RESUMEN
Glucocorticoids play an important role in the treatment of inflammation and immune disorders, despite side effects, which include metabolic derangements such as central adiposity. These studies examine the role of protein phosphatase 5 (Ppp5) in glucocorticoid receptor (GR) complexes which mediate response to glucocorticoids. Mice homozygous for inactivated Ppp5 (Ppp5D274A/D274A) exhibit decreased adipose tissue surrounding the gonads and kidneys compared with wild-type mice. Adipocyte size is smaller, more preadipocytes/stromal cell are present in their gonadal fat tissue and differentiation of preadipocytes to adipocytes is retarded. Glucocorticoid levels are raised and the GR is hyperphosphorylated in adipose tissue of Ppp5D274A/D274A mice at Ser212 and Ser220 (orthologous to human Ser203 and Ser211) in the absence of glucocorticoids. Preadipocyte cultures from Ppp5D274A/D274A mice show decreased down regulation of Delta-like protein-1/preadipocyte factor-1, hyperphosphorylation of extra-cellular signal regulated kinase 2 (ERK2) and increased concentration of (sex determining region Y)-box 9 (SOX9), changes in a pathway essential for preadipocyte differentiation, which leads to decreased concentrations of the transcription factors CEBPß and CEBPα necessary for the later stages of adipogenesis. The data indicate that Ppp5 plays a crucial role in modifying GR-mediated initiation of adipose tissue differentiation, suggesting that inhibition of Ppp5 may potentially be beneficial to prevent obesity during glucocorticoid treatment.
Asunto(s)
Adipocitos/metabolismo , Adipogénesis/genética , Tejido Adiposo/metabolismo , Proteínas Nucleares/genética , Fosfoproteínas Fosfatasas/genética , Receptores de Glucocorticoides/genética , Adipocitos/citología , Adipocitos/efectos de los fármacos , Tejido Adiposo/citología , Tejido Adiposo/efectos de los fármacos , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Diferenciación Celular , Dexametasona/farmacología , Femenino , Regulación de la Expresión Génica , Gónadas/citología , Gónadas/efectos de los fármacos , Gónadas/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Riñón/citología , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Nucleares/deficiencia , PPAR gamma/genética , PPAR gamma/metabolismo , Fosfoproteínas Fosfatasas/deficiencia , Fosforilación , Receptores de Glucocorticoides/metabolismo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Transducción de SeñalRESUMEN
Many pharmaceuticals used to treat cancer target the cell cycle or mitotic spindle dynamics, such as the anti-tumor drug, paclitaxel, which stabilizes microtubules. Here we show that, in cells arrested in mitosis with the spindle toxins, nocodazole, or paclitaxel, the endogenous protein phosphatase 4 (Ppp4) complex Ppp4c-R2-R3A is phosphorylated on its regulatory (R) subunits, and its activity is inhibited. The phosphorylations are blocked by roscovitine, indicating that they may be mediated by Cdk1-cyclin B. Endogenous Ppp4c is enriched at the centrosomes in the absence and presence of paclitaxel, nocodazole, or roscovitine, and the activity of endogenous Ppp4c-R2-R3A is inhibited from G 1/S to the G 2/M phase of the cell cycle. Endogenous γ-tubulin and its associated protein, γ-tubulin complex protein 2, both of which are essential for nucleation of microtubules at centrosomes, interact with the Ppp4 complex. Recombinant γ-tubulin can be phosphorylated by Cdk1-cyclin B or Brsk1 and dephosphorylated by Ppp4c-R2-R3A in vitro. The data indicate that Ppp4c-R2-R3A regulates microtubule organization at centrosomes during cell division in response to stress signals such as spindle toxins, paclitaxel, and nocodazole, and that inhibition of the Ppp4 complex may be advantageous for treatment of some cancers.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Huso Acromático/metabolismo , Tubulina (Proteína)/metabolismo , Secuencia de Aminoácidos , Ciclo Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Células HEK293 , Células HeLa , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Nocodazol/farmacología , Fosfoproteínas Fosfatasas/química , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Mapeo de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Subunidades de Proteína/metabolismo , Huso Acromático/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismoRESUMEN
The liver responds to an increase in blood glucose levels in the postprandial state by uptake of glucose and conversion to glycogen. Liver glycogen synthase (GYS2), a key enzyme in glycogen synthesis, is controlled by a complex interplay between the allosteric activator glucose-6-phosphate (G6P) and reversible phosphorylation through glycogen synthase kinase-3 and the glycogen-associated form of protein phosphatase 1. Here, we initially performed mutagenesis analysis and identified a key residue (Arg(582)) required for activation of GYS2 by G6P. We then used GYS2 Arg(582)Ala knockin (+/R582A) mice in which G6P-mediated GYS2 activation had been profoundly impaired (60-70%), while sparing regulation through reversible phosphorylation. R582A mutant-expressing hepatocytes showed significantly reduced glycogen synthesis with glucose and insulin or glucokinase activator, which resulted in channeling glucose/G6P toward glycolysis and lipid synthesis. GYS2(+/R582A) mice were modestly glucose intolerant and displayed significantly reduced glycogen accumulation with feeding or glucose load in vivo. These data show that G6P-mediated activation of GYS2 plays a key role in controlling glycogen synthesis and hepatic glucose-G6P flux control and thus whole-body glucose homeostasis.
Asunto(s)
Glucosa-6-Fosfato/metabolismo , Glucógeno Sintasa/metabolismo , Hepatocitos/metabolismo , Glucógeno Hepático/biosíntesis , Hígado/metabolismo , Animales , Glucemia/metabolismo , Glucosa/farmacología , Glucógeno Sintasa/genética , Hepatocitos/efectos de los fármacos , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Insulina/farmacología , Hígado/efectos de los fármacos , Ratones , Ratones Transgénicos , Músculo Esquelético/metabolismo , FosforilaciónRESUMEN
Abnormal regulation of brain glycogen metabolism is believed to underlie insulin-induced hypoglycaemia, which may be serious or fatal in diabetic patients on insulin therapy. A key regulator of glycogen levels is glycogen targeted protein phosphatase 1 (PP1), which dephosphorylates and activates glycogen synthase (GS) leading to an increase in glycogen synthesis. In this study, we show that the gene PPP1R3F expresses a glycogen-binding protein (R3F) of 82.8 kDa, present at the high levels in rodent brain. R3F binds to PP1 through a classical 'RVxF' binding motif and substitution of Phe39 for Ala in this motif abrogates PP1 binding. A hydrophobic domain at the carboxy-terminus of R3F has similarities to the putative membrane binding domain near the carboxy-terminus of striated muscle glycogen targeting subunit G(M)/R(GL), and R3F is shown to bind not only to glycogen but also to membranes. GS interacts with PP1-R3F and is hyperphosphorylated at glycogen synthase kinase-3 sites (Ser640 and Ser644) when bound to R3F(Phe39Ala). Deprivation of glucose or stimulation with adenosine or noradrenaline leads to an increased phosphorylation of PP1-R3F bound GS at Ser640 and Ser644 curtailing glycogen synthesis and facilitating glycogen degradation to provide glucose in astrocytoma cells. Adenosine stimulation also modulates phosphorylation of R3F at Ser14/Ser18.
Asunto(s)
Astrocitoma/enzimología , Neoplasias Encefálicas/enzimología , Proteínas Portadoras/fisiología , Espacio Extracelular/fisiología , Glucosa/farmacología , Glucógeno Sintasa/biosíntesis , Fosfoproteínas Fosfatasas/fisiología , Proteína Fosfatasa 1/fisiología , Transducción de Señal/efectos de los fármacos , Adenosina/farmacología , Agonistas alfa-Adrenérgicos/farmacología , Secuencia de Aminoácidos , Animales , Astrocitoma/genética , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Neoplasias Encefálicas/genética , Proteínas Portadoras/genética , Línea Celular Tumoral , ADN/biosíntesis , ADN/genética , Glucógeno/metabolismo , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutagénesis , Norepinefrina/farmacología , Fosfoproteínas Fosfatasas/genética , Fosforilación , Proteína Fosfatasa 1/genética , ARN/biosíntesis , ARN/genética , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismoRESUMEN
Protein phosphorylation is a critical regulatory mechanism in cellular signalling. To this end, PP1 is a major eukaryotic serine/threonine-specific phosphatase whose cellular functions, in turn, depend on complexes it forms with PP1 interacting proteins-PIPs. The importance of the testis/sperm-enriched variant, PP1γ2, in sperm motility and spermatogenesis has previously been shown. Given the key role of PIPs, it is imperative to identify the physiologically relevant PIPs in testis and sperm. Hence, we performed Yeast Two-Hybrid screens of a human testis cDNA library using as baits the different PP1 isoforms and also a proteomic approach aimed at identifying PP1γ2 binding proteins. To the best of our knowledge this is the largest data set of the human testis PP1 interactome. We report the identification of 77 proteins in human testis and 7 proteins in human sperm that bind PP1. The data obtained increased the known PP1 interactome by reporting 72 novel interactions. Confirmation of the interaction of PP1 with 5 different proteins was also further validated by co-immunoprecipitation or protein overlays. The data here presented provides important insights towards the function of these proteins and opens new possibilities for future research. In fact, such diversity in PP1 regulators makes them excellent targets for pharmacological intervention.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Proteína Fosfatasa 1/metabolismo , Testículo/enzimología , Animales , Células COS , Chlorocebus aethiops , ADN Complementario , Biblioteca de Genes , Humanos , Masculino , Unión Proteica , Isoformas de Proteínas , Proteína Fosfatasa 1/genética , Transducción de Señal , Motilidad Espermática/fisiología , Técnicas del Sistema de Dos HíbridosRESUMEN
Activation of 5'-AMP-activated protein kinase (AMPK) is believed to be the mechanism by which the pharmaceuticals, metformin and phenformin, exert their beneficial effects for treatment of type 2 diabetes. These biguanide drugs elevate 5'-AMP, which allosterically activates AMPK and promotes phosphorylation on Thr172 of AMPK catalytic α subunits. Although kinases phosphorylating this site have been identified, phosphatases that dephosphorylate it are unknown. The aim of this study is to identify protein phosphatase(s) that dephosphorylate AMPKα-Thr172 within cells. Our initial data indicated that members of the protein phosphatase Mg/Mn(2+)-dependent [corrected] (PPM) family and not those of the PPP family of protein serine/threonine phosphatases may be directly or indirectly inhibited by phenformin. Using antibodies raised to individual Ppm phosphatases that facilitated the assessment of their activities, phenformin stimulation of cells was found to decrease the Mg(2+)/Mn(2+)-dependent [corrected] protein serine/threonine phosphatase activity of Ppm1E and Ppm1F, but not that attributable to other PPM family members, including Ppm1A/PP2Cα. Depletion of Ppm1E, but not Ppm1A, using lentiviral-mediated stable gene silencing, increased AMPKα-Thr172 phosphorylation approximately three fold in HEK293 cells. In addition, incubation of cells with low concentrations of phenformin and depletion of Ppm1E increased AMPK phosphorylation synergistically. Ppm1E and the closely related Ppm1F interact weakly with AMPK and assays with lysates of cells stably depleted of Ppm1F suggest [corrected] that this phosphatase contributes to dephosphorylation of AMPK. The data indicate that Ppm1E and probably PpM1F are in cellulo AMPK phosphatases and that Ppm1E is a potential anti-diabetic drug target.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Silenciador del Gen , Células HEK293 , Humanos , Fenformina/farmacología , Fosfoproteínas Fosfatasas/genética , Fosforilación , Proteína Fosfatasa 2C , Interferencia de ARNRESUMEN
Type 2 diabetes is characterised by elevated blood glucose concentrations, which potentially could be normalised by stimulation of hepatic glycogen synthesis. Under glycogenolytic conditions, the interaction of hepatic glycogen-associated protein phosphatase-1 (PP1-G(L)) with glycogen phosphorylase a is believed to inhibit the dephosphorylation and activation of glycogen synthase (GS) by the PP1-G(L) complex, suppressing glycogen synthesis. Consequently, the interaction of G(L) with phosphorylase a has emerged as an attractive anti-diabetic target, pharmacological disruption of which could provide a novel mechanism to lower blood glucose levels by increasing hepatic glycogen synthesis. Here we report for the first time the in vivo consequences of disrupting the G(L)-phosphorylase a interaction, using a mouse model containing a Tyr284Phe substitution in the phosphorylase a-binding region of the G(L) protein. The resulting G(L)(Y284F/Y284F) mice display hepatic PP1-G(L) activity that is no longer sensitive to allosteric inhibition by phosphorylase a, resulting in increased GS activity under glycogenolytic conditions, demonstrating that regulation of G(L) by phosphorylase a operates in vivo. G(L)(Y284F/Y284F) and G(L)(Y284F/+) mice display improved glucose tolerance compared with G(L)(+/+) littermates, without significant accumulation of hepatic glycogen. The data provide the first in vivo evidence in support of targeting the G(L)-phosphorylase a interaction for treatment of hyperglycaemia. During prolonged fasting the G(L)(Y284F/Y284F) mice lose more body weight and display decreased blood glucose levels in comparison with their G(L)(+/+) littermates. These results suggest that, during periods of food deprivation, the phosphorylase a regulation of G(L) may prevent futile glucose-glycogen cycling, preserving energy and thus providing a selective biological advantage that may explain the observed conservation of the allosteric regulation of PP1-G(L) by phosphorylase a in mammals.
Asunto(s)
Glucosa/metabolismo , Glucógeno Fosforilasa de Forma Hepática/metabolismo , Glucógeno Hepático/metabolismo , Proteína Fosfatasa 1/metabolismo , Regulación Alostérica , Animales , Peso Corporal , Cruzamientos Genéticos , Ayuno/sangre , Femenino , Marcación de Gen , Prueba de Tolerancia a la Glucosa , Glucógeno Sintasa/metabolismo , Heterocigoto , Humanos , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Fosforilación , Conejos , Pérdida de PesoRESUMEN
Cisplatin (cis-diamminedichloroplatinum) and related chemotherapeutic DNA-crosslinking agents are widely used to treat human cancers. Saccharomyces cerevisiae with separate deletions of the genes encoding the trimeric protein serine/threonine phosphatase (Pph)3p-platinum sensitivity (Psy)4p-Psy2p complex, are more sensitive than the isogenic wild-type (WT) strain to cisplatin. We show here that cisplatin causes an enhanced intra-S-phase cell cycle delay in these three deletion mutants. The C-terminal tail of histone 2AX (H2AX) is hyperphosphorylated in the same mutants, and Pph3p is found to interact with phosphorylated H2AX (gammaH2AX). After cisplatin treatment is terminated, pph3Delta, psy4Delta and psy2Delta mutants are delayed as compared with the WT strain in the dephosphorylation of Rad53p. In contrast, only pph3Delta and psy2Delta cells are more sensitive than WT cells to methylmethanesulfonate, a noncrosslinking DNA-alkylating agent that is known to cause a Rad53p-dependent intra-S-phase cell cycle delay. Dephosphorylation of Rad53p and the recovery of chromosome replication are delayed in the same mutants, but not in psy4Delta cells. By comparison with their mammalian orthologues, the regulatory subunit Psy4p is likely to inhibit Pph3p catalytic activity. The presence of a weak but active Pph3p-Psy2p complex may allow psy4Delta cells to escape from the Rad53p-mediated cell cycle arrest. Overall, our data suggest that the trimeric Pph3p-Psy4p-Psy2p complex may dephosphorylate both gammaH2AX and Rad53p, the differences lying in the more stable interaction of the Pph3 phosphatase with gammaH2AX as opposed to a transient interaction with Rad53p.
