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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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Single label immunolocalization of MAP kinase.  XTC cells were fixed, permeabilized, stained with DAPI (not  shown) and X15 or X15 plus  blocking peptide (35 μM), and  examined by fluorescence and  phase microscopy. The cell  shown is in metaphase.
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Figure 3: Single label immunolocalization of MAP kinase. XTC cells were fixed, permeabilized, stained with DAPI (not shown) and X15 or X15 plus blocking peptide (35 μM), and examined by fluorescence and phase microscopy. The cell shown is in metaphase.

Mentions: Preimmune serum yielded no spindle staining (Fig. 2 G), indicating that the spindle staining observed with affinitypurified X15 was specific. Preincubation of X15 with the peptide against which it was raised markedly decreased the intensity of both spindle and cytoplasmic staining (Fig. 3). Finally, single label immunofluorescence studies with affinity-purified X15 showed spindle staining, confirming that the results seen in the triple label studies were not due to cross-reactivity between secondary antibodies or leakage between the fluorescein and Texas red filters (Fig. 3).


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)

Single label immunolocalization of MAP kinase.  XTC cells were fixed, permeabilized, stained with DAPI (not  shown) and X15 or X15 plus  blocking peptide (35 μM), and  examined by fluorescence and  phase microscopy. The cell  shown is in metaphase.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139774&req=5

Figure 3: Single label immunolocalization of MAP kinase. XTC cells were fixed, permeabilized, stained with DAPI (not shown) and X15 or X15 plus blocking peptide (35 μM), and examined by fluorescence and phase microscopy. The cell shown is in metaphase.
Mentions: Preimmune serum yielded no spindle staining (Fig. 2 G), indicating that the spindle staining observed with affinitypurified X15 was specific. Preincubation of X15 with the peptide against which it was raised markedly decreased the intensity of both spindle and cytoplasmic staining (Fig. 3). Finally, single label immunofluorescence studies with affinity-purified X15 showed spindle staining, confirming that the results seen in the triple label studies were not due to cross-reactivity between secondary antibodies or leakage between the fluorescein and Texas red filters (Fig. 3).

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