Protein Phosphatase 2C Activity Research Articles

Protein phosphatase PP2C in the flagellum of Leishmania major: cloning and characterization

AbstractThe main goal of this work consisted in cloning, purifying and characterizing a protein phosphatase 2C (PP2C) from promastigotes ofLeishmania major. The gene was cloned and amplified by PCR using specific oligonucleotides and the recombinant protein was purified by affinity chromatography. The peak with maximal protein concentration was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and revealed a protein of 44·9 kDa with PP2C activity. This activity was dependent on divalent cations (Mg+2and Mn+2) and was optimal at pH of 8·5, using phosphothreonine as the substrate. Sanguinarine inhibited the activity of the recombinantLmPP2C, while protein tyrosine phosphatase inhibitors had no effect. The recombinantLmPP2C was used to generate polyclonal antibodies. These antibodies recognized a protein of 44·9 kDa in differentLeishmaniaspecies; theLmPP2C was localized in the flagellar pocket and the flagellum of promastigotes.

Tert-Butylhydroquinone mobilizes intracellular-bound zinc to stabilize Nrf2 through inhibiting phosphatase activity.

The nuclear factor erythroid 2-related factor 2 (Nrf2) is required to combat increases in oxidative stress. The chemical compound tert-butylhydroquinone (tBHQ) can downregulate Kelch-like ECH-associated protein 1 (Keap1), a repressor of Nrf2, thus maintaining the stability of Nrf2. tBHQ can also increase intracellular "free" zinc in human bronchial epithelial (16HBE) cells. We aim to investigate whether the intracellular free zinc change plays a role in Nrf2 activation. tBHQ exposure dose-dependently increases intracellular free zinc concentrations within 30 min in 16HBE cells by mobilizing intracellular zinc pools. Active Nrf2 and the antioxidant enzyme heme oxygenase-1 (HO-1) increase at 3 h after tBHQ treatment. Chelating intracellular free zinc with tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) during tBHQ exposure partially abrogates the tBHQ-induced activation of Nrf2 and HO-1 expression, while Keap1 is further decreased. These results indicate that tBHQ-induced stability of Nrf2 is associated with the intracellular free zinc level. Because the activated Nrf2 is phosphorylated, the serine/threonine protein phosphatase activity, which is known to be inhibited by zinc, is assayed. The results showed that tBHQ treatment can suppress cellular protein phosphatase-2A (PP2A) and protein phosphatase-2C (PP2C) activity, which can be abrogated by adding TPEN. This finding is verified in a cell-free protein extract experiment by supplying zinc or by chelating zinc with TPEN. These results provide a novel mechanistic insight into Nrf2 activation in antioxidant enzyme induction involving zinc signaling. The increase of intracellular free zinc may be one mechanism for Nrf2 activation. The inhibition of PP2A and PP2C activity may be involved in Nrf2 phosphorylation modulation.

Inhibition of vascular smooth muscle growth via signaling crosstalk between AMP-activated protein kinase and cAMP-dependent protein kinase

Abnormal vascular smooth muscle (VSM) growth is central in the pathophysiology of vascular disease yet fully effective therapies to curb this growth are lacking. Recent findings from our lab and others support growth control of VSM by adenosine monophosphate (AMP)-based approaches including the metabolic sensor AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA). Molecular crosstalk between AMPK and PKA has been previously suggested, yet the extent to which this occurs and its biological significance in VSM remain unclear. Considering their common AMP backbone and similar signaling characteristics, we hypothesized that crosstalk exists between AMPK and PKA in the regulation of VSM growth. Using rat primary VSM cells (VSMC), the AMPK agonist AICAR increased AMPK activity and phosphorylation of the catalytic Thr172 site on AMPK. Interestingly, AICAR also phosphorylated a suspected PKA-inhibitory Ser485 site on AMPK, and these cumulative events were reversed by the PKA inhibitor PKI suggesting possible PKA-mediated regulation of AMPK. AICAR also increased PKA activity in a reversible fashion. The cAMP stimulator forskolin increased PKA activity and completely ameliorated Ser/Thr protein phosphatase-2C activity, suggesting a potential mechanism of AMPK modulation by PKA since inhibition of PKA by PKI reduced AMPK activity. Functionally, AMPK inhibited serum-stimulated cell cycle progression and cellular proliferation; however, PKA failed to do so. Moreover, AMPK and PKA reduced PDGF-β-stimulated VSMC migration. Collectively, these results show that AMPK is capable of reducing VSM growth in both anti-proliferative and anti-migratory fashion. Furthermore, these data suggest that AMPK may be modulated by PKA and that positive feedback may exist between these two systems. These findings reveal a discrete nexus between AMPK and PKA in VSM and provide basis for metabolically-directed targets in reducing pathologic VSM growth.