Asunto(s)
Antineoplásicos/toxicidad , Cisplatino/toxicidad , Reactivos de Enlaces Cruzados/toxicidad , Daño del ADN , Proteínas Nucleares/fisiología , Fosfoproteínas Fosfatasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Alquilantes/toxicidad , Ciclo Celular/efectos de los fármacos , Cromosomas Fúngicos/metabolismo , Replicación del ADN , Eliminación de Gen , Histonas/metabolismo , Metilmetanosulfonato/toxicidad , Proteínas Nucleares/genética , Fosfoproteínas Fosfatasas/genética , Subunidades de Proteína/genética , Subunidades de Proteína/fisiología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The mechanisms that co-ordinate centrosome maturation and the migration of human cells remain elusive. Protein phosphatase 4 (Ppp4) is a ubiquitous protein serine/threonine phosphatase in eukaryotes that is enriched at centrosomes. HEK293 cells cultures depleted to 30% Ppp4c levels by lentivirus-delivered stable gene silencing were delayed in mitosis at the prometaphase/metaphase boundary and displayed cells with aberrant chromosome organisation and microtubules unconnected to the centrosomes. The levels of alpha- and gamma-tubulin and aurora A were decreased; in mitotic cells, the cytological localisations of polo-like kinase 1, alpha- and gamma-tubulin and aurora A were aberrant and the phosphorylation of Aurora A-Thr 288 was decreased. The novel localisation of endogenous Ppp4 regulatory subunit, R3A, to centrosomes in human mitotic cells suggests that a Ppp4c-R2-R3 trimeric complex mediates centrosome maturation. We demonstrate for the first time that human cells depleted to 30% Ppp4c showed severely decreased migration and exhibit decreased levels of both total beta-actin and filamentous actin in cell extensions, filopodia and lamellopodia-like structures. Our studies show that Ppp4c is required for the organisation of the actin cytoskeleton at the leading edge of human cells during migration. We also demonstrate that the active forms of the RhoGTPases, Rac1 and Cdc42, are substantially decreased in the presence and absence of growth factor in Ppp4c depleted cells, implicating Ppp4c in the regulation of these GTPases. The results suggest that Ppp4c-R2-R3 complexes may co-ordinate centrosome maturation and cell migration via regulation of RhoGTPases and that Ppp4 may be a useful anticancer target.
Asunto(s)
Movimiento Celular , Centrosoma/enzimología , Fosfoproteínas Fosfatasas/deficiencia , Proteínas de Unión al GTP rho/metabolismo , Aurora Quinasas , Caspasas/metabolismo , Recuento de Células , Muerte Celular , Línea Celular , Forma de la Célula , Centriolos/enzimología , Humanos , Lentivirus/metabolismo , Mitosis , Modelos Biológicos , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Cuaternaria de Proteína , Transporte de Proteínas , Tubulina (Proteína)/metabolismoRESUMEN
A prediabetic phenotype of glucose intolerance, insulin resistance and obesity was observed at approximately 12 months of age in mice homozygous for a null allele of the major skeletal muscle glycogen-targeting subunit G(M) of protein phosphatase 1 (PP1) and derived from a 129/Ola donor strain. In this study, backcrossing of these G(M)-/- mice (termed obese G(M)-/- mice) onto two different genetic backgrounds gave rise to lean, glucose-tolerant, insulin-sensitive G(M)-/- mice (termed lean G(M)-/- mice), indicating that at least one variant gene in the 129/Ola background, not present in the C57BL/6 or 129s2/sV background, is required for the development of the prediabetic phenotype of obese mice. Slightly elevated AMP-activated protein kinase alpha2 activity in the skeletal muscle of lean C57BL/6 mice was also observed to a lesser extent in the obese G(M)-/- mice. Normal or slightly raised in vivo glucose transport in lean C57BL/6 G(M)-/- mice compared with decreased glucose transport in the obese G(M)-/- mice supports the tenet that adequate transport of glucose may be a key factor in preventing the development of the prediabetic phenotype. The pH 6.8/pH 8.6 activity ratio of phosphorylase kinase was increased in lean C57BL/6 G(M)-/- mice compared with controls indicating that phosphorylase kinase is an in vivo substrate of PP1-G(M).
Asunto(s)
Glucógeno/metabolismo , Músculo Esquelético/enzimología , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Quinasas Activadas por AMP , Tejido Adiposo/metabolismo , Animales , Cruzamientos Genéticos , Ambiente , Femenino , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Miembro Posterior/enzimología , Isoenzimas/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Complejos Multienzimáticos/metabolismo , Fenotipo , Proteína Fosfatasa 1/química , Proteínas Serina-Treonina Quinasas/metabolismo , DelgadezRESUMEN
The inhibition of hepatic glycogen-associated protein phosphatase-1 (PP1-G(L)) by glycogen phosphorylase a prevents the dephosphorylation and activation of glycogen synthase, suppressing glycogen synthesis when glycogenolysis is activated. Here, we show that a peptide ((280)LGPYY(284)) comprising the last five amino acids of G(L) retains high-affinity interaction with phosphorylase a and that the two tyrosines play crucial roles. Tyr284 deletion abolishes binding of phosphorylase a to G(L) and replacement by phenylalanine is insufficient to restore high-affinity binding. We show that a phosphorylase inhibitor blocks the interaction of phosphorylase a with the G(L) C-terminus, suggesting that the latter interaction could be targeted to develop an anti-diabetic drug.
