A nonproteolytic function of the proteasome is required for the dissociation of Cdc2 and cyclin B at the end of M phase

Abstract

Inactivation of cyclin B-Cdc2 kinase at the exit from M phase depends on the specific proteolysis of the cyclin B subunit, whereas the Cdc2 subunit remains present at nearly constant levels throughout the cell cycle. It is unknown how Cdc2 escapes degradation when cyclin B is destroyed. In Xenopus egg extracts that reproduce the exit from M phase in vitro, we have found that dissociation of the cyclin B-Cdc2 complex occurred under conditions where cyclin B was tethered to the 26S proteasome but not yet degraded. The dephosphorylation of Thr 161 on Cdc2 was unlikely to be necessary for the dissociation of the two subunits. However, the dissociation was dependent on the presence of a functional destruction box in cyclin B. Cyclin B ubiquitination was also, by itself, not sufficient for separation of Cdc2 and cyclin B. The 26S proteasome, but not the 20S proteasome, was capable of dissociating the two subunits. These results indicate that the cyclin B and Cdc2 subunits are separated by the proteasome through a mechanism that precedes proteolysis of cyclin B and is independent of proteolysis. As a result, cyclin B levels decrease on exit from M phase but Cdc2 levels remain constant.

Figure 1

Dissociation of Cdc2 from cyclin B in Xenopus egg CSF extracts. (A) Inhibition of cyclin B destruction by MG115 in Xenopus egg extracts. Calcium (0.6 mM) was added at time zero to all extracts except controls (lanes 1–2). Ten minutes before calcium addition, 0.5 mM MG115 (proteasome inhibitor) or Z-LLH (calpine inhibitor) was added. At the indicated times, aliquots were taken and immunoblotted with antibodies against Xenopus cyclin B2 (top) or PSTAIR (for Cdc2; bottom). The small loss of cyclin B in the presence of MG115 might reflect the leakage of its effect. (B) Inactivation of cyclin B–Cdc2 kinase in the presence of MG115. (Open triangles) Addition of calcium; (Closed circles) addition of MG115; (Open boxes) control. (C) Dissociation of Cdc2 from cyclin B in the presence of MG115. Xenopus egg CSF extracts were activated by calcium in the presence or absence of 0.5 mM MG115. Samples taken at the indicated times were assayed for H1 kinase activity, recovered by Suc1–Sepharose beads, or immediately mixed with Laemmli's sample buffer. Materials retained on Suc1-beads were eluted with 2× Laemmli's sample buffer. Samples were immunoblotted with antibodies against Xenopus cyclin B2 (top and middle) or PSTAIR (bottom). (Lanes 1,2) Control samples without the addition of calcium.

Figure 2

Dephosphorylation of Thr 161 on Cdc2 is not necessary for dissociation from cyclin B. (A) Destruction of cyclin B associated with the T161E mutant of Cdc2. (B) Dissociation of the T161E mutant of Cdc2 from cyclin B. To produce the hcyclin B1–Myc–hCdc2 complex, reticulocyte lysates containing ³⁵S-labeled hcyclin B1 and Myc-tagged hCdc2 were incubated in the presence of ATP and, as a source of CAK, starfish oocyte extracts that had been depleted of endogenous starfish Cdc2 and cyclin B. CSF extracts were mixed with hcyclin B1 that had been complexed with either Myc–hCdc2-wt (lanes 1–3) or Myc–hCdc2-T161E (lanes 4–6), and then activated by calcium addition in the absence (A) or presence (B) of 0.5 mM MG115. Samples taken at the indicated times were directly, or after immunoprecipitation with anti-Myc antibody, resolved by SDS-PAGE and analyzed by a Fuji BAS 2000 phosphorimager.

Figure 3

Dissociation of Cdc2 from cyclin B requires destruction box of cyclin B. (A) Prevention of dissociation of cyclin B–Cdc2 by the N-terminal fragment of cyclin B1 (Nt-B). CSF extracts were activated with calcium addition in the absence (buffer) or presence of either Nt-B(wt) or Nt-B (R36S), which is not recognized by the cyclin B destruction system. Samples taken at the indicated times were assayed for H1 kinase activity, recovered by Suc1–Sepharose, or mixed directly with Laemmli's sample buffer. After SDS-PAGE, samples were immunoblotted with anti-Xenopus cyclin B2 antibody. (B) A functional destruction box is required for dissociation of cyclin B–Cdc2. ³⁵S-Labeled cyclin B derivatives were produced as hcyclin B1/ΔN86 (ΔN86) in which residues 1–86 were deleted from hcyclin B1, and hcyclin B1-Dm (Dm) in which the invariant Arg and Leu residues in the destruction box of human cyclin B1 were mutated to Ala. These proteins, in a complex with Myc-tagged Cdc2, were added to CSF extracts in the absence or presence of MG115. Samples taken at the indicated times were mixed directly with Laemmli's sample buffer or immunoprecipitated with anti-Myc antibody. These samples were analyzed by autoradiography after SDS-PAGE.

Figure 4

Cdc2 retains its association with ubiquitinated cyclin B. (A) Cdc2 is in a complex with cyclin B that has been polyubiquitinated in vitro. Purified GST-cyclin B–Cdc2 complex was incubated with biotinylated ubiquitin, E1, E2 (hE2-C), and, as a source of APC/C, anti-Cdc27 immunoprecipitates from Xenopus egg extracts that were arrested at anaphase by the addition of calcium together with nondegradable cyclin B fragment to CSF extracts. Then, GST–cyclin B was pulled down by glutathione–Sepharose 4B, resolved by SDS-PAGE and transferred to PVDF membrane. The membrane was reacted with ExtraAvidin peroxidase at room temperature for 1 h. Ubiquitinated cyclin B was visualized by ECL (lane 1). Alternatively, Cdc2 was recovered by Suc1–Sepharose, followed by ECL detection of ubiquitination (lane 2). (B) Cdc2 is complexed with cyclin B that is ubiquitinated in Xenopus egg extracts. Biotinylated ubiquitin was added to CSF extracts and, then, cyclin B degradation was induced by the addition of calcium. Samples taken at the indicated times were subjected to immunoprecipitation for Xenopus cyclin B2 or recovery of Cdc2 by Suc1–Sepharose beads. Ubiquitinated cyclins were visualized as described in A. (Asterisk) Non-specific band.

