The level of the glycogen targetting regulatory subunit R5 of protein phosphatase 1 is decreased in the livers of insulin-dependent diabetic rats and starved rats.

Hepatic glycogen synthesis is impaired in insulin-dependent diabetic rats owing to defective activation of glycogen synthase by glycogen-bound protein phosphatase 1 (PP1). The identification of three glycogen-targetting subunits in liver, G(L), R5/PTG and R6, which form complexes with the catalytic subunit of PP1 (PP1c), raises the question of whether some or all of these PP1c complexes are subject to regulation by insulin. In liver lysates of control rats, R5 and R6 complexes with PP1c were found to contribute significantly (16 and 21% respectively) to the phosphorylase phosphatase activity associated with the glycogen-targetting subunits, G(L)-PP1c accounting for the remainder (63%). In liver lysates of insulin-dependent diabetic and of starved rats, the phosphorylase phosphatase activities of the R5 and G(L) complexes with PP1c were shown by specific immunoadsorption assays to be substantially decreased, and the levels of R5 and G(L) were shown by immunoblotting to be much lower than those in control extracts. The phosphorylase phosphatase activity of R6-PP1c and the concentration of R6 protein were unaffected by these treatments. Insulin administration to diabetic rats restored the levels of R5 and G(L) and their associated activities. The regulation of R5 protein levels by insulin was shown to correspond to changes in the level of the mRNA, as has been found for G(L). The in vitro glycogen synthase phosphatase/phosphorylase phosphatase activity ratio of R5-PP1c was lower than that of G(L)-PP1c, suggesting that R5-PP1c may function as a hepatic phosphorylase phosphatase, whereas G(L)-PP1c may be the major hepatic glycogen synthase phosphatase. In hepatic lysates, more than half the R6 was present in the glycogen-free supernatant, suggesting that R6 may have lower affinity for glycogen than R5 and G(L)

Structural and catalytic similarities between nucleotide pyrophosphatases/phosphodiesterases and alkaline phosphatases.

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) generate nucleoside 5'-monophosphates from a variety of nucleotides and their derivatives. Here we show by data base analysis that these enzymes are conserved from eubacteria to higher eukaryotes. We also provide evidence for the existence of two additional members of the mammalian family of ecto-NPPs. Homology searches and alignment-assisted mutagenesis revealed that the catalytic core of NPPs assumes a fold similar to that of a superfamily of phospho-/sulfo-coordinating metalloenzymes comprising alkaline phosphatases, phosphoglycerate mutases, and arysulfatases. Mutation of mouse NPP1 in some of its predicted metal-coordinating residues (D358N or H362Q) or in the catalytic site threonine (T238S) resulted in an enzyme that could still form the nucleotidylated catalytic intermediate but was hampered in the second step of catalysis. We also obtained data indicating that the ability of some mammalian NPPs to auto(de)phosphorylate is due to an intrinsic phosphatase activity, whereby the enzyme phosphorylated on Thr-238 represents the covalent intermediate of the phosphatase reaction. The results of site-directed mutagenesis suggested that the nucleotide pyrophosphatase/phosphodiesterase and the phosphatase activities of NPPs are mediated by a single catalytic site.

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.

Molecular mode of inhibition of glycogenolysis in rat liver by the dihydropyridine derivative, BAY R3401: inhibition and inactivation of glycogen phosphorylase by an activated metabolite.

The racemic prodrug BAY R3401 suppresses hepatic glycogenolysis. BAY W1807, the active metabolite of BAY R3401, inhibits muscle glycogen phosphorylase a and b. We investigated whether BAY R3401 reduces hepatic glycogenolysis by allosteric inhibition or by phosphatase-catalyzed inactivation of phosphorylase. In gel-filtered liver extracts, racemic BAY U6751 (containing active BAY W1807) was tested for inhibition of phosphorylase in the glycogenolytic (in which only phosphorylase a is active) and glycogen-synthetic (for the evaluation of a:b ratios) directions. Phosphorylase inactivation by endogenous phosphatase was also studied. In liver extracts, BAY U6751 (0.9-36 micromol/l) inhibited glycogen synthesis by phosphorylase b (notwithstanding the inclusion of AMP), but not by phosphorylase a. Inhibition of phosphorylase-a-catalyzed glycogenolysis was partially relieved by AMP (500 micromol/l). BAY U6751 facilitated phosphorylase-a dephosphorylation. Isolated hepatocytes and perfused livers were tested for BAY R3401-induced changes in phosphorylase-a:b ratios and glycogenolytic output. Though ineffective in extracts, BAY R3401 (0.25 micromol/l-0.5 mmol/l) promoted phosphorylase-a dephosphorylation in hepatocytes. In perfused livers exposed to dibutyryl cAMP (100 micromol/l) for maximal activation of phosphorylase, BAY R3401 (125 micromol/l) inactivated phosphorylase by 63% but glucose output dropped by 83%. Inhibition of glycogenolysis suppressed glucose-6-phosphate (G6P) levels. Activation of glycogen synthase after phosphorylase inactivation depended on the maintenance of G6P levels by supplementing glucose (50 mmol/l). We conclude that the metabolites of BAY R3401 suppress hepatic glycogenolysis by allosteric inhibition and by the dephosphorylation of phosphorylase a.

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.

Nucleotide pyrophosphatases/phosphodiesterases on the move.

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) release nucleoside 5'-monophosphates from nucleotides and their derivatives. They exist both as membrane proteins, with an extracellular active site, and as soluble proteins in body fluids. The only well-characterized NPPs are the mammalian ecto-enzymes NPP1 (PC-1), NPP2 (autotaxin) and NPP3 (B10; gp130(RB13-6)). These are modular proteins consisting of a short N-terminal intracellular domain, a single transmembrane domain, two somatomedin-B-like domains, a catalytic domain, and a C-terminal nuclease-like domain. The catalytic domain of NPPs is conserved from prokaryotes to mammals and shows remarkable structural and catalytic similarities with the catalytic domain of other phospho-/sulfo-coordinating enzymes such as alkaline phosphatases. Hydrolysis of pyrophosphate/phosphodiester bonds by NPPs occurs via a nucleotidylated threonine. NPPs are also known to auto(de)phosphorylate this active-site threonine, a process accounted for by an intrinsic phosphatase activity, with the phosphorylated enzyme representing the catalytic intermediate of the phosphatase reaction. NPP1-3 have been implicated in various processes, including bone mineralization, signaling by insulin and by nucleotides, and the differentiation and motility of cells. While it has been established that most of these biological effects of NPPs require a functional catalytic site, their physiological substrates remain to be identified.

A capping domain for LRR protein interaction modules.

Leucine-rich repeats (LRR) are protein interaction modules which are present in a large number of proteins with diverse functions. We describe here a novel motif (16-19 residues) downstream of the last, incomplete, LRR in a subfamily of LRR proteins. In the U2A' spliceosomal protein, this motif is folded into a cap that shields the hydrophobic core of the LRRs from the solvent. Modelling of the LRR-cap in the imidazoline-1 candidate receptor, using the known structure of U2A' as template, showed a conservation of the basic structural features.

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.

On the inhibition of hepatic glycogenolysis by fructose. A 31P-NMR study in perfused rat liver using the fructose analogue 2,5-anhydro-D-mannitol.

Inhibition of hormone-stimulated hepatic glycogenolysis by fructose (Fru) has been attributed to accumulation of the competitive inhibitor Fru1P and/or to the associated depletion of the substrate phosphate (Pi). To evaluate the relative importance of either factor, we used the Fru analogue 2,5-anhydro-D-mannitol (aHMol). This analogue is avidly phosphorylated, traps Pi, and inhibits hormone-stimulated glycogenolysis, but it is not a gluconeogenic substrate, and hence does not confound glycogenolytic glucose production. Livers were continuously perfused with dibutyryl-cAMP (100 microM) to clamp phosphorylase in its fully activated a form. We administered aHMol (3.8 mM), and studied changes in glycogenolysis (glucose, lactate and pyruvate output) and in cytosolic Pi and phosphomonoester (PME), using in situ 31P-NMR spectroscopy (n = 4). Lobes of seven livers perfused outside the magnet were extracted for evaluation, by high-resolution 31P-NMR, of the evolution of aHMol1P and of aHMol(1,6)P2. After addition of aHMol, both glycogenolysis and the NMR Pi signal dropped precipitously, while the PME signal rose continuously and was almost entirely composed of aHMol1P. Inhibition of glycogenolysis in excess of the drop in Pi could be explained by continuing accumulation of aHMol1P. A subsequent block of mitochondrial ATP synthesis by KCN (1 mM) caused a rapid increase of Pi. Despite recovery of Pi to values exceeding control levels, glycogenolysis only recovered partially, attesting to the Pi-dependence of glycogenolysis, but also to inhibition by aHMol phosphorylation products. However, KCN resulted in conversion of the major part of aHMol1P into aHMol(1,6)P2. Residual inhibition of glycogenolysis was due to aHMol1P. Indeed, the subsequent withdrawal of aHMol caused a further gradual decrease in the proportion of aHMol1P (being converted into aHMol(1,6)P2, in the absence of de novo aHMol1P synthesis), and this resulted in a gradual de-inhibition of glycogenolysis, in the absence of marked changes in Pi. Glycogenolytic rates were consistently predicted by a model assuming non-saturated Pi kinetics and competition by aHMol1P exclusively: In conclusion, limited Pi availability and the presence of competitive inhibitors are decisive factors in the control of the in situ catalytic potential of phosphorylase a.

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.

Differential regulation of the expression of nucleotide pyrophosphatases/phosphodiesterases in rat liver.

We propose the name nucleotide pyrophosphatases/phosphodiesterases (NPP) for the enzymes that release nucleoside-5'-monophosphates from various pyrophosphate and phosphodiester bonds. Three structurally related mammalian NPPs are known, i.e. NPPalpha (autotaxin), NPPbeta (B10/gp130RB13-6) and NPPgamma (PC-1). We report here that these isozymes have a distinct tissue distribution in the rat but that they are all three expressed in hepatocytes. In FAO rat hepatoma cells only the level of NPPgamma was stimulated by TGF-beta1. In rat liver, the concentration of the transcripts of all three isozymes was found to increase manyfold during the first weeks after birth, but the increased expression of the NPPalpha mRNA was transient. The level of the NPP transcripts transiently decreased after hepatectomy, but NPPalpha mRNA was also lost after sham operation, which suggests that it may belong to the negative acute-phase proteins. The loss of the beta- and gamma-transcripts after hepatectomy was not due to a decreased NPP gene transcription or an increased turnover of the mature transcripts. However, hepatectomy also caused a similar loss of the nuclear pool of the NPPbeta and NPPgamma mRNAs. We conclude that a deficient processing and/or an increased turnover of the NPP pre-mRNAs underlies the hepatectomy-induced decrease of the beta- and gamma-transcripts. A similar loss of nuclear NPPgamma mRNA was also noted after treatment with cycloheximide, indicating that protein(s) with a high turnover control the stability and/or processing of the immature NPPgamma transcript.

Organization and alternate splice products of the gene encoding nuclear inhibitor of protein phosphatase-1 (NIPP-1).

Nuclear inhibitor of protein phosphatase-1 (NIPP-1) is one of two major regulatory subunits of protein phosphatase-1 in mammalian nuclei. We report here the cloning and structural characterization of the human NIPP-1 genes, designated PPP1R8P and PPP1R8 in human gene nomenclature. PPP1R8P (1.2 kb) is a processed pseudogene and was localized by in situ hybridization to chromosome 1p33-32. PPP1R8 is an authentic NIPP-1 gene and was localized to chromosome 1p35. PPP1R8 (25.2 kb) is composed of seven exons and encodes four different transcripts, as determined from cDNA library screening, reverse transcriptase-PCR (RT-PCR) and/or EST (expressed sequence tag) database search analysis. NIPP-1alpha mRNA represents the major transcript in human tissues and various cell lines, and encodes a polypeptide of 351 residues that only differs from the previously cloned calf thymus NIPP-1 by a single residue. The other transcripts, termed NIPP-1beta, gamma and delta, are generated by alternative 5'-splice site usage, by exon skipping and/or by alternative polyadenylation. The NIPP-1beta/delta and NIPP-1gamma mRNAs are expected to encode fragments of NIPP-1alpha that differ from the latter by the absence of the first 142 and 224 residues, respectively. NIPP-1gamma corresponds to 'activator of RNA decay-1' (Ard-1) which, unlike NIPP-1alpha, displays in vitro and endoribonuclease activity and lacks an RVXF consensus motif for interaction with protein phosphatase-1. While the NIPP-1alpha/beta/delta-transcripts were found to be present in various human tissues, the NIPP-1gamma transcript could only be detected in human transformed B-lymphocytes.

