title: Cloning, expression and subcellular localization of the human homolog of p40MO15 catalytic subunit of cdk-activating kinase.
abstract: Transitions of the cell cycle are controlled by cyclin-dependent protein kinases (cdks) whose phosphorylation on the Thr residue included in the conserved sequence YTHEVV dramatically increases the activity. A kinase responsible for this specific phosphorylation, called CAK for cdk-activating kinase, has been recently purified from starfish and Xenopus oocytes and shown to contain the MO15 gene product as a catalytic subunit. In the present paper, we have cloned the human homolog of Xenopus p40MO15 by probing a HeLa cell cDNA library with degenerate oligonucleotides deduced from Xenopus and starfish MO15 sequences. Human and Xenopus MO15 displayed a strong homology showing 86% identity with regard to amino acid sequences. Northern blot analysis of RNA extracts from a series of human tissues as well as from cultured rodent fibroblasts revealed a unique 1.4 kb MO15 mRNA. No variation in the amount of MO15 transcript or protein was found along the entire course of the fibroblast cell cycle. Fluorescence in situ hybridization on human lymphocyte metaphases showed two distinct chromosomal locations of human MO15 gene at 5q12-q13 and 2q22-q24. By using gene tagging and mammalian cell transfection, we demonstrate that the KRKR motif located at the carboxy terminal end of MO15 is required for nuclear targeting of the protein. Mutation of KRKR to NGER retains MO15 in the cytoplasmic compartment, whilst the wild-type protein is detected exclusively in the nucleus. Interestingly, we demonstrate that the nuclear targeting of MO15 is necessary to confer the protein its CAK activity. In contrast to the wild-type, the NLS-mutated MO15 expressed in Xenopus oocytes is unable to generate CAK as long as the nuclear envelope is not broken. The nuclear localization of both the MO15 gene product and CAK activity may imply that cdks activation primarily occurs in the cell nucleus.
authors.affiliation: INSERM U-249-CNRS UPR 9008, Montpellier, France.
authors.forename: J M
authors.initials: JM
authors.lastname: Darbon
authors.forename: A
authors.initials: A
authors.lastname: Devault
authors.forename: S
authors.initials: S
authors.lastname: Taviaux
authors.forename: D
authors.initials: D
authors.lastname: Fesquet
authors.forename: A M
authors.initials: AM
authors.lastname: Martinez
authors.forename: S
authors.initials: S
authors.lastname: Galas
authors.forename: J C
authors.initials: JC
authors.lastname: Cavadore
authors.forename: M
authors.initials: M
authors.lastname: Dorée
authors.forename: J M
authors.initials: JM
authors.lastname: Blanchard
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002874:Chromosome Mapping; D002889:Chromosomes, Human, Pair 2; D002895:Chromosomes, Human, Pair 5; D003001:Cloning, Molecular; D018844:Cyclin-Dependent Kinases; D018076:DNA, Complementary; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014981:Xenopus; D000095043:Cyclin-Dependent Kinase-Activating Kinase
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase