MELANOMA CELL SPREADING ON FIBRONECTIN INDUCED BY 12(S)-HETE INVOLVESBOTH PROTEIN-KINASE-C-DEPENDENT AND PROTEIN-TYROSINE-KINASE-DEPENDENTFOCAL ADHESION FORMATION AND TYROSINE PHOSPHORYLATION OF FOCAL ADHESION KINASE (PP125(FAK))

Our previous work demonstrated that 12(S)-HETE, a lipoxygenase metabol ite of arachidonic acid, promoted B16 amelanotic melanoma (B16a) cell spreading on fibronectin. In the current study, we investigated the bi ochemical mechanisms of the 12(S)-HETE induced response. 12(S)-HETE tr eatment resulted in a time-dependent increase in B16a cell spreading o n fibronectin, which was blocked by either calphostin C or by genistei n but not by H8. Two hours following cell plating, both spontaneous an d 12(S)-HETE promoted cell spreading reached their maximum (nearly 100 %). Spontaneous cell spreading was inhibited by the select 12-lipoxyge nase inhibitor, BHPP, whose inhibitory effect could be overcome by inc reasing doses of exogenous 12(S)-HETE. 12(S)-HETE-treated B16a cells p lated on either fibronectin or cultured on their own extracellular mat rix demonstrated increased vinculin and tyrosine-phosphorylated protei ns, which were colocalized at focal adhesions. The increase in vinculi n localization to focal adhesions appeared to be a post-transcriptiona l process, since 12(S)-HETE treatment did not alter the overall protei n level of vinculin in tumor cells, but resulted in a specific enrichm ent of vinculin to focal adhesions. Pretreatment of B16a cells with ei ther calphostin C or genistein abolished 12(S)-HETE-increased formatio n of vinculin-and phosphotyrosine-containing focal adhesions. Immunobl otting using anti-phosphotyrosine ne anti body 4G10 demonstrated, foll owing 12(S)-HETE stimulation, an increased tyrosine phosphorylation of several proteins in focal adhesions; most prominently, a similar to 1 55 kd protein, a 120-130 kd protein cluster, a 76 kd protein, and a 42 /44 kd complex. Immunoprecipitation with anti-phosphotyrosine antibody PY20 revealed increased tyrosine phosphorylation, post 12(S)-HETE sti mulation, of proteins migrating at 120, 76, and 42/44 kd, of which the 120 kd protein co-migrated with pp125(FAK) .Immunoprecipitation with anti-FAK antibody BC-3 followed by immunoblotting with anti-phosphotyr osine antibody RC20H demonstrated a time-dependent hyperphosphorylatio n of pp125(FAK). Th, present study suggests that 12(S)-HETE promoted m elanoma cell spreading on fibronectin involves tyrosine phosphorylatio n of pp125(FAK) and protein kinase C- and tyrosine kinase-dependent fo cal adhesion formation. (C) 1995 Wiley-Liss, Inc.