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Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics

Abstract

Extracellular signals regulate actin dynamics through small GTPases of the Rho/Rac/Cdc42 (p21) family. Here we show that p21-activated kinase (Pak1) phosphorylates LIM-kinase at threonine residue 508 within LIM-kinase’s activation loop, and increases LIM-kinase-mediated phosphorylation of the actin-regulatory protein cofilin tenfold in vitro. In vivo, activated Rac or Cdc42 increases association of Pak1 with LIM-kinase; this association requires structural determinants in both the amino-terminal regulatory and the carboxy-terminal catalytic domains of Pak1. A catalytically inactive LIM-kinase interferes with Rac-, Cdc42- and Pak1-dependent cytoskeletal changes. A Pak1-specific inhibitor, corresponding to the Pak1 autoinhibitory domain, blocks LIM-kinase-induced cytoskeletal changes. Activated GTPases can thus regulate actin depolymerization through Pak1 and LIM-kinase.

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Figure 1: Activation of LIM-kinase by Pak1 in vitro.
Figure 2: Requirement of Pak1 activation and kinase activity for regulation of LIM-kinase.
Figure 3: Direct interaction of LIM-kinase and Pak1.
Figure 4: Inhibition of Rac- and Cdc42-induced cytoskeletal changes by dominant-negative LIM-kinase.
Figure 5: Inhibition of LIM-kinase-induced actin cytoskeletal changes by the autoinhibitory domain of Pak1.
Figure 6: Inhibition of Pak1(H83,86L)-induced cytoskeletal changes by dominant-negative LIM-kinase(D460N).
Figure 7: Co-localization of Pak and LIM-kinase with actin at membrane ruffles.

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Acknowledgements

We thank L. Blanchoin for cofilin; J. Feramisco for use of the microscopy core facility; A. Newton and A. Edwards for helpful discussions; and C.S. Lazar for technical assistance. G.M.B. thanks J. Bamburg and O. Bernard for providing reagents for preliminary studies. This work was supported by grants DK13149 and CA58689 (to G.N.G.) and GM39434 to (G.M.B.). D.C.E. and L.C.S. were supported by fellowships from the US Army Breast Cancer Research Program (DAMD 17-94-J-4124 and DAMD 17-97-1-7230).

Correspondence and requests for materials should be addressed to G.M.B. or G.N.G.

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Correspondence to Gary M. Bokoch.

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Edwards, D., Sanders, L., Bokoch, G. et al. Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. Nat Cell Biol 1, 253–259 (1999). https://doi.org/10.1038/12963

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