Specificity Profiling of Pak Kinases Allows Identification of Novel Phosphorylation Sites

doi:10.1074/jbc.M700253200

Specificity Profiling of Pak Kinases Allows Identification of Novel Phosphorylation Sites^*

^‡Division of Basic Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, the ^§Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520, and the ^¶Centre for Structural Genomics, Botnar Research Centre, Oxford University, Oxford OX3 7LD, United Kingdom

3 To whom correspondence should be addressed: Division of Basic Science, Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 19111. Tel.: 215-728-3568; Fax: 215-728-3574; E-mail: jeffrey.Peterson{at}fccc.edu.

Abstract

The p21-activated kinases (Paks) serve as effectors of the Rho family GTPases Rac and Cdc42. The six human Paks are divided into two groups based on sequence similarity. Group I Paks (Pak1 to -3) phosphorylate a number of substrates linking this group to regulation of the cytoskeleton and both proliferative and anti-apoptotic signaling. Group II Paks (Pak4 to -6) are thought to play distinct functional roles, yet their few known substrates are also targeted by Group I Paks. To determine if the two groups recognize distinct target sequences, we used a degenerate peptide library method to comprehensively characterize the consensus phosphorylation motifs of Group I and II Paks. We find that Pak1 and Pak2 exhibit virtually identical substrate specificity that is distinct from that of Pak4. Based on structural comparisons and mutagenesis, we identified two key amino acid residues that mediate the distinct specificities of Group I and II Paks and suggest a structural basis for these differences. These results implicate, for the first time, residues from the small lobe of a kinase in substrate selectivity. Finally, we utilized the Pak1 consensus motif to predict a novel Pak1 phosphorylation site in Pix (Pak-interactive exchange factor) and demonstrate that Pak1 phosphorylates this site both in vitro and in cultured cells. Collectively, these results elucidate the specificity of Pak kinases and illustrate a general method for the identification of novel sites phosphorylated by Paks.

Footnotes

↵4 The abbreviations used are: Pak, p21-activated kinase; GST, glutathione S-transferase; PSSM, position-specific scoring matrix; OPS, optimal Pak substrate; PKA, protein kinase A; ROC, receiver operating characteristic; AUC, area under the ROC curve; GTPγS, guanosine 5′-3-O-(thio)triphosphate.
↵5 S. Yokoyama, unpublished results.
↵6 S. W. Deacon and J. R. Peterson, unpublished data.
↵* This research was supported in part by an American Association for Cancer Research-Fox Chase Cancer Center Career Development Award in Translational Cancer Research, by Department of Defense Neurofibromatosis Research Program Grant W81XWH-05-1-0200, and by a grant from the Pennsylvania Department of Health (all to J. R. P.). Additional support was provided by the National Institutes of Health Grant CA006927 and by an appropriation from the Commonwealth of Pennsylvania to Fox Chase Cancer Center. The Structural Genomics Consortium is a registered charity (number 1097737) funded by the Wellcome Trust, GlaxoSmithKline, Genome Canada, the Canadian Institutes of Health Research, the Ontario Innovation Trust, the Ontario Research and Development Challenge Fund, the Canadian Foundation for Innovation, VINNOVA, the Knut and Alice Wallenberg Foundation, the Swedish Foundation for Strategic Research, and Karolinska Institutet. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2 and Fig. 1.
↵1 Supported by funding from NCI, National Institutes of Health (NIH), Grant T32 CA009035.
↵2 Present address: Division of Biology, Kansas State University, Manhattan, KS 66502.
- Received January 9, 2007.
- Revision received March 27, 2007.