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J. Biol. Chem., Vol. 283, Issue 40, 27314-27324, October 3, 2008

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Biochemical and Structural Characterization of the Pak1-LC8 Interaction^*

Christine M. Lightcap, Shangjin Sun^¶, James D. Lear^||, Ulrich Rodeck^**1, Tatyana Polenova^¶2, and John C. Williams³

From the Department of Biochemistry and Molecular Biology and Kimmel Cancer Center, and ^**Department of Dermatology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, the ^¶Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19109, and the ^||Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19716

Pak1 (p21-activated kinase-1) and the dynein light chain, LC8, are overexpressed in breast cancer, and their direct interaction has been proposed to regulate tumor cell survival. These effects have been attributed in part to Pak1-mediated phosphorylation of LC8 at serine 88. However, LC8 is homodimeric, which renders Ser⁸⁸ inaccessible. Moreover, Pak1 does not contain a canonical LC8 binding sequence compared with other characterized LC8 binding sequences. Together, these observations raise the question whether the Pak1/LC8 interaction is distinct (i.e. enabled by a unique interface independent of LC8 dimerization). Herein, we present results from biochemical, NMR, and crystallographic studies that show that Pak1 (residues 212-222) binds to LC8 along the same groove as canonical LC8 interaction partners (e.g. nNOS and BimL). Using LC8 point mutants K36P and T67A, we were able to differentiate Pak1 from canonical LC8 binding sequences and identify a key hydrogen bond network that compensates for the loss of the conserved glutamine in the consensus sequence. We also show that the target binding interface formed through LC8 dimerization is required to bind to Pak1 and precludes phosphorylation of LC8 at Ser⁸⁸. Consistent with this observation, in vitro phosphorylation assays using activated Pak1 fail to phosphorylate LC8. Although these results define structural details of the Pak1/LC8 interaction and suggest a hierarchy of target binding affinities, they do not support the current model whereby Pak1 binds to and subsequently phosphorylates LC8 to promote anchorage-independent growth. Rather, they suggest that LC8 binding modulates Pak1 activity and/or nuclear localization.

Received for publication, January 29, 2008 , and in revised form, July 2, 2008.

The atomic coordinates and structure factors (code 3DVP, 3DVH, and 3DVT) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported, in whole or in part, by National Institutes of Health (National Center for Research Resources) Grant S10-RR022316 (to J. C.). 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 1-3 and Figs. 1-6.

¹ Recipient of a seed grant from the Radiation Therapy Oncology Group.

² Recipient of National Science Foundation Grant NSF-CAREER CHE-0237612.

³ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Thomas Jefferson University, Bluemle Life Science Bldg., Rm. 826, 233 S. 10th St., Philadelphia, PA 19107. Tel.: 215-503-4573; Fax: 215-923-2117; E-mail: jwilliam{at}mail.jci.tju.edu.

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