Dynamic targeting of protein phosphatase 1 within the nuclei of living mammalian cells.

Protein phosphatase 1 (PP1) is expressed in mammalian cells as three closely related isoforms, alpha, beta/delta and gamma1, which are encoded by separate genes. It has yet to be determined whether the separate isoforms behave in a similar fashion or play distinct roles in vivo. We report here on analyses by fluorescence microscopy of functional and fluorescently tagged PP1 isoforms in live cells. PP1alpha and PP1gamma fluorescent protein fusions show largely complimentary localization patterns, particularly within the nucleus where tagged PP1gamma accumulates in the nucleolus, whereas tagged PP1alpha is primarily found in the nucleoplasm. Overexpression of NIPP1 (nuclear inhibitor of PP1), a PP1 targeting subunit that accumulates at interchromatin granule clusters in the nucleoplasm, results in a retargeting of both isoforms to these structures, indicating that steady-state localization is based, at least in part, on relative affinities for various targeting subunits. Photobleaching analyses show that PP1gamma is rapidly exchanging between the nucleolar, nucleoplasmic and cytoplasmic compartments. Fluorescence resonance energy transfer (FRET) analyses indicate that the direct interaction of the two proteins predominantly occurs at or near interchromatin granule clusters. These data indicate that PP1 isoforms are highly mobile in cells and can be dynamically (re)localized through direct interaction with targeting subunits.

hRRN3 is essential in the SL1-mediated recruitment of RNA Polymerase I to rRNA gene promoters.

A crucial step in transcription is the recruitment of RNA polymerase to promoters. In the transcription of human rRNA genes by RNA Polymerase I (Pol I), transcription factor SL1 has a role as the essential core promoter binding factor. Little is known about the mechanism by which Pol I is recruited. We provide evidence for an essential role for hRRN3, the human homologue of a yeast Pol I transcription factor, in this process. We find that whereas the bulk of human Pol I complexes (I alpha) are transcriptionally inactive, hRRN3 defines a distinct subpopulation of Pol I complexes (I beta) that supports specific initiation of transcription. Human RRN3 interacts directly with TAF(I)110 and TAF(I)63 of promoter-selectivity factor SL1. Blocking this connection prevents recruitment of Pol I beta to the rDNA promoter. Furthermore, hRRN3 can be found in transcriptionally autonomous Pol I holoenzyme complexes. We conclude that hRRN3 functions to recruit initiation-competent Pol I to rRNA gene promoters. The essential role for hRRN3 in linking Pol I to SL1 suggests a mechanism for growth control of Pol I transcription.

Characterization of a novel phosphatidylinositol 3-phosphate-binding protein containing two FYVE fingers in tandem that is targeted to the Golgi.

We have identified a novel protein of predicted molecular mass 40 kDa that contains two FYVE domains in tandem and has therefore been named TAFF1 (TAndem FYVE Fingers-1). The protein is expressed predominantly in heart and binds to PtdIns3P specifically, even though the FYVE domains in TAFF1 lacks the first Arg of the consensus sequence R(K/R)HHCR, critical for the PtdIns3P binding of other FYVE domains identified so far. The first Arg is replaced by a Thr and Ser in the N-terminal and C-terminal FYVE domains of TAFF1 respectively. Mutational analysis indicates that both FYVE domains are required for high affinity binding to PtdIns3P. Cell localization studies using a green fluorescent protein fusion show that TAFF1 is localized to the Golgi, and that the Golgi targeting sequence is located within the N-terminal 187 residues and not in either FYVE domain.

Alternative splicing regulates the production of ARD-1 endoribonuclease and NIPP-1, an inhibitor of protein phosphatase-1, as isoforms encoded by the same gene.

ARD-1 is an endoribonuclease identified initially as the product of a human cDNA that complements mutations in rne, a gene that encodes Escherichia coli ribonuclease E. NIPP-1 was identified in bovine nuclear extracts as an inhibitor of protein phosphatase-1. Earlier work has shown that the protein-coding sequence of ARD-1 is identical to the carboxy-terminal third of NIPP-1. However, whether ARD-1 is present in eukaryotes as a distinct entity has been unclear, as neither ARD-1-specific transcripts nor ARD-1 protein were detected in mammalian cells in earlier studies. Here we show that ARD-1 exists in human cells as a discrete protein, and that the ARD-1 and NIPP-1 peptides are isoforms encoded by a single gene and the same alternatively spliced precursor RNA. A retained intron containing multiple translation stop codons that are configured to terminate translation and initiate nonsense-mediated decay, limits the production of cellular ARD-1 protein. Our results establish the process by which functionally disparate ARD-1 and NIPP-1 peptides are generated from the protein-coding sequence of the same gene in human cells.

Nuclear organisation of NIPP1, a regulatory subunit of protein phosphatase 1 that associates with pre-mRNA splicing factors.

Protein phosphatase-1 (PP1) is complexed to many proteins that target it to particular subcellular locations and regulate its activity. Here, we show that 'nuclear inhibitor of PP1' (NIPP1), a major nuclear PP1-binding protein, shows a speckled nucleoplasmic distribution where it is colocalised with pre-mRNA splicing factors. One of these factors (Sm) is also shown to be complexed to NIPP1 in nuclear extracts. Immunodepletion of NIPP1 from nuclear extracts, or addition of a 'dominant negative' mutant lacking a functional PP1 binding site, greatly reduces pre-mRNA splicing activity in vitro. These findings implicate the NIPP1-PP1 complex in the control of pre-mRNA splicing.

Hypertrophy of colonic smooth muscle: structural remodeling, chemical composition, and force output.

The cellular basis of adaptations occurring during the development of megacolon was studied with the lethal spotted mouse model. Age-dependent changes in the length-force characteristics of the colon reach a steady state by 3-4 mo and include an increased relative force development at very short muscle lengths. In megacolon the following occur: 1) structural remodeling expressed as a greater increase in the fraction of maximum force production at short lengths, a shift of optimum length (Lo) to longer lengths, and no change in force per square centimeter; 2) hypertrophy and hyperplasia of both circular and longitudinal muscle; 3) high resting compliance consistent with no disproportionate change in collagen or elastin composition; 4) marked distension so that in situ circumference approximately 1.8 Lo, where active force production is low, and 5) slack length approximately 0.65 Lo, as in normal colon. Biochemical remodeling in megacolon includes disproportionate increases in ATP and phosphocreatine concentration, with 3.5-fold more preformed phosphagen than in normal colon. The myosin concentration is the same in both muscles, but the actin concentration is 1.5-fold greater in megacolon. Most of the cellular changes in megacolon would facilitate high active force output from the muscle at much larger intestinal diameters.

Hypertrophy of colonic smooth muscle: contractile proteins, shortening velocity, and regulation.

Smooth muscle in megacolon was studied in the lethal spotted mouse and its normal sibling. In megacolon, structural remodeling and a very large increase in total protein content are associated with some changes in the contractile protein isoform composition. 1) There is a higher actin concentration in megacolon, primarily caused by a larger proportion of gamma-isoforms. 2) There was no difference in myosin concentration or in SM1/SM2 heavy chains in megacolon and normal muscle contractile proteins. 3) Only LC17a essential light chain is present in both normal and megacolon. 4) The 25- to 50-kDa 5'-insert occurs in 15-20% of the myosin in normal colon, compared with 5- to 10-fold lower amounts in megacolon. In permeabilized muscles there was no significant difference in unloaded shortening velocity (Vo) with maximal thiophosphorylation of the 20-kDa light chains, nor was there significant difference in the force vs. Ca2+ and force vs. myosin light chain phosphorylation relationships. At approximately 60% myosin light chain phosphorylation after Ca2+ activation, Vo of megacolon was approximately two times faster than Vo of normal muscle. These cellular changes largely account for the higher propulsive velocity of the colon in situ. The distribution of myosin and actin isoforms and the lack of a simple relationship between myosin light chain phosphorylation and Vo point to the operation of additional regulatory processes.

Purification and characterisation of p99, a nuclear modulator of protein phosphatase 1 activity.

We have purified a form of protein phosphatase 1 (PP1) from HeLa cell nuclei, in which the phosphatase is complexed to a regulatory subunit termed p99. We report here the cloning and characterisation of the p99 component. p99 mRNA is widely expressed in human tissues and immunofluorescence analysis with anti-p99 antibodies showed a punctate nucleoplasmic staining with additional accumulations within the nucleolus. The C-terminus of p99 contains seven RGG RNA-binding motifs, followed by eleven decapeptide repeats containing six or more of the following conserved residues (GHRPHEGPGG), and finally a putative zinc finger domain. Recombinant p99 suppresses the phosphorylase phosphatase activity of PP1 by > 90% and the canonical PP1-binding motif on p99 (residues 396-401) is unusual in that the phenylalanine residue is replaced by tryptophan.

Phosphorylation of a high molecular weight (approximately 600 kDa) protein regulates catch in invertebrate smooth muscle.

