REGULATION OF LYSOPHOSPHATIDIC ACID-STIMULATED TYROSINE PHOSPHORYLATION OF MITOGEN-ACTIVATED PROTEIN-KINASE BY PROTEIN-KINASE-C-DEPENDENT AND PERTUSSIS-TOXIN-DEPENDENT PATHWAYS IN THE ENDOTHELIAL-CELL LINE EAHY-926

In the endothelial cell line EAhy 926, 1-oleoyl-lysophosphatidic acid (LPA) stimulated the tyrosine phosphorylation of the pp42 isoform of m itogen-activated protein (MAP) kinase. Maximum phosphorylation was obs erved within 5-min of LPA addition, but the response was sustained for up to 120 min. Re-addition of LPA after 60 min stimulated a further s ustained increase in the tyrosine phosphorylation of MAP kinase. In ce lls pretreated with phorbol 12-myristate 13-acetate (PMA; 24 h) or pre incubated with the protein kinase C inhibitor Ro-318220, LPA-induced t yrosine phosphorylation of pp42 MAP kinase was substantially reduced a t 2 min but potentiated at 60 min. Ro-318220 in combination with eithe r PMA or pertussis toxin pretreatment abolished the LPA response at al l time points, suggesting an involvement of protein kinase C in the pe rtussis toxin-sensitive part of the pathway. Agents which raised intra cellular cyclic AMP levels did not affect the initial phase of LPA-sti mulated MAP kinase activation, but abolished the late phase. However, this effect was prevented by Ro-318220, implicating a greater role for protein kinase C than protein kinase A in the regulation of sustained MAP kinase responses. LPA stimulated an increase In the tyrosine phos phorylation of focal adhesion kinase pp125 (pp125(FAK)) in EAhy 926 ce lls which was both protein kinase C- and pertussis toxin-independent. These results are discussed in terms of the pathways regulating both M AP kinase and pp125(FAK) in response to LPA in the EAhy 926 endothelia l cell line.