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* Department of Immunology, and The mechanisms through which the small
GTPases Rac1 and Cdc42 regulate the formation of
membrane ruffles, lamellipodia, and filopodia are currently unknown. The p21-activated kinases (PAKs) are
direct targets of active Rac and Cdc42 which can induce
the assembly of polarized cytoskeletal structures when
expressed in fibroblasts, suggesting that they may play a
role in mediating the effects of these GTPases on cytoskeletal dynamics.
We have examined the subcellular localization of endogenous PAK1 in fibroblast cell lines using specific
PAK1 antibodies. PAK1 is detected in submembranous
vesicles in both unstimulated and stimulated fibroblasts
that colocalize with a marker for fluid-phase uptake. In
cells stimulated with PDGF, in v-Src-transformed fibroblasts, and in wounded cells, PAK1 redistributed
into dorsal and membrane ruffles and into the edges of
lamellipodia, where it colocalizes with polymerized actin. PAK1 was also colocalized with F-actin in membrane ruffles extended as a response to constitutive activation of Rac1. PAK1 appears to precede F-actin in
translocating to cytoskeletal structures formed at the
cell periphery. The association of PAK1 with the actin
cytoskeleton is prevented by the actin filament-disrupting agent cytochalasin D and by the phosphatidylinositol 3-kinase inhibitor wortmannin. Co-immunoprecipitation experiments demonstrate an in vivo interaction
of PAK1 with filamentous (F)-actin in stimulated cells.
Microinjection of a constitutively active PAK1 mutant
into Rat-1 fibroblasts overexpressing the insulin receptor (HIRcB cells) induced the formation of F-actin- and
PAK1-containing structures reminiscent of dorsal ruffles. These data indicate a close correlation between the
subcellular distribution of endogenous PAK1 and the formation of Rac/Cdc42-dependent cytoskeletal structures and support an active role for PAK1 in regulating
cortical actin rearrangements.
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037; and § Department of Medicine, University of California San Diego, La Jolla, California 92037
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