Regulation of cyclooxygenases by protein kinase C. Evidence against the importance of direct enzyme phosphorylation.
Cyclooxygenases (COXs) are key prostaglandin biosynthetic enzymes. While COX-1 expression is largely constitutive, COX-2 is highly regulated by cytokines, growth factors, and tumor promoters, such as the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA). While phosphorylation of transcription factors may regulate COX transcription, the existence of PKC consensus sequences suggests that direct enzyme phosphorylation might also regulate differential expression of the enzymes. Nevertheless, phosphorylation of both human recombinant COX-1 and COX-2 by rat brain PKC in vitro was minimal, as was phosphorylation of peptides based on PKC consensus sequences in COX-1 (less than 4% of the phosphorylation of the PKC-alpha pseudosubstrate peptide). Similarly, phosphorylation of the corresponding COX-2 peptides was not observed using either the phosphocellulose paper absorption method or electrospray mass spectrometry. MEG-01 and NIH 3T3 cells were labeled with [32P]orthophosphate to investigate COX phosphorylation in vivo. COX-2 synthesis was induced by PMA (100 nM) or serum stimulation in NIH 3T3 cells. COX-1 was expressed constitutively in MEG-01 cells. Specific polyclonal antibodies raised against sequences of human COX-1 (Ala24-Cys35) and COX-2 (Asn580-Lys598) were used for immunoprecipitation. Neither COX-1 nor COX-2 was phosphorylated in vivo, irrespective of the presence of a phosphatase inhibitor (1 microM okadaic acid). Although COX-1 and COX-2 are differentially regulated, no differences were observed in terms of susceptibility to phosphorylation by PKC either 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.
Vezza
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Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. garret@spirit.gcrc.upenn.edu
Habib
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Li
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Lawson
J A
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FitzGerald
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D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D011451:Prostaglandin-Endoperoxide Synthases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate