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A role for mitogen-activated protein kinase in the spindle assembly checkpoint in XTC cells.

Wang XM, Zhai Y, Ferrell JE - J. Cell Biol. (1997)

Bottom Line: Cells injected with phosphatase at prometaphase or metaphase exited mitosis in the presence of nocodazole-the chromosomes decondensed and the nuclear envelope re-formed-whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remained arrested in mitosis.Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonized the effects of XCL100.Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Stanford University School of Medicine, California 94305-5332, USA.

ABSTRACT
The spindle assembly checkpoint prevents cells whose spindles are defective or chromosomes are misaligned from initiating anaphase and leaving mitosis. Studies of Xenopus egg extracts have implicated the Erk2 mitogen-activated protein kinase (MAP kinase) in this checkpoint. Other studies have suggested that MAP kinases might be important for normal mitotic progression. Here we have investigated whether MAP kinase function is required for mitotic progression or the spindle assembly checkpoint in vivo in Xenopus tadpole cells (XTC). We determined that Erk1 and/or Erk2 are present in the mitotic spindle during prometaphase and metaphase, consistent with the idea that MAP kinase might regulate or monitor the status of the spindle. Next, we microinjected purified recombinant XCL100, a Xenopus MAP kinase phosphatase, into XTC cells in various stages of mitosis to interfere with MAP kinase activation. We found that mitotic progression was unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain arrested in mitosis after treatment with nocodazole. Cells injected with phosphatase at prometaphase or metaphase exited mitosis in the presence of nocodazole-the chromosomes decondensed and the nuclear envelope re-formed-whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remained arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonized the effects of XCL100. Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells.

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Purification and characterization of the GST–XCL100 fusion protein. (A) Purification of bacterially expressed GST-XCL100.  Full-length GST-XCL100 migrates at ∼67 kD. The prominent band at ∼27 kD in the glutathione agarose purified sample is GST. After  Mono-Q purification, full-length GST-XCL100 was the main band detectable by Coomassie staining, and a single band was detectable  by XCL100 immunoblotting. (B) Activity of GST-XCL100. This panel shows a MAP kinase immunoblot of recombinant nonphosphorylated rat Erk2 (MK), and phosphorylated rat Erk2 (MK-P) incubated with no phosphatase, with GST-XCL100, or with the inactive  GST-XCL100 C260S mutant. (C) In vitro titration of GST-XCL100 activity. Various concentrations of GST-XCL100 were mixed with  250 nM MKK-1* and 1 μM Xenopus Erk2, and the system was allowed to reach steady state. MAP kinase activity was determined by a myelin basic protein phosphorylation assay.
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Figure 4: Purification and characterization of the GST–XCL100 fusion protein. (A) Purification of bacterially expressed GST-XCL100. Full-length GST-XCL100 migrates at ∼67 kD. The prominent band at ∼27 kD in the glutathione agarose purified sample is GST. After Mono-Q purification, full-length GST-XCL100 was the main band detectable by Coomassie staining, and a single band was detectable by XCL100 immunoblotting. (B) Activity of GST-XCL100. This panel shows a MAP kinase immunoblot of recombinant nonphosphorylated rat Erk2 (MK), and phosphorylated rat Erk2 (MK-P) incubated with no phosphatase, with GST-XCL100, or with the inactive GST-XCL100 C260S mutant. (C) In vitro titration of GST-XCL100 activity. Various concentrations of GST-XCL100 were mixed with 250 nM MKK-1* and 1 μM Xenopus Erk2, and the system was allowed to reach steady state. MAP kinase activity was determined by a myelin basic protein phosphorylation assay.

Mentions: Overnight cultures of Escherichia coli transformed with pGEX-XCL100 or pGEX-XCL100 C260S were grown and lysed essentially as described (Wang et al., 1996). The clarified cell extracts were recycled over a 2.5-ml column of glutathione agarose (Sigma Chemical Co.) for 5 h at 4°C. The column was washed with 10 vol of PBS, and fusion proteins were eluted with 50 mM Tris, pH 8.0, containing 15 mM glutathione. Fractions were assessed for MAP kinase phosphatase activity, as described below, and peak fractions were pooled. As has been previously reported, XCL100 and XCL100 C260S manifested substantial proteolytic degradation; much of the glutathione agarose–purified protein was digested back to the GST moiety (see Fig. 4 A; see also Sun et al., 1993). However, we were able to obtain purified full-length XCL100 or XCL100 C260S proteins by anion exchange chromatography. Peak fractions from the glutathione agarose column were applied to a Mono-Q column (Pharmacia Fine Chemicals, Piscataway, NJ) equilibrated with 20 mM Tris, pH 7.8, 20 mM NaCl, and 2 mM DTT. Proteins were eluted from the column with a linear gradient of 20 mM–1 M NaCl. Activity was recovered in the fractions containing 640–660 mM NaCl, pooled, and dialyzed against the microinjection buffer (130 mM KCl, 10 mM NaPipes, pH 7.0, and 1 mM MgCl2 ). Material obtained after the glutathione agarose purification step and after the Mono-Q step exhibited indistinguishable biochemical and biological activities.


A role for mitogen-activated protein kinase in the spindle assembly checkpoint in XTC cells.

Wang XM, Zhai Y, Ferrell JE - J. Cell Biol. (1997)

Purification and characterization of the GST–XCL100 fusion protein. (A) Purification of bacterially expressed GST-XCL100.  Full-length GST-XCL100 migrates at ∼67 kD. The prominent band at ∼27 kD in the glutathione agarose purified sample is GST. After  Mono-Q purification, full-length GST-XCL100 was the main band detectable by Coomassie staining, and a single band was detectable  by XCL100 immunoblotting. (B) Activity of GST-XCL100. This panel shows a MAP kinase immunoblot of recombinant nonphosphorylated rat Erk2 (MK), and phosphorylated rat Erk2 (MK-P) incubated with no phosphatase, with GST-XCL100, or with the inactive  GST-XCL100 C260S mutant. (C) In vitro titration of GST-XCL100 activity. Various concentrations of GST-XCL100 were mixed with  250 nM MKK-1* and 1 μM Xenopus Erk2, and the system was allowed to reach steady state. MAP kinase activity was determined by a myelin basic protein phosphorylation assay.
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Figure 4: Purification and characterization of the GST–XCL100 fusion protein. (A) Purification of bacterially expressed GST-XCL100. Full-length GST-XCL100 migrates at ∼67 kD. The prominent band at ∼27 kD in the glutathione agarose purified sample is GST. After Mono-Q purification, full-length GST-XCL100 was the main band detectable by Coomassie staining, and a single band was detectable by XCL100 immunoblotting. (B) Activity of GST-XCL100. This panel shows a MAP kinase immunoblot of recombinant nonphosphorylated rat Erk2 (MK), and phosphorylated rat Erk2 (MK-P) incubated with no phosphatase, with GST-XCL100, or with the inactive GST-XCL100 C260S mutant. (C) In vitro titration of GST-XCL100 activity. Various concentrations of GST-XCL100 were mixed with 250 nM MKK-1* and 1 μM Xenopus Erk2, and the system was allowed to reach steady state. MAP kinase activity was determined by a myelin basic protein phosphorylation assay.
Mentions: Overnight cultures of Escherichia coli transformed with pGEX-XCL100 or pGEX-XCL100 C260S were grown and lysed essentially as described (Wang et al., 1996). The clarified cell extracts were recycled over a 2.5-ml column of glutathione agarose (Sigma Chemical Co.) for 5 h at 4°C. The column was washed with 10 vol of PBS, and fusion proteins were eluted with 50 mM Tris, pH 8.0, containing 15 mM glutathione. Fractions were assessed for MAP kinase phosphatase activity, as described below, and peak fractions were pooled. As has been previously reported, XCL100 and XCL100 C260S manifested substantial proteolytic degradation; much of the glutathione agarose–purified protein was digested back to the GST moiety (see Fig. 4 A; see also Sun et al., 1993). However, we were able to obtain purified full-length XCL100 or XCL100 C260S proteins by anion exchange chromatography. Peak fractions from the glutathione agarose column were applied to a Mono-Q column (Pharmacia Fine Chemicals, Piscataway, NJ) equilibrated with 20 mM Tris, pH 7.8, 20 mM NaCl, and 2 mM DTT. Proteins were eluted from the column with a linear gradient of 20 mM–1 M NaCl. Activity was recovered in the fractions containing 640–660 mM NaCl, pooled, and dialyzed against the microinjection buffer (130 mM KCl, 10 mM NaPipes, pH 7.0, and 1 mM MgCl2 ). Material obtained after the glutathione agarose purification step and after the Mono-Q step exhibited indistinguishable biochemical and biological activities.

Bottom Line: Cells injected with phosphatase at prometaphase or metaphase exited mitosis in the presence of nocodazole-the chromosomes decondensed and the nuclear envelope re-formed-whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remained arrested in mitosis.Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonized the effects of XCL100.Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Stanford University School of Medicine, California 94305-5332, USA.

ABSTRACT
The spindle assembly checkpoint prevents cells whose spindles are defective or chromosomes are misaligned from initiating anaphase and leaving mitosis. Studies of Xenopus egg extracts have implicated the Erk2 mitogen-activated protein kinase (MAP kinase) in this checkpoint. Other studies have suggested that MAP kinases might be important for normal mitotic progression. Here we have investigated whether MAP kinase function is required for mitotic progression or the spindle assembly checkpoint in vivo in Xenopus tadpole cells (XTC). We determined that Erk1 and/or Erk2 are present in the mitotic spindle during prometaphase and metaphase, consistent with the idea that MAP kinase might regulate or monitor the status of the spindle. Next, we microinjected purified recombinant XCL100, a Xenopus MAP kinase phosphatase, into XTC cells in various stages of mitosis to interfere with MAP kinase activation. We found that mitotic progression was unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain arrested in mitosis after treatment with nocodazole. Cells injected with phosphatase at prometaphase or metaphase exited mitosis in the presence of nocodazole-the chromosomes decondensed and the nuclear envelope re-formed-whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remained arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonized the effects of XCL100. Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells.

Show MeSH
Related in: MedlinePlus