Prostaglandin receptor EP 1 -mediated differential degradation of cyclooxygenases involves a specific lysine residue Almog Spector-Chotiner a , Niva Shraga-Heled a , Rapita Sood a , Gilad Rimon b , Liza Barki-Harrington a,⇑ a Department of Human Biology, Faculty of Natural Sciences, University of Haifa, Mt. Carmel, Haifa 31905, Israel b Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Be’er-Sheva 84105, Israel article info Article history: Received 26 November 2013 Available online 12 December 2013 Keywords: Cyclooxygenase COX-2 COX-1 EP 1 Ubiquitination Degradation abstract The cyclooxygenase (COX) enzyme isoforms COX-1 and COX-2 catalyze the main step in the generation of prostanoids that mediate major physiological functions. Whereas COX-1 is a ubiquitously expressed sta- ble protein, COX-2 is transiently upregulated in many pathologies and is often associated with a poor prognostic outcome. We have recently shown that an interaction of COX-2 with the prostaglandin EP 1 receptor accelerates its degradation via a mechanism that augments its level of ubiquitination. Here we show that the sensitivity of both COX-1 and COX-2 to EP 1 is altered upon modification of one lysine residue. A point mutation of lysine to-arginine in position 432 of COX-2 (K432R) yields an enzyme with decreased sensitivity to EP 1 -mediated degradation. In contrast, insertion of a putative ubiquitination site into the corresponding position of COX-1 (H446K 0 ) yields an enzyme with higher levels of ubiquitination and reduced expression. Furthermore, compared to wild type COX-1, H446K 0 is significantly more sensi- tive to downregulation by EP 1 . Together these data suggest that distinctive ubiquitination of COX-1 and COX-2 may be responsible for their different sensitivity to EP 1 -mediated degradation. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction The cyclooxygenase (COX) enzyme isoforms COX-1 and COX-2 catalyze the rate-limiting step in the conversion of arachidonic acid to prostaglandins that play central roles in cardiovascular, immunological and brain function [1]. Both isoforms share a high degree of structural and catalytic similarities but differ profoundly in their stability [2]. Whereas COX-1 is a constitutively expressed and relatively stable protein (t 1/2 > 24 h), COX-2 is short-lived (t 1/ 2 2–5 h), and its expression is upregulated in many pathologies [3–6]. These differences have led to the formation of the COX-2 hypothesis whereby the ubiquitously expressed COX-1 is responsi- ble for homeostatic functions, while the inducible COX-2 plays a predominant role in pathophysiological conditions (reviewed in [1,7]). Evidences that accumulated over several years suggest that in addition to its catalytic function COX-2 also fulfills non-enzymatic roles in the cell, and therefore regulation of its levels may be as important as inhibiting enzymatic activity. For example, COX-2 was found to directly interact with the tumor suppressor p53 in the nucleus and thereby inhibit it’s activity through a mechanism that does not involve enzymatic activity [8]. Interestingly, while the signaling cascades that lead to the induction of COX-2 are well-characterized [2], the cellular pathways that lead to degrada- tion of COX-1 and COX-2 are partially characterized. Besides the differences in their stability the two isoforms differ in their place of degradation. In the absence of its principal fatty acid substrate arachidonic acid, the mature N-glycosylated COX-2 is shuttled directly from the ER via the ER-associated degradation (ERAD) pathway to the cytosol where it is subsequently degraded by the proteasome [9,10]. Degradation of COX-2 in this pathway is preceded by it polyubiquitination [10–12], but the identity of the specific lysine residues that undergo ubiquitination is not known. In contract to COX-2, under the same conditions COX-1 does not degrade in the proteasome and the location and mechanism of its degradation are not known. Specificity of protein degradation in the proteasome is deter- mined by a large family of ubiquitin-protein E3 ligases that recog- nize different motifs in the substrates. This recognition is modulated by molecular chaperones that render the substrate sus- ceptible for recognition by the ligases via modification or associa- tion with protein substrates [13]. Two proteins have recently emerged as possible molecular chaperons for COX-2 degradation. Caveolin-1 was shown to facilitate the ubiquitination and degrada- tion of COX-2 through an interaction with Derlin-1 [14,15], and we have recently shown that degradation of COX-2 is accelerated fol- lowing its interaction with the G-protein coupled receptor prosta- glandin E 1 (EP 1 ) [11]. We showed that the mechanism by which 0006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2013.12.038 ⇑ Corresponding author. Address: Department of Biology, Faculty of Natural Sciences, University of Haifa, Mt. Carmel, Haifa 31905, Israel. Fax: +972 4 8288763. E-mail address: lbarki@psy.haifa.ac.il (L. Barki-Harrington). Biochemical and Biophysical Research Communications 443 (2014) 738–742 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc