Cell activation mediated by glycosylphosphatidylinositol-anchored or transmembrane forms of CD14

CD14 is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein which functions as a receptor on myeloid cells for ligands derived from microbial pathogens such as lipopolysaccharide (LPS). We have studied the importance of the GPI tail of CD14 in signalling with the promonocytic cell line THP-1 expressing recombinant CD14 in a GPI-anchored form (THP1-wtCD14 cells) or in a transmembrane form (THP1-tmCD14). We found that, like other GPI-anchored molecules, GPI-anchored CD14 was recovered mainly from a Triton X-100-insoluble fraction, whereas transmembrane CD14 was fully soluble in Triton X-100. LPS induced cell activation of THP1-wtCD14 and of THP1-tmCD14 (protein tyrosine kinase phosphorylation, NF-kappaB activation, and cytokine production) in a very similar manner. However, anti-CD14 antibody-induced cross-linking caused a rapid calcium mobilization signal only in GPI-anchored CD14 cells. Studies with pharmacologic inhibitors of intracellular signalling events implicate phospholipase C and protein tyrosine kinases in the genesis of this antibody-induced calcium signal. Our results suggest that GPI anchoring and CD14 targeting to glycolipid-rich membrane microdomains are not required for LPS-mediated myeloid cell activation. GPI anchoring may however be important for other signalling functions, such as those events reflected by antibody cross-linking.     

Syk, but not Lyn, recruitment to B cell antigen receptor and activation following stimulation of CD45- B cells

B cell Ag receptor (BCR) signaling occurs via tyrosine phosphorylation of CD79a and CD79b ITAMs, leading to recruitment and activation of Lyn and Syk tyrosine kinases and subsequent downstream events. CD45 expression is required for BCR triggering of certain of these downstream events, such as calcium mobilization and p21ras activation. However, the site in the BCR signaling cascade at which CD45 impinges is poorly defined. To address this question, we have studied CD45 function in the CD45-deficient (CD45-) and CD45-reconstituted (CD45+) J558L mu m3 plasmacytoma. In both CD45+ and CD45- cells, Ag stimulation led to CD79a and CD79b tyrosine phosphorylation as well as Syk tyrosine phosphorylation, recruitment to the receptors, and activation. In contrast to CD45+ cells, Lyn exhibited high basal tyrosine phosphorylation in the CD45- cells and was not further phosphorylated upon Ag stimulation. Mapping studies indicated that the observed constitutive phosphorylation of Lyn reflects phosphorylation of its C-terminal tyrosine, Y508, at high stoichiometry. Constitutively Y508-phosphorylated Lyn was neither recruited to the BCR nor activated upon Ag stimulation. Moreover, CD79a-ITAM phosphopeptides failed to bind Lyn from the CD45- cells. Thus, Y508 phosphorylation of Lyn occurs in the absence of cellular CD45 expression and appears to render the kinase unable to associate with the phosphorylated receptor complex via its Src homology 2 domain and to participate in signal propagation. Surprisingly, in view of previous findings implicating Src family kinases in ITAM phosphorylation, the data indicate that Ag-induced CD79a and CD79b tyrosine phosphorylation and Syk recruitment and activation can occur in the absence of CD45 expression and, hence, Src-family kinase activation.     

Lovastatin inhibits T-cell antigen receptor signaling independent of its effects on ras

Lovastatin, a cholesterol-lowering drug, has antiproliferative properties that may be related to its inhibition of protein isoprenylation. We examined the effects of lovastatin on signal transduction via the T-cell antigen receptor (TCR). Lovastatin inhibited both proximal and distal TCR-mediated signaling events in a time- and concentration-dependent manner in the human Jurkat T-cell line. Upregulation of CD69 surface expression after TCR stimulation was blocked by lovastatin, although no inhibition of phorbol ester-induced CD69 expression was noted. Proximal TCR-mediated signaling events, including intracellular calcium mobilization, inositol phosphate production, and tyrosine phosphorylation of phospholipase Cgamma1, were similarly inhibited by lovastatin, although global protein tyrosine kinase activity remained intact. In a Jurkat variant transfected with the human type-1 muscarinic receptor, lovastatin also inhibited TCR-mediated calcium mobilization and inositol phosphate production but failed to affect muscarinic receptor-induced responses. Lovastatin, at similar doses, also disrupted post-translational processing of ras and inhibited ras-dependent signals, including phosphorylation and activation of mitogen-associated protein kinase after TCR stimulation. These findings suggest that the antiproliferative properties of lovastatin may be independent of ras and could result from uncoupling protein tyrosine kinases from distinct signal transduction pathways.     

