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.
Localization of p21-Activated Kinase 1 (PAK1) to Pinocytic Vesicles and Cortical Actin Structures in Stimulated Cells
S. Dharmawardhane,L. C. Sanders,Stuart S. Martin,R. H. Daniels,G. Bokoch
Published 1997 in Journal of Cell Biology
ABSTRACT
PUBLICATION RECORD
- Publication year
1997
- Venue
Journal of Cell Biology
- Publication date
1997-09-22
- Fields of study
Biology, Medicine
- Identifiers
- External record
- Source metadata
Semantic Scholar, PubMed
CITATION MAP
EXTRACTION MAP
CLAIMS
CONCEPTS
- constitutively active pak1 mutant
An engineered PAK1 variant designed to signal without normal upstream activation.
Aliases: active PAK1 mutant
- cytochalasin d
An actin filament-disrupting compound used to perturb cortical actin organization.
- dorsal ruffles
Actin-rich ruffled structures that form on the dorsal surface of cells.
Aliases: dorsal ruffling
- f-actin
Filamentous actin, the polymerized form of actin used here as a marker of cortical cytoskeleton.
Aliases: filamentous actin, polymerized actin
- lamellipodia
Broad, sheet-like protrusions at the leading edge of motile cells.
- pak1
p21-activated kinase 1, a Rac/Cdc42 effector kinase examined for its subcellular localization in fibroblasts.
Aliases: p21-activated kinase 1
- submembranous vesicles
Vesicular structures located just beneath the plasma membrane.
- v-src transformation
A transformed fibroblast state driven by expression of the v-Src oncogenic tyrosine kinase.
Aliases: v-Src-transformed fibroblasts
- wortmannin
A phosphatidylinositol 3-kinase inhibitor used to test pathway dependence.
REFERENCES
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