Evidence for site-specific proteolysis of substrate-activated cyclooxygenase-2

Cyclooxygenases (COXs) are the rate limiting enzymes in the production of prostaglandins, soluble lipid mediators of many physiological functions. Recent evidence from our lab shows that exposure of COX-2 to its main substrate arachidonic acid (AA), causes the appearance of lower molecular weight COX-2 immunoreactive bands, suggesting that it undergoes site-specific proteolysis. Given the role that COX-2 plays in tumorigenesis, our goal was to identify the proteolytic products of COX-2 following its activation. For this, we used a system of RAW 267.4 cells where COX-2 levels were induced by overnight treatment with LPS, followed by subsequent treatment with AA. COX-2 was immunoprecipitated, samples were separated by SDS-PAGE and subjected to in-gel trypsin digestion followed by liquid chromatography coupled to mass spectrometry (MS) analysis. We were able to identify 22 putative cleavage sites. Of those, seven appeared in all three biological repeats, all within the catalytic domain of COX-2 (positions 155, 158, 172,177,187, 462, 564). Alignment of the mouse COX-2 with the human protein showed four peptides that are common to both. The presence of two of these peptides, indicating cleavage at positions 158 and 177 (Cosmic). Together, these data suggest that following its activation COX-2 undergoes site-specific proteolysis. The role of these fragments in cellular function is under investigation.