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.

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.

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.

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.

HIV-1 down-regulates CD4 costimulation of TCR/CD3-directed tyrosine phosphorylation through CD4/p56lck dissociation.

One consequence of HIV type 1 (HIV-1) infection is the gradual loss of responsiveness of T lymphocytes to Ags both in vitro and in vivo. It has been suggested that the underlying mechanism that contributes to this T cell dysfunction before CD4+ cell decline involves down-regulation of surface receptors, alterations in intracellular redox status, interference by viral Ags, and later in infection, the absence or alteration of specific cytokine production. In this report, we demonstrate that infection of the T-lymphocytic cell line H9 with the LAI isolate of HIV-1 results in profoundly altered regulation of CD4-induced costimulation of TCR/CD3-directed signaling. TCR/CD3-induced tyrosine phosphorylation of the intracellular enzyme phospholipase-C gamma 1 and the surface receptor/substrates CD5 and CD6 was unaffected by virus infection, whereas augmented responses normally observed after the co-ligation of CD4 with TCR/CD3 on T lymphocytes were absent in HIV-1-infected H9 cells. Costimulation of TCR/CD3-induced signaling via MHC class II molecules was also down-regulated in virally infected cells. TCR/CD3 and HLA-DR receptor expression remained intact in infected cultures for at least 3 wk, whereas CD4 surface expression was gradually lost but maintained for up to 1 wk, suggesting that the absence of costimulation early in infection was not surface receptor density-dependent. In HIV-1-infected cells, CD4 was not physically linked with its associated tyrosine kinase p56lck, whereas normal levels of p56lck were readily recovered from the cellular cytoplasm. Similar observations were noted in cultures of H9 cells infected with a field isolate of HIV-1 obtained from cultured PBMC from an infected donor. HIV-1 infection of T lymphocytes thus down-regulates potentially critical early signal transduction events by a mechanism that appears to involve interference of CD4 receptor association with p56lck. A potential outcome of these biochemical effects may include the limited responsiveness of infected T cells to antigenic stimulation observed during HIV-1 infection.

Evidence for LFA-1/ICAM-1 dependent stimulation of protein tyrosine phosphorylation in human B lymphoid cell lines during homotypic adhesion.

JK32.1 and SKW6.4 are Epstein-Barr virus (EBV)-positive human B cell lines that undergo spontaneous, lymphocyte function-associated antigen 1 (LFA-1) dependent homotypic adhesion in culture. This process is associated with induction of tyrosine phosphoproteins of molecular mass 90, 106, and 120 kDa and could be reproduced when these cells were centrifugationally aggregated. Antibodies to the beta 2 (CD18) chain of LFA-1 interfered with induction of p120, p106, and p90 during cellular aggregation. Response induction was abrogated when cells were incubated with protein tyrosine kinase (PTK) inhibitors (erbstatin, genistein, and geldanomycin) or cytochalasin B prior to aggregation. An in vitro kinase assay did not reveal activation of focal adhesion kinase. Although the role of LFA-1-dependent tyrosine phosphorylation in B cells is uncertain, patients with the leukocyte adhesion defect (LAD) exhibit humoral abnormalities. Moreover, aggregation did not induce specific tyrosine phosphoproteins in an EBV-transformed B cell line from a LAD patient. These results suggest that an LFA-1-dependent PTK pathway may play an important role in human B cell function.

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.

A transmembrane-anchored chimeric focal adhesion kinase is constitutively activated and phosphorylated at tyrosine residues identical to pp125FAK.

Focal adhesion kinase, pp125FAK, is a nonmyristylated cytosolic tyrosine kinase unrelated to protein-tyrosine kinase families categorized to date. The kinase activity and tyrosine phosphorylation of pp125FAK are induced by beta 1 and beta 3 integrin-mediated cell adherence or aggregation. pp125FAK is also a tyrosine phosphorylation substrate in v-src-transformed cells and is localized to focal adhesion contracts of adherent fibroblasts and carcinoma cells. In this report, we have transiently expressed in COS cells a transmembrane-anchored chimeric receptor kinase, CD2FAK, consisting of CD2 and pp125FAK. We analyzed its kinase activity and tyrosine phosphorylation and compared to those of pp125FAK. We found that CD2FAK exhibited constitutive kinase activity and a high basal tyrosine phosphorylation level when COS transfectants were suspended in serum-free media. The kinase activity of CD2FAK was similarly up-regulated upon beta 1 integrin-mediated cell adherence as the endogenous pp125FAK. Both CD2FAK and pp125FAK appeared to be active as autophosphorylating kinases as shown by mutation of the ATP binding site. We determined the major tyrosine phosphorylation site, Tyr397, identical for both the constitutively activated CD2FAK and pp125FAK in response to beta 1 integrin-mediated cell adherence by site-directed mutagenesis. Deletions of the NH2- or the COOH-terminal noncatalytic domain of FAK, including Tyr397 did not lead to abolition of the kinase activity of pp125FAK or CD2FAK. Taken together, CD2FAK exhibits properties of an activated pp125FAK and the kinase activity does not appear to require tyrosine phosphorylation in vitro or in vivo.

