Molecular and Cellular Biology, March 2001, p. 1795-1809, Vol. 21, No. 5
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.5.1795-1809.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Regulates Paxillin
Tyrosine Phosphorylation and Mediates Colony-Stimulating
Factor 1-Induced Morphological Changes in Macrophages
Department of Developmental and Molecular Biology1 and Department of Anatomy and Structural Biology and the Analytical Imaging Facility,2 Albert Einstein College of Medicine, Bronx, New York 10461
Received 11 September 2000/Returned for modification 25 October 2000/Accepted 1 December 2000
Removal of colony-stimulating factor 1 (CSF-1) causes macrophages
to round up and to increase their expression of protein tyrosine
phosphatase
(PTP
). This is accompanied by the disruption of
focal complexes and the formation of ruffles. Here we have overexpressed wild-type (WT) PTP
and a phosphatase-inactive (C325S) mutant in a macrophage cell line in the presence and absence of CSF-1.
In the presence of CSF-1, WT PTP
induces cell rounding and
ruffle formation, while C325S PTP
has no effect. In contrast, in
CSF-1-starved cells, C325S PTP
behaves in a dominant negative fashion, preventing rounding and ruffling. Furthermore, C325S PTP
increases adhesion in cycling cells, while WT PTP
enhances motility. In WT PTP
-overexpressing cells, the
focal contact protein paxillin is selectively depleted from focal
complexes and specifically dephosphorylated on tyrosine. In contrast,
paxillin is hyperphosphorylated in C325S PTP
-expressing cells.
Moreover, a complex containing PTP
, paxillin, and a
paxillin-associated tyrosine kinase, Pyk2, can be
immunoprecipitated from macrophage lysates, and the catalytic domain of
PTP
selectively binds paxillin and Pyk2 in vitro. Although PTP
and Pyk2 do not colocalize with paxillin in focal complexes, all three
proteins are colocalized in dorsal ruffles. The results suggest
that paxillin is dephosphorylated by PTP
in dorsal ruffles, using
Pyk2 as a bridging molecule, resulting in a reduced pool of tyrosine-phosphorylated paxillin available for incorporation into
focal complexes, thereby mediating CSF-1 regulation of macrophage morphology, adhesion, and motility.
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