Regulation of microphthalmia-associated transcription factor MITF protein levels by association with the ubiquitin-conjugating enzyme hUBC9

The basic helix-loop-helix/leucine zipper (bHLH/ZIP) microphthalmia-associa ted transcription factor (MITF) regulates transcription of genes encoding e nzymes essential for melanin biosynthesis in melanocytes and retinal pigmen ted epithelial cells. To determine how MITF activity is regulated, we used the yeast two-hybrid system to identify proteins expressed by human melanom a cells that interact with MITF, The majority of clones that showed positiv e interaction with a 158-amino-acid region of MITF containing the bHLH/ZIP domain (aa 168-325) encoded the ubiquitin conjugating enzyme hUBC9, The ass ociation of MITF with hUBC9 was further confirmed by an in vitro GST pull-d own assay. Although hUBC9 is known to interact preferentially with SENTRIN/ SUMO1, in vitro transcription/translation analysis demonstrated greater as sociation of MITF with ubiquitin than with SENTRIN, Importantly, cotransfec tion of MITF and hUBC9 expression vectors resulted in MITF protein degradat ion. MITF protein was stabilized by the proteasome inhibitor MG132, indicat ing the role of the ubiquitin-proteasome system in MITF degradation. Serine 73, which is located in a region rich in proline, glutamic acid, serine, a nd threonine (PEST), regulates MITF protein stability, since a serine to al anine mutation prevented hUBC9-mediated MITF (S73A) degradation. Furthermor e, 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, hU BC9 is a critical regulator of melanocyte differentiation. (C) 2000 Academi c Press.