Cell cycle-regulated proteolysis of mitotic target proteins - PubMed (original) (raw)
Cell cycle-regulated proteolysis of mitotic target proteins
H Bastians et al. Mol Biol Cell. 1999 Nov.
Free PMC article
Abstract
The ubiquitin-dependent proteolysis of mitotic cyclin B, which is catalyzed by the anaphase-promoting complex/cyclosome (APC/C) and ubiquitin-conjugating enzyme H10 (UbcH10), begins around the time of the metaphase-anaphase transition and continues through G1 phase of the next cell cycle. We have used cell-free systems from mammalian somatic cells collected at different cell cycle stages (G0, G1, S, G2, and M) to investigate the regulated degradation of four targets of the mitotic destruction machinery: cyclins A and B, geminin H (an inhibitor of S phase identified in Xenopus), and Cut2p (an inhibitor of anaphase onset identified in fission yeast). All four are degraded by G1 extracts but not by extracts of S phase cells. Maintenance of destruction during G1 requires the activity of a PP2A-like phosphatase. Destruction of each target is dependent on the presence of an N-terminal destruction box motif, is accelerated by additional wild-type UbcH10 and is blocked by dominant negative UbcH10. Destruction of each is terminated by a dominant activity that appears in nuclei near the start of S phase. Previous work indicates that the APC/C-dependent destruction of anaphase inhibitors is activated after chromosome alignment at the metaphase plate. In support of this, we show that addition of dominant negative UbcH10 to G1 extracts blocks destruction of the yeast anaphase inhibitor Cut2p in vitro, and injection of dominant negative UbcH10 blocks anaphase onset in vivo. Finally, we report that injection of dominant negative Ubc3/Cdc34, whose role in G1-S control is well established and has been implicated in kinetochore function during mitosis in yeast, dramatically interferes with congression of chromosomes to the metaphase plate. These results demonstrate that the regulated ubiquitination and destruction of critical mitotic proteins is highly conserved from yeast to humans.
Figures
Figure 1
Extracts of G0, G1, S, G2, and nocodazole-arrested M phase cells retain their stage-specific differences in D box-dependent cyclin B proteolysis. (A) FR3T3 cells were arrested in G0 by serum starvation and released into the cell cycle by addition of 10% serum. Cells were harvested after 0 (G0), 3 (G1), 16 (S), and 24 (G2) h after release or after growth in the presence of nocodazole [M (nocodazole)]. Cell extracts were prepared, supplemented with an ATP-regenerating system, and incubated for the indicated times with radiolabeled cyclin B or D box-deficient cyclin B Δ86 proteins translated in vitro. Products were analyzed by SDS-PAGE followed by autoradiography. (B) HeLa cells were synchronized in M, G1, S, and G2 as described in MATERIALS AND METHODS. Degradation activity toward radiolabeled cyclin B or cyclin B Δ86 proteins was assayed as described in A. (C) HeLa cell G1 extracts were incubated with DMSO as a control, with 10 mM glucose and 1 μg/μl hexokinase to deplete ATP, with 1 μg/μl methyl ubiquitin to inhibit polyubiquitination, or with 100 μM proteasome inhibitor lactacystin before monitoring the degradation of radiolabeled cyclin proteins as described above. (D) Extracts of HeLa cells synchronized in G1 or S phase were incubated with radiolabeled full-length or C-terminally truncated p27Kip1 translation products and assayed as above.
Figure 2
A protein phosphatase of type 2A is required to maintain cyclin degradation activity in G1. (A) HeLa cell G1 extracts were preincubated with various kinase inhibitors (20 nM staurosporine, 5 mM 6-DMAP, 50 μM olomoucine) or phosphatase inhibitors (inhibitor mix, see MATERIALS AND METHODS; 1 mM sodium orthovanadate, 1 μM OA, 200 nM heatstable inhibitor I-2) as indicated before the addition of radiolabeled cyclin B protein and assayed as in Figure 1. (B) Histone H1 kinase activity in Hela cell G1 extracts treated with kinase or phosphatase inhibitors or both. Hela cell G1 extracts preincubated with DMSO, 20 nM staurosporine (Stsp), 1 μM OA, or 1 μM OA plus 20 nM stauroporine were subjected to histone H1 kinase assays. Phosphorylated histone H1 was resolved on 20% SDS-PAGE followed by autoradiography. (C) OA does not activate a kinase activity that is responsible for inactivating the cyclin B degradation activity in G1 extracts. HeLa cell G1 extracts used in B were preincubated with DMSO (as a control), 20 nM staurosporine (Stsp), 1 μM OA, or 1 μM OA acid plus 20 nM staurosporine before assaying the cyclin B degradation activity as decribed previously.
