title: Proteomics identification of nuclear Ran GTPase as an inhibitor of human VRK1 and VRK2 (vaccinia-related kinase) activities.
abstract: Human vaccinia-related kinase (VRK) 1 is a novel serine-threonine kinase that regulates several transcription factors, nuclear envelope assembly, and chromatin condensation and is also required for cell cycle progression. The regulation of this kinase family is unknown. Mass spectrometry has permitted the identification of Ran as an interacting and regulatory protein of the VRK serine-threonine kinase activities. The stable interaction has been validated by pulldown of endogenous proteins as well as by reciprocal immunoprecipitations. The three members of the VRK family stably interact with Ran, and the interaction was not affected by the bound nucleotide, GDP or GTP. The interaction was stronger with the RanT24N that is locked in its inactive conformation and cannot bind nucleotides. None of the kinases phosphorylated Ran or RCC1. VRK1 does not directly interact with RCC1, but if Ran is present they can be isolated as a complex. The main effect of the interaction of inactive RanGDP with VRK1 is the inhibition of its kinase activity, which was detected by a reduction in VRK1 autophosphorylation and a reduction in phosphorylation of histone H3 in residues Thr-3 and Ser-10. The kinase activity inhibition can be relieved by the interaction with the constitutively active RanGTP or RanL43E, which locks Ran in its GTP-bound active conformation. In this complex, the interaction with VRK proteins does not alter the effect of its guanine exchange factor, RCC1. Ran is a novel negative regulator of nuclear VRK1 and VRK2 kinase activity, which may vary in different subcellular localizations generating an asymmetric intracellular distribution of kinase activity depending on local protein interactions.
authors.affiliation: Programa de Oncología Translacional, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas-Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca E-37007, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Sanz-García
authors.forename: Inmaculada
authors.initials: I
authors.lastname: López-Sánchez
authors.forename: Pedro A
authors.initials: PA
authors.lastname: Lazo
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D017931:DNA Primers / Q000235:genetics; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D040901:Proteomics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D020931:ran GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D017931:DNA Primers; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; C120587:RAN protein, human; C062521:RCC1 protein, human; D011993:Recombinant Fusion Proteins; D017346:Protein Serine-Threonine Kinases; C109890:VRK1 protein, human; C109891:VRK2 protein, human; D020931:ran GTP-Binding Protein