title: Protein ubiquitination is regulated by phosphorylation. An in vitro study.
abstract: Protein ubiquitination has been implicated in ATP-dependent protein turnover and in a number of biological processes in eukaryotic cells. The ubiquitination activating enzyme, E1, and ubiquitin carrier protein, E2, are two essential enzymes in the protein ubiquitination machinery. Using purified E1 and E2 from rabbit reticulocytes and various protein kinases, which include cAMP-dependent protein kinase, protein kinase C, and protein tyrosine kinase, we demonstrated that E1 is phosphorylated by protein kinase C, with a stoichiometry of 0.65 mol of phosphate/mol of E1, and one of the E2 isoforms, E2(32kDa), is phosphorylated by protein tyrosine kinase to 2 eq of phosphate/mol of protein. Phosphorylation of E1 causes a 2-fold enhancement of its activity as monitored by ubiquitin-dependent ATP in equilibrium PPi exchange. When 1 eq of phosphate was incorporated into E2(32kDa), a 2.4-fold activation was also observed for its activity to catalyze the ubiquitination of histone H2A. The regulatory significance of this finding is discussed.
authors.affiliation: Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: S K
authors.initials: SK
authors.lastname: Kong
authors.forename: P B
authors.initials: PB
authors.lastname: Chock
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D006657:Histones / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: D006657:Histones; D007527:Isoenzymes; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D011494:Protein Kinases; D008025:Ligases