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

EZH2 is a Polycomb group (PcG) protein that promotes the late-stage development of cancer by silencing a specific set of genes, at least in part through trimethylation of associated histone H3 on Lys 27 (H3K27). Nuclear inhibitor of protein phosphatase-1 (NIPP1) is a ubiquitously expressed transcriptional repressor that has binding sites for the EZH2 interactor EED. Here, we examine the contribution of NIPP1 to EZH2-mediated gene silencing. Studies on NIPP1-deficient cells disclose a widespread and essential role of NIPP1 in the trimethylation of H3K27 by EZH2, not only in the onset of this trimethylation during embryonic development, but also in the maintenance of this repressive mark in proliferating cells. Consistent with this notion, EZH2 and NIPP1 silence a common set of genes, as revealed by gene-expression profiling, and NIPP1 is associated with established Polycomb target genes and with genomic regions that are enriched in Polycomb targets. Furthermore, most NIPP1 target genes are trimethylated on H3K27 and the knockdown of either NIPP1 or EZH2 is often associated with a loss of this modification. Our data reveal that NIPP1 is required for the global trimethylation of H3K27 and is implicated in gene silencing by EZH2.

The gene encoding the prostatic tumor suppressor PSP94 is a target for repression by the Polycomb group protein EZH2.

PSP94, for prostatic secretory protein of 94 amino acids, is secreted by the prostate gland and functions as a suppressor of tumor growth and metastasis. The expression of PSP94 is lost in advanced, hormone-refractory prostate cancer and this correlates with an increased expression of the Polycomb protein EZH2 (enhancer of zeste homolog 2), which represses transcription via trimethylation of histone H3 on Lys27 (H3K27). We show here that these events are causally related and that the MSMB gene, which encodes PSP94, is trimethylated on H3K27 in androgen-refractory, but not in androgen-sensitive prostate cancer cells. Chromatin immunoprecipitation experiments confirmed an association of EZH2 with the MSMB gene. The RNAi-mediated knockdown of EZH2 resulted in a loss of H3K27 trimethylation and an increased expression of the MSMB gene. Conversely, the overexpression of EZH2 was associated with a decreased expression of the MSMB gene. We also demonstrate that MSMB is additionally repressed in androgen-refractory prostate cancer cells by the hypoacetylation of histone H3K9 and the hypermethylation of a CpG island in the promoter region. Our data disclose a hitherto unexplored link between the putative oncogene EZH2 and the tumor suppressor PSP94, and show that MSMB is silenced by EZH2 in advanced prostate cancer cells.

Progressive versus constant tapered shaft design using NiTi rotary instruments.

AIM: To evaluate the influence of a progressive versus constant tapered shaft design on canal preparation by NiTi rotary techniques. METHODOLOGY: A XMCT-scanner and custom-made software were used to nondestructively analyse the mesial canals of 10 extracted mandibular molars in 3D with a spatial resolution of 12.5 microm. Specimens (n = 10 per group) were scanned before and after preparation using ProTaper (progressive tapered) or K3 (constant tapered) files. Numerical values for volume, curvature, dentine removal and centring ratio were obtained in addition to a visual inspection for canal aberrations. Data were analysed by (multiway factorial) anova, Wilcoxon tests and t-tests. RESULTS: The volume of total dentine removal (mean +/- SD) was 1.21 +/- 0.66 mm(3) (ProTaper) and 1.06 +/- 0.23 mm(3) (K3) (P > 0.05), and the amount of dentine removal at all separate horizontal regions examined was comparable for both groups. The mean linear dentine removal (transportation) was in the range of 8-212 microm (ProTaper) and 4-187 microm (K3). The resultant centring ratio varied from 0.01 to 0.24 (ProTaper) and from 0.01 to 0.17 (K3), whilst different straightening patterns were observed. A centre displacement towards the furcation coronally was most pronounced for the ProTaper group whereas a centre displacement towards the outer side of the curvature more apically was only observed for the K3 group. No severe canal aberrations were found. CONCLUSIONS: The progressive tapered shaft design of the ProTaper instrument was less influenced by the mid-root curvature than the constant tapered design of the K3 instrument thereby providing a good centred apical preparation. However, ProTaper instruments tended to transport towards the furcation in the coronal region.

Smooth flexible versus active tapered shaft design using NiTi rotary instruments.

AIM: The aim of this study was to evaluate the influence of a smooth flexible versus active tapered shaft design on canal preparation by NiTi rotary techniques. METHODOLOGY: A XMCT-scanner (SkyScan 1072) and developed software (Bergmans et al. 2001) were used to nondestructively analyze the mesial canals of 10 extracted mandibular molars in 3D with a spatial resolution of 30 microm. Specimens (n = 10 per group) were scanned before (PRE) and after (POST) preparation using Lightspeed (smooth flexible) or GT-rotary (active tapered) files. Numerical values for volumes, dentine removal (net) transportation and centring ability were obtained in addition to a visual inspection on canal aberrations. Data were analyzed by Shapiro Wilk test, multiway factorial anova, Tukey-Kramer test, Wilcoxon test and t-test. RESULTS: Results indicated that the active tapered shaft removed significantly more dentine in the middle to apical portion of the root compared to the smooth flexible design. Both groups demonstrated some straightening, but no significant differences were found with respect to instrument types. However, absolute values for net transportation and centering ratio were small and no canal aberrations could be found. CONCLUSIONS: The smooth flexible shaft design did not improve the morphological characteristics of canal preparation by NiTi rotary instruments when compared with the active tapered design. Therefore, system selection should be based upon other criteria.

The C-terminus of NIPP1 (nuclear inhibitor of protein phosphatase-1) contains a novel binding site for protein phosphatase-1 that is controlled by tyrosine phosphorylation and RNA binding.

Nuclear inhibitor of protein phosphatase-1 (NIPP1; 351 residues) is a nuclear RNA-binding protein that also contains in its central domain two contiguous sites of interaction with the catalytic subunit of protein phosphatase-1 (PP1(C)). We show here that mutation of these phosphatase-interaction sites did not completely abolish the ability of NIPP1 to bind and inhibit PP1(C). This could be accounted for by an additional inhibitory phosphatase-binding site in the C-terminal region (residues 311-351), with an inhibitory core corresponding to residues 331-337. Following mutation of all three PP1(C)-binding sites in the central and C-terminal domains, NIPP1 no longer interacted with PP1(C). Remarkably, while both NIPP1 domains inhibited the phosphorylase phosphatase activity of PP1(C) independently, mutation of either domain completely abolished the ability of NIPP1 to inhibit the dephosphorylation of myelin basic protein. The inhibitory potency of the C-terminal site of NIPP1 was decreased by phosphorylation of Tyr-335 and by the addition of RNA. Tyr-335 could be phosphorylated by tyrosine kinase Lyn, but only in the presence of RNA. In conclusion, NIPP1 contains two phosphatase-binding domains that function co-operatively but which are controlled independently. Our data are in agreement with a shared-site model for the interaction of PP1(C) with its regulatory subunits.

Nuclear and subnuclear targeting sequences of the protein phosphatase-1 regulator NIPP1.

NIPP1 is a nuclear subunit of protein phosphatase-1 (PP1) that colocalizes with pre-mRNA splicing factors in speckles. We report here that the nuclear and subnuclear targeting of NIPP1, when expressed in HeLa cells or COS-1 cells as a fusion protein with the enhanced-green-fluorescent protein (EGFP), are mediated by distinct sequences. While NIPP1-EGFP can cross the nuclear membrane passively, the active transport to the nucleus is mediated by two independent nuclear localization signals in the central domain of NIPP1, which partially overlap with binding site(s) for PP1. Furthermore, the concentration of NIPP1-EGFP in the nuclear speckles requires the 'ForkHead-Associated' domain in the N terminus. This domain is also required for the nuclear retention of NIPP1 when active transport is blocked. Our data imply that the nuclear and subnuclear targeting of NIPP1 are controlled independently.

