Dominant-negative cyclin-selective ubiquitin carrier protein E2-C/UbcH10 blocks cells in metaphase

Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2362-7. doi: 10.1073/pnas.94.6.2362.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Bivalvia
  • Carrier Proteins / chemistry
  • Carrier Proteins / metabolism*
  • Cell Cycle*
  • Cyclins / metabolism
  • DNA Primers
  • Female
  • HeLa Cells
  • Humans
  • Kinetics
  • Ligases / biosynthesis
  • Ligases / chemistry
  • Ligases / metabolism*
  • Metaphase
  • Mitosis
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oocytes / physiology
  • Point Mutation
  • Polymerase Chain Reaction
  • Ranidae
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae
  • Sequence Homology, Amino Acid
  • Ubiquitin-Conjugating Enzymes*

Substances

  • Carrier Proteins
  • Cyclins
  • DNA Primers
  • Recombinant Proteins
  • ubiquitin carrier proteins
  • UBE2C protein, human
  • Ubiquitin-Conjugating Enzymes
  • Ligases

Associated data

  • GENBANK/U73379