NOM1 targets protein phosphatase I to the nucleolus

Protein phosphatase I (PP1) is an essential eukaryotic serine/threonine phosphatase required for many cellular processes, including cell division, signaling, and metabolism. In mammalian cells there are three major isoforms of the PP1 catalytic subunit (PP1alpha, PP1beta, and PP1gamma) that are over 90% identical. Despite this high degree of identity, the PP1 catalytic subunits show distinct localization patterns in interphase cells; PP1alpha is primarily nuclear and largely excluded from nucleoli, whereas PP1gamma and to a lesser extent PP1beta concentrate in the nucleoli. The subcellular localization and the substrate specificity of PP1 catalytic subunits are determined by their interaction with targeting subunits, most of which bind PP1 through a so-called "RVXF" sequence. Although PP1 targeting subunits have been identified that direct PP1 to a number of subcellular locations and/or substrates, no targeting subunit has been identified that localizes PP1 to the nucleolus. Identification of nucleolar PP1 targeting subunit(s) is important because all three PP1 isoforms are included in the nucleolar proteome, enzymatically active PP1 is present in nucleoli, and PP1gamma is highly concentrated in nucleoli of interphase cells. In this study, we identify NOM1 (nucleolar protein with MIF4G domain 1) as a PP1-interacting protein and further identify the NOM1 RVXF motif required for its binding to PP1. We also define the NOM1 nucleolar localization sequence. Finally, we demonstrate that NOM1 can target PP1 to the nucleolus and show that a specific NOM1 RVXF motif and the NOM1 nucleolar localization sequence are required for this targeting activity. We therefore conclude that NOM1 is a PP1 nucleolar targeting subunit, the first identified in eukaryotic cells.     

Repo-Man recruits PP1 gamma to chromatin and is essential for cell viability

Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase regulating many cellular processes. PP1alpha and -gamma are closely related isoforms with distinct localization patterns, shown here by time-lapse microscopy of stably expressed fluorescent protein fusions. A pool of PP1gamma is selectively loaded onto chromatin at anaphase. Using stable isotope labeling and proteomics, we identified a novel PP1 binding protein, Repo-Man, which selectively recruits PP1gamma onto mitotic chromatin at anaphase and into the following interphase. This approach revealed both novel and known PP1 binding proteins, quantitating their relative distribution between PP1alpha and -gamma in vivo. When overexpressed, Repo-Man can also recruit PP1alpha to chromatin. Mutating Repo-Man's PP1 binding domain does not disrupt chromatin binding but abolishes recruitment of PP1 onto chromatin. RNA interference-induced knockdown of Repo-Man caused large-scale cell death by apoptosis, as did overexpression of this dominant-negative mutant. The data indicate that Repo-Man forms an essential complex with PP1gamma and is required for the recruitment of PP1 to chromatin.     

Determinants of the nucleolar targeting of protein phosphatase-1

The ubiquitously expressed protein Ser/Thr phosphatase-1 isoforms PP1alpha, PP1beta and PP1gamma1 are dynamically targeted to distinct, but overlapping cellular compartments by associated proteins. Within the nucleus of HeLa cells, EGFP-tagged PP1gamma1 and PP1beta were predominantly targeted to the nucleoli, while PP1alpha showed a more diffuse distribution. Using PP1 chimaeras and point mutants we show here that a single N-terminal residue, i.e., Gln20 for PP1alpha, Arg19 for PP1beta and Arg20 for PP1gamma1 accounts for their distinct subnuclear distribution. Our data also suggest that the N-terminus of PP1beta and PP1gamma1 harbours an interaction site for one or more nucleolar interactors.     

Protein phosphatase-1 inhibitor-3 is co-localized to the nucleoli and centrosomes with PP1gamma1 and PP1alpha, respectively

In this study, we show that protein phosphatase-1 (PP1) inhibitor-3 (Inh3) is localized to the nucleoli and centrosomes in interphase HEK 293 cells. Inh3 exhibited a specific co-localization to the nucleoli with PP1gamma1, and to the centrosomes with PP1alpha. These findings indicate that Inh3 may act as a modulator of PP1 functions in the processes of cytokinesis, as well as of nucleolar events. The specificity of the interaction of Inh3 with the PP1 isoforms was also demonstrated in vitro, where Inh3 co-immunoprecipitated with PP1alpha and PP1gamma1, but not with PP1beta. The nuclear localization signal of Inh3 was identified as a N-terminal basic cluster (33RKRK36), while nucleolar localization was shown to be dependent on a C-terminal basic cluster (94HRKGRRR100). The importance of the individual basic residues was quantitatively assessed by site-directed mutagenesis and a novel use of laser scanning cytometry.     

Association of protein phosphatase 1 gamma 1 with spinophilin suppresses phosphatase activity in a Parkinson disease model

Sustained nigrostriatal dopamine depletion increases the serine/threonine phosphorylation of multiple striatal proteins that play a role in corticostriatal synaptic plasticity, including Thr(286) phosphorylation of calcium/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha). Mechanisms underlying these changes are unclear, but protein phosphatases play a critical role in the acute modulation of striatal protein phosphorylation. Here we show that dopamine depletion for periods ranging from 3 weeks to 10 months significantly reduces the total activity of protein phosphatase (PP) 1, but not of PP2A, in whole lysates of rat striatum, as measured using multiple substrates, including Thr(286)-autophosphorylated CaMKIIalpha. Striatal PP1 activity is partially inhibited by a fragment of the PP1-binding protein neurabin-I, Nb-(146-493), because of the selective inhibition of the PP1gamma(1) isoform. The fraction of PP1 activity that is insensitive to Nb-(146-493) was unaffected by dopamine depletion, demonstrating that dopamine depletion specifically reduces the activity of PP1 isoforms that are sensitive to Nb-(146-493) (i.e. PP1gamma(1)). However, total striatal levels of PP1gamma(1) or any other PP1 isoform were unaffected by dopamine depletion, and our previous studies showed that total levels of the PP1 regulatory/targeting proteins DARPP-32, spinophilin, and neurabin were also unchanged. Rather, co-immunoprecipitation experiments demonstrated that dopamine depletion increases the association of PP1gamma(1) with spinophilin in striatal extracts. In combination, these data demonstrate that striatal dopamine depletion inhibits a specific synaptic phosphatase by increasing PP1gamma(1) interaction with spinophilin, perhaps contributing to hyperphosphorylation of synaptic proteins and disruptions of synaptic plasticity and/or dendritic morphology.     

Serine/threonine protein phosphatase type 1gamma1 is required for the completion of cytokinesis in human A549 lung carcinoma cells

In lower eukaryotic organisms, the loss of serine/threonine protein phosphatase type 1 (PP1) results in growth arrest after the onset of mitosis. In humans, four highly homologous isoforms of PP1 (PP1alpha, PP1delta, PP1gamma1, and PP1gamma2) have been identified. Determining the roles of these phosphatases, however, has proven difficult due to the lack of subtype-specific inhibitors. In this study, we developed chimeric antisense 2'-O-(2-methoxy)ethylphosphothioate oligonucleotides targeting human PP1gamma1 that specifically inhibit PP1gamma1 gene expression. Two potent antisense oligonucleotides (ISIS 14435 and 14439; IC(50) approximately 50 nM) were then employed to elucidate the cellular functions of PP1gamma1 during cell cycle progression. In A549 cells, the inhibition of PP1gamma1 expression resulted in a dose-dependent inhibition of cellular proliferation, with growth arrest occurring after approximately 36-48 h, when PP1gamma1 mRNA expression was inhibited by >85%. Fluorescence-activated cell sorter analysis revealed that ISIS 14435/14439-induced growth arrest was associated with an increase in the number of cells containing 4N DNA. Immunostaining of treated cells revealed that the inhibition of PP1gamma1 expression had no apparent effect on the formation of mitotic spindles. However, decreased expression was associated with the failure of cell division in a late stage of cytokinesis and the formation of dikaryons.     

S6K1-mediated disassembly of mitochondrial URI/PP1gamma complexes activates a negative feedback program that counters S6K1 survival signaling

S6 kinase 1 (S6K1) acts to integrate nutrient and growth factor signals to promote cell growth but also cell survival as a mitochondria-tethered protein kinase that phosphorylates and inactivates the proapoptotic molecule BAD. Here we report that the prefoldin chaperone URI represents a mitochondrial substrate of S6K1. In growth factor-deprived or rapamycin-treated cells, URI forms stable complexes with protein phosphatase (PP)1gamma at mitochondria, thereby inhibiting the activity of the bound enzyme. Growth factor stimulation induces disassembly of URI/PP1gamma complexes through S6K1-mediated phosphorylation of URI at serine 371. This activates a PP1gamma-dependent negative feedback program that decreases S6K1 activity and BAD phosphorylation, thereby altering the threshold for apoptosis. These findings establish URI and PP1gamma as integral components of an S6K1-regulated mitochondrial pathway dedicated, in part, to oppose sustained S6K1 survival signaling and to ensure that the mitochondrial threshold for apoptosis is set in accord with nutrient and growth factor availability.     

Isolation of human mitotic protein phosphatase complexes: identification of a complex between protein phosphatase 1 and the RNA helicase Ddx21

Metazoan mitosis requires remodelling of sub-cellular structures to ensure proper division of cellular and genetic material. Faults often lead to genomic instability, cell cycle arrests and disease onset. These key structural changes are under tight spatial-temporal and post-translational control, with crucial roles for reversible protein phosphorylation. The phosphoprotein phosphatases PP1 and PP2A are paramount for the timely execution of mitotic entry and exit but their interaction partners and substrates are still largely unresolved. High throughput, mass-spectrometry based studies have limited sensitivity for the detection of low-abundance and transient complexes, a typical feature of many protein phosphatase complexes. Moreover, the limited timeframe during which mitosis takes place reduces the likelihood of identifying mitotic phosphatase complexes in asynchronous cells. Hence, numerous mitotic protein phosphatase complexes still await identification. Here we present a strategy to enrich and identify serine/threonine protein phosphatase complexes at the mitotic spindle. We thus identified a nucleolar RNA helicase, Ddx21/Gu, as a novel, direct PP1 interactor. Furthermore, our results place PP1 within the toposome, a Topoisomerase II alpha (TOPOIIα) containing complex with a key role in mitotic chromatin regulation and cell cycle progression, possibly via regulated protein phosphorylation. This study provides a strategy for the identification of further mitotic PP1 partners and the unravelling of PP1 functions during mitosis.     

Cytostatin, an inhibitor of cell adhesion to extracellular matrix, selectively inhibits protein phosphatase 2A

Cytostatin, which is isolated from a microbial cultured broth as a low molecular weight inhibitor of cell adhesion to extracellular matrix (ECM), has anti-metastatic activity against B16 melanoma cells in vivo. In this study, we examined a target of cytostatin inhibiting cell adhesion to ECM. Cytostatin inhibited tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin upon B16 cell adhesion to fibronectin. While the amount of FAK was not affected by cytostatin, electrophoretically slow-migrating paxillin appeared. Alkaline phosphatase treatment diminished cytostatin-induced slow-migrating paxillin. Furthermore, cytostatin increased intracellular serine/threonine-phosphorylated proteins and was found to be a selective inhibitor of protein phosphatase 2A (PP2A). Cytostatin inhibited PP2A with an IC(50) of 0.09 microgram/ml in a non-competitive manner against a substrate, p-nitrophenyl phosphate, but it had no apparent effect on other protein phosphatases including PP1, PP2B and alkaline phosphatase even at 100 microgram/ml. On the contrary, dephosphocytostatin, a cytostatin analogue, without inhibitory effect on PP2A did not affect B16 cell adhesion including FAK and paxillin. These results indicate that cytostatin inhibits cell adhesion through modification of focal contact proteins such as paxillin by inhibiting a PP2A type protein serine/threonine phosphatase. This is the first report that describes a drug with anti-metastatic ability that inhibits PP2A selectively.     

Leucine carboxyl methyltransferase-1 is necessary for normal progression through mitosis in mammalian cells

Protein phosphatase 2A (PP2A) is a multifunctional phosphatase that plays important roles in many cellular processes including regulation of cell cycle and apoptosis. Because PP2A is involved in so many diverse processes, it is highly regulated by both non-covalent and covalent mechanisms that are still being defined. In this study we have investigated the importance of leucine carboxyl methyltransferase-1 (LCMT-1) for PP2A methylation and cell function. We show that reduction of LCMT-1 protein levels by small hairpin RNAs causes up to a 70% reduction in PP2A methylation in HeLa cells, indicating that LCMT-1 is the major mammalian PP2A methyltransferase. In addition, LCMT-1 knockdown reduced the formation of PP2A heterotrimers containing the Balpha regulatory subunit and, in a subset of the cells, induced apoptosis, characterized by caspase activation, nuclear condensation/fragmentation, and membrane blebbing. Knockdown of the PP2A Balpha regulatory subunit induced a similar amount of apoptosis, suggesting that LCMT-1 induces apoptosis in part by disrupting the formation of PP2A(BalphaAC) heterotrimers. Treatment with a pan-caspase inhibitor partially rescued cells from apoptosis induced by LCMT-1 or Balpha knockdown. LCMT-1 knockdown cells and Balpha knockdown cells were more sensitive to the spindle-targeting drug nocodazole, suggesting that LCMT-1 and Balpha are important for spindle checkpoint. Treatment of LCMT-1 and Balpha knockdown cells with thymidine dramatically reduced cell death, presumably by blocking progression through mitosis. Consistent with these results, homozygous gene trap knock-out of LCMT-1 in mice resulted in embryonic lethality. Collectively, our results indicate that LCMT-1 is important for normal progression through mitosis and cell survival and is essential for embryonic development in mice.     

A sds22 homolog that is associated with the testis-specific serine/threonine protein phosphatase 1gamma2 in rat testis

Two cDNAs sequences (1320 bp and 1180 bp) of the 55-kDa subunit associated with a testis-specific serine/threonine protein phosphatase 1gamma2 (PP1gamma2) were cloned. They were the same up to 1180 bp, suggesting that they may be generated by alternative splicing. Sequence studies showed that the 1320 bp-cDNA is a homolog of the human sds22alpha(1) (thus, named rat sds22alpha(1)). The 1180 bp-cDNA is a new splice-variant since its sequence at the 3' end has not been identified in human sds22 genes (named rat sds22alpha(3)). The 1320 bp-cDNA is ubiquitously expressed in various tissues including the immature testis. However, the expression of 1180 bp-cDNA was only observed in the testis after puberty. This expression pattern matches very well with that of PP1gamma2, suggesting that 1180 bp-cDNA may encode the 55-kDa subunit to associate with PP1gamma2 in rat testis and is involved in spermatogenesis by controlling PP1gamma2 activity.     

Juxtamembrane localization of the protein phosphatase-1 inhibitor protein PHI-1 in smooth muscle cells

Protein phosphorylation regulates many fundamental processes and protein phosphatase-1 (PP1) is a major phosphatase that determines the levels of Ser/Thr phosphorylation. Regulatory subunits and inhibitor phosphoproteins control PP1 activity. PHI-1 is a member of a family of PP1 inhibitor phosphoproteins that was discovered based on sequence similarity to the known inhibitor CPI-17. To learn more about PHI-1 we determined the tissue distribution of PHI-1 in embryonic and adult tissues, and examined its cellular localization by immunohistochemistry. In the embryo PHI-1 appeared first in the heart at E10, and by E15 it was detected in multiple tissues. Expression in adult tissues was strikingly different, with PHI-1 detected primarily in smooth muscles in the intestine, blood vessels, and male and female genitourinary tracts. PHI-1 also was highly expressed in the endothelial layer of blood vessels. Both PHI-1 and CPI-17 are expressed predominantly in adult smooth muscles. Whereas CPI-17 staining was diffuse PHI-1 was concentrated along the cell membrane in distinct foci, detected by confocal and electron microscopy. The common tissue distribution but different cellular localization of PHI-1 and CPI-17 suggest distinctive physiological roles for these two PP1 inhibitors.     

Phosphatase PP2A enhances MCL-1 protein half-life in multiple myeloma cells

Multiple myeloma (MM), a treatable but incurable malignancy, is characterized by the growth of clonal plasma cells in protective niches in the bone marrow. MM cells depend on expression of BCL-2 family proteins, in particular MCL-1, for survival. The regulation of MCL-1 is complex and cell type-dependent. Unraveling the exact mechanism by which MCL-1 is overexpressed in MM may provide new therapeutic strategies for inhibition in malignant cells, preferably limiting side effects in healthy cells. In this study, we reveal that one cause of overexpression could be stabilization of the MCL-1 protein. We demonstrate this in a subset of MM and diffuse large B cell lymphoma (DLBCL) cell lines and MM patient samples. We applied a phosphatase siRNA screen to identify phosphatases responsible for MCL-1 stabilization in MM, and revealed PP2A as the MCL-1 stabilizing phosphatase. Using the PP2A inhibitor okadaic acid, we validated that PP2A dephosphorylates MCL-1 at Ser159 and/or Thr163, and thereby stabilizes MCL-1 in MM cells with long MCL-1 half-life, but not in DLBCL cells. Combined kinase and phosphatase inhibition experiments suggest that the MCL-1 half-life in MM is regulated by the counteracting functions of JNK and PP2A. These findings increase the understanding of the mechanisms by which MCL-1 is post-translationally regulated, which may provide novel strategies to inhibit MCL-1 in MM cells.Subject terms: Myeloma, Preclinical research     

Proteomic characterization of protein phosphatase 1 complexes in ischemia-reperfusion and ischemic tolerance

Serine/threonine protein phosphatase 1 (PP1) regulates multiple cellular processes. Protein phosphorylation-dephosphorylation is largely altered during ischemia and subsequent reperfusion. The brain is particularly vulnerable to stress resulting from ischemia-reperfusion (IR), however, the acquisition of ischemic tolerance (IT) protects against IR stress. We studied PP1 complexes in response to IR stress and IT in brain using proteomic characterization of PP1 complexes in animal models of IR and IT. PP1alpha and PP1gamma were immunoprecipitated and resolved by 2-D. DIGE analysis detected 14 different PP1-interacting proteins that exhibited significant changes in their association with PP1alpha or PP1gamma. These proteins were identified by MALDI-TOF MS. Seven had the PP1-binding RVxF motif. IR altered the interaction of heat shock cognate 71 kDa-protein, creatine kinase B, and dopamine- and cAMP-regulated phosphoprotein 32 kDa (DARPP32) with both PP1alpha and PP1gamma, and the interaction of phosphodiesterase-6B, transitional ER ATPase, lamin-A, glucose-regulated 78 kDa-protein, dihydropyrimidinase-related protein-2, gamma-enolase, neurofilament-L, and ubiquitin ligase SIAH2 with PP1gamma. IT prevented most of the IR-induced effects. This study identifies novel PP1alpha- and PP1gamma-interacting proteins and reveals an in vivo modularity of PP1 holoenzymes in response to physiological ischemic stress. It supports a potential role of PP1 in IR stress and as a target of the endogenous protective mechanisms induced by IT.     