Asunto(s)
Glucógeno Fosforilasa de Forma Hepática/metabolismo , Indoles/farmacología , Fenilbutiratos/farmacología , Tirosina/metabolismo , Secuencia de Aminoácidos , Animales , Calorimetría , Glucógeno Fosforilasa de Forma Hepática/química , Glucógeno Fosforilasa de Forma Hepática/genética , Humanos , Ratones , Datos de Secuencia Molecular , Mutación/genética , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Conejos , Ratas , Termodinámica , Volumetría , Tirosina/genéticaRESUMEN
Adrenaline and insulin are the major hormones regulating glycogen metabolism in skeletal muscle. We have investigated the effects of these hormones on the rate-limiting enzymes of glycogen degradation and synthesis (phosphorylase and glycogen synthase respectively) in GM-/- mice homozygous for a null allele of the major skeletal muscle glycogen targeting subunit (GM) of protein phosphatase 1 (PP1). Hyperphosphorylation of Ser14 in phosphorylase, and Ser7, Ser640 and Ser640/644 of GS, in the skeletal muscle of GM-/- mice compared with GM+/+ mice indicates that the PP1-GM complex is the major phosphatase that dephosphorylates these sites in vivo. Adrenaline caused a 2.4-fold increase in the phosphorylase (-/+AMP) activity ratio in the skeletal muscle of control mice compared to a 1.4 fold increase in GM-/- mice. Adrenaline also elicited a 67% decrease in the GS (-/+G6P) activity ratio in control mice but only a small decrease in the skeletal muscle of GM-/- mice indicating that GM is required for the full response of phosphorylase and GS to adrenaline. PP1-GM activity and the amount of PP1 bound to GM decreased 40% and 45% respectively, in response to adrenaline in control mice. The data support a model in which adrenaline stimulates phosphorylation of phosphorylase Ser14 and GS Ser7 in GM+/+ mice by both kinase activation and PP1-GM inhibition and the phosphorylation of GS Ser640 and Ser640/644 by PP1-GM inhibition alone. Insulin decreased the phosphorylation of GS Ser640 and Ser640/644 and stimulated the GS (-/+G6P) activity ratio by approximately 2-fold in the skeletal muscle of either GM-/- and or control mice, but the low basal and insulin stimulated GS activity ratios in GM-/- mice indicate that PP1-GM is essential for maintaining normal basal and maximum insulin stimulated GS activity ratios in vivo.
Asunto(s)
Epinefrina/metabolismo , Glucógeno/metabolismo , Músculo Esquelético/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Glucógeno Sintasa/metabolismo , Insulina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Músculo Esquelético/enzimología , Miocardio/metabolismo , Mapeo Peptídico , Fosfoproteínas Fosfatasas/genética , Fosforilación , Proteína Fosfatasa 1RESUMEN
PP1 (protein phosphatase 1) is a ubiquitously expressed serine/threonine-specific protein phosphatase whose activity towards different substrates appears to be mediated via binding to specific proteins that play critical regulatory and targeting roles. In the present paper we report the cloning and characterization of a new protein, termed SARP (several ankyrin repeat protein), which is shown to interact with all isoforms of PP1 by a variety of techniques. A region encompassing a consensus PP1-binding motif in SARP (K354VHF357) modulates endogenous SARP-PP1 activity in mammalian cells. This SARP-PP1 interaction motif lies partially within the first ankyrin repeat in contrast with other proteins [53BP2 (p53 binding protein 2), MYPT1/M(110)/MBS (myosin binding protein of PP1) and TIMAP (transforming growth factor beta inhibited, membrane-associated protein)], where a PP1-binding motif precedes the ankyrin repeats. Alternative mRNA splicing produces several isoforms of SARP from a single human gene at locus 11q14. SARP1 and/or SARP2 (92-95 kDa) are ubiquitously expressed in all tissues with high levels in testis and sperm, where they are shown to interact with both PP1gamma1 and PP1gamma2. SARP3 (65 kDa) is most abundant in brain where SARP isoforms interact with both PP1alpha and PP1gamma1. SARP is highly abundant in the nucleus of mammalian cells, consistent with the putative nuclear localization signal at the N-terminus. The presence of a leucine zipper near the C-terminus of SARP1 and SARP2, and the binding of mammalian DNA to SARP2, suggests that SARP1 and SARP2 may be transcription factors or DNA-associated proteins that modulate gene expression.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas del Ojo/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Repetición de Anquirina , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Clonación Molecular , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas del Ojo/química , Proteínas del Ojo/genética , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Leucina Zippers , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Fosfatasa 1 , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
