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).
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing / metabolism
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Blood Proteins / chemistry
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Blood Proteins / metabolism*
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Cell Line
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Down-Regulation / drug effects
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GTPase-Activating Proteins / metabolism*
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Gene Silencing / drug effects
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Glucose / pharmacology
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Humans
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Mass Spectrometry
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Membrane Proteins
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Neurofibromin 1 / metabolism*
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Proteasome Endopeptidase Complex / metabolism
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Protein Binding / drug effects
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Protein Stability / drug effects
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Protein Structure, Tertiary
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RNA, Small Interfering / metabolism
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Recombinant Fusion Proteins / metabolism
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Saccharomyces cerevisiae / drug effects
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Saccharomyces cerevisiae / metabolism*
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Saccharomyces cerevisiae Proteins / chemistry
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Saccharomyces cerevisiae Proteins / metabolism*
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Ubiquitination / drug effects
Substances
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Adaptor Proteins, Signal Transducing
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Blood Proteins
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FAF2 protein, human
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GPB1 protein, S cerevisiae
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GTPase-Activating Proteins
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Gpb2 protein, S cerevisiae
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IRA2 protein, S cerevisiae
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Membrane Proteins
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Neurofibromin 1
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RNA, Small Interfering
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Recombinant Fusion Proteins
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Saccharomyces cerevisiae Proteins
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Proteasome Endopeptidase Complex
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Glucose