Hyperphosphorylation of PP2A in colorectal cancer and the potential therapeutic value showed by its forskolin-induced dephosphorylation and activation

BackgroundThe tumor suppressor protein phosphatase 2A (PP2A) is frequently inactivated in human cancer and phosphorylation of its catalytic subunit (p-PP2A-C) at tyrosine-307 (Y307) has been described to inhibit this phosphatase. However, its molecular and clinical relevance in colorectal cancer (CRC) remains unclear.Methodsp-PP2A-C Y307 was determined by immunoblotting in 7 CRC cell lines and 35 CRC patients. CRC cells were treated with the PP2A activator forskolin alone or combined with the PP2A inhibitor okadaic acid, 5-fluorouracil and oxaliplatin. We examined cell growth, colonosphere formation, caspase activity and AKT and ERK activation.ResultsPP2A-C was found hyperphosphorylated in CRC cell lines. Forskolin dephosphorylated and activated PP2A, impairing proliferation and colonosphere formation, and inducing activation of caspase 3/7 and changes in AKT and ERK phosphorylation. Moreover, forskolin showed additive effects with 5-fluorouracil and oxaliplatin treatments. Analysis of p-PP2A-C Y307 in primary tumors confirmed the presence of this alteration in a subgroup of CRC patients.ConclusionsOur data show that PP2A-C hyperphosphorylation is a frequent event that contributes to PP2A inhibition in CRC. Antitumoral effects of forskolin-mediated PP2A activation suggest that the analysis of p-PP2A-C Y307 status could be used to identify a subgroup of patients who would benefit from treatments based on PP2A activators.     

Sperm PP1gamma2 is regulated by a homologue of the yeast protein phosphatase binding protein sds22

Serine/threonine phosphatase PP1gamma2 is a testis-specific protein phosphatase isoform in spermatozoa. This enzyme appears to play a key role in motility initiation and stimulation. Catalytic activity of PP1gamma2 is higher in immotile compared with motile spermatozoa. Inhibition of PP1gamma2 activity causes both motility initiation and motility stimulation. Protein phosphatases, in general, are regulated by their binding proteins. The objective of this article is to understand the mechanisms by which PP1gamma2 is regulated, first by identifying its regulatory proteins. We had previously shown that a portion of bovine sperm PP1gamma2 is present in the cytosolic fraction of sperm sonicates. We purified PP1gamma2 from soluble bovine sperm extracts by immunoaffinity chromatography. Gel electrophoresis of the purified enzyme showed that it was complexed to a protein 43 M(r) x 10(-3) in size. Microsequencing revealed that this protein is a mammalian homologue of sds22, which is a yeast PP1 binding protein. Phosphatase activity measurements showed that PP1gamma2 complexed to sds22 is catalytically inactive. The complex cannot be activated by limited proteolysis. The complex is unable to bind to microcystin sepharose. This suggests that sds22 may block the microcystin binding site in PP1gamma2. A proportion of PP1gamma2 in sperm extracts, which is presumably not complexed to sds22, is catalytically active. Fluorescence immunocytochemistry was used to determine the intrasperm localization of PP1gamma2 and sds22. Both proteins are present in the tail. They are also present in distinct locations in the head. Our data suggest that PP1gamma2 binding to sds22 inhibits its catalytic activity. Mechanisms regulating sds22 binding to PP1gamma2 are likely to be important in understanding the biochemical basis underlying development and regulation of sperm function.     

Proteomic studies of PP2A-B56gamma1 phosphatase complexes reveal phosphorylation-regulated partners in cardiac local signaling

Defects of kinase-phosphatase signaling in cardiac myocytes contribute to human heart disease. The activity of one phosphatase, PP2A, is governed by B targeting subunits, including B56gamma1, expressed in heart cells. As the role of PP2A/B56gamma1 on the heart function remains largely unknown, this study sought to identify protein partners through unbiased, affinity purification-based proteomics combined with the functional validation. The results reveal multiple interactors that are localized in strategic cardiac sites to participate in Ca2+ homeostasis and gene expression, exemplified by the Ca pump, SERCA2a, and the splicing factor ASF/SF2. These results are corroborated by confocal imaging where adenovirally overexpressed B56gamma1 is found in z-line/t-tubule region and nuclear speckles. Importantly, overexpression of B56gamma1 in cultured myocytes dramatically impairs cell contractility. These results provide a global view of B56gamma1-regulated local signaling and heart function.