Inhibition of tyrosine phosphorylation prevents T-cell receptor-mediated signal transduction.

Abstract

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...

Herbimycin A blocks IL‐1‐induced NF‐κB DNA‐binding activity in lymphoid cell lines

“…Similarly to these results, a differential effect of herbimycin A has previously been reported for the T-cell receptor-and PMA-mediated IL-2 production and IL-2 receptor expression in human peripheral T cells [29].…”

Hsp90 Is Essential for the Synthesis and Subsequent Membrane Association, But Not the Maintenance, of the Src-Kinase p56lck

“…Previous studies have reported that prolonged (4 -16 h) treatment of cells with inhibitors of Hsp90 reduces the levels of Lck protein and kinase activity (June et al, 1990;Hartson et al, 1996). To determine whether Hsp90 activity is required for the synthesis of Lck or for maintenance of the mature protein, we examined the effect of Hsp90 inhibition on newly synthesized and mature Lck separately.…”

The Role of Protein Kinases in Antigen-activation of Peripheral Blood Mononuclear Cells of Schistosoma mansoni Infected Individuals

“…Engagement of the TCR with antigen or anti-TCR monoclonal antibody induces at least three rapid biochemical changes that are of probable significance in signal transduction: an increase in the level of free intracellular calcium (Imboden et al 1985), the activation of protein kinase C (Chen et al 1986, Nel et al 1986, and an increase in tyrosine phosphorylation (Campbell & Sefton 1990, June et al 1990, Lane et al 1990.…”

“…It is cur-rently accepted that the signal transduction cascade is initiated by activation of protein tyrosine kinases (PTK) (Samelson et al 1986, June et al 1990, Chan et al 1994, which results in the phosphorylation of a number of cellular proteins, such as phospholipase C-g1 (PLC) (Weiss et al 1991). Activated PLC, in turn, catalyzes the hydrolysis of membrane phosphoinositides to generate inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol, second messengers responsible for the mobilization of cytoplasmic free calcium (Ca 2+ ) and activation of protein kinase C (PKC), respectively (Imboden et al 1985).…”