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Published online 25 December 2000. doi:10.1083/jcb.151.7.1449
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© The Rockefeller University Press, 0021-9525/2000//1449 $5.00
The Journal of Cell Biology, Volume 151, Number 7, , 2000 1449-1458

Original Article

Temporal and Spatial Distribution of Activated Pak1 in Fibroblasts



Mary Ann Sellsa, Amanda Pfaffa, and Jonathan Chernoffa

a Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111.(215) 728-3616(215) 728-5319

j_chernoff{at}fccc.edu

p21-activated kinases (Paks) are effectors of the small GTPases Cdc42 and Rac, and are thought to mediate some of the cytoskeletal and transcriptional activities of these proteins. To localize activated Pak1 in cells, we developed an antibody directed against a phosphopeptide that is contained within the activation loop of Pak1. This antibody specifically recognizes the activated form of Pak1. Immunofluorescence analysis of NIH-3T3 cells coexpressing activated Cdc42 or Rac1 plus wild-type Pak1 shows that activated Pak1 accumulates at sites of focal adhesion, throughout filopodia and within the body and edges of lamellipodia. Platelet-derived growth factor stimulation of NIH-3T3 cells shows a pattern of Pak1 activation similar to that observed with Rac1. During closure of a fibroblast monolayer wound, Pak1 is rapidly activated and localizes to the leading edge of motile cells, then gradually tapers off as the wound closes. The activation of Pak1 by wounding is blocked by inhibitors of phosphatidylinositol 3-kinase, and Src family kinases, but not by an inhibitor of the epidermal growth factor receptor. These findings indicate that activated Pak1, and by extension, probably activated Cdc42 or Rac, accumulates at sites of cortical actin remodeling in motile fibroblasts.

Key Words: p21-activated kinase • motility • GTPases • fibroblasts • phosphorylation

© 2000 The Rockefeller University Press

Abbreviations used in this paper: CA, constitutive-active; Erk, extracellular signal–regulated kinase; GC, germinal center; HA, hemagglutinin; KD, kinase-dead; Pak, p21-activated kinase; PI3 kinase, phosphatidylinositol 3-kinase; RMLC, regulatory myosin light chain.


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