The RasGAP Proteins Ira2 and Neurofibromin Are Negatively Regulated by Gpb1 in Yeast and ETEA in Humans

Abstract

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. To understand how these proteins are regulated, we utilized an unbiased proteomics approach to identify Ira2 and neurofibromin binding partners. We demonstrate that the Gpb1/Krh2 protein binds and negatively regulates Ira2 by promoting its ubiquitin-dependent proteolysis. We extended our findings to show that in mammalian cells, the ETEA/UBXD8 protein directly interacts with and negatively regulates neurofibromin. ETEA contains both UBA and UBX domains. Overexpression of ETEA downregulates 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. Silencing of ETEA expression increases neurofibromin levels and downregulates Ras activity. These findings provide evidence for conserved ubiquitination pathways regulating the RasGAP proteins Ira2 (in yeast) and neurofibromin (in humans).

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Articles of Significant Interest Selected from This Issue by the Editors

We thank Cynthia Mysinger, Amy Young, and Erika Woodbury for critical reading of the manuscript. We thank all of the members of the McCormick laboratory for thoughtful discussion and comments.

The UCSF Mass Spectrometry Facility (A. L. Burlingame, Director) was supported by the Biomedical Research Technology Program of the National Center for Research Resources, NIH NCRR RR015804, NIH NCRR RR001614, and NIH NCRR RR012961. Frank McCormick was supported, in part, by ONYX Pharmaceuticals and UC Discovery grant bio-02-17 10242. Vernon T. Phan is the recipient of the Young Investigator Award from the Children's Tumor Foundation.