title: Rheb protein binds CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase) protein in a GTP- and effector domain-dependent manner and influences its cellular localization and carbamoyl-phosphate synthetase (CPSase) activity.
abstract: Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis.
authors.affiliation: From the Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641, Japan and satot@pharm.kitasato-u.ac.jp.
authors.forename: Tatsuhiro
authors.initials: T
authors.lastname: Sato
authors.affiliation: From the Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641, Japan and.
authors.forename: Hitomi
authors.initials: H
authors.lastname: Akasu
authors.affiliation: From the Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641, Japan and.
authors.forename: Wataru
authors.initials: W
authors.lastname: Shimono
authors.affiliation: From the Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641, Japan and.
authors.forename: Chisa
authors.initials: C
authors.lastname: Matsu
authors.affiliation: From the Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641, Japan and.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Fujiwara
authors.affiliation: From the Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641, Japan and.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Shibagaki
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, Molecular Biology Institute, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California, 90095.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Heard
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, Molecular Biology Institute, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California, 90095.
authors.forename: Fuyuhiko
authors.initials: F
authors.lastname: Tamanoi
authors.affiliation: From the Division of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641, Japan and.
authors.forename: Seisuke
authors.initials: S
authors.lastname: Hattori
mesh_terms: D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism*; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000378:metabolism*; D049109:Cell Proliferation / Q000235:genetics; D004080:Dihydroorotase / Q000378:metabolism*; D006801:Humans; D008247:Lysosomes / Q000378:metabolism / Q000473:pathology; D000076222:Mechanistic Target of Rapamycin Complex 1; D051379:Mice; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011741:Pyrimidine Nucleosides / Q000096:biosynthesis*; D000076205:Ras Homolog Enriched in Brain Protein; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C033626:CAD trifunctional enzyme; D046912:Multiprotein Complexes; D009479:Neuropeptides; D011741:Pyrimidine Nucleosides; C490211:RHEB protein, human; C504241:RHEBL1 protein, human; D000076205:Ras Homolog Enriched in Brain Protein; D001221:Aspartate Carbamoyltransferase; C546842:MTOR protein, human; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D004080:Dihydroorotase; D020559:Monomeric GTP-Binding Proteins; D018631:ras Proteins; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)
keywords: CAD; Cell Growth; Insulin; Mammalian Target of Rapamycin (mTOR); Nucleoside/Nucleotide Biosynthesis; Rheb; Rheb2; Tuberous Sclerosis Complex (TSC)