NIPP1-mediated interaction of protein phosphatase-1 with CDC5L, a regulator of pre-mRNA splicing and mitotic entry.

NIPP1 is a regulatory subunit of a species of protein phosphatase-1 (PP1) that co-localizes with splicing factors in nuclear speckles. We report that the N-terminal third of NIPP1 largely consists of a Forkhead-associated (FHA) protein interaction domain, a known phosphopeptide interaction module. A yeast two-hybrid screening revealed an interaction between this domain and a human homolog (CDC5L) of the fission yeast protein cdc5, which is required for G(2)/M progression and pre-mRNA splicing. CDC5L and NIPP1 co-localized in nuclear speckles in COS-1 cells. Furthermore, an interaction between CDC5L, NIPP1, and PP1 in rat liver nuclear extracts could be demonstrated by co-immunoprecipitation and/or co-purification experiments. The binding of the FHA domain of NIPP1 to CDC5L was dependent on the phosphorylation of CDC5L, e.g. by cyclin E-Cdk2. When expressed in COS-1 or HeLa cells, the FHA domain of NIPP1 did not affect the number of cells in the G(2)/M transition. However, the FHA domain blocked beta-globin pre-mRNA splicing in nuclear extracts. A mutation in the FHA domain that abolished its interaction with CDC5L also canceled its anti-splicing effects. We suggest that NIPP1 either targets CDC5L or an associated protein for dephosphorylation by PP1 or serves as an anchor for both PP1 and CDC5L.

Mapping of the RNA-binding and endoribonuclease domains of NIPP1, a nuclear targeting subunit of protein phosphatase 1.

NIPP1 (351 residues) is a major regulatory and RNA-anchoring subunit of protein phosphatase 1 in the nucleus. Using recombinant and synthetic fragments of NIPP1, the RNA-binding domain was mapped to the C-terminal residues 330-351. A synthetic peptide encompassing this sequence equalled intact NIPP1 in RNA-binding affinity and could be used to dissociate NIPP1 from the nuclear particulate fraction. An NIPP1 fragment consisting of residues 225-351 (Ard1/NIPP1gamma), that may be encoded by an alternatively spliced transcript in transformed B-lymphocytes, displayed a single-strand Mg(2+)-dependent endoribonuclease activity. However, full-length NIPP1 and NIPP1(143-351) were not able to cleave RNA, indicating that the endoribonuclease activity of NIPP1 is restrained by its central domain. The endoribonuclease activity was also recovered in the RNA-binding domain, NIPP1(330-351), but with a 30-fold lower specific activity. Thus, the endoribonuclease catalytic site and the RNA-binding site both reside in the C-terminal 22 residues of NIPP1. The latter domain does not conform to any known nucleic-acid binding motif.

Identification of MYPT1 and NIPP1 as subunits of protein phosphatase 1 in rat liver cytosol.

Various studies have provided evidence for the existence of spontaneously active cytosolic species of protein phosphatase 1, but these enzymes have never been purified and characterized. We have used chromatography on microcystin-Sepharose and Resource Q to purify cytosolic protein phosphatases from rat liver. Two of the isolated enzymes were identified by Western analysis and peptide sequencing as complexes of the catalytic subunit of protein phosphatase 1 and either the inhibitor NIPP1 or the myosin-binding subunit MYPT1, which reportedly is not present in chicken liver. In contrast, PCR cloning revealed the expression of two MYPT1 splice variants in rat liver.

Molecular determinants of nuclear protein phosphatase-1 regulation by NIPP-1.

NIPP-1 is a subunit of the major nuclear protein phosphatase-1 (PP-1) in mammalian cells and potently inhibits PP-1 activity in vitro. Using yeast two-hybrid and co-sedimentation assays, we mapped a PP-1-binding site and the inhibition function to the central one-third domain of NIPP-1. Full-length NIPP-1 (351 residues) and the central domain, NIPP-1(143-217), were equally potent PP-1 inhibitors (IC50 = 0.3 nM). Synthetic peptides spanning the central domain of NIPP-1 further narrowed the PP-1 inhibitory function to residues 191-200. A second, noninhibitory PP-1-binding site was identified by far-Western assays with digoxygenin-conjugated catalytic subunit (PP-1C) and included a consensus RVXF motif (residues 200-203) found in many other PP-1-binding proteins. The substitutions, V201A and/or F203A, in the RVXF motif, or phosphorylation of Ser199 or Ser204, which are established phosphorylation sites for protein kinase A and protein kinase CK2, respectively, prevented PP-1C-binding by NIPP-1(191-210) in the far-Western assay. NIPP-1(191-210) competed for PP-1 inhibition by full-length NIPP-1(1-351), inhibitor-1 and inhibitor-2, and dissociated PP-1C from inhibitor-1- and NIPP-1(143-217)-Sepharose but not from full-length NIPP-1(1-351)-Sepharose. Together, these data identified some of the key elements in the central domain of NIPP-1 that regulate PP-1 activity and suggested that the flanking sequences stabilize the association of NIPP-1 with PP-1C.

Structure and splice products of the human gene encoding sds22, a putative mitotic regulator of protein phosphatase-1.

sds22 is a regulatory subunit of protein phosphatase-1 that is required for the completion of mitosis in yeast. It consists largely of 11 tandem leucine-rich repeats of 22 residues that are expected to mediate interactions with other polypeptides, including protein phosphatase-1. In this paper, we report on the structure of the human gene encoding sds22, designated PPP1R7. This gene (33 kb) comprises 11 exons, but these do not coincide with the sequences encoding the leucine-rich repeats. Up to six splice variants can be generated by exon skipping and alternative polyadenylation, as revealed by expressed sequence tag database analysis, RT-PCR and Northern blot analysis. The sds22 transcripts are expected to encode four different polypeptides. sds22alpha1 corresponds to the variant cloned previously from human brain [Renouf et al. (1995) FEBS Lett. 375, 75-78]. Sds22beta1 is truncated within the ninth repeat and has a short and different C-terminus. Both variants also exist without the sequence corresponding to exon 2, and these are termed sds22alpha2 and sds22beta2. The 5'-flanking region of PPP1R7 contains two NF-Y-binding CCAAT boxes near the transcription start site and potential binding sites for the transcription factors c-Myb, Ik-2 and NF-1, which are conserved in the mouse gene.

NIPP-1, a nuclear inhibitory subunit of protein phosphatase-1, has RNA-binding properties.

NIPP-1 is a nuclear inhibitory subunit of protein phosphatase-1 with structural similarities to some proteins involved in RNA processing. We report here that baculovirus-expressed recombinant NIPP-1 displays RNA-binding properties, as revealed by North-Western analysis, by UV-mediated cross-linking, by RNA mobility-shift assays, and by chromatography on poly(U)-Sepharose. NIPP-1 preferentially bound to U-rich sequences, including RNA-destabilizing AUUUA motifs. NIPP-1 also associated with single-stranded DNA, but had no affinity for double-stranded DNA. The binding of NIPP-1 to RNA was blocked by antibodies directed against the COOH terminus of NIPP-1, but was not affected by prior phosphorylation of NIPP-1 with protein kinase A or casein kinase-2, which decreases the affinity of NIPP-1 for protein phosphatase-1. The catalytic subunit of protein phosphatase-1 did not bind to poly(U)-Sepharose, but it bound very tightly after complexation with NIPP-1. These data are in agreement with a function of NIPP-1 in targeting protein phosphatase-1 to RNA.

