PNUTS, a protein Phosphatase 1 (PP1) NUclear Targeting Subunit: Characterization of its PP1 and RNA-binding domains and regulation by phosphorylation

Young Mi Kim, Takuo Watanabe, Patrick B. Allen, Young Myoung Kim, Shin Jeong Lee, Paul Greengard, Angus C. Nairn, Young-Guen Kwon

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)13819-13828
Number of pages10
JournalJournal of Biological Chemistry
Volume278
Issue number16
DOIs
Publication statusPublished - 2003 Apr 18

Fingerprint

Protein Phosphatase 1
Phosphorylation
RNA
Cyclic AMP-Dependent Protein Kinases
Carrier Proteins
Poly G
RNA-Binding Motifs
Poly C
Ribonucleotides
Poly U
Proteins
HEK293 Cells
Escherichia coli Proteins
Single-Stranded DNA
Glutathione Transferase
Homopolymerization
Phosphoric Monoester Hydrolases
Escherichia coli
Fusion reactions
Cells

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Kim, Young Mi ; Watanabe, Takuo ; Allen, Patrick B. ; Kim, Young Myoung ; Lee, Shin Jeong ; Greengard, Paul ; Nairn, Angus C. ; Kwon, Young-Guen. / PNUTS, a protein Phosphatase 1 (PP1) NUclear Targeting Subunit : Characterization of its PP1 and RNA-binding domains and regulation by phosphorylation. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 16. pp. 13819-13828.
@article{64cc1857c3104913a60de33d245a4336,
title = "PNUTS, a protein Phosphatase 1 (PP1) NUclear Targeting Subunit: Characterization of its PP1 and RNA-binding domains and regulation by phosphorylation",
abstract = "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.",
author = "Kim, {Young Mi} and Takuo Watanabe and Allen, {Patrick B.} and Kim, {Young Myoung} and Lee, {Shin Jeong} and Paul Greengard and Nairn, {Angus C.} and Young-Guen Kwon",
year = "2003",
month = "4",
day = "18",
doi = "10.1074/jbc.M209621200",
language = "English",
volume = "278",
pages = "13819--13828",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "16",

}

PNUTS, a protein Phosphatase 1 (PP1) NUclear Targeting Subunit : Characterization of its PP1 and RNA-binding domains and regulation by phosphorylation. / Kim, Young Mi; Watanabe, Takuo; Allen, Patrick B.; Kim, Young Myoung; Lee, Shin Jeong; Greengard, Paul; Nairn, Angus C.; Kwon, Young-Guen.

In: Journal of Biological Chemistry, Vol. 278, No. 16, 18.04.2003, p. 13819-13828.

Research output: Contribution to journalArticle

TY - JOUR

T1 - PNUTS, a protein Phosphatase 1 (PP1) NUclear Targeting Subunit

T2 - Characterization of its PP1 and RNA-binding domains and regulation by phosphorylation

AU - Kim, Young Mi

AU - Watanabe, Takuo

AU - Allen, Patrick B.

AU - Kim, Young Myoung

AU - Lee, Shin Jeong

AU - Greengard, Paul

AU - Nairn, Angus C.

AU - Kwon, Young-Guen

PY - 2003/4/18

Y1 - 2003/4/18

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0037515628&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037515628&partnerID=8YFLogxK

U2 - 10.1074/jbc.M209621200

DO - 10.1074/jbc.M209621200

M3 - Article

C2 - 12574161

AN - SCOPUS:0037515628

VL - 278

SP - 13819

EP - 13828

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 16

ER -