The binding ofantigen to the multicomponent T-cell receptor (TCR) activates several signal transduction pathways via coupling mechanisms that are poorly understood. One event that follows antigen receptor engagement is the activation of inositol phospholipid-specific phospholipase C (PLC). TCR activation by antigen, lectins, or anti-TCR monoclonal antibody has also been showu to cause increases in tyrosine phosphorylation of TCR-C and other substrates, suggesting stimulation of protein tyrosine kinase (PTK) activity. A critical question is whether these two pathways, PLC and PTK, are independently activated or whether one initiates and/or regulates the other. In the former case, PLC activation could be coupled to the TCR via a GTP-binding protein (G protein).We have reported, however, that tyrosine phosphorylation of intracellular substrates precedes detection of PLC activation and intracellular calcium elevation, suggesting that inositol phospholipid turnover in T cells is initiated by a PTIK pathway.In this study, we test this hypothesis by treating T cells with the drug herbimycin A. We demonstrate that this agent inhibits substrate tyrosine phosphorylation, TCR-mediated inositol phospholipid hydrolysis, and calcium elevation. In contrast, under these conditions G-protein-mediated PLC activity, as tested by addition of aluminum fluoride, remains intact. Furthermore, whereas herbimycin treatment prevents TCRmediated interleukin 2 production and interleukin 2 receptor expression, phorbol ester-induced effects are substantially resistant to herbimycin. The drug thus appears to abrogate TCR-mediated signaling without affecting distal signaling mechanisms.Triggering of the T-cell receptor (TCR) for antigen activates multiple biochemical pathways. One event that follows antigen receptor engagement is the activation of inositol phospholipid-specific phospholipase C (PLC) (reviewed in refs. 1 and 2) with the generation of the second messengers diacylglycerol and inositol 1,4,5-trisphosphate (3). Treatment of T cells with phorbol ester and calcium ionophore, agents whose effects mimic these second messengers, has been shown to reproduce many features of TCR stimulation, leading to the conclusion that the signal transduction pathway mediated by PLC is important for T-cell activation.TCR stimulation has also been shown to cause increases in the tyrosine phosphorylation of several substrates in both murine and human T cells (4-8). Immunoblots using specific anti-phosphotyrosine antibodies have revealed increased tyrosine phosphorylation on multiple proteins, including those of 145, 135, 100, 75, and 40 kDa after ligation of the human TCR with anti-CD3 monoclonal antibody. In a previous report, we demonstrated that tyrosine phosphorylation of these proteins is rapid, with the earliest increase in phosphorylation detectable at 5 sec and with maximal stimulation by 90 sec (7).Herbimycin A is a benzoquinonoid ansamycin antibiotic that was found to reverse oncogenic transformation induced by pp6v-src (9,10). Subsequen...

StlmmaryCD28 is an adhesion receptor expressed as a 44-kD dimer on the surface of a major subset of human T cells. The CD28 receptor regulates the production of multiple lymphokines, including interleukin 2 (Ib2), by activation of a signal transduction pathway that is poorly understood. Here we show that ligation of CD28 by a monoclonal antibody (mAb) or by a natural ligand, B7/BB1, induces protein tyrosine phosphorylation that is distinct from T cell receptor (TCR)-induced tyrosine phosphorylation. CD28-induced protein tyrosine phosphorylation was greatly enhanced in cells that had been preactivated by ligation of the TCR, or by pretreatment with phorbol esters. Rapid and prolonged tyrosine phosphorylation of a single substrate, ppl00, was induced in T cells after interaction with B7/BB1 presented on transfected Chinese hamster ovary (CHO) cells. Anti-B7 mAb inhibited B7/BB1 receptor-induced tyrosine phosphorylation, indicating that B7-CD28 interaction was required. CD28-induced tyrosine phosphorylation was independent of the TCR because it occurred in a variant of the Jurkat T cell line that does not express the TCR. Herbimycin A, a protein tyrosine kinase inhibitor, could prevent CD28-induced tyrosine phosphorylation and CD28-induced I1.-2 production in normal T cells. The simultaneous crosslinking of CD28 and CD45, a tyrosine phosphatase, could prevent tyrosine phosphorylation of ppl00. These results suggest that specific tyrosine phosphorylation, particularly of ppl00, occurs directly as a result ofCD28 ligand binding and is involved in transducing the signal delivered through CD28 by accessory cells that express the B7/BB1 receptor. Thus, this particular form of signal transduction may be relevant to lymphokine production and, potentially may provide a means to study the induction of self-tolerance, given the putative role of the costimulatory signal in the induction of T cell activation or anergy.

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