Two distinct pathways for cyclooxygenase-2 protein degradation.
Cyclooxygenases (COX-1 and COX-2) are N-glycosylated, endoplasmic reticulum-resident, integral membrane proteins that catalyze the committed step in prostanoid synthesis. COX-1 is constitutively expressed in many types of cells, whereas COX-2 is usually expressed inducibly and transiently. The control of COX-2 protein expression occurs at several levels, and overexpression of COX-2 is associated with pathologies such as colon cancer. Here we have investigated COX-2 protein degradation and demonstrate that it can occur through two independent pathways. One pathway is initiated by post-translational N-glycosylation at Asn-594. The N-glycosyl group is then processed, and the protein is translocated to the cytoplasm, where it undergoes proteasomal degradation. We provide evidence from site-directed mutagenesis that a 27-amino acid instability motif (27-IM) regulates posttranslational N-glycosylation of Asn-594. This motif begins with Glu-586 8 residues upstream of the N-glycosylation site and ends with Lys-612 near the C terminus at Leu-618. Key elements of the 27-IM include a helix involving residues Glu-586 to Ser-596 with Asn-594 near the end of this helix and residues Leu-610 and Leu-611, which are located in an apparently unstructured downstream region of the 27-IM. The last 16 residues of the 27-IM, including Leu-610 and Leu-611, appear to promote N-glycosylation of Asn-594 perhaps by causing this residue to become exposed to appropriate glycosyl transferases. A second pathway for COX-2 protein degradation is initiated by substrate-dependent suicide inactivation. Suicide-inactivated protein is then degraded. The biochemical steps have not been resolved, but substrate-dependent degradation is not inhibited by proteasome inhibitors or inhibitors of lysosomal proteases. The pathway involving the 27-IM occurs at a constant rate, whereas degradation through the substrate-dependent process is coupled to the rate of substrate turnover.
Mbonye
Uri R
UR
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Yuan
Chong
C
Harris
Clair E
CE
Sidhu
Ranjinder S
RS
Song
Inseok
I
Arakawa
Toshiya
T
Smith
William L
WL
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D051545:Cyclooxygenase 1 / Q000235:genetics / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D016415:Sequence Alignment; D012756:Sheep; D015398:Signal Transduction; D013379:Substrate Specificity
D016861:Cyclooxygenase Inhibitors; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human