ROLE OF GUANINE-NUCLEOTIDE-BINDING PROTEIN AND TYROSINE KINASE IN PLATELET-ACTIVATING-FACTOR ACTIVATION OF PHOSPHOLIPASE-C IN A431 CELLS - PROPOSAL FOR DUAL MECHANISMS

A431 cells, a human epidermoid carcinoma, possess specific [H-3]platel et-activating factor (PAF) and [H-3]WEB 2086 binding sites indicating the presence of PAF receptors. PAF-stimulated PLC as determined by the increase in inositol phosphate levels. Pretreatment of A431 cells wit h genistein, a putative tyrosine kinase inhibitor, abolished the abili ty of PAF to activate PLC, whereas pretreatment with staurosporine, a protein kinase C inhibitor, potentiated the ability of PAF to activate PLC. Pretreatment of A431 cells with phorbol-12-myristate-1 3-acetate , a protein kinase C activator, blocked PAF-stimulated PLC. Overnight exposure of cells to pertussis toxin (PT) partially blocked the abilit y of PAF to stimulate PLC. Based on these observations the involvement of PT-sensitive and -insensitive guanine nucleotide-binding protein(s ) (G-protein) as well as the role of tyrosine kinase in the activation of PLC by PAF was considered further. PT treatment of A431 cell membr anes obliterated PAF-stimulated GTPase and indicated that PT-insensiti ve membrane-associated G-proteins were not involved in PAF actions. In alpha-toxin permeabilized cells, PT blocked GTP-gamma-S potentiation of PLC activation by PAF, thus suggesting that PT-insensitive G-protei ns were not involved in PAF activation of PLC in A431 cells. PAF stimu lated tyrosine kinase activity as observed with the increase in radioa ctivity associated with proteins immunoprecipitated with polyclonal an tibodies to phosphotyrosine residues. This increase was blocked by PAF receptor antagonists, CV 6209 and TCV 309, and by pretreatment with g enistein. PAF also activated the phosphorylation of pp60c-src and Src associated proteins in A431 cells. It is concluded that a dual mechani sm for the activation of PLC by the PAF receptor operates. One mechani sm functions through a membrane-associated PT-sensitive G-protein, whe reas the other pathway is independent of G-protein mediation. Interest ingly, tyrosine kinase activation is common for both mechanisms.