The transmembrane protein-tyrosine phosphatase CD45 is associated with decreased insulin receptor signaling

Overexpression of the transmembrane protein-tyrosine phosphatase (PTPase) CD45 in nonhematopoietic cells results in decreased signaling through growth factor receptor tyrosine kinases. Consistent with these data, insulin receptor signaling is increased when the CD45-related PTPase LAR is reduced by antisense suppression in a rat hepatoma cell line. To test whether the hematopoietic cell-specific PTPase CD45 functions in a manner similar to LAR by negatively modulating insulin receptor signaling in hematopoietic cells, the insulin-responsive human multiple myeloma cell line U266 was isolated into two subpopulations that differed in CD45 expression. In CD45 nonexpressing (CD45-) cells, insulin receptor autophosphorylation was increased by 3-fold after insulin treatment when compared to CD45 expressing (CD45+) cells. This increase in receptor autophosphorylation was associated with similar increases in insulin-dependent tyrosine kinase activation. These receptor level effects were paralleled by postreceptor responses. Insulin-dependent tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) and Shc was 3-fold greater in CD45- cells. In addition, insulin-dependent IRS-1/phosphatidylinositol 3-kinase association and MAP kinase activation in CD45- cells were also 3-fold larger. While expression of CD45 was associated with a decrease in the responsiveness of early insulin receptor signaling, interleukin 6-dependent activation of mitogen-activated protein kinase kinase and mitogen-activated protein kinase was equivalent between CD45- and CD45+ cells. These observations indicate that CD45 can function as a negative modulator of growth factor receptor tyrosine kinases in addition to its well-established role as an activator of src family tyrosine kinases.     

Interphase cytogenetic (in situ hybridization) analysis of astrocytomas using archival, formalin-fixed, paraffin-embedded tissue and nonfluorescent light microscopy

The effect of CD4 expression on the activation threshold of mouse T lymphocytes has been analysed. To do this, the authors studied the response to antigen and other T cell receptor (TCR) ligands in a series of CD4- mutants obtained from the SR.D10 clone. This non-tumour clone spontaneously arose from the Th2 clone D10.G4.1, and characteristically shows a low threshold for antigen activation as well as reactivity to syngeneic antigen presenting cells (APC). Although SR.D10 CD4- mutant cells can be stimulated by antigen, they need higher antigen concentration or more APC than SR.D10 or CD4 transfectants to yield optimal antigen responses. Furthermore, CD4- clones are not activated by syngeneic APC or by clonotypic antibodies. These effects do not correlate with changes in the expression of cell surface molecules implicated in antigen recognition, like TCR/CD3, CD2, LFA-1, or CD45, or with lower p56lck or p59fyn activity in the mutant cells. Since inhibition experiments using anti-CD4 antibodies have previously shown that activation of the CD4+ T cell clone D10.G4.1 by antigen or alloantigens is largely dependent on CD4, our results indicate that activation by antigen-plus self MHC may become CD4-independent if the activation threshold is lowered enough, e.g. in cells like SR.D10. Expression of CD4 further lowers the activation threshold of the cells, allowing the detection of low-affinity TCR reactivities like those directed at self MHC. Moreover, by using anti-TCR/CD3 antibodies, the authors have confirmed the importance of CD4-associated tyrosine kinase activity in early TCR/CD3 signalling in this Th2 cell line, as (1) upon TCR/CD3 ligation, tyrosine phosphorylation is detected only in those CD3 chains co-precipitating with CD4; and (2) CD4 expression is needed for efficient early tyrosine phosphorylation and detectable p56lck-TCR co-precipitation.     

Association of phosphatidylinositol 3-kinase with a specific sequence of the T cell receptor zeta chain is dependent on T cell activation

