Regulation of the ubiquitin-conjugating enzyme hHR6A by CDK-mediated phosphorylation

• Published in: The EMBO journal 2002-04, Vol.21 (8), p.2009-2018
• Main Authors: Sarcevic, Boris, Mawson, Amanda, Baker, Rohan T, Sutherland, Robert L
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding Sites; CDC2-CDC28 Kinases; Cell Cycle; Cell Division; CHO Cells; Cricetinae; Cyclin A - genetics; Cyclin A - metabolism; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases - genetics; Cyclin-Dependent Kinases - metabolism; EMBO06; EMBO31; G2 Phase; hHR6A protein; Histones - metabolism; Humans; Ligases - genetics; Ligases - metabolism; Mitosis; Molecular Sequence Data; Mutation; Phosphorylation; Protein Serine-Threonine Kinases - genetics; Protein Serine-Threonine Kinases - metabolism; Rad6; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Serine - metabolism; ubiquitin; Ubiquitin - metabolism; Ubiquitin-Conjugating Enzymes
• ISSN: 0261-4189; 1460-2075; 1460-2075
• DOI: 10.1093/emboj/21.8.2009

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 G 2 /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.