Taken together, these findings suggest that the interaction between beta1 and filamin, which in turn can bind actin, provides a mechanism for the interaction of this cell surface receptor with cytoskeletal proteins and that this interaction plays a role in normal receptor function.

Interleukin-2 induces beta2-integrin-dependent signal transduction involving the focal adhesion kinase-related protein B (fakB).

beta2 integrin molecules are involved in a multitude of cellular events, including adhesion, migration, and cellular activation. Here, we studied the influence of beta2 integrins on interleukin-2 (IL-2)-mediated signal transduction in human CD4(+) T cell lines obtained from healthy donors and a leukocyte adhesion deficiency (LAD) patient. We show that IL-2 induces tyrosine phosphorylation of a 125-kDa protein and homotypic adhesion in beta2 integrin (CD18)-positive but not in beta2-integrin-negative T cells. EDTA, an inhibitor of integrin adhesion, blocks IL-2-induced tyrosine phosphorylation of the 125-kDa protein but not other proteins in beta2-integrin-positive T cells. Likewise, a beta2 integrin (CD18) antibody selectively inhibits induction of the 125-kDa phosphotyrosine protein, whereas cytokine-mediated tyrosine phosphorylation of other proteins is largely unaffected. Immunoprecipitation experiments indicate that the IL-2-induced 125-kDa phosphotyrosine protein is the focal adhesion kinase-related protein B (fakB). Thus, IL-2 induces strong tyrosine phosphorylation of fakB in beta2-integrin-positive but not in beta2-integrin-negative T cells, and CD18 mAb selectively blocks IL-2-induced fakB-tyrosine phosphorylation in beta2-integrin-positive T cells. In parallel experiments, IL-2 does not induce or augment tyrosine phosphorylation of p125(FAK). In conclusion, our data indicate that IL-2 induces beta2-integrin-dependent signal transduction events involving the tyrosine kinase substrate fakB.

Mitogenic properties of a bispecific single-chain Fv-Ig fusion generated from CD2-specific mAb to distinct epitopes.

The combination of anti-CD2 mAb 9.6 and 9-1, specific for distinct epitopes, induces proliferation of resting human T cells. The mitogenic activity of this mAb mixture depends upon accessory cells and the 9-1 mAb Fc domain. To further study the functional properties of these mAb, their variable regions were cloned and expressed as monospecific single-chain Fv (scFv) proteins fused to the human IgG1 Fc domain (scFvIg). A novel bispecific scFvIg was constructed by cloning the two monospecific scFv binding sites in tandem, with the 9.6 scFv placed N-terminal to the 9-1 scFvIg. Monospecific scFvIg binding to CD2 was comparable to that of the corresponding parental mAb, while the bispecific scFvIg exhibited binding activity similar to that of the 9-1 scFvIg. The combination of 9.6 scFvIg and 9-1 mAb was mitogenic, whereas mixtures including the 9-1 scFvIg were non-stimulatory, confirming the unique properties of the 9-1 IgG3 Fc. Without the IgG3 tail, the bispecific 9.6/9-1 scFvIg was directly mitogenic and was a more potent mitogen than the mAb mixture, but was accessory cell dependent. Unlike the combination of mAb, the bispecific reagent did not directly mobilize calcium in T cells. In comparison to the mAb mixture, bispecific 9.6/9-1 scFvIg-mediated stimulation of a mixed lymphocyte reaction was significantly more resistant to inhibition of the CD28 co-stimulatory pathway by the inhibitor CTLA-4-Ig. These results show that expression of the 9.6 and 9-1 binding sites together on a bispecific scFvIg increased the mitogenic properties of the mAb and altered the degree of accessory cell signals required for T cell activation.

Wiskott-Aldrich syndrome/X-linked thrombocytopenia: WASP gene mutations, protein expression, and phenotype.

Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT), caused by mutations of the WAS protein (WASP) gene, represent different phenotypes of the same disease. To demonstrate a phenotype/genotype correlation, we determined WASP gene mutations in 48 unrelated WAS families. Mutations included missense (20 families) and nonsense (eight) mutations located mostly in exons 1 to 4, and splice-site mutations (seven) and deletions and insertions (13) located preferentially in exons 7 to 11. Both genomic DNA and cDNA were sequenced and WASP expression was measured in cell lysates using peptide-specific rabbit anti-WASP antibodies. WASP was expressed in hematopoietic cell lines including bone marrow-derived CD34+ cells. Missense mutations located in exons 1 to 3 caused mild disease in all but one family and permitted WASP expression, although frequently at decreased concentration. Missense mutations affecting exon 4 were associated with classic WAS and, with one exception, barely detectable WASP. Nonsense mutations caused classic WAS and lack of protein. Insertions, deletions, and splice-site mutations resulted in classic WAS and absent, unstable, truncated, or multiply spliced protein. Using affinity precipitation, WASP was found to bind to Src SH3-containing proteins Fyn, Lck, PLC-gamma, and Grb2, and mutated WASP, if expressed, was able to bind to Fyn-glutathione S-transferase (GST) fusion protein. We conclude that missense mutations affecting the PH domain (exons 1 to 3) of WASP inhibit less important functions of the protein and result in a mild phenotype, and that missense mutations affecting exon 4 and complex mutations affecting the 3' portion of WASP interfere with crucial functions of the protein and cause classic WAS.

