title: Dominant-negative cyclin-selective ubiquitin carrier protein E2-C/UbcH10 blocks cells in metaphase.
abstract: Destruction of mitotic cyclins by ubiquitin-dependent proteolysis is required for cells to complete mitosis and enter interphase of the next cell cycle. In clam eggs, this process is catalyzed by a cyclin-selective ubiquitin carrier protein, E2-C, and the cyclosome/anaphase promoting complex (APC), a 20S particle containing cyclin-selective ubiquitin ligase activity. Here we report cloning a human homolog of E2-C, UbcH10, which shares 61% amino acid identity with clam E2-C and can substitute for clam E2-C in vitro. Dominant-negative clam E2-C and human UbcH10 proteins, created by altering the catalytic cysteine to serine, inhibit the in vitro ubiquitination and destruction of cyclin B in clam oocyte extracts. When transfected into mammalian cells, mutant UbcH10 inhibits the destruction of both cyclin A and B, arrests cells in M phase, and inhibits the onset of anaphase, presumably by blocking the ubiquitin-dependent proteolysis of proteins responsible for sister chromatid separation. Thus, E2-C/UbcH10-mediated ubiquitination is involved in both cdc2 inactivation and sister chromatid separation, processes that are normally coordinated during exit from mitosis.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: F M
authors.initials: FM
authors.lastname: Townsley
authors.forename: A
authors.initials: A
authors.lastname: Aristarkhov
authors.forename: S
authors.initials: S
authors.lastname: Beck
authors.forename: A
authors.initials: A
authors.lastname: Hershko
authors.forename: J V
authors.initials: JV
authors.lastname: Ruderman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D049872:Bivalvia; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002453:Cell Cycle; D016213:Cyclins / Q000378:metabolism; D017931:DNA Primers; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008025:Ligases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D008677:Metaphase; D008938:Mitosis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011898:Ranidae; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D044763:Ubiquitin-Conjugating Enzymes
chemical_list: D002352:Carrier Proteins; D016213:Cyclins; D017931:DNA Primers; D011994:Recombinant Proteins; C046288:ubiquitin carrier proteins; C105156:UBE2C protein, human; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases