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J. Biol. Chem., Vol. 279, Issue 37, 38912-38920, September 10, 2004

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Overlapping Binding Sites in Protein Phosphatase 2A for Association with Regulatory A and -4 (mTap42) Subunits^*

Todd D. Prickett and David L. Brautigan

From the Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, Virginia 22908

Diverse functions of protein Ser/Thr phosphatases depend on the distribution of the catalytic subunits among multiple regulatory subunits. In cells protein phosphatase 2A catalytic subunit (PP2Ac) mostly binds to a scaffold subunit (A subunit or PR65); however, PP2Ac alternatively binds to -4, a subunit related to yeast Tap42 protein, which also associates with phosphatases PP4 or PP6. We mapped -4 binding to PP2Ac to the helical domain, residues 19-165. We mutated selected residues and transiently expressed epitope-tagged PP2Ac to assay for association with A and -4 subunits by co-precipitation. The disabling H118N mutation at the active site or the presence of the active site inhibitor microcystin-LR did not interfere with binding of PP2Ac to either the A subunit or -4, showing that these are allosteric regulators. Positively charged side chains Lys⁴¹, Arg⁴⁹, and Lys⁷⁴ on the back surface of PP2Ac are unique to PP2Ac, compared with phosphatases PP4, PP6, and PP1. Substitution of one, two, or three of these residues with Ala produced a progressive loss of binding to the A subunit, with a corresponding increase in binding to -4. Conversely, mutation of Glu⁴² in PP2Ac essentially eliminated PP2Ac binding to -4, with an increase in binding to the A subunit. Reciprocal changes in binding because of mutations indicate competitive distribution of PP2Ac between these regulatory subunits and demonstrate that the mutated catalytic subunits retained a native conformation. Furthermore, neither the Lys⁴¹-Arg⁴⁹-Lys⁷⁴ nor Glu⁴² mutations affected the phosphatase-specific activity or binding to microcystin-agarose. Binding of PP2Ac to microcystin and to -4 increased with temperature, consistent with an activation energy barrier for these interactions. Our results reveal that the A subunit and -4 (mTap42) require charged residues in separate but overlapping surface regions to associate with the back side of PP2Ac and modulate phosphatase activity.

Received for publication, February 9, 2004 , and in revised form, July 13, 2004.

^* This work was supported by NCI, National Institutes of Health Grant CA-77584. 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: Center for Cell Signaling, University of Virginia School of Medicine, P. O. Box 800577, West Complex MSB 7196, Charlottesville, VA 22908. Tel.: 434-924-5892; Fax: 434-243-2829; E-mail: db8g{at}virginia.edu.

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