TY - JOUR
T1 - Degradation of the endoplasmic reticulum–anchored transcription factor MyRF by the ubiquitin ligase SCFFbxw7 in a manner dependent on the kinase GSK-3
AU - Nakayama, Shogo
AU - Yumimoto, Kanae
AU - Kawamura, Atsuki
AU - Nakayama, Keiichi I.
N1 - Funding Information:
This work was supported in part by Japan Society for the Promotion of Science KAKENHI Grants 25221303 and 17H06301 (to K. I. N.) and 15H01186 (to K. Y.). The authors declare that they have no conflicts of interest with the contents of this article. We thank T. Kitamura for pMX-puro; K. Tsunematsu, T. Takami, Y. Okabe, and other laboratory members for technical assistance and discussion; A. Ohta for help with preparation of the manuscript; and Laboratory for Technical Support, Medical Institute of Bioregulation, Kyushu University, for technical support. Computations were performed in part on the National Institute of Genetics supercomputer at National Institute of Genetics, Research Organization of Information and Systems.
Funding Information:
This work was supported in part by Japan Society for the Promotion of Sci-ence KAKENHI Grants 25221303 and 17H06301 (to K. I. N.) and 15H01186 (to K. Y.). The authors declare that they have no conflicts of interest with the contents of this article. The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) DRA005955. All data are available in ProteomeXchange under the accession number PXD008705. All data are available in jPOSTrepo under the accession number JPST000373. 1To whom correspondence should be addressed: Tel.: 81-92-642-6815; Fax: 81-92-642-6819; E-mail: nakayak1@bioreg.kyushu-u.ac.jp.
Publisher Copyright:
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2018/4/13
Y1 - 2018/4/13
N2 - 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 polyubiqui-tylation 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.
AB - 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 polyubiqui-tylation 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.
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U2 - 10.1074/jbc.RA117.000741
DO - 10.1074/jbc.RA117.000741
M3 - Article
C2 - 29472293
AN - SCOPUS:85045377203
VL - 293
SP - 5705
EP - 5714
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 15
ER -