Regulation of STRA13 by the von Hippel-Lindau tumor suppressor protein, hypoxia, and the UBC9/ubiquitin proteasome degradation pathway

In this study, we focus on different modes of regulation of STRA13, a human ortholog of the mouse basic helix-loop-helix transcriptional factor, previ ously identified by us as a new von Hippel-Lindau tumor suppressor gene (VH L) target. The gene was overexpressed in VHL-deficient cell lines and tumor s, specifically clear cell renal carcinomas and hemangioblastomas, Introduc tion of wild type VHL transgene into clear cell renal carcinoma restored lo w level expression of STRA13, Overexpression was also detected in many comm on malignancies with an intact VHL gene, suggesting the existence of anothe r, VHL-independent pathway of STRA13 regulation, Similar to many other von Hippel-Lindau tumor-suppressor protein (pVHL) targets, the expression of ST RA13 on the mRNA level was hypoxia-sensitive, indicating oxygen-dependent r egulation of the gene, presumably through the pVHL/hypoxia-inducible factor 1 (HIF-1) pathway. The yeast two-hybrid screening revealed interaction of the STRA13 protein with the human ubiquitin-conjugating enzyme (UBC9) prote in, the specificity of which was confirmed in mammalian cells. By adding th e proteasome inhibitor acetyl-leucinyl-leucinyl-norleucinal, we demonstrate d that the 26 S proteasome pathway regulates the stability of pSTRA13, Go-e xpression of STRA13 and UBC9 led to an increase of the pSTRA13 ubiquitinati on and subsequent degradation. These data established that UBC9/STRA13 asso ciation in cells is of physiological importance, presenting direct proof of UBC9 involvement in the ubiquitin-dependent degradation of pSTRA13, Hypoxi a treatment of mammalian cells transiently expressing STRA13 protein showed that stability of pSTRA13 is not affected by hypoxia or VHL, Thus, STRA13, a new pVHL target, is regulated in cells on multiple levels. We propose th at STRA13 may play a critical role in carcinogenesis, since it is a potent transcriptional regulator, abundant in a variety of common tumors.