ABSTRACT
NIPP1 is a ubiquitously expressed nuclear protein that represses the transcription of targeted genes. Here we show that the transcriptional repression by NIPP1 is alleviated by the RNAi-mediated knockdown of EED and EZH2, two core components of the Polycomb Repressive Complex 2 (PRC2), and by the overexpression of a catalytically dead mutant of the histone methyltransferase EZH2. NIPP1 is present in a complex with EED and EZH2 in vivo and has distinct binding sites for these proteins. These data disclose an essential role for the PRC2 complex in the transcriptional repression by NIPP1.
Subject(s)
DNA-Binding Proteins/metabolism , Endoribonucleases/metabolism , Multiprotein Complexes/metabolism , Phosphoprotein Phosphatases/metabolism , RNA-Binding Proteins/metabolism , Repressor Proteins/metabolism , Transcription Factors/metabolism , Transcription, Genetic/physiology , Binding Sites/genetics , Cell Line, Tumor , DNA-Binding Proteins/genetics , Endoribonucleases/genetics , Enhancer of Zeste Homolog 2 Protein , Humans , Multiprotein Complexes/genetics , Phosphoprotein Phosphatases/genetics , Polycomb Repressive Complex 2 , Polycomb-Group Proteins , Protein Binding/genetics , RNA-Binding Proteins/genetics , Repressor Proteins/genetics , Transcription Factors/geneticsABSTRACT
Protein Ser/Thr phosphatase-1 (PP1) is a ubiquitous eukaryotic enzyme that controls numerous cellular processes by the dephosphorylation of key regulatory proteins. PP1 is expressed in various cellular compartments but is most abundant in the nucleus. We have examined the determinants for the nuclear localization of enhanced green fluorescent protein-tagged PP1 in COS1 cells. Our studies show that PP1gamma(1) does not contain a functional nuclear localization signal and that its nuclear accumulation does not require Sds22, which has previously been implicated in the nuclear accumulation of PP1 in yeast (Peggie, M. W., MacKelvie, S. H., Bloecher, A., Knatko, E. V., Tatchell, K., and Stark, M. J. R. (2002) J. Cell Sci. 115, 195-206). However, the nuclear targeting of PP1 isoforms was alleviated by the mutation of their binding sites for proteins that interact via an RVXF motif. Moreover, one of the mutants with a cytoplasmic accumulation and decreased affinity for RVXF motifs (PP1gamma(1)-F257A) could be re-targeted to the nucleus by the overexpression of nuclear interactors (NIPP1 (nuclear inhibitor of PP1) and PNUTS (PP1 nuclear targeting subunit)) with a functional RVXF motif. Also, the addition of a synthetic RVXF-containing peptide to permeabilized cells resulted in the loss of nuclear enhanced green fluorescent protein-PP1gamma(1). Finally, NIPP1(-/-) mouse embryos showed a nuclear hyperphosphorylation on threonine, consistent with a role for NIPP1 in the nuclear targeting and/or retention of PP1. Our data suggest that both the nuclear translocation and the nuclear retention of PP1 depend on its binding to interactors with an RVXF motif.
Subject(s)
Active Transport, Cell Nucleus , Intracellular Signaling Peptides and Proteins/physiology , Phosphoprotein Phosphatases/metabolism , Amino Acid Motifs , Animals , Binding Sites , COS Cells , Cell Nucleus/metabolism , Dose-Response Relationship, Drug , Endoribonucleases/physiology , Glutathione Transferase/metabolism , Green Fluorescent Proteins/metabolism , HeLa Cells , Histidine/chemistry , Humans , Immunohistochemistry , Immunoprecipitation , Intracellular Signaling Peptides and Proteins/chemistry , Ligands , Mice , Microscopy, Fluorescence , Mutagenesis, Site-Directed , Mutation , Nuclear Localization Signals , Phosphorylation , Protein Binding , Protein Isoforms , Protein Phosphatase 1 , Protein Transport , RNA-Binding Proteins/physiology , Rabbits , Rats , Recombinant Proteins/chemistry , Threonine/chemistryABSTRACT
NIPP1 (nuclear inhibitor of protein phosphatase 1) is a ubiquitously expressed nuclear scaffold protein that has been implicated in both transcription and RNA processing. Among its protein ligands are a protein kinase, a protein phosphatase, two splicing factors, and a transcriptional regulator, and the binding of these proteins to NIPP1 is tightly regulated by phosphorylation. To study the function of NIPP1 in vivo, we have used homologous recombination to generate mice that are deficient in NIPP1. NIPP1(-/+) mice developed normally. However, NIPP1(-/-) embryos showed severely retarded growth at embryonic day 6.5 (E6.5) and were resorbed by E8.5. This early embryonic lethality was not associated with increased apoptosis but correlated with impaired cell proliferation. Blastocyst outgrowth experiments and the RNA interference-mediated knockdown of NIPP1 in cultured cells also revealed an essential role for NIPP1 in cell proliferation. In further agreement with this function, no viable NIPP1(-/-) cell lines were obtained by derivation of embryonic stem (ES) cells from blastocysts of NIPP1(-/+) intercrosses or by forced homogenotization of heterozygous ES cells at high concentrations of Geneticin. We conclude that NIPP1 is indispensable for early embryonic development and cell proliferation.
