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Two Distinct Pathways for Cyclooxygenase-2 Protein Degradation

2008; Elsevier BV; Volume: 283; Issue: 13 Linguagem: Inglês

10.1074/jbc.m710137200

1083-351X

Uri Mbonye, Chong Yuan, Clair Harris, Ranjinder S. Sidhu, In‐Seok Song, Toshiya Arakawa, William L. Smith,

Monoclonal and Polyclonal Antibodies Research

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. 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. Cyclooxygenases (COX-1 and COX-2) 2The abbreviations used are:COXcyclooxygenaseAAarachidonic acidKIFkifunensine19-aathe unique C-terminal 19-amino acid insert of COX-227-IM27-amino acid instability motifERendoplasmic reticulumERADER-associated degradationGERARDglycoprotein ERADLPSlipopolysaccharideCHXcycloheximideManmannoseHis6hexahistadine-taggedUPSTRM8the 8-amino acid segment immediately upstream of Asn-594 in COX-2huCOXhuman COXmuCOXmurine COXovCOXovine COXDMEMDulbecco's modified Eagle's mediumFBflurbiprofen. 2The abbreviations used are:COXcyclooxygenaseAAarachidonic acidKIFkifunensine19-aathe unique C-terminal 19-amino acid insert of COX-227-IM27-amino acid instability motifERendoplasmic reticulumERADER-associated degradationGERARDglycoprotein ERADLPSlipopolysaccharideCHXcycloheximideManmannoseHis6hexahistadine-taggedUPSTRM8the 8-amino acid segment immediately upstream of Asn-594 in COX-2huCOXhuman COXmuCOXmurine COXovCOXovine COXDMEMDulbecco's modified Eagle's mediumFBflurbiprofen. catalyze the committed step in prostanoid synthesis (1Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2444) Google Scholar, 2Rouzer C.A. Marnett L.J. Chem. Rev. 2003; 103: 2239-2304Crossref PubMed Scopus (203) Google Scholar, 3van der Donk W.A. Tsai A.L. Kulmacz R.J. Biochemistry. 2002; 41: 15451-15458Crossref PubMed Scopus (137) Google Scholar). These enzymes are multiply N-glycosylated ER-resident proteins that exist as homodimers and exhibit ∼60% primary structure identity (1Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2444) Google Scholar, 2Rouzer C.A. Marnett L.J. Chem. Rev. 2003; 103: 2239-2304Crossref PubMed Scopus (203) Google Scholar, 3van der Donk W.A. Tsai A.L. Kulmacz R.J. Biochemistry. 2002; 41: 15451-15458Crossref PubMed Scopus (137) Google Scholar, 4Xiao G. Chen W. Kulmacz R.J. J. Biol. Chem. 1998; 273: 6801-6811Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 5Yuan C. Rieke C.J. Rimon G. Wingerd B.A. Smith W.L. 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The mature forms of the COX isoforms are very similar in structure except that COX-2 has a unique 19-residue insertion (19-aa; residues Asn-594 to Lys-612) near its C terminus. cyclooxygenase arachidonic acid kifunensine the unique C-terminal 19-amino acid insert of COX-2 27-amino acid instability motif endoplasmic reticulum ER-associated degradation glycoprotein ERAD lipopolysaccharide cycloheximide mannose hexahistadine-tagged the 8-amino acid segment immediately upstream of Asn-594 in COX-2 human COX murine COX ovine COX Dulbecco's modified Eagle's medium flurbiprofen. cyclooxygenase arachidonic acid kifunensine the unique C-terminal 19-amino acid insert of COX-2 27-amino acid instability motif endoplasmic reticulum ER-associated degradation glycoprotein ERAD lipopolysaccharide cycloheximide mannose hexahistadine-tagged the 8-amino acid segment immediately upstream of Asn-594 in COX-2 human COX murine COX ovine COX Dulbecco's modified Eagle's medium flurbiprofen. The principal endogenous fatty acid substrate of COX-1 and COX-2 is arachidonic acid (AA), which is mobilized from the sn-2-position of membrane phospholipids upon the activation of phospholipase A2s through the actions of bradykinin, thrombin, growth factors, calcium ionophore (A23187), or cytokines (1Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2444) Google Scholar, 2Rouzer C.A. Marnett L.J. Chem. Rev. 2003; 103: 2239-2304Crossref PubMed Scopus (203) Google Scholar, 9Leslie C.C. Biochem. Cell Biol. 2004; 82: 1-17Crossref PubMed Scopus (107) Google Scholar, 10Lin L.L. Lin A.Y. DeWitt D.L. J. Biol. Chem. 1992; 267: 23451-23454Abstract Full Text PDF PubMed Google Scholar, 11Murakami M. Matsumoto R. Urade Y. Austen K.F. Arm J.P. J. Biol. Chem. 1995; 270: 3239-3246Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 12Miyakawa T. Kojima M. Ui M. Biochem. 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Prostaglandin endoperoxide H2 is a common substrate for downstream terminal prostanoid synthases that form various prostanoids. COX-1 is a stable protein that is constitutively expressed in resting cells of many tissues, notably in platelets and vesicular gland (1Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2444) Google Scholar, 18Mbonye U.R. Wada M. Rieke C.J. Tang H.Y. Dewitt D.L. Smith W.L. J. Biol. Chem. 2006; 281: 35770-35778Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 19Tanaka N. Sato T. Fujita H. Morita I. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 607-612Crossref PubMed Scopus (30) Google Scholar, 20Miyamoto T. Ogino N. Yamamoto S. Hayaishi O. J. Biol. Chem. 1976; 251: 2629-2636Abstract Full Text PDF PubMed Google Scholar). In contrast, COX-2 is a stimulus-inducible protein whose expression is short-lived in epithelial, endothelial, smooth muscle, and fibroblast cells (18Mbonye U.R. Wada M. Rieke C.J. 