The RasGAP proteins Ira2 and neurofibromin are negatively regulated by Gpb1 in yeast and ETEA in humans.
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).
Phan
Vernon T
VT
UCSF Helen Diller Family, Comprehensive Cancer Center, 2340 Sutter Street, San Francisco, CA 94115, USA.
Ding
Vivianne W
VW
Li
Fenglei
F
Chalkley
Robert J
RJ
Burlingame
Alma
A
McCormick
Frank
F
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D015536:Down-Regulation / Q000187:drug effects; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D020868:Gene Silencing / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins; D025542:Neurofibromin 1 / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects
D048868:Adaptor Proteins, Signal Transducing; D001798:Blood Proteins; C468782:FAF2 protein, human; C476991:GPB1 protein, S cerevisiae; D020690:GTPase-Activating Proteins; C476990:Gpb2 protein, S cerevisiae; C064960:IRA2 protein, S cerevisiae; D008565:Membrane Proteins; D025542:Neurofibromin 1; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D046988:Proteasome Endopeptidase Complex; D005947:Glucose