Mutational activation of pp60(c-src) leads to a tumorigenic phenotype in a preneoplastic Syrian hamster embryo cell line.

Previous studies indicated that overexpression of wild-type avian c-src cannot induce neoplastic transformation of NIH 3T3 cells. In this study, we isolated and characterized novel spontaneously derived transforming mutants of avian pp60(c-src) from a Syrian hamster embryo-derived cell line, 10W, transfected with the avian c-src gene. Seventeen independently derived transfected 10W cell clones were injected into athymic nude mice. After a latency period, tumors eventually arose and were established in culture. The tumorigenic phenotype was always accompanied by the presence of the avian c-src DNA and functional expression of pp60(c-src). However, most of the tumor-derived cell lines expressed an electrophoretically altered form of pp60(c-src), suggesting mutations in src. Consistent with this hypothesis, DNAs isolated from the tumor-derived lines, but not the parental 10W cell lines, morphologically transformed NIH 3T3 cells in a focus-forming assay. We characterized pp60(c-src) in detail from three of the tumor-derived lines: 4AT, 4BT, and E2T. Two of these lines contained mutations within the exogenous c-src coding region. Line 4AT has an internal repeat of 29 amino acids immediately following Gln-513, which disrupts the spacing between the end of the kinase domain and Tyr-527, the negative regulatory site in pp60(c-src). Line 4BT has a 5-bp deletion following Phe-520, which results in loss of Tyr-527. However, the DNA sequence of the coding region of pp60(c-src) from a third line, E2T, was completely wild type. Cyanogen bromide cleavage analyses of the altered pp60(c-src) from lines 4AT and 4BT showed that Tyr-527, the site of negative regulation of c-src, is not phosphorylated, but Tyr-416, the site of in vitro autophosphorylation, is phosphorylated. However, in line E2T, Tyr-527 was phosphorylated, and Tyr-416 was phosphorylated to a lesser extent. Additionally, two proteins that indicate activation of src, p85 cortactin and p120(cas), are phosphorylated in at least six of the tumor-derived cell lines, although to a lesser extent in line E2T. These results suggest that dephosphorylation of Tyr-527 and phosphorylation of Tyr-416 correlate with activation of pp60(c-src) in the tumor-derived lines 4AT and 4BT, respectively. However, in line E2T, the high levels of pp60(c-src), in combination with a partial activation of the pp60(c-src) protein as indicated by phosphorylation of Tyr-416, appear to be involved in the neoplastic process, rather than mutation.

The human p167 gene encodes a unique structural protein that contains centrosomin A homology and associates with a multicomponent complex.

The characterization of novel cytoplasmic, structural, and enzymatic proteins has been enhanced by a panel of monoclonal antibodies specific for protein substrates of transforming and nontransforming c-Src mutants. These protein substrates have included the focal adhesion kinase (FAK), cortactin, AFAP-110, p120CAS, and p130CAS. The monoclonal antibody 4G8 was generated as part of this panel of antibodies and was used to isolate the human gene for a 167-kD polypeptide. The cDNA sequence is 5,238 nucleotides in length with a predicted open reading frame consisting of 1,382 amino acids. The polypeptide is largely hydrophilic and highly charged. The central region of p167 has 88% identity with the entire 278-amino-acid encoded sequence of the murine centrosomin A gene. The carboxyl third of p167 contains a unique cluster of 10 amino acid repeats with the consensus sequence (A/M)DDDRGPRRG. The p167 protein was found primarily in the cytoplasm of lymphocytes and is part of a multicomponent protein complex with prominent members of 167, 120, 64, 45, 40, 38, and 25 kD. Finally, we illustrate the conservation of p167 and its associated complex, and demonstrate its expression in different human tissues and cell types. The data suggest that p167 is novel and has an important cellular function as a cytoplasmic structural protein.

The integrin-triggered rescue of T lymphocyte apoptosis is blocked in HIV-1-infected individuals.