The anticancer agents cisplatin and oxaliplatin are widely used in the treatment of human neoplasias. A genome-wide screen in Saccharomyces cerevisiae previously identified PPH3 and PSY2 among the top 20 genes conferring resistance to these anticancer agents. The mammalian orthologue of Pph3p is the protein serine/threonine phosphatase Ppp4c, which is found in high molecular mass complexes bound to a regulatory subunit R2. We show here that the putative S. cerevisiae orthologue of R2, which is encoded by ORF YBL046w, binds to Pph3p and exhibits the same unusually high asymmetry as mammalian R2. Despite the essential function of Ppp4c-R2 in microtubule-related processes at centrosomes in higher eukaryotes, S. cerevisiae diploid strains with homozygous deletion of YBL046w and two or one functional copies of the TUB2 gene were viable and no more sensitive to microtubule-depolymerizing drugs than the control strain. The protein encoded by YBL046w exhibited a predominantly nuclear localization. These studies suggest that the centrosomal function of Ppp4c-R2 is not required or may be performed by a different phosphatase in yeast. Homozygous diploid deletion strains of S. cerevisiae, pph3Delta, ybl046wDelta and psy2Delta, were all more sensitive to cisplatin than the control strain. The YBL046w gene therefore confers resistance to cisplatin and was termed PSY4 (platinum sensitivity 4). Ppp4c, R2 and the putative orthologue of Psy2p (termed R3) are shown here to form a complex in Drosophila melanogaster and mammalian cells. By comparison with the yeast system, this complex may confer resistance to cisplatin in higher eukaryotes.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Resistencia a Antineoplásicos/genética , Fosfoproteínas Fosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Animales , Antineoplásicos Alquilantes/farmacología , Benomilo/farmacología , Sitios de Unión , Proteínas de Ciclo Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Quinasa de Punto de Control 2 , Relación Dosis-Respuesta a Droga , Drosophila/citología , Homocigoto , Humanos , Metilmetanosulfonato/farmacología , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/clasificación , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/aislamiento & purificación , Unión Proteica , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/efectos de los fármacos , Homología de Secuencia de AminoácidoRESUMEN
Microcystins produced by cyanobacterial 'blooms' in reservoirs and lakes pose significant public health problems because they are highly toxic due to potent inhibition of protein serine/threonine phosphatases in the PPP family. A dehydrobutyrine (Dhb)-containing microcystin variant [Asp3, ADMAdda5, Dhb7]microcystin-HtyR isolated from Nostoc sp. was found to potently inhibit PP1, PP2A, PPP4 and PPP5 with IC50 values similar to those of microcystin-LR. However, in contrast to microcystin-LR, which forms a covalent bond with a cysteine residue in these protein phosphatases, Asp,ADMAdda,Dhb-microcystin-HtyR did not form any covalent interaction with PP2A. Since the LD50 for Asp,ADMAdda,Dhb-microcystin-HtyR was 100 microg kg(-1) compared to 50 microg kg(-1) for microcystin-LR, the data indicate that the non-covalent inhibition of protein phosphatases accounts for most of the harmful effects of microcystins in vivo. A 3-amino-6-hydroxy-2-piperidone containing cyclic peptide, nostocyclin, also isolated from Nostoc sp., was non-toxic and exhibited more than 500-fold less inhibitory potency towards PP1, PP2A, PPP4 and PPP5, consistent with the conclusion that potent inhibition of one or more these protein phosphatases underlies the toxicity of microcystins, both lacking and containing Dhb.
