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A more recent version of this article appeared on May 1, 2002
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Submitted on September 19, 2001
Revised on January 30, 2002
Accepted on February 6, 2002
1 Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, New York, USA
* Corresponding author. E-mail address: Turnerce{at}upstate.edu.
The precise temporal-spatial regulation of the p21-activated serine-threonine kinase PAK at the plasma membrane is required for proper cytoskeletal reorganization and cell motility. However, the mechanism by which PAK localizes to focal adhesions has not yet been elucidated. Indirect binding of PAK to the focal adhesion protein paxillin via the Arf-GAP protein Paxillin Kinase Linker (PKL) and PIX/Cool suggested a mechanism. In this report we demonstrate an essential role for a paxillin-PKL interaction in the recruitment of activated PAK to focal adhesions. Similar to PAK, expression of activated Cdc42 and Rac1, but not RhoA, stimulated the translocation of PKL from a generally diffuse localization to focal adhesions. Expression of the PAK regulatory domain (PAK1-329) or the Autoinhibitory Domain (AID 83-149) induced PKL, PIX and PAK localization to focal adhesions indicating a role for PAK scaffold activation. We show PIX but not NCK binding to PAK is necessary for efficient focal adhesion localization of PAK and PKL consistent with a PAK-PIX-PKL linkage. Although PAK activation is required, it is not sufficient for localization. The PKL amino-terminus, containing the PIX binding site, but lacking paxillin-binding subdomain 2 (PBS2), was unable to localize to focal adhesions and also abrogatedPAK localization. An identical result was obtained following PKL
PBS2 expression. Finally, neither PAK nor PKL were capable of localizing to focal adhesions in cells overexpressing paxillinLD4 confirming a requirement for this motif in recruitment of the PAK/PIX/PKL complex to focal adhesions. These results suggest a GTP-Cdc42/GTP-Rac triggered multistep activation cascade leading to the stimulation of the adaptor function of PAK which through interaction with PIX provokes a functional PKL PBS2-paxillin LD4 association and consequent recruitment to focal adhesions. This mechanism is likely critical for the correct subcellular positioning of PAK thereby influencing the ability of PAK to coordinate cytoskeletal reorganization associated with changes in cell shape and motility.
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