THE PLANT CELL, Vol 7, Issue 4 431-445, Copyright © 1995 by American Society of Plant Biologists
The Timing of Protein Kinase Activation Events in the Cascade That Regulates Mitotic Progression in Tradescantia Stamen Hair Cells
S. M. Wolniak and P. M. Larsen
Department of Botany, University of Maryland, College Park, Maryland 20742
Stamen hair cells of the spiderwort plant Tradescantia virginiana exhibit
unusually predictable rates of progression through mitosis, particularly
from the time of nuclear envelope breakdown (NEBD) through the initiation
of cytokinesis. The predictable rate of progression through prometaphase
and metaphase has made these cells a useful model system for the
determination of the timing of regulatory events that trigger entry into
anaphase. A number of studies suggest that the elevation of one or more
protein kinase activities is a necessary prerequisite for entry into
anaphase. The current experiments employ two strategies to test when these
elevations in protein kinase activity actually occur during metaphase. In
perfusions, we added the protein kinase inhibitors K-252a, staurosporine,
or calphostin C to living stamen hair cells for 10-min intervals at known
times during prometaphase or metaphase and monitored the subsequent rate of
progression into anaphase. Metaphase transit times were altered as a
function of the time of addition of K-252a or staurosporine to the cells;
metaphase transit times were extended significantly by treatments initiated
in prometaphase through early metaphase and again late in metaphase.
Transit times were normal after treatments initiated in mid-metaphase, ~15
to 21 min after NEBD. Calphostin C had no significant effect on the
metaphase transit times. In parallel, cells were microinjected with known
quantities of a general-purpose protein kinase substrate peptide,
VRKRTLRRL, at predefined time points during prometaphase and metaphase. At
a cytosolic concentration of 100 nM to 1 [mu]M, the peptide doubled or
tripled the metaphase transit times when injected into the cytosol of
mitotic cells within the first 4 min after NEBD, at any point from 7.5 to 9
min after NEBD, at any point from 14 to 16 min after NEBD, at 21 min after
NEBD, or at 24 min after NEBD. At the concentration used and during these
brief intervals, the peptide appeared to act as a competitive inhibitor to
reveal inflection points when protein kinase activation was occurring or
when endogenous substrate levels approached levels of the peptide. The
timing of these inflection points coincides with the changes in protein
kinase activities during prometaphase and metaphase, as indicated by our
perfusions of cells with the broad spectrum kinase inhibitors.
Collectively, our results suggest that the cascade that culminates in
anaphase is complex and involves several successive protein kinase
activation steps punctuated by the activation of one or more protein
phosphatases in mid-metaphase.
