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Molecular and Cellular Biology, August 2001, p. 5179-5189, Vol. 21, No. 15
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.15.5179-5189.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Conformational Switch and Role of Phosphorylation in PAK Activation

Gretel Buchwald,1 Eva Hostinova,1,dagger Markus G. Rudolph,1,Dagger Astrid Kraemer,1 Albert Sickmann,2 Helmut E. Meyer,2 Klaus Scheffzek,1,§ and Alfred Wittinghofer1,*

Max-Planck-Institut für Molekulare Physiologie, 44227 Dortmund,1 and Institut für Physiologische Chemie, Proteinstrukturlabor, Ruhr-Universität Bochum, 44780 Bochum,2 Germany

Received 20 September 2000/Returned for modification 27 October 2000/Accepted 27 April 2001

p21-activated protein kinases (PAKs) are involved in signal transduction processes initiating a variety of biological responses. They become activated by interaction with Rho-type small GTP-binding proteins Rac and Cdc42 in the GTP-bound conformation, thereby relieving the inhibition of the regulatory domain (RD) on the catalytic domain (CD). Here we report on the mechanism of activation and show that proteolytic digestion of PAK produces a heterodimeric RD-CD complex consisting of a regulatory fragment (residues 57 to 200) and a catalytic fragment (residues 201 to 491), which is active in the absence of Cdc42. Cdc42-GppNHp binds with low affinity (Kd 0.6 µM) to intact kinase, whereas the affinity to the isolated regulatory fragment is much higher (Kd 18 nM), suggesting that the difference in binding energy is used for the conformational change leading to activation. The full-length kinase, the isolated RD, and surprisingly also their complexes with Cdc42 behave as dimers on a gel filtration column. Cdc42-GppNHp interaction with the RD-CD complex is also of low affinity and does not dissociate the RD from the CD. After autophosphorylation of the kinase domain, Cdc42 binds with high (14 nM) affinity and dissociates the RD-CD complex. Assuming that the RD-CD complex mimics the interaction in native PAK, this indicates that the small G protein may not simply release the RD from the CD. It acts in a more subtle allosteric control mechanism to induce autophosphorylation, which in turn induces the release of the RD and thus full activation.

* Corresponding author. Mailing address: Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Str. 11, 44227 Dortmund, Germany. Phone: 49-231-133-2100. Fax: 49-231-133-2199. E-mail: Alfred.Wittinghofer{at}mpi-dortmund.mpg.de.

dagger Present address: Institute of Molecular Biology, Bratislava, Slovakia.

Dagger Present address: Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037.

§ Present address: EMBL, 69117 Heidelberg, Germany.

Molecular and Cellular Biology, August 2001, p. 5179-5189, Vol. 21, No. 15
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.15.5179-5189.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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