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 [3H]platelet-activating factor (PAF) and [3H]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 with genistein, a putative tyrosine kinase inhibitor, abolished the ability 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-13-acetate, a protein kinase C activator, blocked PAF-stimulated PLC. Overnight exposure of cells to pertussis toxin (PT) partially blocked the ability 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 membranes obliterated PAF-stimulated GTPase and indicated that PT-insensitive 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-proteins were not involved in PAF activation of PLC in A431 cells. PAF stimulated tyrosine kinase activity as observed with the increase in radioactivity associated with proteins immunoprecipitated with polyclonal antibodies to phosphotyrosine residues. This increase was blocked by PAF receptor antagonists, CV 6209 and TCV 309, and by pretreatment with genistein. PAF also activated the phosphorylation of pp60c-src and Src associated proteins in A431 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Thurston
A W
AW
Department of Pharmacology, School of Medicine, University of Missouri-Columbia.
Rhee
S G
SG
Shukla
S D
SD
D002294:Carcinoma, Squamous Cell / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000032:analysis; D043562:Receptors, G-Protein-Coupled; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000187:drug effects* / Q000378:metabolism
D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; D004815:Epidermal Growth Factor; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D010738:Type C Phospholipases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins