UCSF

The Neurofibromatosis type 1 (NF1) gene encodes the GTPase activating protein (GAP) neurofibromin which negatively regulates Ras activity. The yeast Saccharomyces cerevisiae has two neurofibromin homologs, Ira1 and Ira2. Similar to mammalian cells, mutations or deletions of the IRA genes result in hyperactive Ras. I utilized an unbiased proteomics approach to investigate Ira2 and neurofibromin binding partners and their involvement in regulating Ras signaling. I demonstrated that the Gpb1 protein negatively regulates Ira2 by promoting Ira2 proteolysis. Loss and gain of function experiments showed the Gpb1 protein is essential for Ira2 function. Whereas deletion of GPB1 increased Ira2 protein levels, overexpression of Gpb1 destabilized Ira2. In addition, the purified Gpb1complex can ubiquitinate Ira2 in vitro. I demonstrated that Gpb1 is required for the Rpn1 proteasome base subunit to trigger Ira2 proteolysis. In addition, I showed that the deubiquitination enzyme Ubp6 interacts with Ira2 and antagonizes Gpb1-mediated degradation of Ira2. Finally, I showed that the serine/threonine kinase CK2 binds and phosphorylates Ira2, preventing Ira2 from protein degradation.

I extended the findings to the mammalian system to show that the ETEA/UBXD8 protein directly interacts and negatively regulates neurofibromin. ETEA contains both UBA and UBX domains. Similar to Gpb1 negative regulation of Ira2 in yeast, ETEA over-expression down-regulates neurofibromin in human cells. Purified ETEA, but not a mutant of ETEA that lacks the UBX domain, ubiquitinates the neurofibromin GAP-related domain in vitro. Importantly, ETEA shares approximately 18% homology with Gpb1 N-terminal domain, including amino acid sequence homology in the UBA and UBX domains. Silencing of ETEA increases neurofibromin levels and downregulates Ras activities. These findings provide evidence for conserved ubiquitination pathways regulating the RasGAP proteins Ira2 in yeast and neurofibromin in humans.