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Molecular and Cellular Biology, August 2001, p. 5179-5189, Vol. 21, No. 15
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.
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


*
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.
Present address: Institute of Molecular Biology, Bratislava, Slovakia.
Present address: Department of Molecular Biology, The Scripps
Research Institute, La Jolla, CA 92037.
§
Present address: EMBL, 69117 Heidelberg, Germany.
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