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

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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) band the stimulatory guanine nucleotide analogue guanosine 5''-[.gamma.-thio]triphosphate (GTP[S]). Application of a pseudo-substrate inhibitor of PKC indicated that CD3.gamma.-chain phopshorylation induced in response to all three agonists was mediated by PKC. PHA and GTP[S] also stimulated inositol phosholipid turnover and inositol phosphate accumulation. The kinetic and concentration-dependence of PHA-induced inositol phosholipid hydrolysis correlated with PHA-induced CD3.gamma. phosphorylation, usggesting that PHA may regulate CD3.gamma. phopsphorylation via diacylglycerol produced as a conseuqence of inositol phospholipid hydrolysis. However, there was an inconsistency in that PHA induced greater (< 200%) levels of inositol phospholipid turnover than did GTP[S] concentration for inositol phoshate production, but only 10% of maximal CD3.gamma.-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 lymphocytes.