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© The Rockefeller University Press, 0021-9525/1997//433 $5.00
The Journal of Cell Biology, Volume 137, Number 2, , 1997 433-443

Article

A Role for Mitogen-activated Protein Kinase in the Spindle Assembly Checkpoint in XTC Cells



Xiao Min Wang*, Ye Zhai{ddagger}, and James E. Ferrell, Jr.*

* Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305-5332; and {ddagger} Preuss Laboratory Molecular Neuro-oncology, Brain Tumor Research Center, University of California at San Francisco, San Francisco, California 94143-0520

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.

1. Abbreviations used in this paper: DAPI, 4',6-diamidino-2-phenylindole; GST, glutathione-S-transferase; MAP kinase, mitogen-activated protein kinase; XTC, Xenopus tadpole cell line.

Please address all correspondence to James E. Ferrell, Jr., Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305-5332. Tel.: (415) 725-0765. Fax: (415) 725-2952. e-mail: ferrell{at}cmgm.stanford.edu


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