Human Homologs of Ubc6p Ubiquitin-conjugating Enzyme and Phosphorylation of HsUbc6e in Response to Endoplasmic Reticulum Stress

Xinli Bai,Johanna M. Rommens,Ray S. Oh

doi:10.1074/jbc.m601843200

Xinli Bai, Johanna M. Rommens + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m601843200

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Abstract

Ubiquitin-conjugating enzyme Ubc6p is a tail-anchored protein that is localized to the cytoplasmic face of the endoplasmic reticulum (ER) membrane and has been implicated in the degradation of many misfolded membrane proteins in yeast. We have undertaken characterization studies of two human homologs, hsUbc6 and hsUbc6e. Both possess tail-anchored protein motifs, display high conservation in their catalytic domains, and are functional ubiquitin-conjugating enzymes as determined by in vitro thiol-ester assay. Both also display induction by the unfolded protein response, a feature of many ER-associated degradation (ERAD) components. Post-translational modification involving phosphorylation of hsUbc6e was observed to be ER-stress-related and dependent on signaling of the PRK-like ER kinase (PERK). The phosphorylation site was mapped to Ser-184, which resides within the uncharacterized region linking the highly conserved catalytic core and the C-terminal transmembrane domain. Phosphorylation of hsUbc6e also did not alter stability, subcellular localization, or interaction with a partner ubiquitin-protein isopeptide ligase. Assays of hsUbc6e(S184D) and hsUbc6e(S184E), which mimic the phosphorylated state, suggest that phosphorylation may reduce capacity for forming ubiquitin-enzyme thiol-esters. The occurrence of two distinct Ubc6p homologs in vertebrates, including one with phosphorylation modification in response to ER stress, emphasizes diversity in function between these Ub-conjugating enzymes during ERAD processes.

Highlights

Their participation in the quality control of non-membrane proteins has been suggested (30, 31). These homologs have yet to be characterized in detail, and it is unknown how they are distinguished functionally. We investigated these homologs and have identified a phosphorylation modification of hsUbc6e that occurs in response to endoplasmic reticulum (ER) stress
HsUbc6 and hsUbc6e Are Up-regulated with Distinct Responses during unfolded protein response (UPR)—Given their predicted role in the ER-associated degradation (ERAD) of misfolded proteins, we examined whether the Ubc6 homologs are regulated by the UPR in Panc-1 secretory cells
ER Stress-induced Phosphorylation of mmUbc6e Requires PRK-like ER kinase (PERK) Signaling—It was considered that hsUbc6e may be a target of PERK, in unstressed Panc-1 cells was detected as a single band; how- an ER transmembrane serine/threonine protein kinase that is ever, when protein expression was induced by tunicamycin activated during ER stress to inhibit global protein synthesis via treatment, the upper band appeared

Summary

Introduction

HsUbc6e Is Phosphorylated in Response to ER Stress—To characterize the human Ubc6 homologs, N-terminal c-myc epitope fusion plasmids were constructed for transient expression in HEK293 cells. ER Stress-induced Phosphorylation of mmUbc6e Requires PERK Signaling—It was considered that hsUbc6e may be a target of PERK, in unstressed Panc-1 cells was detected as a single band; how- an ER transmembrane serine/threonine protein kinase that is ever, when protein expression was induced by tunicamycin activated during ER stress to inhibit global protein synthesis via treatment, the upper band appeared.

Results

Conclusion