The T cell antigen receptor (TCR).CD3 complex contains several distinct but related signal transduction modules termed "Reth motifs": one each in the cytoplasmic domains of CD3-gamma, -delta, and -epsilon chains and three in the CD3-zeta polypeptide (zeta A, zeta B, and zeta C). Cross-linking of individual motifs expressed in chimeric molecules leads to early and late T cell activation events. Although the activated T cell receptor associates with nonreceptor tyrosine kinases, the sites of interaction with kinases and other potential effector molecules have not been fully mapped. Here we show that phosphatidylinositol 3-kinase (PI 3-kinase) preferentially associated with the zeta chain membrane proximal motif zeta A. Maximal PI 3-kinase/zeta A association occurred following TCR.CD3 activation and was dependent upon phosphorylation of both tyrosine residues in zeta A. The association of PI 3-kinase was specific for zeta A and could be ranked zeta A > zeta C > zeta B. Phosphorylation of the zeta A motif on tyrosine residues occurred in response to TCR.CD3 cross-linking in vivo. These results indicate that T cell activation leads to assembly of an intracellular signaling complex: recruitment of a tyrosine kinase, phosphorylation of zeta A, and association of PI 3-kinase. These data also support a model in which different Reth motifs of the TCR.CD3 complex recruit distinct signal transduction molecules. Thus, the subdomains of the T cell antigen receptor zeta chain may serve different roles during T cell maturation and antigen-driven activation.     

Pyk2 is differentially regulated by beta1 integrin- and CD28-mediated co-stimulation in human CD4+ T lymphocytes

Beta1 integrins can provide T cell co-stimulation, but little is known concerning their downstream signaling pathways. We found that Pyk2, a focal adhesion kinase-related tyrosine kinase, is regulated by beta1 integrin signaling in human T cells. Stimulation of Jurkat T cells with the alpha4beta1 integrin ligand VCAM-1 results in Pyk2 tyrosine phosphorylation, and combined stimulation with VCAM-1 and anti-CD3 mAb induces rapid and sustained synergistic Pyk2 phosphorylation. Studies with mAb suggest that in synergistic CD3- and alpha4beta1 integrin-mediated Pyk2 tyrosine phosphorylation, a major contribution of CD3-derived signals is independent of their effects on regulating integrin adhesion. Analysis of resting human CD4+ T cells confirmed the ability of CD3-derived signals to synergize with beta1 integrin-dependent signals in the induction of Pyk2 tyrosine phosphorylation. In addition, although CD28-mediated co-stimulatory signals were able to synergize with CD3-mediated signals in inducing ERK and JNK activation and secretion of IL-2 in the primary T cells, they did not contribute to the induction of Pyk2 phosphorylation. Taken together, these results indicate a potential role for Pyk2 in T cell co-stimulation mediated specifically by beta1 integrins.     

Involvement of tyrosine phosphorylation and protein kinase C in the activation of phospholipase D by H2O2 in Swiss 3T3 fibroblasts

We have investigated the mechanisms involved in H2O2-mediated phospholipase D (PLD) activation in Swiss 3T3 fibroblasts. In the presence of vanadate, H2O2 induced tyrosine phosphorylation of PLD as well as the platelet-derived growth (PDGF) factor receptor, protein kinase Calpha (PKCalpha), and a 62-kDa protein in rat brain PLD1 (rPLD1) immune complexes. PDGF also induced tyrosine phosphorylation of PLD, but this was abolished by catalase, indicating that it was mediated by H2O2 generation. Interestingly, PLD was found to be constitutively associated with the PDGF receptor and PKCalpha. Stimulation by H2O2 showed a concentration- and time-dependent tyrosine phosphorylation of the proteins in rPLD1 immunoprecipitates and activation of PLD in the cells. Pretreatment of the cells with the protein-tyrosine kinase inhibitors genistein and herbimycin A resulted in a concentration-dependent inhibition of H2O2-induced tyrosine phosphorylation and PLD activation. Activation of PLD by H2O2 was also inhibited dose-dependently by the PKC inhibitors Ro 31-8220 and calphostin C. Down-regulation of PKC by prolonged treatment with 4beta-phorbol 12-myristate 13-acetate also abolished H2O2-stimulated PLD activity. H2O2 or vanadate alone did not induce tyrosine phosphorylation of proteins in the rPLD1 immune complex or PLD activation. Reduction of intracellular H2O2 levels by pretreatment of the cells with catalase dramatically abrogated tyrosine phosphorylation of proteins in the rPLD1 immune complex and PLD activation, suggesting the potential role of intracellular H2O2 in H2O2-mediated PLD signaling. Taken together, these results suggest that both protein-tyrosine kinase(s) and protein kinase C participate in H2O2-induced PLD activation in Swiss 3T3 cells.     

