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J. Biol. Chem., Vol. 279, Issue 50, 52150-52159, December 10, 2004

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Molecular Determinants of Substrate Recognition in Hematopoietic Protein-tyrosine Phosphatase^*

Zhonghui Huang, Bo Zhou, and Zhong-Yin Zhang

From the Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461

The extracellular signal-regulated protein kinase 2 (ERK2) plays a central role in cellular proliferation and differentiation. Full activation of ERK2 requires dual phosphorylation of Thr¹⁸³ and Tyr¹⁸⁵ in the activation loop. Tyr¹⁸⁵ dephosphorylation by the hematopoietic protein-tyrosine phosphatase (HePTP) represents an important mechanism for down-regulating ERK2 activity. The bisphosphorylated ERK2 is a highly efficient substrate for HePTP with a k_cat/K_m of 2.6 x 10⁶ M^–1 s^–1. In contrast, the k_catK/_m values for the HePTP-catalyzed hydrolysis of Tyr(P) peptides are 3 orders of magnitude lower. To gain insight into the molecular basis for HePTP substrate specificity, we analyzed the effects of altering structural features unique to HePTP on the HePTP-catalyzed hydrolysis of p-nitrophenyl phosphate, Tyr(P) peptides, and its physiological substrate ERK2. Our results suggest that substrate specificity is conferred upon HePTP by both negative and positive selections. To avoid nonspecific tyrosine dephosphorylation, HePTP employs Thr¹⁰⁶ in the substrate recognition loop as a key negative determinant to restrain its protein-tyrosine phosphatase activity. The extremely high efficiency and fidelity of ERK2 dephosphorylation by HePTP is achieved by a bipartite protein-protein interaction mechanism, in which docking interactions between the kinase interaction motif in HePTP and the common docking site in ERK2 promote the HePTP-catalyzed ERK2 dephosphorylation (20-fold increase in k_cat/K_m) by increasing the local substrate concentration, and second site interactions between the HePTP catalytic site and the ERK2 substrate-binding region enhance catalysis (20-fold increase in k_cat/K_m) by organizing the catalytic residues with respect to Tyr(P)¹⁸⁵ for optimal phosphoryl transfer.

Received for publication, July 12, 2004 , and in revised form, August 20, 2004.

^* This work was supported by National Institutes of Health Grant CA69202 and by the G. Harold and Leila Y. Mathers Charitable Foundation. 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.

To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-4288; Fax: 718-430-8922; E-mail: zyzhang{at}aecom.yu.edu.

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