Figure 3
Nuclei from HeLa cells synchronized in S and G2 phase contain an inhibitor of cyclin B degradation. (A) An inhibitory activity for cyclin B degradation resides in S and G2 phase nuclei. HeLa cells were synchronized in early mitosis (nocodazole), G1, S, or G2, and nuclear and cytoplasmic extracts were prepared. These extracts (20 μg) or extraction buffer as control were mixed with total cell extracts derived from G1 cells (200 μg). Degradation activity toward cyclin B and cyclin B Δ86 was assayed as in Figure 1. (B) Dose dependency of nuclear inhibitory activity. Different amounts of protein of S phase nuclear extracts were added to HeLa cell G1 extracts, and destruction activity of radiolabeled cyclin B was assayed. (C) The S phase nuclear component responsible for inactivating cyclin B degradation in G1 extracts is not a kinase. HeLa cell G1 extracts were coincubated with buffer (as control), S phase nuclear extract (20 μg), 20 nM staurosporine (Stsp), or S phase nuclear extract pretreated with 20 nM staurosporine. Degradation activity toward cyclin B and cyclin B Δ86 was assayed. (D) S phase nuclear extracts treated with kinase inhibitor exhibit no kinase activity. Nuclear extracts derived from HeLa cells synchronized in S phase used in C were preincubated with DMSO (as control) or 20 nM staurosporine (Stsp), and histone H1 kinase activity was assayed (left panel). The same assay was performed using anti-cyclin A immunoprecipitates from S phase nuclear extracts (right panel).
Figure 4
D box-dependent in vitro degradation of cyclin A, cyclin B, Xenopus geminin H, and S. pombe Cut2p is active in G1 and inactive in S phase. HeLa cells were synchronized in G1 or S phase. Cell extracts were prepared, supplemented with an ATP-regenerating system and 2.5 μM purified recombinant wild-type UbcH10, and then assayed for their destruction activity toward various in vitro translated proteins as indicated.
Figure 5
The in vitro degradation of cyclin A, cyclin B, Xenopus geminin H, and S. pombe Cut2p involves the function of the “cyclin-selective Ubc” UbcH10. (A) HeLa G1 cell extracts were supplemented with buffer, 2.5 μM purified recombinant wild-type UbcH10 (-WT), or 2.5 μM purified recombinant dominant negative UbcH10 (-DN) and assayed for destruction activity toward radiolabeled cyclin A, cyclin B, Xenopus geminin H, and S. pombe Cut2p as described in Figure 1. (B) As a control, G1 extracts were supplemented with either 2.5 μM wild-type human Ubc3/CDC34 or 2.5 μM dominant negative human Ubc3/CDC34, and degradation of cyclin B was monitored.
Figure 6
Microinjection of purified dominant negative UbcH10 protein delays anaphase onset in PtK1 cells in vivo. Phase-contrast images are shown of PtK1 cells injected either buffer (A–C), wild-type UbcH10 protein (D–F), dominant negative UbcH10 (G–I), dominant negative UbcH5b (K–M), or dominant negative human Ubc3/CDC34 (N–P). All cells were injected in prophase (2–6 min before capture of the image), and time was determined when chromosomes aligned at the metaphase plate (metaphase) and separated subsequently (anaphase). Bar, 5 μm.
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References
- Adams PD, Lopez P, Sellers WR, Kaelin WG. Fluorescence-activated cell sorting of transfected cells. Methods Enzymol. 1997;283:59–72. - PubMed
- Alessi F, Quarta S, Savio M, Riva F, Rossi L, Stivala LA, Scovassi AI, Meijer L, Prosperi E. The cyclin-dependent kinase inhibitors olomoucine and roscovitine arrest human fibroblasts in G1 phase by specific inhibition of CDK2 kinase activity. Exp Cell Res. 1998;245:8–18. - PubMed
- Amon A, Irniger S, Nasmyth K. Closing the cell cycle circle in yeast: G2 cyclin proteolysis initiated at mitosis persists until the activation of G1 cyclins in the next cycle. Cell. 1994;77:1037–1050. - PubMed
- Arvand A, Bastians H, Welford S, Ruderman J, Denny C. EWS/FLI1 up regulates E2-C, a cyclin-selective ubiquitin conjugating enzyme involved in cyclin B destruction. Oncogene. 1998;17:2039–2045. - PubMed
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