The Protein Phosphatase-1 (PP1) Regulator, Nuclear Inhibitor of PP1 (NIPP1), Interacts with the Polycomb Group Protein, Embryonic Ectoderm Development (EED), and Functions as a Transcriptional Repressor

Abstract

The nuclear protein NIPP1 (nuclear inhibitor of protein Ser/Thr phosphatase-1) interacts with the splicing factors SAP155 and CDC5L and is involved in a late step of spliceosome assembly. In addition, NIPP1 is an interactor of protein phosphatase-1 and a COOH-terminal NIPP1 fragment displays an RNase E like endoribonuclease activity. A yeast two-hybrid screening resulted in the identification of the Polycomb group protein EED (embryonic ectoderm development), an established transcriptional repressor, as a novel NIPP1 interactor. NIPP1 only interacted with full-length EED, whereas two EED interaction domains were mapped to the central and COOH-terminal thirds of NIPP1. The NIPP1-EED interaction was potentiated by the binding of (d)Grich nucleic acids to the central domain of NIPP1. Nucleic acids also decreased the potency of NIPP1 as an inhibitor of PP1, but they did not prevent the formation of a ternary NIPP1⅐EED⅐PP1 complex. EED had no effect on the function of NIPP1 as a splicing factor or as an endoribonuclease. However, similar to EED, NIPP1 acted as a transcriptional repressor of targeted genes and this NIPP1 effect was mediated by the EED interaction domain. Also, the histone deacetylase 2 was present in a complex with NIPP1. Our data are in accordance with a role for NIPP1 as a DNA-targeting protein for EED and associated chromatin-modifying enzymes.NIPP1 1 (39 kDa, 351 residues) is a ubiquitously expressed nuclear protein that was originally discovered as a potent and specific inhibitor of protein Ser/Thr phosphatase-1 (PP1), hence its name nuclear inhibitor of PP1 (1). NIPP1 contains binding sites for PP1 in its central and COOH-terminal domains (2,3) and is complexed to 30 -50% of the nuclear pool of PP1 (4). The NIPP1-PP1 holoenzyme in nuclear extracts is inactive but can be activated by the phosphorylation of NIPP1 with protein kinase A (Ser-199), protein kinase CK2 (Ser-204), or protein tyrosine kinases of the Src family (Tyr-335) (3, 5-7). More recently, we demonstrated that NIPP1 is required for a late step in the assembly of spliceosomes (8), which are the complexes of small nuclear RNAs and proteins that catalyze pre-mRNA splicing. The splicing function of NIPP1 depends on the NH 2 -terminal third (residues 1-142) of NIPP1, which largely consists of a forkhead-associated (FHA) domain, and on residues 225-329 in the COOH-terminal domain of NIPP1. The FHA domain of NIPP1 interacts with phosphorylated forms of the splicing factors SAP155 (9) and CDC5L (10), which are components of the U2 small nuclear ribonucleoprotein particle and U4/U5/U6 tri-small nuclear ribonucleoprotein particles, respectively. It is not known how residues 225-329 of NIPP1 contribute to spliceosome assembly. The extreme COOH terminus of NIPP1 (residues 329 -351) does not appear to be involved in spliceosome assembly but has binding sites for PP1 and for A/U-rich single-stranded nucleic acids (3, 11). In addition, a synthetic peptide corresponding to residues 329 -351 as well as NIPP1-(225-351), which may exist as a splice va...

The transcriptional repressor NIPP1 is an essential player in EZH2-mediated gene silencing

“…For example, the NIPP1 ligand and U2 snRNP component SAP155 was recently reported to be required for PcG-mediated gene silencing and to interact with PRC1 components (Isono et al, 2005). Since NIPP1 interacts with PRC2 components (Jin et al, 2003;Roy et al, 2007) as well as with phosphorylated forms of SAP155 (Boudrez et al, 2002), it is tempting to speculate that NIPP1 contributes to interactions between PRC1 and PRC2.…”

“…Since NIPP1 is part of a macromolecular complex that contains EZH2 and EED (Roy et al, 2007), has distinct interaction sites for EED (Jin et al, 2003) and EZH2 (Roy et al, 2007) and is also a nucleic acid-binding protein (Jagiello et al, 1997;Jin et al, 1999), this leads to the enticing hypothesis that NIPP1 plays a role in the targeting of the PRC2 complex to at least a large subset of its target genes. FGF5 ID2 RPS6KC1 TMEM27 AGPAT 4 down C14ORF39 C20ORF103 NMU AGR2 LASS6 LPGAT1 SLC40A1 COL2A1 GJB2 YAF 2 EFCBP1 SLC35F3 STC2 IMPDH1 FZD6 MYT1 WNT1 KCNA1 MSMB Figure 3.…”

“…Furthermore, NIPP1 is required for a late step of spliceosome assembly in nuclear splicing extracts. Interestingly, NIPP1 also interacts with the PRC2 component EED and functions as a transcriptional repressor in transient transfection assays by a mechanism that does not require functional interaction sites for CDC5L, SAP155, MELK or PP1 (Jin et al, 2003).…”

Deactylase Inhibitors Disrupt Cellular Complexes Containing Protein Phosphatases and Deacetylases

“…The indirect recruitment of HDAC2 to a nuclear PP1 complex is also consistent with the recent identification of a multiprotein complex containing NIPP1, a known PP1-binding protein, and the polycomb group protein, EED, also identified as a NIPP1-binding protein. Because EED also bound HDAC2, this generated an HDAC⅐phosphatase complex that potentially repressed the transcription of EED-regulated genes (30). Finally, the recruitment of other phosphatases, such as PP2A, to the DNA-bound CREB complex must also occur via proteins other than HDAC1 because no direct association of HDAC1 with PP2A could be demonstrated.…”

Protein Phosphatase 1 Regulates the Histone Code for Long-Term Memory

“…In yeast, Caenorhabditis elegans, and Xenopus during mitosis, PP1 controls histone 3 (H3) dephosphorylation and can thereby reverse the action of Aurora protein kinases (Hsu et al, 2000;Murnion et al, 2001). In dividing kidney cells, PP1 also influences histone acetylation, through its ability to form a complex with HDAC1 and recruit it to the chromatin (Canettieri et al, 2003;Jin et al, 2003;Brush et al, 2004). Little is known, however, about the role of PP1 in the control of histone PTMs in postmitotic cells such as adult neurons, in particular in the context of memory formation.…”