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J. Biol. Chem., Vol. 280, Issue 43, 36372-36379, October 28, 2005

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A Single Pair of Acidic Residues in the Kinase Major Groove Mediates Strong Substrate Preference for P-2 or P-5 Arginine in the AGC, CAMK, and STE Kinase Families^*

Guozhi Zhu, Koichi Fujii1, Yin Liu, Vlad Codrea, Juan Herrero, and Stephen Shaw2

From the Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892 and the AxCell Biosciences, Newtown, Pennsylvania 18940

Most basophilic serine/threonine kinases preferentially phosphorylate substrates with Arg at P-3 but vary greatly in additional strong preference for Arg at P-2 or P-5. The structural basis for P-2 or P-5 preference is known for two AGC kinases (family of protein kinases A, G, and C) in which it is mediated by a single pair of acidic residues (PEN+1 and YEM+1). We sought a general understanding of P-2 and P-5 Arg preference. The strength of Arg preference at each position was assessed in 15 kinases using a new degenerate peptide library approach. Strong P-2 or P-5 Arg preference occurred not only in AGC kinases (7 of 8 studied) but also in calmodulin-dependent protein kinase (CAMK, 1 of 3) and Ste20 (STE) kinases (2 of 4). Analysis of sequence conservation demonstrated almost perfect correlation between (a) strong P-2 or P-5 Arg preference and (b) acidic residues at both PEN+1 and YEM+1. Mutation of two kinases (PKC- and p21-activated kinase 1 (PAK1)) confirmed critical roles of both PEN+1 and YEM+1 residues in determining strong R-2 Arg preference. PAK kinases were unique in having exceptionally strong Arg preference at P-2 but lacking strong Arg preference at P-3. Preference for Arg at P-2 was so critical to PAK recognition that PAK1 activity was virtually eliminated by mutating the PEN+1 or YEM+1 residues. The fact that this specific pair of acidic residues has been repeatedly and exclusively used by evolution for conferring strong Arg preference at two different substrate positions in three different kinase families implies it is uniquely well suited to mediate sufficiently good substrate binding without unduly restricting product release.

Received for publication, May 6, 2005 , and in revised form, August 1, 2005.

^* This work was supported by the Intramural Research Program of the NCI, National Institutes of Health. 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 Figs. S1-S5.

¹ Present address: First Dept. of Internal Medicine, University of Occupational and Environmental Health, Kitakyushu, Fukuoka 807-8555, Japan.

² To whom correspondence should be addressed: Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-435-6499; Fax: 301-496-0887; E-mail: sshaw{at}nih.gov.

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