ZAP-70 tyrosine kinase, CD45, and T cell receptor involvement in UV- and H2O2-induced T cell signal transduction.

Several mammalian responses to UV irradiation, including the activation of NF-kappa B, are believed to involve tyrosine phosphorylation. UV irradiation and H2O2 treatment of T lymphocytes induce protein tyrosine phosphorylation and Ca2+ signals similar to those observed following biological stimulation. We have examined the role of cell surface molecules in these responses. Normal T lymphocytes whose surface expression of CD3 was depleted showed impaired UV-induced tyrosine phosphorylation and Ca2+ signals. Similarly, Jurkat T cell lines deficient in CD3 or CD45 expression also gave impaired UV responses. However, all these cell types still gave strong Ca2+ and tyrosine phosphorylation responses to H2O2. The T cell tyrosine kinase ZAP-70 was found to be highly responsive to UV and H2O2 treatment. ZAP-70 responsiveness to UV required expression of both CD3 and CD45, whereas only CD3 was required for the response to H2O2. UV-induced activation of NF-kappa B was blocked by CD3 depletion, indicating the importance of such cell surface molecules in biological responses to UV. In nonlymphoid cells, the epidermal growth factor receptor displayed increased tyrosine phosphorylation within seconds of UV irradiation. These results suggest that UV-induced signal transduction is mediated via cell surface receptors that normally respond to biological stimulation, whereas H2O2 is able to partially bypass this requirement.

Human T and B lymphocytes express a structurally conserved focal adhesion kinase, pp125FAK.

Clustering of beta 1-integrins on adherent cells with antibodies or ligands results in increased tyrosine phosphorylation and activation of a novel focal adhesion tyrosine kinase, pp125FAK. The genes encoding pp125FAK have been cloned previously from both chicken and mouse cDNA libraries, and the deduced amino acid sequences are nearly identical (94%). Two synthetic peptides derived from sequences at the carboxyl terminus of chicken pp125FAK were conjugated to ovalbumin to generate rabbit heteroantisera. Human pp125FAK was immunodetected in both T and B lymphocytes with these antisera. A basal state of pp125FAK tyrosine phosphorylation was observed in T and B lymphocytes, and its expression level was in general augmented among human T- and B-cell leukemia/lymphoma lines. Additionally, the full-length sequence of human T-cell pp125FAK (huT-FAK) was derived from a Jurkat T-cell cDNA library. huT-FAK is structurally identical with both mouse and chicken FAK, and shares 95% amino acid identity with chicken pp125FAK and has 97% homology with the mouse sequence. This high degree of evolutionary conservation between species suggests that pp125FAK is likely to have a crucial function in the cell. Expression of the full-length huT-FAK gene in COS cells showed an immunologically indistinct human pp125FAK protein compared with the endogenous primate pp125FAK. Taken together, the data indicate that this structurally conserved human T-cell pp125FAK likely functions in T- and B-cell lineages, and its altered expression in human lymphocyte tumor cell lines may contribute to their transformed phenotype.

Beta 2-integrin LFA-1 signaling through phospholipase C-gamma 1 activation.

One of the beta 2-integrins found on hematopoietic cells is lymphocyte function-associated antigen 1 (LFA-1), a lymphocyte/myeloid cell-specific receptor that binds to members of the intercellular adhesion molecule (ICAM) family on antigen-presenting cells. Stimulation of LFA-1 with antibodies or purified ICAMs induces augmentation of T-cell antigen receptor (TCR)-directed T-cell responsiveness. In the present study, LFA-1 was shown to be linked to the tyrosine kinase signaling pathway that stimulates tyrosine phosphorylation and activation of phospholipase C-gamma 1 (PLC-gamma 1). Integrin beta-chain (CD18) crosslinking independently induced downstream mobilization of intracellular Ca2+ and potently costimulated TCR-induced Ca2+ flux with an increase in both amplitude and kinetics. beta 2-Integrin signaling through this pathway was completely inhibited by herbimycin A and was prevented by TCR modulation. Coligation of the TCR via antibody and LFA-1 with a counter-receptor in the form of a soluble ICAM-1/Rg fusion protein resulted in prolonged tyrosine phosphorylation of PLC-gamma 1. Monoclonal antibodies to both the alpha chain (CD11a) and the beta chain (CD18) of LFA-1 induced Ca2+ mobilization to different levels, suggesting epitope specificity for activation potential. In addition to PLC-gamma 1, tyrosine phosphorylation of an 80-kDa protein substrate was augmented following CD18 crosslinking but was not TCR-dependent. The beta 2-integrin LFA-1 on T cells is therefore directly linked to a tyrosine kinase pathway that stimulates signaling by phosphatidylinositol-specific PLC-gamma 1.

Tyrosine phosphorylation of CD19 in pre-B and mature B cells.

