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Originally published In Press as doi:10.1074/jbc.M209621200 on February 6, 2003

J. Biol. Chem., Vol. 278, Issue 16, 13819-13828, April 18, 2003
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PNUTS, a Protein Phosphatase 1 (PP1) Nuclear Targeting Subunit
CHARACTERIZATION OF ITS PP1- AND RNA-BINDING DOMAINS AND REGULATION BY PHOSPHORYLATION*

Young-Mi KimDagger , Takuo Watanabe§, Patrick B. Allen§, Young-Myoung Kim||, Shin-Jeong LeeDagger , Paul Greengard§, Angus C. NairnDagger **, and Young-Guen KwonDagger Dagger Dagger

From the Dagger  Department of Biochemistry, College of Natural Sciences and || Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chunchon, Kangwon-Do 200-701, Korea and § Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021

PNUTS, Phosphatase 1 NUclear Targeting Subunit, is a recently described protein that targets protein phosphatase 1 (PP1) to the nucleus. In the present study, we characterized the biochemical properties of PNUTS. A variety of truncation and site-directed mutants of PNUTS was prepared and expressed either as glutathione S-transferase fusion proteins in Escherichia coli or as FLAG-tagged proteins in 293T cells. A 50-amino acid domain in the center of PNUTS mediated both high affinity PP1 binding and inhibition of PP1 activity. The PP1-binding domain is related to a motif found in several other PP1-binding proteins but is distinct in that Trp replaces Phe. Mutation of the Trp residue essentially abolished the ability of PNUTS to bind to and inhibit PP1. The central PP1-binding domain of PNUTS was an effective substrate for protein kinase A in vitro, and phosphorylation substantially reduced the ability of PNUTS to bind to PP1 in vitro and following stimulation of protein kinase A in intact cells. In vitro RNA binding experiments showed that a C-terminal region including several RGG motifs and a novel repeat domain rich in His and Gly interacted with mRNA and single-stranded DNA. PNUTS exhibited selective binding for poly(A) and poly(G) compared with poly(U) or poly(C) ribonucleotide homopolymers, with specificity being mediated by distinct regions within the domain rich in His and Gly and the domain containing the RGG motifs. Finally, a PNUTS-PP1 complex was isolated from mammalian cell lysates using RNA-conjugated beads. Together, these studies support a role for PNUTS in protein kinase A-regulated targeting of PP1 to specific RNA-associated complexes in the nucleus.

* This work was supported by Grant D00359 from the Korea Research Foundation and Vascular System Research Center (to Y.-G. K.), Grant 981-0508-038-2 from the Korea Science and Engineering Foundation (to Y.-G. K.), and by United States Public Health Grant MH40899 (to A. C. N. and P. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Biochemistry and Molecular Vascular Biology, Kanazawa University Graduate School of Medical Science, 13-1, Takara-machi, Kanazawa 920-8640, Japan.

** Present address and to whom correspondence may be addressed: Dept. of Psychiatry, Yale University School of Medicine, New Haven, CT 06508. Tel.: 203-974-7725; Fax: 203-974-7724; E-mail: angus.nairn@yale.edu.

Dagger Dagger To whom correspondence may be addressed: Dept. of Biochemistry, College of Natural Sciences, Kangwon National University, Chunchon, Kangwon-Do 200-701, Korea. Tel.: 82-33-250-8517; Fax: 82-33-242-0459; E-mail: ygkwon@kangwon.ac.kr.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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