Regulation of the cyclin B degradation system by an inhibitor of mitotic proteolysis - PubMed (original) (raw)
Regulation of the cyclin B degradation system by an inhibitor of mitotic proteolysis
E Vorlaufer et al. Mol Biol Cell. 1998 Jul.
Free PMC article
Abstract
The initiation of anaphase and exit from mitosis depend on the anaphase-promoting complex (APC), which mediates the ubiquitin-dependent proteolysis of anaphase-inhibiting proteins and mitotic cyclins. We have analyzed whether protein phosphatases are required for mitotic APC activation. In Xenopus egg extracts APC activation occurs normally in the presence of protein phosphatase 1 inhibitors, suggesting that the anaphase defects caused by protein phosphatase 1 mutation in several organisms are not due to a failure to activate the APC. Contrary to this, the initiation of mitotic cyclin B proteolysis is prevented by inhibitors of protein phosphatase 2A such as okadaic acid. Okadaic acid induces an activity that inhibits cyclin B ubiquitination. We refer to this activity as inhibitor of mitotic proteolysis because it also prevents the degradation of other APC substrates. A similar activity exists in extracts of Xenopus eggs that are arrested at the second meiotic metaphase by the cytostatic factor activity of the protein kinase mos. In Xenopus eggs, the initiation of anaphase II may therefore be prevented by an inhibitor of APC-dependent ubiquitination.
Figures
Figure 1
The PP1 inhibitor I-2 does not prevent activation of mitotic cyclin B proteolysis. (A) Dose–response curve showing the effect of different concentrations of I-2 on the dephosphorylation of32P-labeled glycogen phosphorylase A in_Xenopus_ interphase extracts. (B) Dose–response curve showing the effect of different concentrations of OA on the phosphatase activity in Xenopus interphase extracts containing 10 μM I-2 (determined as in A). (C) The stability of35S-labeled cyclin B was analyzed in extracts entering mitosis in the absence or presence of 10 μM I-2. Entry into mitosis was triggered by addition of nondegradable recombinant cyclin Δ90 at time zero, and samples taken at the indicated time points were analyzed by SDS-PAGE and phosphorimaging.
Figure 2
OA inhibits cyclin B proteolysis if added before entry into mitosis. H1 kinase activity, the phosphorylation-induced mobility shift of 35S-labeled Cdc25 and of Cdc27, and the degradation of 35S-labeled cyclin B (Cyc B) were analyzed in extracts treated either with buffer (A) or with 1 μM OA (B and C), which was added either at time zero (B) or 25 min later (C). Δ90 was added to all reactions at time zero. Samples were taken at the indicated time points and analyzed by kinase assays and by SDS-PAGE followed by immunoblotting (anti-Cdc27) or phosphorimaging (H1 kinase, Cdc25, and Cyc B). The time points of buffer and OA addition are marked by arrows.
Figure 3
OA does not efficiently block the mitotic activation of the APC, but activates an IMP. (A and B) Time course showing the ability of different APC immunoprecipitates to ubiquitinate 125I-labeled-cyclin B 13–110 (Cyc B) in a reconstituted system containing purified E1, UBC4, and UBCx. APC was isolated from an interphase extract (APCi), from a mitotic Δ90 extract (APCm), and from an extract treated simultaneously with Δ90 and 1 μM OA (APCOA). Samples were analyzed by SDS-PAGE and phosphorimaging (B), and the ubiquitination activities were expressed as percentage of cyclin B converted into conjugates (A). (C and D) The stability of125I-labeled cyclin B1 1–102 (1–102-CycB) was analyzed in APC-depleted extracts supplemented with a mitotic APC fraction. Interphase extracts were either treated with Δ90 alone (−OA) or simultaneously with Δ90 and 1 μM OA (+OA) for 45 min before the APC depletion and reconstitution. Samples were analyzed as above (D), and cyclin B levels were quantitated (C). (E) Cdc27 immunoblot of the extracts used in C and D to control for the immunodepletion of APC. Lanes 1 and 3, Δ90 extract before and after depletion with Cdc27 antibodies, respectively; lanes 2 and 4, extract treated with Δ90 and OA at time zero, before and after Cdc27 immunodepletion, respectively. A protein band from a different portion of the same blot, which nonspecifically cross-reacts with Cdc27 antisera, is shown as a loading control.
Figure 4
Inhibition of cyclin B proteolysis in OA-treated extracts is overcome by Ca2+ treatment. The electrophoretic mobility shift of 35S-labeled Cdc25 and the stability of35S-labeled cyclin B were analyzed in extracts entering mitosis, induced by addition of Δ90. The extracts contained 1 μM OA and were either treated with 0.5 mM CaCl2 after 20 min (right panel; indicated by arrows) or not (left panel). Samples were taken at the indicated time points and analyzed by SDS-PAGE and phosphorimaging.
