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© The Rockefeller University Press, 0021-9525/1997//1265 $5.00
The Journal of Cell Biology, Volume 138, Number 6, , 1997 1265-1278

Article

Localization of p21-Activated Kinase 1 (PAK1) to Pinocytic Vesicles and Cortical Actin Structures in Stimulated Cells



Suranganie Dharmawardhane*, Luraynne C. Sanders*, Stuart S. Martin§, R. Hugh Daniels*, and Gary M. Bokoch{ddagger}

* Department of Immunology, and {ddagger} 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

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.

Abbreviations used in this paper: F-actin, filamentous actin; MBP, myelin basic protein; PI, phosphatidylinositol.

Please address all correspondence to Dr. Gary M. Bokoch, Department of Immunology-IMM14, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037. Tel.: (619) 784-8217; Fax: (619) 784-8218.

The authors gratefully acknowledge the technical assistance of Yan Wang, M.D., and Benjamin P. Bohl. We also thank Dr. Malcolm Wood and George Klier for their assistance with confocal microscopy. Dr. Pamela Maher (TSRI) graciously provided highly purified Swiss 3T3 nuclei for these studies, and anti–LAMP-2 antibody was a kind gift of Dr. Bruce Granger (Montana State University, Bozeman, MT). Secretarial services provided by Ms. Antonette Lestelle and helpful discussions with Drs. Sandra Schmid and Klaus Hahn (both of TSRI) are greatly appreciated.


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