Guanine nucleotide regulation of inositol phospholipid hydrolysis and CD3-antigen phosphorylation in permeabilized T lymphocytes

Abstract

A method of membrane permeabilization of T lymphocytes with the bacterial cytotoxin streptolysin O has allowed the effect of guanine nucleotide analogues on phosphatidylinositol metabolism and protein kinase C (PKC) activation to be investigated. The data demonstrate that, in permeabilized cells, phosphorylation of the gamma subunit of the CD3 antigen can be induced in response to the PKC activator phorbol 12,13-dibutyrate, the polyclonal mitogen phytohaemagglutinin (PHA) and the stimulatory guanine nucleotide analogue guanosine 5′-[gamma-thio]triphosphate (GTP[S]). Application of a pseudo-substrate inhibitor of PKC indicated that CD3gamma-chain phosphorylation induced in response to all three agonists was mediated by PKC. PHA and GTP[S] also stimulated inositol phospholipid turnover and inositol phosphate accumulation. The kinetics and concentration-dependence of PHA-induced inositol phospholipid hydrolysis correlated with PHA-induced CD3gamma phosphorylation, suggesting that PHA may regulate CD3gamma phosphorylation via diacylglycerol produced as a consequence of inositol phospholipid hydrolysis. However, there was an inconsistency in that PHA induced greater (greater than 200%) levels of inositol phospholipid turnover than did GTP[S], but much weaker (less than 50%) levels of CD3-antigen phosphorylation. There was also a discrepancy between GTP[S] effects on phosphatidylinositol turnover and PKC activation, in that the half-maximal GTP[S] concentration for inositol phosphate production and CD3gamma-chain phosphorylation was 0.75 microM and 75 microM respectively. Moreover, 10 microM-GTP[S] induced maximal inositol phosphate production, but only 10% of maximal CD3gamma-chain phosphorylation. The data are consistent with the idea that other signal-transduction pathways, in addition to those involving inositol phosphate production, exist for the regulation of PKC in T lymphocytes.