title: Smurf1 directly targets hPEM-2, a GEF for Cdc42, via a novel combination of protein interaction modules in the ubiquitin-proteasome pathway.
abstract: Smurf1, a member of HECT-type E3 ubiquitin ligases, regulates cell polarity and protrusive activity by inducing ubiquitination and subsequent proteasomal degradation of the small GTPase RhoA. We report here that hPEM-2, a guanine nucleotide exchange factor for the small GTPase Cdc42, is a novel target of Smurf1. Pulse-chase labeling and a ubiquitination experiment using MG132, a proteasomal inhibitor, indicate that Smurf1 induces proteasomal degradation of hPEM-2 in cells. GST pull-down assays with heterologously expressed firefly luciferase-fusion proteins that include partial sequences of hPEM-2 reveal that part of the PH domain (residues 318-343) of hPEM-2 is sufficient for binding to Smurf1. In contrast, the hPEM-2 binding domain in Smurf1 was mapped to the C2 domain. Although it has been reported that the binding activities of some C2 domains to target proteins are regulated by Ca2+, Smurf1 interacts with hPEM-2 in a Ca2+-independent manner. Our discovery that hPEM-2 is, in addition to RhoA, a target protein of Smurf1 suggests that Smurf1 plays a crucial role in the spatiotemporal regulation of Rho GTPase family members.
authors.affiliation: Graduate School of Science and Technology, Chiba University, 648 Matsudo, Matsudo, Chiba 271-8510, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Yamaguchi
authors.forename: Osamu
authors.initials: O
authors.lastname: Ohara
authors.forename: Akikazu
authors.initials: A
authors.lastname: Ando
authors.forename: Takahiro
authors.initials: T
authors.lastname: Nagase
mesh_terms: D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D064067:Rho Guanine Nucleotide Exchange Factors; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: C577410:ARHGEF9 protein, human; D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors; D025801:Ubiquitin; C498342:SMURF1 protein, human; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; D020742:rhoA GTP-Binding Protein; D002118:Calcium