Cross-linking of B cell surface immunoglobulins (sIg) results in activation of mature B cells and stimulates a molecular signaling mechanism for antigen-specific B cell expansion and differentiation. This signaling pathway is dependent on tyrosine (Tyr) phosphorylation and results in the activation of sIg-associated src family kinases and p72SYK. Rapid Tyr phosphorylation occurs on multiple protein substrates. Here we show that activation of B cells by cross-linking sIg results in an increase in Tyr phosphorylation of the lineage-restricted B cell surface antigen CD19, and show that it is a major substrate of activated Tyr kinase following sIg stimulation. Lower levels of constitutive CD19 Tyr phosphorylation occurred in most sIg+ mature B cell lines examined and in normal dense tonsillar B cells. We also find that when CD19 is Tyr-phosphorylated it becomes competent to interact with SH2 domains suggesting a mechanism whereby, following B cell activation, CD19 could be linked to intracellular signaling pathways. In sIg- pre-B cell lines, CD19 was expressed but was not constitutively phosphorylated on tyrosine. Upon CD19 cross-linking, Tyr phosphorylation of CD19 was induced in sIg- pre-B cell lines. CD19 cross-linking also directly induced Tyr phosphorylation of CD19 and other substrates in mature B cells. The ability of CD19 to signal in the absence of sIg expression may provide important stimulation in pre-B cell development.

CD4, CD8 and the role of CD45 in T-cell activation.

CD4, CD8 and CD45 regulate the coupling of the T-cell receptor complex (CD3-TCR) to tyrosine kinase activation and phosphorylation of key substrates such as phospholipase C gamma 1. CD4 and CD8 contribute to activation signals through their cytoplasmic association with p56lck. Expression of the zeta-chain is required for functional synergy of the T-cell receptor with CD4 in the activation of phospholipase C gamma 1, which probably reflects an interaction between p56lck and zeta-associated kinase ZAP-70. CD45 expression is required for CD3-TCR signaling. CD45 may positively regulate signaling by dephosphorylating the carboxyl-terminal tyrosine of p56lck and p59fyn, and negatively regulate signaling by dephosphorylation of other TCR-associated substrates directly. One ligand for CD45 receptor has been identified as the B cell CD22 molecule. The positive and negative effects of CD45 are sensitive to the composition of CD45 in receptor complexes, and may be regulated by specific associations of CD45 isoforms with other receptors such as CD3-TCR, CD2 and CD4.

The effect of 1-chloro-2,4-dinitrobenzene exposure on antigen receptor (CD3)-stimulated transmembrane signal transduction in purified subsets of human peripheral blood lymphocytes.

The intracellular low-molecular-weight thiol glutathione (GSH) is an important scavenger of free radicals and plays a role in the maintenance of the redox status of protein sulfhydryl groups. We have previously shown that human peripheral blood lymphocytes sorted on their basal GSH content proliferate proportionately to their GSH levels, and that an early event in lymphocyte activation appeared to be dependent on GSH. We have now analyzed transmembrane signal transduction in cells treated with 1-chloro-2,4-dinitrobenzene (CDNB), a GSH-depleting agent. Transmembrane signal transduction was measured as changes in intracellular free calcium and in protein tyrosine phosphorylation after stimulation with anti-CD3 monoclonal antibody. The results show a CDNB dose-dependent reduction in GSH content, the magnitude of intracellular free calcium mobilization, and the extent of tyrosine phosphorylation of several proteins, including phospholipase C-gamma 1. This suggests a role for GSH and/or protein thiol redox status in one of the earliest events controlling the ability of lymphocytes to respond to important proliferative signals in their environment and implies that agents which deplete lymphocyte GSH may be immunosuppressive through effects on CD3/T cell receptor-dependent transmembrane signal transduction.

Lymphocyte lineage-restricted tyrosine-phosphorylated proteins that bind PLC gamma 1 SH2 domains.

Epidermal growth factor (EGF) or platelet-derived growth factor binding to their receptor on fibroblasts induces tyrosine phosphorylation of PLC gamma 1 and stable association of PLC gamma 1 with the receptor protein tyrosine kinase. Similarly in lymphocytes, cross-linking of antigen receptors induces the formation of molecular complexes incorporating PLC gamma 1; however, associated kinase activity is thought to be mediated through cytoplasmic protein tyrosine kinase(s). In this report, we generated a fusion protein containing the SH2 domains of human PLC gamma 1 and human IgG1 heavy chain constant region to identify lymphocyte phosphoprotein-binding PLC gamma 1 SH2 domains following cellular activation. As in EGF- or platelet-derived growth factor-stimulated fibroblasts, PLC gamma 1 is coprecipitated in activated lymphocytes, complexed with associated tyrosine-phosphorylated proteins. One of these, a 35/36-kDa protein found prominently in T cells and at lower levels in B cells, bound to the fusion protein in immunoprecipitation experiments. The fusion protein showed lineage restricted association with a 74-kDa phosphoprotein in T cells and a 93-kDa phosphoprotein in B cells. It bound to activated EGF receptor in fibroblasts as expected, and protein tyrosine kinase activity was precipitated from EGF-stimulated cells. However, PLC gamma 1-associated protein tyrosine kinase activity was not detected in activated lymphocytes. These data suggest that lymphocyte PLC gamma 1 SH2-binding proteins are cell lineage specific and may be transiently associated with activated PLC gamma 1.