Document Detail

Regulation of microphthalmia-associated transcription factor MITF protein levels by association with the ubiquitin-conjugating enzyme hUBC9.
MedLine Citation:
PMID:  10694430     Owner:  NLM     Status:  MEDLINE    
The basic helix-loop-helix/leucine zipper (bHLH/ZIP) microphthalmia-associated transcription factor (MITF) regulates transcription of genes encoding enzymes essential for melanin biosynthesis in melanocytes and retinal pigmented epithelial cells. To determine how MITF activity is regulated, we used the yeast two-hybrid system to identify proteins expressed by human melanoma cells that interact with MITF. The majority of clones that showed positive interaction with a 158-amino-acid region of MITF containing the bHLH/ZIP domain (aa 168-325) encoded the ubiquitin conjugating enzyme hUBC9. The association of MITF with hUBC9 was further confirmed by an in vitro GST pull-down assay. Although hUBC9 is known to interact preferentially with SENTRIN/SUMO1, in vitro transcription/translation analysis demonstrated greater association of MITF with ubiquitin than with SENTRIN. Importantly, cotransfection of MITF and hUBC9 expression vectors resulted in MITF protein degradation. MITF protein was stabilized by the proteasome inhibitor MG132, indicating the role of the ubiquitin-proteasome system in MITF degradation. Serine 73, which is located in a region rich in proline, glutamic acid, serine, and threonine (PEST), regulates MITF protein stability, since a serine to alanine mutation prevented hUBC9-mediated MITF (S73A) degradation. Furthermore, we identified lysine 201 as a potential ubiquitination site. A lysine to arginine mutation abolished MITF (K201R) degradation by hUBC9 in vivo. Our experiments indicate that by targeting MITF for proteasome degradation, hUBC9 is a critical regulator of melanocyte differentiation.
W Xu; L Gong; M M Haddad; O Bischof; J Campisi; E T Yeh; E E Medrano
Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Experimental cell research     Volume:  255     ISSN:  0014-4827     ISO Abbreviation:  Exp. Cell Res.     Publication Date:  2000 Mar 
Date Detail:
Created Date:  2000-04-24     Completed Date:  2000-04-24     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0373226     Medline TA:  Exp Cell Res     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  135-43     Citation Subset:  IM    
Copyright Information:
Copyright 2000 Academic Press.
Roy M. and Phyllis Gough Huffington Center on Aging, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza M320 and VAMC, Houston, Texas, 77030, USA.
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MeSH Terms
DNA-Binding Proteins / physiology*
Gene Expression Regulation*
Helix-Loop-Helix Motifs / physiology
Ligases / physiology*
Melanocytes / physiology*
Microphthalmia-Associated Transcription Factor
Pigment Epithelium of Eye / physiology*
Transcription Factors / physiology
Tumor Cells, Cultured
Ubiquitin-Conjugating Enzymes*
Ubiquitins / physiology
Grant Support
Reg. No./Substance:
0/DNA-Binding Proteins; 0/MITF protein, human; 0/Microphthalmia-Associated Transcription Factor; 0/Transcription Factors; 0/Ubiquitins; EC 6.-/Ligases; EC Enzymes; EC enzyme UBC9

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