MAP kinase is required for the spindle assembly checkpoint but is dispensable for the normal M phase entry and exit in Xenopus egg cell cycle extracts - PubMed (original) (raw)

MAP kinase is required for the spindle assembly checkpoint but is dispensable for the normal M phase entry and exit in Xenopus egg cell cycle extracts

K Takenaka et al. J Cell Biol. 1997.

Abstract

In Xenopus laevis egg cell cycle extracts that mimic early embryonic cell cycles, activation of MAP kinase and MAP kinase kinase occurs in M phase, slightly behind that of maturation promoting factor. To examine the possible role of MAP kinase in the in vitro cell cycle, we depleted the extracts of MAP kinase by using anti-Xenopus MAP kinase antibody. Like in the mock-treated extracts, the periodic activation and deactivation of MPF occurred normally in the MAP kinase-depleted extracts, suggesting that MAP kinase is dispensable for the normal M phase entry and exit in vitro. It has recently been reported that microtubule depolymerization by nocodazole treatment can block exit from mitosis in the extracts if enough sperm nuclei are present, and that the addition of MAP kinase-specific phosphatase MKP-1 overcomes this spindle assembly checkpoint, suggesting the involvement of MAP kinase in the checkpoint signal transduction. We show here that the spindle assembly checkpoint mechanism cannot operate in the MAP kinase-depleted extracts. But, adding recombinant Xenopus MAP kinase to the MAP kinase-depleted extracts restored the spindle assembly checkpoint. These results indicate unambiguously that classical MAP kinase is required for the spindle assembly checkpoint in the cell cycle extracts. In addition, we show that strong activation of MAP kinase by the addition of a constitutively active MAP kinase kinase kinase in the absence of sperm nuclei and nocodazole, induced mitotic arrest in the extracts. Therefore, activation of MAP kinase alone is sufficient for inducing the mitotic arrest in vitro.

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Figures

Figure 1

Activation of MAP kinase and MAPKK in M phase of cell cycle extracts. Xenopus egg cell cycle extracts were incubated with 100 sperm nuclei/μl. Samples were taken and assayed for histone H1 kinase activity (A). MAPKK activities were assayed as the activity to phosphorylate kinase-negative recombinant MAP kinase (B, closed circle), and the activity to activate the ability of wild-type recombinant MAP kinase to phosphorylate MBP (B, open circle). Interphase (I) and mitotic (M) extracts were immunoblotted with anti-phosphotyrosine antibody PY20 (C). An arrow indicates MAP kinase. MAPKK activities of the same extracts were assayed as the activity to phosphorylate kinase-negative MAP kinase (KNMAPK; D) and the activity to activate MAP kinase (E). Assays were done in crude extracts (left) or with anti-MAPKK antibody immunoprecipitates (right). The same extracts were immunoblotted with anti-MAPKK antibody (F).

Figure 1

Figure 7

Mitotic arrest by Ste11ΔN in cell cycle extracts. Cell cycle extracts were incubated with 5,000 sperm nuclei/μl with or without Ste11ΔN (100 μg/ml). Samples were withdrawn at 10 min intervals and assayed for histone H1 kinase activity (A), MBP kinase activity (B), and MAP kinase activity in MBP-containing gels (C).

Figure 2

Effect of MAP kinase depletion on the normal cell cycle progress. Xenopus egg cell cycle extracts were subjected to immunodepletion with anti-MAP kinase antiserum or with preimmune serum (mock treatment), as described in Materials and Methods. (A) Total protein and MAP kinase were visualized by Coomassie brilliant blue staining and immunoblotting with anti-MAP kinase antibody, respectively. Extracts were untreated (lanes 1 and 6), mock treated (lanes 2 and 7), or treated with anti-MAP kinase antiserum (lanes 3 and 8), and the remaining extracts were analyzed. The precipitate with the protein A–Toyopearl beads in mock treatment (lanes 4 and 9) or in anti-MAP kinase antiserum treatment (lanes 5 and 10) was analyzed. An arrowhead indicates MAP kinase. (B) MPF activities were monitored as histone H1 kinase activities. Mock-treated extracts (mock) or MAP kinasedepleted extracts (αMAPK) were incubated with indicated concentrations of sperm nuclei (Sp). Samples were withdrawn at 10 min intervals. Assayed reactions were subjected to SDS-PAGE and autoradiographed.