Figure 5
An IMP exists in CSF extracts. (A and B) Time course showing the ability of different APC immunoprecipitates to ubiquitinate 125I-labeled-cyclin B 13–110 (Cyc B) in a reconstituted system containing purified E1, UBC4, and UBCx. APC was isolated from an interphase extract (APCi), from a CSF extract (APCCSF), and from a mitotic Δ90 extract (APCm). Samples were analyzed by SDS-PAGE and phosphorimaging (B), and the ubiquitination activities were expressed as percentage of cyclin B converted into conjugates (A). (C and D) The stability of 125I-labeled cyclin B 13–110 (Cyc B) was analyzed in APC-depleted Δ90 and CSF extracts supplemented with a mitotic APC fraction. Samples were analyzed as above (D), and cyclin B levels were quantitated (C). (E) Cdc27 immunoblot of the extracts used in C and D to control for the immunodepletion of APC. Lanes 1 and 2, Δ90 extract before and after depletion with Cdc27 antibodies, respectively; lanes 3 and 4, CSF extract before and after Cdc27 immunodepletion, respectively. A protein band from a different portion of the same blot, which nonspecifically cross-reacts with Cdc27 antisera, is shown as a loading control.
Figure 6
The steady-state levels of cyclin B–ubiquitin conjugates are lowered in OA-treated extracts. (A and B) The conversion of 125I-labeled cyclin B 13–110 (Cyc B) into ubiquitin conjugates was followed in LLnL-treated extracts (50 μg/ml) induced to enter mitosis by addition of Δ90 in the absence (−OA) or presence (+OA) of 1 μM OA. Samples were taken at the indicated time points and analyzed by SDS-PAGE and phosphorimaging (B). The ubiquitination activity was expressed as the percentage of cyclin B converted into conjugates.
Figure 7
OA stabilizes different APC substrates. The electrophoretic mobility shift of 35S-labeled Cdc25 caused by activating phosphorylation and the stability of35S-labeled cyclin B, 35S-labeled Pds1, or35S-labeled geminin were analyzed in extracts entering mitosis either in the presence (+OA) or absence of 1 μM OA (−OA). Δ90 cyclin was added to all reactions at time zero. Samples were withdrawn at the indicated time points and analyzed by SDS-PAGE and phosphorimaging.
Figure 8
Activation of MAP kinase by OA and malE-mos correlates with the inhibition of cyclin B degradation. The electrophoretic mobility shift of MAP kinase that accompanies its activation and the stability of 35S-labeled cyclin B were examined in extracts entering mitosis, induced by addition of Δ90 at time zero. The extracts contained either 1 μM OA (added at time 0 or after 25 min), 20 μg/ml malE-mos (added at time 0 or after 25 min), or DMSO as a control (buffer). 35S-labeled cyclin B, Δ90, and ubiquitin were added a second time after 65 min. This was done to exclude the possibility that addition of OA or malE-mos after 25 min did not prevent cyclin B proteolysis, because the degradation reactions were completed before these reagents could activate MAP kinase. Samples were taken at the indicated time points and analyzed by SDS-PAGE and immunoblotting with Erk2 antibodies (anti-MAP kinase) or phosphorimaging (Cyc B). The slower-migrating bands representing active MAP kinase are indicated by arrowheads. The time points of35S-labeled cyclin B addition are marked by arrows.
Figure 9
Inhibition of malE-mos- but not OA-induced MAP kinase activation allows cyclin B proteolysis. The electrophoretic mobility shift of MAP kinase caused by activating phosphorylation and the stability of 35S-labeled cyclin B were examined in extracts that were induced to enter mitosis by addition of Δ90 cyclin. The extracts contained either 20 μg/ml malE-mos or 1 μM OA and either 400 μM PD98059 or buffer. Samples were taken at the indicated time points and analyzed by SDS-PAGE and immunoblotting with Erk2 antibodies (anti-MAP kinase) or phosphorimaging (Cyc B). The positions of the slower-migrating bands representing active MAP kinase are indicated by arrowheads. Note that an activated form of MAP kinase cannot be detected in extracts treated with OA and PD98059.
Figure 10
Model for the mechanism of the CSF arrest and its artificial induction by OA. The model proposes that both mos and OA can activate an IMP, which prevents the initiation of anaphase II in unfertilized Xenopus eggs.
References
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