REGULATION OF CYCLOOXYGENASES BY PROTEIN-KINASE-C - EVIDENCE AGAINST THE IMPORTANCE OF DIRECT ENZYME PHOSPHORYLATION

Cyclooxygenases (COXs) are key prostaglandin biosynthetic enzymes. Whi le COX-1 expression is largely constitutive, COX-2 is highly regulated by cytokines, growth factors, and tumor promoters, such as the protei n kinase C (PKC) activator, phorbol la-myristate 13-acetate (PMA). Whi le phosphorylation of transcription factors may regulate COX transcrip tion, the existence of PKC consensus sequences suggests that direct en zyme phosphorylation might also regulate differential expression of th e enzymes, Nevertheless, phosphorylation of both human recombinant COX -1 and COX-2 by rat brain PKC in vitro was minimal, as was phosphoryla tion of peptides based on PKC consensus sequences in COX-1 (less than 4% of the phosphorylation of the PKC-alpha pseudosubstrate peptide), S imilarly, phosphorylation of the corresponding COX-2 peptides was not observed using either the phosphocellulose paper absorption method or electrospray mass spectrometry, MEG-01 and NTH 3T3 cells were labeled with [P-32]orthophosphate to investigate COX phosphorylation in vivo. COX-2 synthesis was induced by PMA (100 nM) or serum stimulation in NI H 3T3 cells, COX-1 was expressed constitutively in MEG-01 cells. Speci fic poly clonal antibodies raised against sequences of human COX-1 (Al a(24)-Cys(35)) and COX-2 (Asn(588)-Lys(598)) were used for immunopreci pitation, Neither COX-1 nor COX-2 was phosphorylated in vice, irrespec tive of the presence of a phosphatase inhibitor (1 mu M okadaic acid), Although COX-1 and COX-2 are differentially regulated, no differences were observed in terms of susceptibility to phosphorylation by PKC ei ther in vitro or in vivo. Despite regulated expression of COX-2 by PMA and the existence of consensus sequences for PKC phosphorylation, it appears that it is an unfavorable substrate for this enzyme.