Figure 2

Figure 3

Effect of MAP kinase depletion on microtubule depolymerization-induced mitotic arrest. Mock-treated extracts (mock) or MAP kinase–depleted extracts (αMAPK) were incubated with 9,000 sperm nuclei/μl in the presence or absence of 10 μg/ml nocodazole (Noc). Samples were withdrawn at 10 min intervals, assayed for histone H1 kinase activity (A and B), MBP kinase activity (C), or immunoblotted with anti-MAP kinase antibody (D).

Figure 4

Mitotic arrest by microtubule depolymerization in extracts insufficiently depleted of MAP kinase. (A) Cell cycle extracts which had been insufficiently immunodepleted of MAP kinase (B, lane 2) were incubated with 9,000 sperm nuclei/μl and nocodazole. Samples were withdrawn at 10 min intervals and assayed for histone H1 kinase activity. (B) Extracts which had been untreated (lane 1), insufficiently depleted of MAP kinase (lane 2), and depleted sufficiently of MAP kinase (lane 3) were immunoblotted with anti-MAP kinase antibody. The samples for lanes 2 and 3 were from the extracts assayed in A and Fig. 3 (αMAPK), respectively.

Figure 5

Rescue of mitotic arrest by microtubule depolymerization by addition of purified recombinant Xenopus MAP kinase in the MAP kinase-depleted extracts. MAP kinase-depleted extracts were incubated with 9,000 sperm nuclei/μl and 10 μg/ml nocodazole without (A) or with (B) histidine-tagged recombinant wild-type Xenopus MAP kinase. Samples were withdrawn at 10 min intervals and assayed for histone H1 kinase activity.

Figure 6

Ste11ΔN is a constitutively active MAPKK-K. (A) Xenopus oocyte extracts were incubated with or without 100 μg/ml recombinant Ste11ΔN. Samples were withdrawn at the indicated times and subjected to the kinase detection assay within MBP-containing gels. An arrowhead indicates MAP kinase. (B) Xenopus oocyte extracts were incubated with various concentrations of Ste11ΔN for 2 h. And then, MAPKK was immunoprecipitated and assayed for the activity to phosphorylate recombinant kinase negative MAP kinase. (C) Xenopus oocyte extracts were incubated with 100 μg/ml Ste11ΔN. Ste11ΔN was then immunoprecipitated from samples withdrawn at the indicated times and assayed for the activity to activate the ability of recombinant MAPKK to phosphorylate KNMAPK.

Figure 6

Figure 8

Effect of sperm nuclei concentration on the Ste11ΔNinduced mitotic arrest. Cell cycle extracts were incubated without or with various concentrations of sperm nuclei (Sp) in the presence or absence of Ste11ΔN (100 μg/ml). Samples were withdrawn at 10 min intervals and assayed for histone H1 kinase activity.

Figure 9

Ste11ΔN induces mitotic arrest through activation of MAP kinase. Mock-treated extracts (mock) or MAP kinase–depleted extracts (αMAPK) were incubated with 5,000 sperm nuclei/μl in the presence (+Ste11ΔN) or absence (no addition) of Ste11ΔN (100 μg/ml). Samples were withdrawn at 10 min intervals and assayed for histone H1 kinase activity (A) and MBP kinase activity (B). MAPKK was immunoprecipitated from the extracts in the presence of Ste11ΔN at 0 and 60 min, and the activity to phosphorylate KNMAPK was measured (C).

Figure 9

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MAP kinase is required for the spindle assembly checkpoint but is dispensable for the normal M phase entry and exit in Xenopus egg cell cycle extracts - PubMed (original) (raw)