PNUTS, a protein phosphatase 1 (PP1) nuclear targeting subunit. Characterization of its PP1- and RNA-binding domains and regulation by phosphorylation.
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.
Kim
Young-Mi
YM
Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chunchon, Kangwon-Do 200-701, Korea.
Watanabe
Takuo
T
Allen
Patrick B
PB
Kim
Young-Myoung
YM
Lee
Shin-Jeong
SJ
Greengard
Paul
P
Nairn
Angus C
AC
Kwon
Young-Guen
YG
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology* / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry* / Q000302:isolation & purification; D016716:PC12 Cells; D010455:Peptides / Q000737:chemistry; D010649:Phenylalanine / Q000737:chemistry; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D054645:Protein Phosphatase 1; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012685:Sepharose / Q000494:pharmacology; D014158:Transcription, Genetic; D014364:Tryptophan / Q000737:chemistry
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C110225:PPP1R10 protein, human; D010455:Peptides; D011108:Polymers; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D010649:Phenylalanine; D012313:RNA; D014364:Tryptophan; D012685:Sepharose; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1