HIV infection is associated with a disease status-dependent impairment of Ag-specific T cell responses, resulting in anergy or unchecked apoptotic cell death. beta1 integrins play an important role in the induction of T lymphocyte responses to antigenic challenge by providing a T cell costimulatory signal, and have been shown to rescue various cell types from undergoing apoptosis. We examined the integrin-triggered cell survival signal and associated pathways in CD3+ T cells derived from 69 HIV-1-infected individuals in comparison with healthy controls. We found beta1 integrin-mediated costimulation of TCR-induced T cell proliferation and protection from aberrant cell death to be absent in the majority of patients with AIDS, but intact in asymptomatic, infected individuals. The lack of integrin-mediated rescue may be partly due to an early impairment of TCR/integrin-costimulated secretion of IFN-gamma, a type 1 lymphokine that protects against TCR-induced apoptosis of T cells from HIV-seropositive donors, but not loss of integrin expression. The mechanism of integrin hyporesponsiveness appeared to correlate with a failure of the integrin-generated signal to induce pp125FAK mRNA and protein expression. Protein kinase C activation in CD3+ T cells following integrin stimulation was also impaired in HIV-infected individuals, mostly among the symptomatic/AIDS patients. Protein kinase C inactivation in T cells was shown to have a destabilizing effect in vitro on pp125FAK mRNA that contains an AUUUA motif in the 3'-untranslated region, a consensus sequence for the AU-rich elements responsible for mRNA destabilization. These aberrant changes in pp125FAK expression may have direct significance to the overall immunopathogenesis during infection with HIV-1.

Focal adhesion kinase-related fakB is regulated by the integrin LFA-1 and interacts with the SH3 domain of phospholipase C gamma 1.

Signal transduction through integrin molecules expressed on platelets and nonlymphoid cells involves activation of the intracellular focal adhesion kinase ppI25FAK (FAK) to phosphorylate substrate proteins on tyrosine residues. Similar mechanisms are also functional in T-lymphocytes through the beta 1-integrin VLA-4. A putative FAK-related phosphoprotein (fakB) was identified that is responsive to intracellular signals induced through ligation of antigen receptors on both T- and B-lymphocytes, and whose induced tyrosine phosphorylation is augmented by TCR costimulation through the adhesion/costimulatory receptors CD2 and CD4. In this report, fakB is shown to respond to extracellular signals through the beta 2-integrin LFA-1 in the absence of primary signals through the TCR. Protein-protein complex formation was observed involving an association between fakB, phospholipase C gamma 1 (PLC gamma 1), and the tyrosine phosphoprotein pp35-36. Evidence is provided here that fakB interacts with PLC gamma 1 through its SH3 domain. The association between fakB and PLC gamma 1 does not appear to require T-cell activation, whereas the induced tyrosine phosphorylation of the protein complex components occurs following engagement of LFA-1. These data indicate that the beta2-integrin LFA-1 expressed on T-lymphocytes stimulates a novel, FAK-related molecule that may function in the interplay between adhesion receptors and intracellular signaling enzymes responsible for downstream second messenger generation.

A major tyrosine-phosphorylated protein of Trypanosoma brucei is a nucleolar RNA-binding protein.

We have previously identified a set of tyrosine-phosphorylated proteins with apparent molecular masses of 44-46 kDa as some of the major tyrosine phosphorylated species in the protozoan parasite Trypanosoma brucei. We now show that these molecules, herein named Nopp44/46, are localized in the nucleolus. Using monoclonal antibodies, we have isolated Nopp44/46 cDNA clones from expression libraries. Sequence analysis reveals that the predicted amino acid sequence of the molecule is composed of an N-terminal unique region, an internal acidic region, and C-terminal repeat region. Analysis of the cDNA clones and genomic Southern analysis indicated that Nopp44/46 belongs to a multigene family in which different gene copies are very similar but vary in the number of repeats. Interestingly, the repetitive amino acid sequence motif contains multiple RGG (Arg-Gly-Gly) boxes characteristic of RNA-binding proteins. In vitro binding experiments demonstrated that Nopp44/46 is indeed capable of binding nucleic acids. Competition experiments with different RNA homopolymers demonstrated that Nopp44/46 preferentially binds to poly(U). These studies suggest that Nopp44/46 may play a role in RNA metabolism in trypanosomes and raise the possibility that tyrosine phosphorylation may regulate the process.

ZAP-70 and p72syk are signaling response elements through MHC class II molecules.