Specific features of glycogen metabolism in the liver.

Although the general pathways of glycogen synthesis and glycogenolysis are identical in all tissues, the enzymes involved are uniquely adapted to the specific role of glycogen in different cell types. In liver, where glycogen is stored as a reserve of glucose for extrahepatic tissues, the glycogen-metabolizing enzymes have properties that enable the liver to act as a sensor of blood glucose and to store or mobilize glycogen according to the peripheral needs. The prime effector of hepatic glycogen deposition is glucose, which blocks glycogenolysis and promotes glycogen synthesis in various ways. Other glycogenic stimuli for the liver are insulin, glucocorticoids, parasympathetic (vagus) nerve impulses and gluconeogenic precursors such as fructose and amino acids. The phosphorolysis of glycogen is mainly mediated by glucagon and by the orthosympathetic neurotransmitters noradrenaline and ATP. Many glycogenolytic stimuli, e.g. adenosine, nucleotides and NO, also act indirectly, via secretion of eicosanoids from non-parenchymal cells. Effectors often initiate glycogenolysis cooperatively through different mechanisms.

Growth-related expression of the ectonucleotide pyrophosphatase PC-1 in rat liver.

Plasma cell differentiation antigen-1 (PC-1) is a 5'-ectonucleotide pyrophosphatase that has been implicated in various processes including insulin- and nucleotide-mediated signaling and cell growth. We show here that the expression of both PC-1 mRNA and protein in rat liver and in hepatoma cells is strictly growth-related. Thus, the level of PC-1 in FAO hepatoma cells increased with the cell density. PC-1 was not expressed in the neonatal rat liver, but gradually appeared in the first weeks of age, to reach adult levels around the weaning period. Furthermore, PC-1 protein and mRNA largely disappeared from the liver within 24 hours following a hepatectomy of 70%, but re-appeared in the later phases (3-15 days) of the ensuing regeneration period. An equally rapid loss of PC-1 protein and mRNA could also be provoked in normal livers by the administration of the translational inhibitor, cycloheximide, but the transcriptional inhibitors, actinomycin D and alpha-amanitin, did not show these effects. Nuclear run-on assays revealed that the loss of PC-1 mRNA after hepatectomy or after the administration of cycloheximide was not caused by a decreased transcription of the PC-1 gene, suggesting that the level of PC-1 is controlled by an mRNA-stabilizing protein that is lost after hepatectomy and has a high turnover.

Loss of the hepatic glycogen-binding subunit (GL) of protein phosphatase 1 underlies deficient glycogen synthesis in insulin-dependent diabetic rats and in adrenalectomized starved rats.

Hepatic glycogen synthesis is impaired in insulin-dependent diabetic rats and in adrenalectomized starved rats, and although this is known to be due to defective activation of glycogen synthase by glycogen synthase phosphatase, the underlying molecular mechanism has not been delineated. Glycogen synthase phosphatase comprises the catalytic subunit of protein phosphatase 1 (PP1) complexed with the hepatic glycogen-binding subunit, termed GL. In liver extracts of insulin-dependent diabetic and adrenalectomized starved rats, the level of GL was shown by immunoblotting to be substantially reduced compared with that in control extracts, whereas the level of PP1 catalytic subunit was not affected by these treatments. Insulin administration to diabetic rats restored the level of GL and prolonged administration raised it above the control levels, whereas re-feeding partially restored the GL level in adrenalectomized starved rats. The regulation of GL protein levels by insulin and starvation/feeding was shown to correlate with changes in the level of the GL mRNA, indicating that the long-term regulation of the hepatic glycogen-associated form of PP1 by insulin, and hence the activity of hepatic glycogen synthase, is predominantly mediated through changes in the level of the GL mRNA.