Ufd1 phosphorylation at serine 229 negatively regulates endoplasmic reticulum-associated degradation by inhibiting the interaction of Ufd1 with VCP

• Published in: Biochemical journal Vol. 476; no. 18; p. 2561
• Main Authors: Nguyen, Quynh-Anh Thi, Choi, Juyong, Yang, Jin Kuk, Lee, Sang Yoon
• Format: Journal Article
• Language: English
• Published: England 20.09.2019
• Subjects: Amino Acid Motifs; Amino Acid Substitution; Cyclic AMP-Dependent Protein Kinases; Endoplasmic Reticulum-Associated Degradation; HEK293 Cells; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; Mutation, Missense; Phosphorylation - genetics; Serine - genetics; Serine - metabolism; Valosin Containing Protein - genetics; Valosin Containing Protein - metabolism; ubiquitin fusion degradation protein 1; valosin-containing protein; protein kinase A; phosphorylation; endoplasmic reticulum-associated degradation; SHP box
• ISSN: 1470-8728
• DOI: 10.1042/BCJ20190254

Misfolded proteins in the endoplasmic reticulum (ER) are removed through multistep processes termed ER-associated degradation (ERAD). Valosin-containing protein (VCP) plays a crucial role in ERAD as the interaction of ubiquitin fusion degradation protein 1 (Ufd1) with VCP via its SHP box motif ( F-S-G-S-G-N-R-L ) is required for ERAD. However, the mechanisms by which the VCP-Ufd1 interaction is regulated are not well understood. Here, we found that the serine 229 residue located in the Ufd1 SHP box is phosphorylated and by cyclic adenosine monophosphate-dependent protein kinase A (PKA), with this process being enhanced by either forskolin (an adenylyl cyclase activator) or calyculin A (a protein phosphatase inhibitor). Moreover, a phosphomimetic mutant (S229D) of Ufd1 as well as treatment by forskolin, calyculin A, or activated PKA strongly reduced Ufd1 binding affinity for VCP. Consistent with this, the Ufd1 S229D mutant significantly inhibited ERAD leading to the accumulation of ERAD substrates such as a tyrosinase mutant (C89R) and 3-hydroxy-3-methylglutaryl coenzyme A reductase. However, a non-phosphorylatable Ufd1 mutant (S229A) retained VCP-binding ability and was less effective in blocking ERAD. Collectively, our results support that Ufd1 S229 phosphorylation status mediated by PKA serves as a key regulatory point for the VCP-Ufd1 interaction and functional ERAD.