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J. Biol. Chem., Vol. 281, Issue 47, 35770-35778, November 24, 2006

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The 19-amino Acid Cassette of Cyclooxygenase-2 Mediates Entry of the Protein into the Endoplasmic Reticulum-associated Degradation System^*

Uri R. Mbonye, Masayuki Wada, Caroline J. Rieke, Hui-Yuan Tang, David L. DeWitt, and William L. Smith¹

From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824 and the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109

Cyclooxygenase (COX) isoforms catalyze the committed step in prostaglandin biosynthesis. The primary structures of COX-1 and COX-2 are very similar except that COX-2 has a 19-amino acid (19-AA) segment of unknown function located just inside its C terminus. Here we provide evidence that the major role of the 19-AA cassette is to mediate entry of COX-2 into the ER-associated degradation system that transports ER proteins to the cytoplasm. COX-1 is constitutively expressed in many cells, whereas COX-2 is usually expressed inducibly and transiently. In murine NIH/3T3 fibroblasts, we find that COX-2 protein is degraded with a half-life (t) of about 2 h, whereas COX-1 is reasonably stable (t >12 h); COX-2 degradation is retarded by 26 S proteasome inhibitors. Similarly, COX-1 expressed heterologously in HEK293 cells is quite stable (t > 24 h), whereas COX-2 expressed heterologously is degraded with a t of 5h, and its degradation is slowed by proteasome inhibitors. A deletion mutant of COX-2 was prepared lacking 18 residues of the 19-AA cassette. This mutant retains native COX-2 activity but, unlike native COX-2, is stable in HEK293 cells. Conversely, inserting the COX-2 19-AA cassette near the C terminus of COX-1 yields a mutant ins594–612 COX-1 that is unstable (t 3 h). Mutation of Asn-594, an N-glycosylation site at the beginning of the 19-AA cassette, stabilizes both COX-2 and ins594–612 COX-1; nonetheless, COX mutants that are glycosylated at Asn-594 but lack the remainder of the 19-amino acid cassette (i.e. del597–612 COX-2 and ins594–596 COX-1) are stable. Thus, although glycosylation of Asn-594 is necessary for COX-2 degradation, at least part of the remainder of the 19-AA insert is also required. Finally, kifunensine, a mannosidase inhibitor that can block entry of ER proteins into the ER-associated degradation system, retards COX-2 degradation.

Received for publication, August 30, 2006

^* This work was supported by National Institutes of Health Grant GM68848. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biological Chemistry, University of Michigan Medical School, 5416 Medical Science Bldg. I, 1301 E. Catherine St., Ann Arbor, MI 48109-0606. Tel.: 734-647-6180; Fax: 734-764-3509; E-mail: smithww{at}umich.edu.

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