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At the 3′-untranslated region of COX-2 mRNA are multiple AUUUA elements that target mRNAs for rapid exonuclease cleavage (21Tanabe T. Tohnai N. Prostaglandins Other Lipid Mediat. 2002; 68: 95-114Crossref PubMed Scopus (369) Google Scholar, 26Chen C.Y. Shyu A.B. Mol. Cell Biol. 1994; 14: 8471-8482Crossref PubMed Scopus (224) Google Scholar). COX-1 mRNA lacks these 3′-untranslated region AU-rich elements, and COX-1 mRNA is stable in megakaryocytes and vascular endothelial cells (27Xu X.M. Tang J.L. Chen X. Wang L.H. Wu K.K. J. Biol. Chem. 1997; 272: 6943-6950Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 28Duquette M. Laneuville O. J. Biol. Chem. 2002; 277: 44631-44637Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 29Xu X.M. Tang J.L. Hajibeigi A. Loose-Mitchell D.S. Wu K.K. Am. J. Physiol. 1996; 270: C259-C264Crossref PubMed Google Scholar). We have recently reported that the C-terminal 19-aa of COX-2 causes the enzyme to undergo proteasomal degradation via the endoplasmic reticulum-associated degradation (ERAD) pathway (18Mbonye U.R. Wada M. Rieke C.J. Tang H.Y. Dewitt D.L. Smith W.L. J. Biol. Chem. 2006; 281: 35770-35778Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In the present study, we set out to examine what specific features of the C-terminal 19-aa, in addition to the Asn-594 glycosylation site, are critical for targeting the enzyme to the ERAD system. The role of the helical region upstream of the Asn-594 glycosylation site in regulating Asn-594 was also investigated. Our findings enabled us to identify a C-terminal 27-amino acid destabilizing motif (27-IM) of COX-2 that regulates the glycosylation of the enzyme at Asn-594 and controls, at least in part, the timing and extent of its degradation. In the course of our studies on COX-2 degradation, we discovered that N594A human COX (huCOX)-2, which lacks a functional 27-IM, was degraded rapidly when AA was added to cells expressing this mutant. This led us to examine the impact of COX catalysis on the protein stabilities of COX-1 and COX-2. We have found that the rate of COX-2 protein degradation is enhanced by AA in NIH/3T3 and HEK293 cells in a proteasome-independent manner. We provide evidence that substrate-dependent degradation of COX-2 requires a functional COX active site and proceeds from the substrate-induced inactive form(s) of the enzyme. Materials—Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), ponasterone A, and tetracycline were obtained from Invitrogen. Bovine calf serum was from Hyclone. Arachidonic acid, (R/S)-flurbiprofen, (S)-flurbiprofen, (R)-flurbiprofen, and NS-398 were from Cayman Chemicals. (S)-flurbiprofen and NS-398 are time-dependent, irreversible inhibitors of COX activity, whereas (R)-flurbiprofen is a competitive COX inhibitor. Cycloheximide, puromcyin, bacterial lipopolysaccharide (LPS), and GSH were purchased from Sigma. Calcium ionophore (A23187), bradykinin, and MG132 were purchased from Calbiochem. Endoglycosidase H was purchased from Roche Applied Science. [1-14C]AA (55 mCi/mmol) was from American Radiolabeled Chemicals. Microsomal PGES-1 was kindly provided by Dr. Michael Garavito (Michigan State University). Construction of Plasmids for Transfection—Recombinant ovine COX (ovCOX)-1 and huCOX-2 cDNA were subcloned into the tetracycline-inducible vector pcDNA5/FRT/TO (Invitrogen). After subcloning, the QuikChange™ site-directed mutagenesis kit (Stratagene) was used to create the following C-terminal mutants: N594A huCOX-2, del595-612 huCOX-2, del602-612 huCOX-2, del607-612 huCOX-2, P607A/T608A huCOX-2, V609A huCOX-2, L610A huCOX-2, L611A huCOX-2, K612A huCOX-2, V591P T592G huCOX-2, ovCOX-1 UPSTRM8 huCOX-2, murine COX (muCOX)-2 UPSTRM8 huCOX-2, ins594-612 ovCOX-1, ins594-612 (N594A) ovCOX-1, ins586-612 ovCOX-1, and ins586-612 (N594A) ovCOX-1. Recombinant ovCOX-1 was also subcloned into the ecdysone-inducible pIND vector (Invitrogen) and used to develop the following constructs: ins594-596 ovCOX-1, ins595-597 ovCOX-1, and ins594-601 ovCOX-1. The COX-2 mutants, scrambled insert 595-612 (Sins595-612) huCOX-2 and Sins597-612 huCOX-2, were created from the cDNA template for native huCOX-2 by overlap extension PCR and then subcloned into pcDNA5/FRT/TO using BamHI and XhoI sites. Correct cDNA orientation and mutations were confirmed by sequencing. Recombinant ovCOX-1 cDNA and N-terminal His6 ovCOX-1 cDNA were subcloned into pIND (Invitrogen). cDNAs for huCOX-2, ovCOX-1, and N-terminal His6 muCOX-2 were subcloned into pcDNA5/FRT/TO (Invitrogen). Cell Culture and Transfection—NIH/3T3 fibroblasts at early passage ( 24 h) than native huCOX-2 (data not shown). These results suggested that there is primary structure information within the C-terminal 16-amino acid segment of 19-aa, perhaps within the last six residues of the insertion, that is essential for mediating COX-2 protein degradation. To identify which of the last 6 amino acids of 19-aa were important for COX-2 degradation, we performed alanine scanning