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J. Biol. Chem., Vol. 277, Issue 35, 32282-32293, August 30, 2002
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From the Polo kinases play critical roles for proper
M-phase progression. They are characterized by the presence of two
regions of homology in the C-terminal non-catalytic domain, termed
polo-box 1 (PB1) and polo-box 2 (PB2). Here we demonstrate that both
PB1 and PB2 are required for targeting the catalytic activity of Plk1 to centrosomes, midbody, and kinetochores. Expression of either kinase-inactive PLK1/K82M or the C-terminal
plk1
A Spindle Checkpoint Arrest and a Cytokinesis Failure by the
Dominant-negative Polo-box Domain of Plk1 in U-2 OS Cells*,
,
,**
Laboratory of Metabolism, § Basic
Research Laboratory, ¶ Laboratory of Cellular Carcinogenesis and
Tumor Promotion, Center for Cancer Research, NCI, National
Institutes of Health, Bethesda, Maryland 20892-4258 and
Department of Genetics, Cell Biology, and Development,
University of Minnesota, Minneapolis, Minnesota 55455
N induced a pre-anaphase arrest with
elevated Cdc2 and Plk1 activity. Prophase-arrested cells exhibited
randomly oriented spindle structures, whereas metaphase cells exhibited
aberrant bipolar spindles with Mad2 localization at kinetochores of
misaligned chromosomes. Microtubule nucleation activity of centrosomes
was not compromised. In vivo time-lapse studies revealed
that expression of plk1N resulted in
repeated cycles of bipolar spindle formation and disruption, suggestive
of a defect in spindle stability. A prolonged arrest frequently led to
the generation of micronucleated cells in the absence of
sisterchromatid separation and centrosome duplication, indicating that
micronucleation is not a result of accumulated cytokinesis failures.
Interestingly, bypass of the mitotic arrest by dominant-negative
spindle checkpoint components led to a failure in completion of
cytokinesis. We propose that, in mammalian cells, the
polo-box-dependent Plk1 activity is required for proper
metaphase/anaphase transition and for cytokinesis.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains videos of cells
depicted in Fig. 4, A, B
(DsRed-centrin + EGFP-plk1), and C
(upper and lower) and Fig. 5D.
**
To whom correspondence should be addressed: Laboratory of
Metabolism, Center for Cancer Research, NCI/NIH, 9000 Rockville Pike,
Bldg. 37, Rm. 3D25, Bethesda, MD 20892-4258. Tel.: 301-496-9635; Fax:
301-496-8419; E-mail: kyunglee@pop.nci.nih.gov.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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