Regulation of the ubiquitin-conjugating enzyme hHR6A by CDK-mediated phosphorylation.
Cell cycle progression in eukaryotes is mediated by phosphorylation of protein substrates by the cyclin-dependent kinases (CDKs). We screened a cDNA library by solid-phase phosphorylation and isolated hHR6A as a CDK2 substrate. hHR6A is the human homologue of the product of the Saccharomyces cerevisiae RAD6/UBC2 gene, a member of the family of ubiquitin-conjugating enzymes. hHR6A is phosphorylated in vitro by CDK-1 and -2 on Ser120, a residue conserved in all hHR6A homologues, resulting in a 4-fold increase in its ubiquitin-conjugating activity. In vivo, hHR6A phosphorylation peaks during the G2/M phase of cell cycle transition, with a concomitant increase in histone H2B ubiquitylation. Mutation of Ser120 to threonine or alanine abolished hHR6A activity, while mutation to aspartate to mimic phosphorylated serine increased hHR6A activity 3-fold. Genetic complementation studies in S.cerevisiae demonstrated that hHR6A Ser120 is critical for cellular proliferation. This is the first study to demonstrate regulation of UBC function by phosphorylation on a conserved residue and suggests that CDK-mediated phosphorylation of hHR6A is an important regulatory event in the control of cell cycle progression.
Sarcevic
Boris
B
Cancer Research Program, Garvan Institute of Medical Research, St Vincent's Hospital, Darlinghurst, NSW, 2010, Australia. b.sarcevic@garvan.org.au
Mawson
Amanda
A
Baker
Rohan T
RT
Sutherland
Robert L
RL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D042846:CDC2-CDC28 Kinases; D016466:CHO Cells; D002453:Cell Cycle; D002455:Cell Division; D006224:Cricetinae; D019925:Cyclin A / Q000235:genetics / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D016195:G2 Phase; D006657:Histones / Q000378:metabolism; D006801:Humans; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D008938:Mitosis; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes
D019925:Cyclin A; D006657:Histones; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D012694:Serine; C471606:RAD6 protein, S cerevisiae; C471545:UBE2A protein, human; D044763:Ubiquitin-Conjugating Enzymes; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D008025:Ligases