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J. Biol. Chem., Vol. 282, Issue 30, 21838-21847, July 27, 2007

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Expression of Human Protein Phosphatase-1 in Saccharomyces cerevisiae Highlights the Role of Phosphatase Isoforms in Regulating Eukaryotic Functions^*

Jennifer A. Gibbons¹, Lukasz Kozubowski, Kelly Tatchell, and Shirish Shenolikar²

From the Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 and the Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130

Human (PP1) isoforms, PP1, PP1, PP11, and PP12, differ in primary sequences at N and C termini that potentially bind cellular regulators and define their physiological functions. The GLC7 gene encodes the PP1 catalytic subunit with >80% sequence identity to human PP1 and is essential for viability of Saccharomyces cerevisiae. In yeast, Glc7p regulates glycogen and protein synthesis, actin cytoskeleton, gene expression, and cell division. We substituted human PP1 for Glc7p in yeast to investigate the ability of individual isoforms to catalyze Glc7p functions. S. cerevisiae expressing human PP1 isoforms were viable. PP1-expressing yeast grew more rapidly than strains expressing other isoforms. On the other hand, PP1-expressing yeast accumulated less glycogen than PP1-or PP11-expressing yeast. Yeast expressing human PP1 were indistinguishable from WT yeast in glucose derepression. However, unlike WT yeast, strains expressing human PP1 failed to sporulate. Analysis of chimeric PP1/ subunits highlighted a critical role for their unique N termini in defining PP1 and PP1 functions in yeast. Biochemical studies established that the differing association of PP1 isoforms with the yeast glycogen-targeting subunit, Gac1p, accounted for their differences in glycogen synthesis. In contrast to human PP1 expressed in Escherichia coli, enzymes expressed in yeast displayed in vitro biochemical properties closely resembling PP1 from mammalian tissues. Thus, PP1 expression in yeast should facilitate future structure-function studies of this protein serine/threonine phosphatase.

Received for publication, February 12, 2007 , and in revised form, May 31, 2007.

^* This work was supported in part by National Institutes of Health Grants DK52054 (to S. S.) and GM47789 (to K. T.). 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. 1–3.

¹ Supported in part by National Institutes of Health Toxicology Training Grant T32ES07031.

² To whom correspondence should be addressed: Molecular Pharmacology, Pfizer Global Research and Development, 2800 Plymouth Rd., Ann Arbor, MI 48105. Tel.: 734-622-7302; Fax: 734-622-1565; E-mail: Shirish.Shenolikar{at}Pfizer.com.

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