Abstract
Constitutive photomorphogenic 1 (COP1) is the ubiquitin E3 ligase that mediates degradation of c-Jun protein upon Erk1/2 inactivation. It remains unknown how this protein degradation pathway is regulated. In this study, we investigated the roles of protein phosphatases, ubiquitin-conjugating E2 enzymes (UBE2), and an intrinsic motif of c-Jun in regulating this degradation pathway. By using pharmacological inhibitors and/or gene knockdown techniques, we identified protein phosphatase 1 (PP1) and PP2A as the phosphatases and UBE23d as the UBE2 promoting c-Jun degradation, triggered by Erk1/2 inactivation. In addition, we report that the C-terminus of c-Jun protein facilitates its degradation. The addition of a C-terminal tag or deletion of the last four amino acid residues from the C-terminus of c-Jun protects it from degradation under Erk1/2-inactivating conditions. Taken together, this study reveals that the Erk1/2 inactivation-triggered and COP1-mediated c-Jun degradation is extrinsically and intrinsically regulated, providing a new understanding of the mechanisms underlying this protein degradation pathway.
Highlights
Division of Biotechnology Review and Research II, Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, U.S Food and Drug Administration, Silver Spring, MD 20993, USA
Since the phosphorylation states are generally determined by the net balance between the activities of kinases and phosphatases [19], we hypothesized that phosphatase(s) may play a critical role in regulating the process leading to Constitutive photomorphogenic 1 (COP1) activation and degradation of its substrates upon MKK1/2–Erk1/2 inactivation
In contrast to experiments involving endogenous c-Jun, levels of ectopically over-expressed c-Jun incorporating a C-terminal tGFP tag were not modulated by treatment with the proteasome inhibitor, MG132, and/or the Erk1/2 activation inhibitor, lethal toxin (LT) (Figure 4A). These results indicate that c-Jun-tGFP is resistant to proteasome-mediated protein degradation via the Erk1/2 inactivation-induced and COP1-dependent degradation pathway
Summary
Division of Biotechnology Review and Research II, Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, U.S Food and Drug Administration, Silver Spring, MD 20993, USA. Constitutive photomorphogenic 1 (COP1) is the ubiquitin E3 ligase that mediates degradation of c-Jun protein upon Erk1/2 inactivation. It remains unknown how this protein degradation pathway is regulated. This study reveals that the Erk1/2 inactivation-triggered and COP1-mediated c-Jun degradation is extrinsically and intrinsically regulated, providing a new understanding of the mechanisms underlying this protein degradation pathway. COP1 was originally identified during the study of the COP/DET/FUS loci in plants, in which it was found to function as a ubiquitin E3 ligase that promotes the degradation of photoreceptors and photomorphogenic transcription factors such as HY5, HYH, LAF1, and HFR1, thereby repressing plant photomorphogenesis. As an E3 ligase, COP1 plays a major role in the protein ubiquitination process, which is a cascade catalyzed by three major enzyme components—a ubiquitin-activating enzyme
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