title: Phosphorylation of RanGAP1 stabilizes its interaction with Ran and RanBP1.
abstract: Ran is a nuclear Ras-like GTPase that is required for various nuclear events including the bi-directional transport of proteins and ribonucleoproteins through the nuclear pore complex, spindle formation, and reassembly of the nuclear envelope. One of the key regulators of Ran is RanGAP1, a Ran specific GTPase activating protein. The question of whether a mechanism exists for controlling nucleocytoplasmic transport through the regulation of RanGAP1 activity continues to be debated. Here we show that RanGAP1 is phosphorylated in vivo and in vitro. Serine-358 (358S) was identified as the major phosphorylation site, by MALDI-TOF-MS spectrometry. Site directed mutagenesis at this position abolished the phosphorylation. Experiments using purified recombinant kinase and specific inhibitors such as DRB and apigenin strongly suggest that casein kinase II (CK2) is the responsible kinase. Although the phosphorylation of 358S of RanGAP1 did not significantly alter its GAP activity, the phosphorylated wild type RanGAP1, but not a mutant harboring a mutation at the phosphorylation site 358S, efficiently formed a stable ternary complex with Ran and RanBP1 in vivo, suggesting that the 358S phosphorylation of RanGAP1 affects the Ran system.
authors.affiliation: Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Eri
authors.initials: E
authors.lastname: Takeda
authors.forename: Miki
authors.initials: M
authors.lastname: Hieda
authors.forename: Jun
authors.initials: J
authors.lastname: Katahira
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Yoneda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D047310:Apigenin / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D025941:Protein Interaction Mapping; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D025842:SUMO-1 Protein; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020931:ran GTP-Binding Protein / Q000378:metabolism*
chemical_list: D020690:GTPase-Activating Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; C120587:RAN protein, human; C092317:RANGAP1 protein, human; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; C091775:ran-binding protein 1; D010768:Phosphoserine; D004004:Dichlororibofuranosylbenzimidazole; D047310:Apigenin; D020931:ran GTP-Binding Protein