Molecular Cloning of a New Member of the p21-Cdc42/Rac-activated Kinase (PAK) Family

doi:10.1074/jbc.270.42.25070

Molecular Cloning of a New Member of the p21-Cdc42/Rac-activated Kinase (PAK) Family (*)

From the (1)Glaxo-IMCB Group, Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge, Singapore 0511 and the
(2) Department of Neurochemistry, Institute of Neurology, 1 Wakefield St., London WC1N 1PJ, United Kingdom

^§ To whom correspondence should be addressed:
Glaxo-IMCB Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge, Singapore 0511.
Tel.: 65-772-6167; Fax: 65-774-0742.

Abstract

A number of “target” proteins for the Rho family of small GTP-binding proteins have now been identified, including the protein kinases ACK and p65 (Manser, E., Leung, T., Salihuddin, H., Zhao, Z.-S., and Lim, L.(1994) Nature 367, 40-46). The purified serine/threonine kinase p65 has been shown to be directly activated by GTP-Rac1 or GTP-Cdc42. Here we report the cDNA sequence encoding a new brain-enriched PAK isoform β-PAK, which shares 79% amino acid identity with the previously described α-isoform. Their mRNAs are differentially expressed in the brain, with α-PAK mRNA being particularly abundant in motor-associated regions. In vitro translation products of the α- and β-PAK cDNAs exhibited relative molecular masses of 68,000 and 65,000, respectively, by SDS-polyacrylamide analysis. A specific β-PAK peptide sequence was obtained from rat brain-purified p65. Recombinant α- and β-PAKs exhibited an increase in kinase activity mediated by GTP-p21 induced autophosphorylation. Cdc42 was a more potent activator in vitro of α-PAK kinase, and the fully activated enzyme is 300 times more active than the unphosphorylated form. Interestingly the down-regulation in the binding of p21s to recombinant β-PAK and brain p65, which is observed upon kinase activation does not occur with recombinant α-PAK.

Footnotes

↵* This work was supported by the Glaxo-Singapore Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U33314[GenBank].
↵¹ The abbreviations used are:

GAP

GTPase-activating protein

GST

glutathione S-transferase

PAGE

polyacrylamide gel electrophoresis

PAK

p21 (Cdc42/Rac1)-activated protein kinase

PCR

polymerase chain reaction

PVDF

polyvinylidene difluoride

MBP

myelin basic protein

GTPS

guanosine 5′-O-(thiotriphosphate).
↵²F. Cvrckova, C. Di Virgilio, E. Manser, J. R. Pringle, and K. Nasmyth, in press.
↵³M. Teo, E. Manser, and L. Lim, in press.
↵⁴E. Manser, C. Chong, T. Leung, and L. Lim, unpublished observations.
↵⁵Z.-S. Zhao, T. Leung, E. Manser, and L. Lim, in press.
- Received June 12, 1995.
- Revision received August 14, 1995.