The translation initiation factor eIF2beta is an interactor of protein phosphatase-1.

It is reasonably well understood how the initiation of translation is controlled by reversible phosphorylation of the eukaryotic translation initiation factors eIF2alpha, eIF2Bepsilon and eIF4E. Other initiation factors, including eIF2beta, are also established phosphoproteins but the physiological impact of their phosphorylation is not known. Using a sequence homology search we found that the central region of eIF2beta contains a putative PP1-(protein phosphatase-1) binding RVxF-motif.

Proteolytic maturation and activation of autotaxin (NPP2), a secreted metastasis-enhancing lysophospholipase D.

Autotaxin (NPP2) is an extracellular protein that is upregulated in various malignancies, including breast and lung cancer. It potently stimulates cell proliferation, cell motility and angiogenesis, which is accounted for by its intrinsic lysophospholipase-D activity that generates the lipid mediators lysophosphatidic acid and sphingosine-1-phosphate. Based on its structural similarities with the better characterized nucleotide pyrophosphatase/phosphodiesterase NPP1, it has always been assumed that NPP2 is also synthesized as a type-II integral membrane protein and that extracellular NPP2 is generated from this membrane precursor.

The nuclear scaffold protein NIPP1 is essential for early embryonic development and cell proliferation.

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.

Substrate-specifying determinants of the nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2.

The nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2/autotaxin are structurally related eukaryotic ecto-enzymes, but display a very different substrate specificity. NPP1 releases nucleoside 5'-monophosphates from various nucleotides, whereas NPP2 mainly functions as a lysophospholipase D. We have used a domain-swapping approach to map substrate-specifying determinants of NPP1 and NPP2.

Inhibition of spliceosome assembly by the cell cycle-regulated protein kinase MELK and involvement of splicing factor NIPP1.

NIPP1 is a ubiquitous nuclear protein that is required for spliceosome assembly. We report here that the phosphothreonine-binding Forkhead-associated domain of NIPP1 interacts with the cell cycle-regulated protein Ser/Thr kinase MELK (maternal embryonic leucine zipper kinase). The NIPP1-MELK interaction was critically dependent on the phosphorylaton of Thr-478 of MELK and was increased in lysates from mitotically arrested cells.

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.

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.

Degeneracy and function of the ubiquitous RVXF motif that mediates binding to protein phosphatase-1.

Most interactors of protein phosphatase-1 (PP1) contain a variant of a so-called "RVXF" sequence that binds to a hydrophobic groove of the catalytic subunit. A combination of sequence alignments and site-directed mutagenesis has enabled us to further define the consensus sequence for this degenerate motif as [RK]-X(0-1)-[VI]-[P]-[FW], where X denotes any residue and [P] any residue except Pro. Naturally occurring RVXF sequences differ in their affinity for PP1, and we show by swapping experiments that this binding affinity is an important determinant of the inhibitory potency of the regulators NIPP1 and inhibitor-1.

Binding of the concave surface of the Sds22 superhelix to the alpha 4/alpha 5/alpha 6-triangle of protein phosphatase-1.

Functional studies of the protein phosphatase-1 (PP1) regulator Sds22 suggest that it is indirectly and/or directly involved in one of the most ancient functions of PP1, i.e. reversing phosphorylation by the Aurora-related protein kinases.

Phosphorylation-dependent interaction between the splicing factors SAP155 and NIPP1.

NIPP1 is a ubiquitously expressed nuclear protein that functions both as a regulator of protein Ser/Thr phosphatase-1 and as a splicing factor. The N-terminal part of NIPP1 consists of a phosphothreonine-interacting Forkhead-associated (FHA) domain. We show here that the FHA domain of NIPP1 interacts in vitro and in vivo with a TP dipeptide-rich fragment of the splicing factor SAP155/SF3b(155), a component of the U2 small nuclear ribonucleoprotein particle.

Regulator-driven functional diversification of protein phosphatase-1 in eukaryotic evolution.

We have used the (nearly) completed eukaryotic genome sequences to trace the evolution of thirteen families of established vertebrate regulators of type-1 protein phosphatases (PP1). Two of these families are present in all lineages of the eukaryotic crown and therefore qualify as candidate primordial regulators that determined the surface of PP1. The set of regulators of PP1 has continued to expand ever since, often in response to functional innovations in different eukaryotic lineages.

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%).

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)).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.