Asunto(s)
Proteínas Bacterianas/química , Toxinas Bacterianas/química , Inhibidores Enzimáticos/química , Nostoc/química , Péptidos Cíclicos/química , Fosfoproteínas Fosfatasas/química , Aminobutiratos/química , Activación Enzimática , Humanos , Toxinas Marinas , Microcistinas , Fosfoproteínas Fosfatasas/antagonistas & inhibidoresRESUMEN
Protein phosphatase 4 (Ppp4) is a ubiquitous serine/threonine phosphatase in the PPP family that is now recognised to regulate a variety of cellular functions independently of protein phosphatase 2A (PP2A). Regulatory subunits (R1 and R2) have been identified in mammals that interact with the catalytic subunit of Ppp4 (Ppp4c) and control its activity. Ppp4c-R2 complexes play roles in organelle assembly; not only are they essential for maturation of the centrosome, but they are also involved in spliceosomal assembly via interaction with the survival of motor neurons (SMNs) complex. Several cellular signalling routes, including NF-kappaB and the target of rapamycin (TOR) pathways appear to be regulated by Ppp4. Emerging evidence indicates that Ppp4 may play a role in the DNA damage response and that Ppp4c-R1 complexes decrease the activity of a histone deacetylase, implicating Ppp4 in the regulation of chromatin activities. Antitumour agents, cantharidin and fostriecin, potently inhibit the activity of Ppp4. Orthologues of mammalian Ppp4 subunits in Saccharomyces cerevisiae confer resistance to the anticancer, DNA-binding drugs, cisplatin and oxaliplatin.
Asunto(s)
Fosfoproteínas Fosfatasas/metabolismo , Animales , Antineoplásicos/farmacología , Núcleo Celular/metabolismo , Humanos , Orgánulos/metabolismo , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/clasificación , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 2 , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transducción de SeñalRESUMEN
Stimulation of glycogen-targeted protein phosphatase 1 (PP1) activity by insulin contributes to the dephosphorylation and activation of hepatic glycogen synthase (GS) leading to an increase in glycogen synthesis. The glycogen-targeting subunits of PP1, GL and R5/PTG, are downregulated in the livers of diabetic rodents and restored by insulin treatment. We show here that the mammalian gene PPP1R3E encodes a novel glycogen-targeting subunit of PP1 that is expressed in rodent liver. The phosphatase activity associated with R3E is slightly higher than that associated with R5/PTG and it is downregulated in streptozotocin-induced diabetes by 60-70% and restored by insulin treatment. Surprisingly, although mRNA for R3E is most highly expressed in rat liver and heart muscle, with only low levels in skeletal muscle, R3E mRNA is most abundant in human skeletal muscle and heart tissues with barely detectable levels in human liver. This species-specific difference in R3E mRNA expression has similarities to the high level of expression of GL mRNA in human but not rodent skeletal muscle. The observations imply that the mechanisms by which insulin regulates glycogen synthesis in liver and skeletal muscle are different in rodents and humans.
Asunto(s)
Glucógeno/metabolismo , Insulina/farmacología , Fosfoproteínas Fosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Secuencia de Consenso , ADN Complementario , Diabetes Mellitus Experimental/enzimología , Biblioteca de Genes , Humanos , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 1 , Subunidades de Proteína/metabolismo , Ratas , Ratas Wistar , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Ppp5 (protein phosphatase 5) is a serine/threonine protein phosphatase that has been conserved throughout eukaryotic evolution. In mammalian cells, FLAG-tagged Ppp5 and endogenous Ppp5 are found to interact with endogenous Hsp (heat-shock protein) 70, as well as Hsp90. Incubation of cells with arachidonic acid or the microtubule-depolymerizing agent, nocodazole, causes loss of interaction of Hsp70 and Hsp90 with FLAG-tagged Ppp5 and increase of Ppp5 activity. In response to the same treatments, endogenous Ppp5 undergoes proteolytic cleavage of the N- and C-termini, with the subsequent appearance of high-molecular-mass species. The results indicate that Ppp5 is activated by proteolysis on dissociation from Hsps, and is destroyed via the proteasome after ubiquitination. Cleavage at the C-terminus removes a nuclear localization sequence, allowing these active cleaved forms of Ppp5 to translocate to the cytoplasm. The response of Ppp5 to arachidonic acid and nocodazole suggests that Ppp5 may be required for stress-related processes that can sometimes cause cell-cycle arrest, and leads to the first description for in vivo regulation of Ppp5 activity.