Identification of sds22 as an inhibitory subunit of protein phosphatase-1 in rat liver nuclei.

sds22 was originally identified in yeast as a regulator of protein phosphatase-1 that is essential for the completion of mitosis. We show here that a structurally related mammalian polypeptide (41.6 kDa) is part of a 260-kDa species of protein phosphatase-1. This holoenzyme, designated PP-1N(sds22), could be immunoprecipitated with sds22 antibodies and was retained by microcystin-Sepharose. PP-1N(sds22) is a latent phosphatase, but its activity could be revealed by the proteolytic destruction of the noncatalytic subunit(s). PP-1N(sds22) accounted for only 5-10% of the total activity of PP-1 in rat liver nuclear extracts. A synthetic 22-mer peptide, corresponding to a leucine-rich repeat of sds22, specifically inhibited the catalytic subunit of PP-1, showing that at least part of the latency stems from the interaction of the sds22 repeat(s) with PP-1C.

Phosphorylation, subcellular localization, and membrane orientation of the Alzheimer's disease-associated presenilins.

Presenilins 1 and 2 are unglycosylated proteins with apparent molecular mass of 45 and 50 kDa, respectively, in transfected COS-1 and Chinese hamster ovary cells. They colocalize with proteins from the endoplasmic reticulum and the Golgi apparatus in transfected and untransfected cells. In COS-1 cells low amounts of intact endogeneous presenilin 1 migrating at 45 kDa are detected together with relative larger amounts of presenilin 1 fragments migrating between 18 and 30 kDa. The presenilins have a strong tendency to form aggregates (mass of 100-250 kDa) in SDS-polyacrylamide gel electrophoresis, which can be partially resolved when denatured by SDS at 37 degrees C instead of 95 degrees C. Sulfation, glycosaminoglycan modification, or acylation of the presenilins was not observed, but both proteins are posttranslationally phosphorylated on serine residues. The mutations Ala-246 --> Glu or Cys-410 --> Tyr that cause Alzheimer's disease do not interfere with the biosynthesis or phosphorylation of presenilin 1. Finally, using low concentrations of digitonin to selectively permeabilize the cell membrane but not the endoplasmic reticulum membrane, it is demonstrated that the two major hydrophilic domains of presenilin 1 are oriented to the cytoplasm. The current investigation documents the posttranslational modifications and subcellular localization of the presenilins and indicates that postulated interactions with amyloid precursor protein metabolism should occur in the early compartments of the biosynthetic pathway.

Properties and phosphorylation sites of baculovirus-expressed nuclear inhibitor of protein phosphatase-1 (NIPP-1).

NIPP-1 is the RNA-binding subunit of a major species of protein phosphatase-1 in the nucleus. We have expressed nuclear inhibitor of protein phosphatase-1 (NIPP-1) in Sf9 cells, using the baculovirus-expression system. The purified recombinant protein was a potent (Ki = 9.9 +/- 0.3 pM) and specific inhibitor of protein phosphatase-1 and was stoichiometrically phosphorylated by protein kinases A and CK2. At physiological ionic strength, phosphorylation by these protein kinases drastically decreased the inhibitory potency of free NIPP-1. Phosphorylation of NIPP-1 in a heterodimeric complex with the catalytic subunit of protein phosphatase-1 resulted in an activation of the holoenzyme without a release of NIPP-1. Sequencing and phosphoamino acid analysis of tryptic phosphopeptides enabled us to identify Ser178 and Ser199 as the phosphorylation sites of protein kinase A, whereas Thr161 and Ser204 were phosphorylated by protein kinase CK2. These residues all conform to consensus recognition sites for phosphorylation by protein kinases A or CK2 and are clustered near a RVXF sequence that has been identified as a motif that interacts with the catalytic subunit of protein phosphatase-1.

Characterization of a ribosomal inhibitory polypeptide of protein phosphatase-1 from rat liver.

