The 19-amino acid cassette of cyclooxygenase-2 mediates entry of the protein into the endoplasmic reticulum-associated degradation system.
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(1/2)) of about 2 h, whereas COX-1 is reasonably stable (t(1/2) > 12 h); COX-2 degradation is retarded by 26 S proteasome inhibitors. Similarly, COX-1 expressed heterologously in HEK293 cells is quite stable (t(1/2) > 24 h), whereas COX-2 expressed heterologously is degraded with a t(1/2) of approximately 5 h, 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(1/2) approximately 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.
Mbonye
Uri R
UR
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA.
Wada
Masayuki
M
Rieke
Caroline J
CJ
Tang
Hui-Yuan
HY
Dewitt
David L
DL
Smith
William L
WL
D000595:Amino Acid Sequence; D000818:Animals; D051546:Cyclooxygenase 2 / Q000737:chemistry* / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D041681:NIH 3T3 Cells; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D051546:Cyclooxygenase 2; D010100:Oxygen