A ROLE FOR MITOGEN-ACTIVATED PROTEIN-KINASE IN THE SPINDLE ASSEMBLY CHECKPOINT IN XTC CELLS

The spindle assembly checkpoint prevents cells whose spindles are defe ctive or chromosomes are misaligned from initiating anaphase and leavi ng mitosis. Studies of Xenopus egg extracts have implicated the Erk2 m itogen-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 fun ction is required for mitotic progression or the spindle assembly chec kpoint in vivo in Xenopus tadpole cells (XTC). We determined that Erk1 and/or Erk2 are present in the mitotic spindle during prometaphase an d metaphase, consistent with the idea that MAP kinase might regulate o r monitor the status of the spindle. Next, we microinjected purified r ecombinant XCL100, a Xenopus MAP kinase phosphatase, into XTC cells in various stages of mitosis to interfere progression was unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain a rrested in mitosis after treatment with nocodazole. Cells injected wit h phosphatase at prometaphase or metaphase exited mitosis in the prese nce of nocodazole-the chromosomes decondensed and the nuclear envelope re-formed-whereas cells injected with buffer or a catalytically inact ive XCL100 mutant protein remained arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-l, which opposes XCL100's eff ects on MAP kinase, antagonized the effects of XCL100. Since the only known targets of MAP kinase kinase-l are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly c heckpoint in XTC cells.