Figure 5

Cyclin B that has dissociated from Cdc2 remains associated with the 26S proteasome. (A) Cyclin B dissociated from Cdc2 is in high molecular weight complexes. CSF extracts were treated with calcium in the presence of MG115 (MG115-treated extracts), and then fractionated by gel filtration on a Superose 6 column in the presence of 1 mM ATP. Each fraction was tested on immunoblots for the presence of Xenopus cyclin B2 and Cdc2 (PSTAIR). As a control, untreated CSF extracts (MII extracts) were applied to the same column. (B) Cyclin B dissociated from Cdc2 co-elutes with the 26S proteasome. High molecular weight fractions in A containing cyclin B2, but not Cdc2, were pooled and refractionated in the presence of 1 mM ATP by ion-exchange chromatography on Mono Q Sepharose. After elution with increasing concentrations of KCl, each fraction was tested for peptidase activity against Suc-LLVY-AMC, and analyzed by immunoblots with antibodies against Xenopus cyclin B2, the Xenopus 20S proteasome, human MSS1 and p58 (both for the 19S particle), and human Cdc27 (for APC/C). (C) Cyclin B dissociated from Cdc2 is co-precipitated with the 26S proteasome. CSF extracts and MG115-treated extracts were subjected to immunoprecipitation with anti-Xenopus 20S proteasome antibody. The precipitates were probed with antibodies against cyclin B2, PSTAIR (Cdc2), the 20S proteasome, and S5a.

Figure 6

Dissociation of cyclin B–Cdc2 requires the proteasome. (A) Immunodepletion of the 26S and 20S proteasomes from Xenopus egg extracts. CSF extracts (100 μL) were immunodepleted with anti-Xenopus 20S proteasome or with control rabbit IgG, and then tested for the peptidase activity (right) against Suc-LLVY-AMC in the presence of SDS, which fully activates the 20S peptidase activity. Remaining proteins after the immunodepletion were assayed by immunoblots (left) for components of the 20S proteasome, MSS1 and TBP1 (both for the 19S particle). Note that immunodepletion of the 20S proteasome also removed the 26S proteasome from CSF extracts. (Asterisk) Non-specific band. (B) Dissociation of cyclin B–Cdc2 is prevented in the proteasome-depleted CSF extracts. The 20S/26S proteasome-depleted or mock-depleted CSF extracts were treated with calcium in the presence of 0.5 mM MG115. Samples taken at the indicated times were mixed directly with Laemmli's sample buffer or recovered by Suc1–Sepharose beads for immunoblots with anti-cyclin B2 antibody (top, middle). Separate samples were processed for assay of histone H1 kinase activity (bottom). (Closed circles) 20S/26S proteasome-depleted extracts; (Open squares) mock depleted.

Figure 7

Dissociation of cyclin B–Cdc2 in proteasome-depleted extracts is restored by the addition of immunopurified 26S, but not 20S, proteasomes. (A) Immunoprecipitates of the 26S or 20S proteasomes. For the 26S proteasome immunoprecipitates, CSF extracts were diluted in buffer A containing 2 mM ATP, incubated for 60 min at 37°C in the same buffer supplemented with an ATP-regenerating system and then subjected to immunoprecipitation with anti-20S proteasome antibody. The 20S proteasomes were immunoprecipitated from CSF extracts that were diluted in buffer A without ATP and then incubated for 60 min at 37°C with an ATP-depleting system (see Materials and Methods). The 26S or 20S proteasome immunoprecipitates were assayed by proteasomal peptidase activity against Suc-LLVY-AMC in the presence or absence of 0.05% SDS (right). Alternatively, the same immunoprecipitates were assayed by immunoblots for the presence of TBP1, S5a, and MSS1 (components of the 19S regulatory particle), and components of the 20S proteasome. (B) Addition of the 26S proteasome to the proteasome-depleted extracts restores dissociation of cyclin B–Cdc2. The 20S/26S proteasome-depleted extracts (see Fig. 6A) were mixed with the 26S proteasome immunoprecipitates (●), the 20S proteasome immunoprecipitates (▵), or control beads (□), and then activated by 0.6 mM calcium in the presence of MG115. Samples were taken at indicated times and analyzed as described in Fig. 6B.

Figure 8

A model for release of Cdc2 from cyclin B by the 26S proteasome. First, cyclin B complexed with Cdc2 is polyubiquitinated by an APC/C-dependent pathway at exit from M phase. Second, the lid of the 19S regulatory particle recognizes and tethers polyubiquitinated cyclin B that remains associated with Cdc2. Third, the cyclin B–Cdc2 complex is unfolded and dissociated by the chaperone-like activity of the base of the 19S regulatory particle with the aid of the α ring of the 20S proteasome. The translocation of unfolded cyclin B to the 20S proteasome may be required for further unfolding of cyclin B, and both processes may be coupled to each other. Finally, the unfolded cyclin B is translocated and degraded in the hollow center (β rings of the 20S proteasome) in which all catalytic sites are located. The inhibitory step by MG115 in the present study is indicated by the proteasome inhibitor.