Abstract
The ubiquitin-proteasome system regulates the abundance of many cellular proteins by mediating their targeted degradation. We previously developed a method-differential proteomics-based identification of ubiquitylation substrates (DiPIUS)-for the comprehensive identification of substrates for a given F-box protein subunit of SCF-type ubiquitin ligases. We have now applied DiPIUS to the F-box protein Fbxw7 in three cell lines (mHepa, Neuro2A, and C2C12) and thereby identified myelin regulatory factor (MyRF), an endoplasmic reticulum-anchored transcription factor that is essential for myelination of nerves in the central nervous system, as a candidate substrate of Fbxw7 specifically in mHepa cells. Co-immunoprecipitation analysis confirmed that the NH2-terminal cytoplasmic domain of MyRF interacted with Fbxw7 in these cells. Furthermore, an in vitro ubiquitylation assay revealed that MyRF undergoes polyubiquitylation in the presence of purified recombinant SCFFbxw7 In addition, the stability of MyRF in mHepa cells was increased by mutation of a putative phosphodegron sequence or by exposure of the cells to an inhibitor of glycogen synthase kinase-3 (GSK-3). We found that MyRF mRNA is not restricted to the central nervous system but is instead distributed widely among mouse tissues. Furthermore, with the use of RNA sequencing in mHepa cells overexpressing or depleted of MyRF, we identified many novel potential target genes of MyRF. Our results thus suggest that Fbxw7 controls the transcription of MyRF target genes in various tissues through regulation of MyRF protein stability in a manner dependent on MyRF phosphorylation by GSK-3.
Highlights
The ubiquitin-proteasome system regulates the abundance of many cellular proteins by mediating their targeted degradation
To comprehensively identify the substrates targeted by a given F-box protein for degradation, we previously developed a differential proteomics approach termed differential proteomics– based identification of ubiquitylation substrates (DiPIUS) [7]
Fbxw7␣ mediates the nuclear degradation of many transcription factors, some of which are localized at the endoplasmic reticulum (ER) membrane under basal conditions but undergo cleavage and translocation to the nucleus in response to specific cues
Summary
To comprehensively identify the substrates targeted by a given F-box protein for degradation, we previously developed a differential proteomics approach termed DiPIUS [7]. B, cycloheximide chase analysis of MyRF in mHepa cells stably expressing HA-tagged MyRF with or without Fbxw7␣. Cycloheximide chase analysis revealed that this mutant was more stable than WT or other CPD mutant proteins in mHepa cells (Fig. 3, C and D). Cells stably expressing WT or CPD mutant forms of HA-tagged MyRF (C) or those expressing HA-MyRF(WT) (E) were incubated for the indicated times in the presence of cycloheximide (100 g/ml) either without (C) or with 1 M BIO or DMSO vehicle (E). These results suggested that Fbxw depletion leads to the accumulation of MyRF(N) in OPCs and thereby promotes their maturation into oligodendrocytes
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