12(S)-HYDROXYEICOSATETRAENOIC ACID AND 13(S)-HYDROXYOCTADECADIENOIC ACID REGULATION OF PROTEIN-KINASE C-ALPHA IN MELANOMA-CELLS - ROLE OF RECEPTOR-MEDIATED HYDROLYSIS OF INOSITOL PHOSPHOLIPIDS

Protein kinase C (PKC) isoenzymes are essential components of cell sig naling. In this study, we investigated the regulation of PKC-alpha in murine B16 amelanotic melanoma (B16a) cells by the monohydroxy fatty a cids 12(S)-hydroxyeidosatetraenoic acid [12(S)-HETE] and 13(S)-hydroxy octadecadienoic acid [13(S)-HODE]. 12(S)-HETE induced a translocation of PKC-alpha to the plasma membrane and focal adhesion plaques, leadin g to enhanced adhesion of B16a cells to the matrix protein fibronectin . However, 13(S)-HODE inhibited these 12(S)-HETE effects on PKC-alpha. A receptor-mediated mechanism of action for 12(S)-HETE and 13(S)HODE is supported by the following findings. First, 12(S)HETE triggered a r apid increase in cellular levels of diacylglycerol and inositol trisph osphate in B16a cells. 13(S)-HODE blocked the 12(S)-HETE-induced burst s of both second messengers. Second, the 12(5)-HETE-increased adhesion of B16a cells to fibronectin was sensitive to inhibition by a phospho lipase C inhibitor and pertussis toxin. Finally, a high-affinity bindi ng site (K-d = 1 nM) for 12(S)-HETE was detected in B16a cells, and bi nding of 12(S)-HETE to B16a cells was effectively inhibited by 13(S)-H ODE (IC50 = 4 nM). In summary, our data provide evidence that regulati on of PKC-alpha by 12(S)-HETE and 13(S)-HODE may be through a guanine nucleotide-binding protein-linked receptor-mediated hydrolysis of inos itol phospholipids.