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J. Biol. Chem., Vol. 280, Issue 51, 42219-42226, December 23, 2005

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Phosphorylation of WASP by the Cdc42-associated Kinase ACK1

DUAL HYDROXYAMINO ACID SPECIFICITY IN A TYROSINE KINASE^*

Noriko Yokoyama, Julie Lougheed, and W. Todd Miller1

From the Department of Physiology and Biophysics, School of Medicine, Stony Brook University, New York 11794 and Exelixis Inc., South San Francisco, California 94083

ACK1 is a nonreceptor tyrosine kinase that associates specifically with Cdc42. Relatively few ACK1 substrates and interacting proteins have been identified. In this study, we demonstrated that ACK1 phosphorylates the Wiskott-Aldrich syndrome protein (WASP), a Cdc42 effector that plays an important role in the formation of new actin filaments. ACK1 and WASP interact in intact cells, and overexpression of ACK1 promotes WASP phosphorylation. Phosphorylation of WASP in vitro was enhanced by the addition of Cdc42 or phosphatidylinositol 4,5-biphosphate, presumably due to release of the autoinhibitory interactions in WASP. Surprisingly, when we mapped the sites of WASP phosphorylation, we found that ACK1 possesses significant serine kinase activity toward WASP (directed at Ser-242), as well as tyrosine kinase activity directed at Tyr-256. A serine peptide derived from the Ser-242 WASP phosphorylation site is also a substrate for ACK1. ACK1 expressed in bacteria retained its serine kinase activity, eliminating the possibility of contamination with a copurifying kinase. Serine phosphorylation of WASP enhanced the ability of WASP to stimulate actin polymerization in mammalian cell lysates. Thus, the tyrosine kinase ACK1 acts as a dual specificity kinase toward this substrate. In contrast to other dual specificity kinases that more closely resemble Ser/Thr kinases, ACK1 is a tyrosine kinase with an active site that can accommodate both types of hydroxyamino acids in substrates.

Received for publication, June 27, 2005 , and in revised form, October 27, 2005.

^* This work was supported by National Institutes of Health Grant CA 28146 (to W. T. M.). 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.

¹ To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Basic Science Tower, T-6, School of Medicine, Stony Brook University, Stony Brook, NY 11794-8661. Tel.: 631-444-3533; Fax: 631-444-3432; E-mail: todd.miller{at}stonybrook.edu.

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