Ligation of major histocompatibility complex (MHC) class II antigens expressed on antigen-activated human CD4+ T-lymphocytes induces early signal transduction events including the activation of tyrosine kinases, the tyrosine phosphorylation of phospholipase-C gamma 1 and the mobilization of intracellular calcium. Similar responses have been observed in B-cells following stimulation of MHC class II molecules, including the increased production of intracellular cAMP. In this report, we demonstrate that the ZAP-70 tyrosine kinase is a responsive signaling element following cross-linking of HLA-DR in class II+ T-cells, and that the homologous tyrosine kinase p72syk is stimulated in B-cells following ligation of class II antigens. Antibody mediated co-ligation of the T-cell antigen receptor (TCR/CD3) with class II molecules resulted in augmented tyrosine phosphorylation of ZAP-70. Comparable to antibody induced receptor ligation, bacterial superantigen (SEA and SEB) treatment of HLA-DR+ T-cells stimulated ZAP-70 tyrosine phosphorylation, consistent with class II transmembrane signaling by ligation of HLA-DR and V beta in cis. Modulation of the TCR/CD3 led to abrogation of class II induced ZAP-70 tyrosine phosphorylation, but did not result in sequestering of ZAP-70 from the cellular cytoplasm. Hyperphosphorylated ZAP-70 was associated with TCR/CD3 zeta-chain following cross-linking of HLA-DR, suggesting a mechanism for the TCR/CD3-dependence of class II induced signals in alloantigen-activated human T-cells. In both tonsillar B-lymphocytes and B-cell leukemia lines, p72syk was rapidly phosphorylated on tyrosine residues following HLA-DR cross-linking. Tyrosine phosphorylation of p72syk induced through ligation of either the B-cell antigen receptor or class II molecules was potently inhibited by herbimycin A. MHC class II ligation on B-lymphocytes resulted in cell death, which was both qualitatively distinct from Fas-induced apoptosis and partially protected by herbimycin A pretreatment. Thus, ligation of MHC class II molecules expressed on human lymphocytes stimulates the ZAP-70/p72syk family of tyrosine kinases, leading functionally to a tyrosine kinase-dependent pathway of receptor-induced cell death.

Ligation of the T-cell antigen receptor (TCR) induces association of hSos1, ZAP-70, phospholipase C-gamma 1, and other phosphoproteins with Grb2 and the zeta-chain of the TCR.

Signaling by the T-cell antigen receptor (TCR) involves both phospholipase C (PLC)-gamma 1 and p21ras activation. While failing to induce Shc/Grb2 association, ligation of the TCR/CD3 receptor in Jurkat T-cells induced hSos1-Grb2 complexes. In addition to hSos1, Grb2 participates in the formation of a tyrosine phosphoprotein complex that includes 145-, 95-, 70-, 54-, and 36-38-kDa proteins. p145 was identified as PLC-gamma 1 and p70 as the protein tyrosine kinase, ZAP-70. Although of the same molecular weight, p95 was not recognized by an anti-serum to p95 Vav. The SH2 domains of Grb2 and PLC-gamma 1 were required for the formation of this protein complex. In anti-CD3-treated cells, Grb2 redistributed from the cytosol to a particulate cell compartment along with p36/p38, ZAP-70, and PLC-gamma 1. Part of the Grb2 complex associated with the particulate compartment could be extracted with Nonidet P-40, while the rest was Nonidet P-40 insoluble. In both the detergent-soluble and -insoluble fractions, Grb2 coimmunoprecipitated with the zeta-chain of the TCR. Taken together, these results indicate that anti-CD3 induces Grb2-hSos1-PLC-gamma 1-p36/p38-ZAP70 complexes, which localize in the vicinity of TCR-zeta.

Activation of murine T-cells via phospholipase-C gamma 1-associated protein tyrosine phosphorylation is reduced with aging.

Cross-linking of the T-cell receptor (CD3) induces activation of tyrosine kinases and the subsequent phosphorylation of intracellular protein substrates. We examined whether early events in signal transduction through CD3 or CD3 x CD4 receptor ligation were altered in aged murine T-lymphocytes. Both calcium mobilization and tyrosine phosphorylation of phospholipase C gamma 1 (PLC gamma 1) were decreased in T-lymphocytes from old mice. In addition, there was less tyrosine phosphorylation of a 35/36 kDa protein both in whole cell lysates and in PLC gamma 1 immunoprecipitates from old mice. This 35/36 kDa phosphoprotein binds specifically to the SH2 domains of PLC gamma 1. Using a fusion protein containing the SH2 domains of PLC gamma 1 and human IgG1 heavy chain, we identified three additional proteins that bind to the SH2 domains which were tyrosine phosphorylated following CD3 x CD4 ligation to a lesser degree with age. The tyrosine phosphorylation of two phosphoproteins binding to a fusion protein consisting of the SH2 domains of GAP (ras GTPase-activating protein) and human IgG1 heavy chain was also reduced with aging. The observed binding to SH2 domains was thiol redox sensitive. Thus, decreases in antioxidants with age may be responsible for inhibitory effects on PLC gamma 1-phosphatidylinositol signaling through redox regulation of tyrosine phosphoproteins.