Dominant-negative cyclin-selective ubiquitin carrier protein E2-C/UbcH10 blocks cells in metaphase - PubMed (original) (raw)

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

F M Townsley et al. Proc Natl Acad Sci U S A. 1997.

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.

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Figures

Figure 1

Figure 1

(A) Nucleotide and amino acid sequence of human UbcH10. (B) Comparison of the amino acid sequences of human UbcH10, clam E2-C, frog UBC-x, and a candidate yeast homolog.

Figure 2

Figure 2

Stimulation of cyclin-ubiquitin ligation by recombinant human E2-C, UbcH10. Cyclin-ubiquitin ligation was assayed as described in the text. Recombinant, purified human UbcH10 (○) or clam E2-C (•) was added at the concentrations indicated.

Figure 3

Figure 3

Inhibition of cyclin-ubiquitin ligation by C→S mutants of human UbcH10 and clam E2-C. (A) All incubations contained 0.25 μM wild-type clam E2-C. Where indicated, increasing concentrations of recombinant, purified C→S mutants of human UbcH10 (○) or clam E2-C (•) were added. Cyclin-ubiquitin ligation was assayed as described in the text. (B) Recombinant, purified human UbcH10 was added at the concentrations indicated in the absence (○, Control) or presence (•) of human UbcH10 C→S mutant (1 μM).

Figure 4

Figure 4

The degradation of endogenous cyclin B in mitotic clam oocyte extracts is inhibited by C→S mutants of human UbcH10 and clam E2-C. Incubations contained in a volume of 12 μl: 10 μl extract of early M-phase clam oocytes (75 min postfertilization, see ref. 7), 2 mM MgCl2, 1 mM ATP, 20 mM creatine phosphate, 50 μg/ml creatine kinase, 0.5 mM DTT, 250 μM emetine, and recombinant E2-C derivatives as specified. After incubation at 16°C for the time periods indicated, samples of 2 μl were withdrawn into SDS electrophoresis sample buffer. The samples were separated on a 10% polyacrylamide gel, blotted on nitrocellulose, probed with an antiserum directed against clam cyclin B (25), and detected by ECL. (A) Effects of human UbcH10 and clam E2-C C→S mutants on the degradation of clam cyclin B. (B) Reversal of the effects of the C→S mutant by wild-type human UbcH10. The various recombinant Ubc derivatives were added at the concentrations indicated. The position of cyclin B is indicated. The identity of the crossreactive protein that migrates ahead of cyclin B is not known. WT, wild type.

Figure 5

Figure 5

Effect of dominant-negative human UbcH10 on cell cycle progression in mammalian somatic tissue culture cells. (A) COS cells were mock-transfected (control) or transfected with AU1-tagged wild-type (WT) or dominant-negative (mutant) UbcH10, and after 48 h were fixed and stained with AU1 antibodies and Hoechst 33342.Two hundred cells each were scored for the percent cells in interphase vs. M phase. (B) Immunoblots of total protein extracts from cells transfected with either wild-type or dominant-negative AU1-tagged UbcH10, reacted with cyclin A or B antibodies. (C) Morphologies of cells transfected with either wild-type (Left) or dominant-negative UbcH10 (Right). Cells were stained with AU1 antibodies (Top) to identify transfected cells, and with Hoechst dye (Bottom) to monitor chromosomes.

Figure 6

Figure 6

Hoechst-stained fields of cells from embryos injected with RNA encoding wild-type (striped bars) or dominant-negative mutant (black bars) clam E2-C. Fertilized Xenopus eggs were injected with 10 ng RNA encoding wild-type or dominant-negative clam E2-C and cultured to late blastula stage. Cells were fixed and stained with Hoechst 33342. (A) Two hundred cells in M phase were scored for percent in prophase, metaphase, and anaphase/telophase. (B) A field of Hoechst-stained chromosomes from embryos injected with wild-type (Left) and dominant-negative mutant (Right) RNA.

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