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J. Biol. Chem., Vol. 276, Issue 39, 36163-36167, September 28, 2001
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From the Department of Physiology, University of Tennessee Health
Science Center, Memphis, Tennessee 38163 and the
We have previously shown that tyrosine
phosphorylation of the actin-regulatory protein villin is accompanied
by the redistribution of phosphorylated villin and a concomitant
decrease in the F-actin content of intestinal epithelial cells. The
temporal and spatial correlation of these two events suggested that
tyrosine phosphorylation of villin may be involved in the rearrangement
of the microvillar cytoskeleton. This hypothesis was investigated by
analyzing the effects of tyrosine phosphorylation of villin on the
kinetics of actin polymerization by reconstituting in vitro
the tyrosine phosphorylation of villin and its association with actin.
Full-length recombinant human villin was phosphorylated in
vitro by expression in the TKX1-competent cells that carry an
inducible tyrosine kinase gene. The actin-binding properties
of villin were examined using a co-sedimentation assay. Phosphorylation
of villin did not change the stoichiometry (1:2) but decreased the
binding affinity (4.4 µM for unphosphorylated
versus 0.6 µM for phosphorylated) of villin for actin. Using a pyrene-actin-based fluorescence assay, we
demonstrated that tyrosine phosphorylation had a negative effect on
actin nucleation by villin. In contrast, tyrosine phosphorylation
enhanced actin severing by villin. Electron microscopic analysis showed
complementary morphological changes. Phosphorylation inhibited the
actin bundling and enhanced the actin severing functions of villin.
Taken together our data show that tyrosine phosphorylation of villin
decreases the amount of villin bound to actin filaments, inhibits the
actin-polymerizing properties of villin, and promotes the
actin-depolymerizing functions instead. These observations suggest a
role for tyrosine phosphorylation in modulating the microvillar
cytoskeleton in vivo by villin in response to specific
physiological stimuli.
Tyrosine Phosphorylation of Villin Regulates the Organization of
the Actin Cytoskeleton*
, and Department of Biophysics and Biophysical Chemistry, Johns
Hopkins University, Baltimore, Maryland 21205
*
This work has been supported by grants from the American
Digestive Health Foundation (Industry Research Scholar award) and the
NIDDK, National Institutes of Health (DK-54755) (to S. K.).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.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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