Protein-tyrosine kinase and protein-tyrosine phosphatase (PTPase) activities are essential for T-cell antigen receptor-mediated signalng. To assess the functional consequences of alteration of the levels of tyrosine phosphorylation in normal human T cells, the effects of vanadate and hydrogen peroxide were studied. In combination, these agents induced tyrosine phosphorylation of cellular substrates, elevated cytosolic free calcium, and induced interleukin 2 receptor (IL-2R) a chain expression but not IL-2 secretion. However, anti-CD28 antibody in combination with vanadate and hydrogen peroxide induced IL-2 secretion, consistent with the requirement for a costimulatory signal in the induction of this gene. The effects of vanadate and hydrogen peroxide were enhanced in the absence of the T-cell PTPase, CD45. Thus, acute pharmacologic manipulation of the level of tyrosine phosphorylation in normal T cells correlates with partial, but not full, activation of these cells; in concert with a costimulatory signal provided by perturbation of the CD28 molecule, the complete program of activation is initiated. These agents should prove useful in dissecting signaling pathways involved in the regulation of genes critical to the immune response.In contrast to the growth factor class of receptors that display intrinsic protein-tyrosine kinase (PTK) activity, many immunologically relevant, oligomeric receptors including the T-cell antigen receptor (TCR) and Fc receptors lack intrinsic kinase activity. Nonetheless, stimulation of these receptors results in protein-tyrosine phosphorylation of cellular substrates (reviewed in refs. 1-10), and pharmacologic inhibition of tyrosine kinases prevents both the early biochemical and late functional events that occur after receptor stimulation (11)(12)(13)(14). Thus, PTK activity appears to be necessary for T-cell activation, but is PTK function alone sufficient to initiate any or all of the events involved in this process? Expression of PTKs in T-cell lines can enhance TCRmediated interleukin 2 (IL-2) induction or in the case of v-src can induce constitutive but suboptimal IL-2 secretion (15)(16)(17). However, the limitation oftransfection studies is that the consequences of acute alteration of the levels of tyrosine phosphorylation in normal T cells cannot be addressed.Vanadate is a well-documented inhibitor of proteintyrosine phosphatases (PlPases), but its effects on intact cells are variable (18)(19)(20). By contrast, the combination of vanadate and H202 generates the compound pervanadate, the efficacy of which has been demonstrated to be far greater than that of vanadate (21)(22)(23) (24). Two-color fluorescence measurements were performed on a FACScan IV flow cytometer (Becton Dickinson) to assess purity. The murine thymoma cell line, BW5147.3, and its CD45-counterpart were grown in RPMI 1640 medium supplemented with glutamine, antibiotics, and 10% fetal bovine serum.Anti-phosphotyrosine [anti-Tyr(P)l Immunobtig. Stock solutions of H202 and sodium orthovanadate, Na3VO4, wer...

Ligand binding to the T-cell antigen receptor results in phosphatidylinositol hydrolysis and the resultant activation of protein kinase C, as well as the activation of a receptor-coupled protein-tyrosine kinase. As a model for tyrosine kinase activation in T cells, we used retroviral gene transfer to express the v-src oncogene in an antigen-specific murine T-cell hybridoma. Clones that expressed v-src mRNA demonstrated constitutive tyrosine phosphorylation of several cellular substrates, including the {chain of the T-cell receptor, and constitutive interleukin 2 production. Thus, expression of a constitutively active protein-tyrosine kinase such as pp6Ovsrc appears to be sufficient to induce the expression of at least one gene critical to the process of T-cell activation.The biochemical events that occur following perturbation of the T-cell antigen receptor (TCR) include the activation of protein kinase C (PKC) (reviewed in ref.