About 4% of the spontaneous phosphorylase phosphatase activity in a rat liver extract was associated with the ribosomal fraction and stemmed from both protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A). However, after repeated washing, only PP-1 remained bound to the ribosomes. The activity of ribosome-associated PP-1 (PP-1R) was partially latent and could be increased 2-3-fold by incubation with trypsin and an additional 50% by incubation with low concentrations of exogenous type-1 catalytic subunit. In contrast, incubation of the ribosomal fraction with MgATP resulted in a 50% drop in the activity of PP-1R. We have purified from a ribosomal extract a basic polypeptide (pI > or = 10.5) of 23 kDa that potently inhibited PP-1. This ribosomal inhibitor of PP-1, termed RIPP-1, was at least 30-times less efficient in inhibiting other major Ser/Thr protein phosphatases (PP-2A, PP-2B and PP-2C). RIPP-1 was identified as a non-competitive inhibitor of PP-1 with a substrate-dependent potency. The lowest Ki (approximately 20 nM) was obtained with phosphorylase and myelin basic protein as substrates. Besides instantaneously inhibiting the type-1 catalytic subunit, RIPP-1 also converted the catalytic subunit in a time-dependent manner (t 1/2 = 45 min at 25 degrees C) into a less active conformation. Unlike the inhibition, this slow inactivation was not reversed by the removal of RIPP-1. We propose that RIPP-1 accounts, at least in part, for the latency of PP-1R.

Molecular cloning of NIPP-1, a nuclear inhibitor of protein phosphatase-1, reveals homology with polypeptides involved in RNA processing.

NIPP-1 was originally isolated as a potent and specific nuclear inhibitory polypeptide (16-18 kDa) of protein phosphatase-1. We report here the cDNA cloning of NIPP-1 from bovine thymus and show that the native polypeptide consists of 351 residues and has a calculated mass of 38.5 kDa. The bacterially expressed central third of NIPP-1 completely inhibited the type-1 catalytic subunit, but displayed a reduced inhibitory potency after phosphorylation by protein kinase A and casein kinase 2. Translation of NIPP-1 mRNA in reticulocyte lysates resulted in the accumulation of both intact NIPP-1 and a smaller polypeptide generated by alternative initiation at the codon corresponding to Met143. A data base search showed that the COOH terminus of NIPP-1 is nearly identical to the human ard-1 protein (13 kDa), which has been implicated in RNA processing (Wang, M., and Cohen, S. N. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10591-10595). Comparison of the cDNAs encoding ard-1 and NIPP-1 suggests that their mRNAs are generated by alternative splicing of the same pre-mRNA. Western blotting with antibodies against the COOH terminus of NIPP-1, however, showed a single polypeptide of 47 kDa, which was enriched in the nucleus. Northern analysis revealed a single transcript of 2.2 kilobases in bovine thymus and of 2.4 kilobases in various human tissues.

Molecular cloning of a human polypeptide related to yeast sds22, a regulator of protein phosphatase-1.

sds22 is a regulatory polypeptide of protein phosphatase-1 that is required for the completion of mitosis in both fission and budding yeast. We report here the cDNA cloning of a human polypeptide that is 46% identical to yeast sds22. The human homolog of sds22 consists of 360 residues, has a calculated molecular mass of 41.6 kDa and shows a tandem array of 11 leucine-rich repeat structures of 22 residues. Northern analysis revealed a major transcript of 1.39 kb in all 8 investigated human tissues. sds22 was detected by western analysis in both the cytoplasm and the nucleus of rat liver cells as a polypeptide of 44 kDa.

Subunit structure and regulation of protein phosphatase-1 in rat liver nuclei.

The activity of protein phosphatase-1 in rat liver nuclei (PP-1N) was decreased by up to 97% by associated inhibitory polypeptides, depending on the assay and extraction conditions. These inhibitors were rapidly degraded by endogenous proteases, resulting in the accumulation of active heat-stable intermediates. Two major species of PP-1N could be differentiated by fractionation of a nuclear extract. PP-1NR111 contained, besides the delta-isoform of the catalytic subunit, an inhibitory polypeptide of 111 kDa. PP-1NR41 was found to be an inactive heterodimer between the delta-isoform of the catalytic subunit and NIPP-1, a nuclear inhibitor of PP-1, which in its undegraded form is heat labile and migrates during SDS-polyacrylamide gel electrophoresis as a polypeptide of 41 kDa. Native hepatic NIPP-1 displayed a reduced affinity for the catalytic subunit after phosphorylation by protein kinase A in vitro and after glucagon-induced phosphorylation in vivo.