The CD4-associated tyrosine kinase p56lck is required for lymphocyte chemoattractant factor-induced T lymphocyte migration

Lymphocyte chemoattractant factor (LCF) is a polypeptide cytokine which induces both cell motility and activation of T lymphocytes. These LCF-induced events demonstrate an absolute requirement for the cell surface expression of CD4. Because many CD4-mediated T lymphocyte activation events have been demonstrated to require the association of the src-related tyrosine kinase p56lck with the cytoplasmic domain of CD4, we examined the role of p56lck in LCF-induced lymphocyte migration in a murine T cell hybridoma line expressing transfected human CD4. LCF induces the catalytic activity of CD4 associated p56lck at chemoattractant concentrations of cytokine. Hybridoma cells that express CD4 with cytoplasmic point mutations which uncouple the CD4-lck association lack both lck enzymatic activity and chemotactic responses to LCF. The enzymatic activity of lck however does not appear to be required for CD4-mediated migratory signal. First, the protein tyrosine kinase inhibitor herbimycin A blocked LCF-induced p56lck activation but had no effect on the LCF-induced motile response. Second, T cell hybridomas expressing a chimeric receptor combining the extracellular domain of human CD4 and murine p56lck which lacked the kinase domain had a normal LCF-induced motile response. We conclude from these observations that CD4-lck coupling is essential for LCF-induced T lymphocyte migration but the motile response is independent of the enzymatic activity of CD4-associated p56lck.     

Structural basis for activation of human lymphocyte kinase Lck upon tyrosine phosphorylation

Regulation through phosphorylation is a characteristic of signalling pathways and the lymphocyte kinase Lck (p56lck) both performs phosphorylation and is affected by it. Lck is a Src-family tyrosine kinase expressed in T lymphocytes, where it participates in the cellular immune response. Like all Src homologues, it comprises SH3, SH2 and kinase domains. Lck associates through its distinctive amino-terminal segment with the cytoplasmic tails of either T-cell co-receptor, CD4 or CD8-alpha. Activated Lck phosphorylates T-cell receptor zeta-chains, which then recruit the ZAP70 kinase to promote T-cell activation. Lck is activated by autophosphorylation at Tyr 394 in the activation loop and it is inactive when Tyr 505 near the carboxy terminus is phosphorylated and interacts with its own SH2 domain. Here we report the crystal structure of the Lck tyrosine kinase domain (LCKK) in its activated state at 1.7 A resolution. The structure reveals how a phosphoryl group at Tyr 394 generates a competent active site. Comparisons with other kinase structures indicate that tyrosine phophophorylation and ligand binding may in general elicit two distinct hinge-like movements between the kinase subdomains. From modelling studies, we suggest a basis for inhibition by phosphorylation at Tyr 505.     

T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells

The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin-mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1-mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR-CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1-dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.     

Regulation of cytotoxic T lymphocyte-associated molecule-4 by Src kinases

Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) is a cell surface receptor expressed on activated T cells that can inhibit T cell responses induced by activation of the TCR and CD28. Studies with phosphorylated peptides based on the CTLA-4 intracellular domain have suggested that tyrosine phosphorylation of CTLA-4 may regulate its interactions with cytoplasmic proteins that could determine its intracellular trafficking and/or signal transduction. However, the kinase(s) that phosphorylate CTLA-4 remain uncharacterized. In this report, we show that CTLA-4 can associate with the Src kinases Fyn and Lck and that transfection of Fyn or Lck, but not the unrelated kinase ZAP70, can induce tyrosine phosphorylation of CTLA-4 on residues Y201 and Y218. A similar pattern of tyrosine phosphorylation was found in pervanadate-treated Jurkat T cells stably expressing CTLA-4. Phosphorylation of CTLA-4 Y201 in Jurkat cells correlated with cell surface accumulation of CTLA-4. CTLA-4 phosphorylation induced the association of CTLA-4 with the tyrosine phosphatase SHP-2, but not with phosphatidylinositol 3-kinase. In contrast, Lck-induced phosphorylation of CD28 resulted in the recruitment of phosphatidylinositol 3-kinase, but not SHP-2. These findings suggest that phosphorylation of CD28 and CTLA-4 by Lck activates distinct intracellular signaling pathways. The association of CTLA-4 with Src kinases and with SHP-2 results in the formation of a CTLA-4 complex with the potential to regulate T cell activation.     

Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase

We have previously described a type of selective T cell deficiency (STD) characterized by persistent infections reminiscent of severe combined immunodeficiency. We show here that STD patients carry a mutation of zap-70, resulting in loss of the activity of this kinase. The thymi of zap-70-/- patients show the presence of CD4+CD8+ cells in the cortex; however, only CD4, not CD8, single-positive cells are present in the medulla. Peripheral CD4+ T cells from the zap-70-/- patients exhibit markedly reduced tyrosine phosphorylation, fail to produce interleukin-2, and do not proliferate in response to T cell receptor stimulation by mitogens or antigens. Thus, Zap-70 kinase appears to be indispensable for the development of CD8 single-positive T cells as well as for signal transduction and function of single-positive CD4 T cells.     

Novel mechanism by which hemoglobin induces constriction of cerebral arteries

Since oxyhemoglobin (OxyHb) is implicated in the pathogenesis of cerebral vasospasm, we have investigated the role of protein tyrosine phosphorylation in OxyHb-mediated signalling in canine cerebral arteries and cultured canine cerebrovascular smooth muscle cells. OxyHb produced a contraction of basilar artery preparations, which was reversed by genistein, an inhibitor of tyrosine kinases, and PD098059, an inhibitor of mitogen-activated protein kinase. In cerebrovascular smooth muscle cells, OxyHb induced tyrosine phosphorylation of 42, 46, 54-60 and 80-100 kDa proteins with a time-course which paralleled the contractile action of OxyHb, suggesting that these events might be functionally linked. The 42 and 60 kDa proteins were immunologically related to the mitogen-activated protein kinase, extracellular signal regulated protein kinase (ERK2), and to p60c-Src (c-Src), respectively. The increase in protein tyrosine phosphorylation was attenuated by genistein, and the phosphorylation of the 42 kDa protein (ERK2) was inhibited by PD098059. These results suggest that OxyHb-mediated signalling utilizes a protein tyrosine kinase-based mechanism.     

Ligand-like effects induced by anti-c-erbB-2 antibodies do not correlate with and are not required for growth inhibition of human carcinoma cells

The c-erbB-2 gene encodes a M(r) 185,000 tyrosine kinase receptor (p185) with extensive homology to the epidermal growth factor receptor. We have conducted mechanistic studies with several anti-p185 monoclonal antibodies (TAb 250, -255, -257, -260, and -263) directed against the extracellular domain of p185 utilizing the SKBR-3, BT-474, and SKOV-3 cancer cell lines. Several of these antibodies exhibited ligand-mimicking properties: they induced tyrosine phosphorylation of p185; increased the catalytic activity of the receptor substrate phospholipase C-gamma 1; exhibited time- and pH-dependent internalization; induced receptor down-regulation; and increased the turnover of the p185 protein delta 3-fold. However, there was not a universal correlation between the antibody-mediated ligand-like effects and growth inhibition. TAb 250 inhibited BT-474 cells but did not alter p185 phosphotyrosine content or increase receptor turnover in these cells. TAb 260 increased p185 protein turnover but did not affect proliferation of the SKOV-3 cell line. Furthermore, blockade of TAb 250-induced receptor phosphorylation with the tyrosine kinase inhibitor tyrphostin 50864-2 did not abrogate TAb 250-mediated growth inhibition of SKBR-3 cells. These data suggest that ligand-like effects mediated by p185 antibodies are not critical for the growth inhibition of c-erbB-2-overexpressing carcinoma cells.     

TCR activation inhibits chemotaxis toward stromal cell-derived factor-1: evidence for reciprocal regulation between CXCR4 and the TCR

Stromal cell-derived factor-1 (SDF-1), a C-X-C family chemokine, is a potent T lymphocyte chemoattractant. We investigated the effects of T cell activation on the chemotactic response to SDF-1. Anti-CD3 Ab stimulation of either Jurkat T cells or murine peripheral CD4+ T lymphocytes produced a dramatic inhibition of SDF-1-induced chemotaxis. In contrast, the SDF-1 responses of Jurkat clones with deficiencies in key TCR signaling components (Lck, CD45, and TCR-beta), were only marginally reduced by anti-CD3 stimulation. Similar to PMA treatment, which abolished both CXCR4 receptor expression and the chemotactic response of Jurkat cells to SDF-1, anti-CD3 Ab treatment reduced cell surface expression of CXCR4 to 65% of the control value, an effect that was blocked by protein kinase C inhibitors. Our data suggest that initial T cell activation events inhibit the response of Jurkat T cells to CXCR4 stimulation. In contrast, SDF-1 treatment resulted in a reduction of tyrosine phosphorylation of the TCR downstream effectors, ZAP-70, SLP-76, and LAT (linker for activation of T cells), suggesting that this chemokine potentially regulates the threshold for T cell activation.