A unique property of smooth muscle is its ability to maintain force with a very low expenditure of energy. This characteristic is highly expressed in molluscan smooth muscles, such as the anterior byssus retractor muscle (ABRM) of Mytilus edulis, during a contractile state called 'catch'. Catch occurs following the initial activation of the muscle, and is characterized by prolonged force maintenance in the face of a low [Ca2+]i, high instantaneous stiffness, a very slow cross-bridge cycling rate, and low ATP usage. In the intact muscle, rapid relaxation (release of catch) is initiated by serotonin, and mediated by an increase in cAMP and activation of protein kinase A. We sought to determine which proteins undergo a change in phosphorylation on a time-course that corresponds to the release of catch in permeabilized ABRM. Only one protein consistently satisfied this criterion. This protein, having a molecular weight of approximately 600 kDa and a molar concentration about 30 times lower than the myosin heavy chain, showed an increase in phosphorylation during the release of catch. Under the mechanical conditions studied (rest, activation, catch, and release of catch), changes in phosphorylation of all other proteins, including myosin light chains, myosin heavy chain and paramyosin, are minimal compared with the cAMP-induced phosphorylation of the approximately 600 kDa protein. Under these conditions, somewhat less than one mole of phosphate is incorporated per mole of approximately 600 kDa protein. Inhibition of A kinase blocked both the cAMP-induced increase in phosphorylation of the protein and the release of catch. In addition, irreversible thiophosphorylation of the protein prevented the development of catch. In intact muscle, the degree of phosphorylation of the protein increases significantly when catch is released with serotonin. In muscles pre-treated with serotonin, a net dephosphorylation of the protein occurs when the muscle is subsequently put into catch. We conclude that the phosphorylation state of the approximately 600 kDa protein regulates catch.

Thiophosphorylation of the 130-kDa subunit is associated with a decreased activity of myosin light chain phosphatase in alpha-toxin-permeabilized smooth muscle.

Pretreatment of alpha-toxin-permeabilized smooth muscle with ATP gamma S (adenosine 5'-O-(thiotriphosphate)) under conditions resulting in minimal (< 1%) thiophosphorylation of the myosin light chain increases the subsequent calcium sensitivity of force output and myosin light chain phosphorylation. The change in calcium sensitivity results at least in part from a 5-fold decrease in myosin light chain phosphatase activity. One of the few proteins thiophosphorylated under these conditions is the 130-kDa subunit of myosin light chain phosphatase. These results suggest that thiophosphorylation of this subunit leads to a decrease in the activity of the phosphatase, and that phosphorylation and dephosphorylation of the subunit may play a role in regulating myosin light chain phosphatase activity.

Metabolic characteristics of alpha-toxin-permeabilized smooth muscle.

Rabbit portal veins were permeabilized using Staphylococcus aureus alpha-toxin, and adenosinetriphosphatase (ATPase) was measured as the formation of [3H]ADP, [3H]AMP, and [3H]adenosine from [3H]ATP in the solution bathing the muscle. The resting ATPase (1.96 +/- 0.15 mM/min, n = 13) is approximately 5-10 times higher than that measured in Triton X-100-permeabilized muscles (0.28 +/- 0.01 mM/min, n = 4), with nucleotide accumulating as ADP, AMP, and adenosine. The ATPase activity is also seen when the intact muscle is incubated in a Krebs solution containing 1 mM MgATP (2.76 +/- 0.10 mM/min, n = 73). This suggests that it is due primarily to an ecto-ATPase. The ectoenzyme is capable of hydrolyzing both ATP and ADP, and in both cases there is a higher rate at 3 than at 1 mM nucleotide. The high resting ATPase compromises the control of nucleotide concentrations within the permeabilized tissue even in the presence of an ATP-regenerating system consisting of phosphocreatine (PCr, 35mM) and creatine kinase (1 mg/ml). Treatment of the intact muscle with the ectonucleotidase inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) followed by alpha-toxin permeabilization and inclusion of sodium azide in subsequent solutions reduces the ecto-ATPase by approximately 70%. Addition of PCr and creatine kinase then results in the maintenance of high [ATP] and low [ADP] in the muscle, and importantly, there are no significant changes in [ATP], [ADP], [adenosine/AMP], or the ADP-to-ATP ratio upon activation of the muscle in pCa 4.5. In general, the force output in high Ca2+ increased as the metabolic profile of the muscle improved. When ATPase was measured as the appearance of [32P]Pi from [32P]PCr and [gamma-32P]ATP, the alpha-toxin-permeabilized muscle subjected to the above treatment showed only approximately 30% higher total ATPase under activated conditions compared with the freeze-glycerinated Triton-treated portal vein. The suprabasal ATPase is similar in both preparations. We conclude that the reduction of the basal ATPase by the DIDS-azide treatment permits both rigorous control of nucleotide contents and accurate measurement of ATPase activity in alpha-toxin-permeabilized smooth muscle.