ABSTRACT
Our previous work demonstrated that 12(S)-HETE, a lipoxygenase metabolite of arachidonic acid, promoted B16 amelanotic melanoma (B16a) cell spreading on fibronectin. In the current study, we investigated the biochemical mechanisms of the 12(S)-HETE induced response. 12(S)-HETE treatment resulted in a time-dependent increase in B16a cell spreading on fibronectin, which was blocked by either calphostin C or by genistein but not by H8. Two hours following cell plating, both spontaneous and 12(S)-HETE promoted cell spreading reached their maximum (nearly 100%). Spontaneous cell spreading was inhibited by the select 12-lipoxygenase inhibitor, BHPP, whose inhibitory effect could be overcome by increasing doses of exogenous 12(S)-HETE. 12(S)-HETE-treated B16a cells plated on either fibronectin or cultured on their own extracellular matrix demonstrated increased vinculin and tyrosine-phosphorylated proteins, which were colocalized at focal adhesions. The increase in vinculin localization to focal adhesions appeared to be a post-transcriptional process, since 12(S)-HETE treatment did not alter the overall protein level of vinculin in tumor cells, but resulted in a specific enrichment of vinculin to focal adhesions. Pretreatment of B16a cells with either calphostin C or genistein abolished 12(S)-HETE-increased formation of vinculin- and phosphotyrosine-containing focal adhesions. Immunoblotting using antiphosphotyrosine antibody 4G10 demonstrated, following 12(S)-HETE stimulation, an increased tyrosine phosphorylation of several proteins in focal adhesions; most prominently, a approximately 155 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 stimulation, of proteins migrating at 120, 76, and 42/44 kd, of which the 120 kd protein co-migrated with pp125FAK. Immunoprecipitation with anti-FAK antibody BC-3 followed by immunoblotting with anti-phosphotyrosine antibody RC20H demonstrated a time-dependent hyperphosphorylation of pp125FAK. The present study suggests that 12(S)-HETE promoted melanoma cell spreading on fibronectin involves tyrosine phosphorylation of pp125FAK and protein kinase C- and tyrosine kinase-dependent focal adhesion formation.