Evolution of Abscisic Acid Synthesis and Signaling Mechanisms

The plant hormone abscisic acid (ABA) mediates seed dormancy, controls seedling development and triggers tolerance to abiotic stresses, including drought. Core ABA signaling components consist of a recently identified group of ABA receptor proteins of the PYRABACTIN RESISTANCE (PYR)/REGULATORY COMPONENT OF ABA RECEPTOR (RCAR) family that act as negative regulators of members of the PROTEIN PHOSPHATASE 2C (PP2C) family. Inhibition of PP2C activity enables activation of SNF1-RELATED KINASE 2 (SnRK2) protein kinases, which target downstream components, including transcription factors, ion channels and NADPH oxidases. These and other components form a complex ABA signaling network. Here, an in depth analysis of the evolution of components in this ABA signaling network shows that (i) PYR/RCAR ABA receptor and ABF-type transcription factor families arose during land colonization of plants and are not found in algae and other species, (ii) ABA biosynthesis enzymes have evolved to plant- and fungal-specific forms, leading to different ABA synthesis pathways, (iii) existing stress signaling components, including PP2C phosphatases and SnRK kinases, were adapted for novel roles in this plant-specific network to respond to water limitation. In addition, evolutionarily conserved secondary structures in the PYR/RCAR ABA receptor family are visualized.

Infusion of a Lipid Emulsion Modulates AMPK and Related Proteins in Rat Liver, Muscle, and Adipose Tissues

The primary objective of this study was to investigate the impact of lipid oversupply on the AMPK pathway in skeletal muscle, liver, and adipose tissue. Male Wistar rats were infused with lipid emulsion (LE) or phosphate-buffered saline for 5 h/day for 6 days. Muscles exposed to LE for 6 days exhibited increased AMPK and acetyl-CoA carboxylase (ACC) phosphorylation, along with a greater association between AMPK and Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK). No differences in muscle protein phosphatase 2C (PP2C) activity, LKB1 phosphorylation or AMPK and LKB1 association were observed. Muscle ACCbeta, and adiponectin receptor 1 (AdipoR1) mRNA levels and PPARgamma-co-activator 1alpha (PGC1alpha) protein levels were also increased in LE-treated rats. In contrast, AMPK and ACC phosphorylation decreased and PP2C activity increased in rat livers exposed to LE. Hepatic mRNA levels of ACCalpha, PPARalpha, AdipoR1, AdipoR2, and sterol regulatory element-binding protein-1c (SREBP1c) were also reduced after LE infusion. In adipose tissue, there was no significant alteration in AMPK or ACC phosphorylation. These results demonstrate that following lipid oversupply the AMPK pathway was enhanced in rat skeletal muscle while diminished in the liver and was unchanged in adipose tissue. CaMKK in skeletal muscle and PP2C in the liver, at least in part, appear to mediate these alterations. Alterations in AMPK pathway in the liver induced metabolic defects associated with lipid oversupply.

Rice XB15, a Protein Phosphatase 2C, Negatively Regulates Cell Death and XA21-Mediated Innate Immunity

Perception of extracellular signals by cell surface receptors is of central importance to eukaryotic development and immunity. Kinases that are associated with the receptors or are part of the receptors themselves modulate signaling through phosphorylation events. The rice (Oryza sativa L.) XA21 receptor kinase is a key recognition and signaling determinant in the innate immune response. A yeast two-hybrid screen using the intracellular portion of XA21, including the juxtamembrane (JM) and kinase domain as bait, identified a protein phosphatase 2C (PP2C), called XA21 binding protein 15 (XB15). The interaction of XA21 and XB15 was confirmed in vitro and in vivo by glutathione-S-transferase (GST) pull-down and co-immunoprecipitation assays, respectively. XB15 fusion proteins purified from Escherichia coli and from transgenic rice carry PP2C activity. Autophosphorylated XA21 can be dephosphorylated by XB15 in a temporal- and dosage-dependent manner. A serine residue in the XA21 JM domain is required for XB15 binding. Xb15 mutants display a severe cell death phenotype, induction of pathogenesis-related genes, and enhanced XA21-mediated resistance. Overexpression of Xb15 in an XA21 rice line compromises resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae. These results demonstrate that Xb15 encodes a PP2C that negatively regulates the XA21-mediated innate immune response.

ABA-Hypersensitive Germination3Encodes a Protein Phosphatase 2C (AtPP2CA) That Strongly Regulates Abscisic Acid Signaling during Germination among Arabidopsis Protein Phosphatase 2Cs

The phytohormone abscisic acid (ABA) regulates physiologically important developmental processes and stress responses. Previously, we reported on Arabidopsis (Arabidopsis thaliana) L. Heynh. ahg mutants, which are hypersensitive to ABA during germination and early growth. Among them, ABA-hypersensitive germination3 (ahg3) showed the strongest ABA hypersensitivity. In this study, we found that the AHG3 gene is identical to AtPP2CA, which encodes a protein phosphatase 2C (PP2C). Although AtPP2CA has been reported to be involved in the ABA response on the basis of results obtained by reverse-genetics approaches, its physiological relevance in the ABA response has not been clarified yet. We demonstrate in vitro and in vivo that the ahg3-1 missense mutation causes the loss of PP2C activity, providing concrete confirmation that this PP2C functions as a negative regulator in ABA signaling. Furthermore, we compared the effects of disruption mutations of eight structurally related PP2C genes of Arabidopsis, including ABI1, ABI2, HAB1, and HAB2, and found that the disruptant mutant of AHG3/AtPP2CA had the strongest ABA hypersensitivity during germination, but it did not display any significant phenotypes in adult plants. Northern-blot analysis clearly showed that AHG3/AtPP2CA is the most active among those PP2C genes in seeds. These results suggest that AHG3/AtPP2CA plays a major role among PP2Cs in the ABA response in seeds and that the functions of those PP2Cs overlap, but their unique tissue- or development-specific expression confers distinct and indispensable physiological functions in the ABA response.

Allosteric Activation of Protein Phosphatase 2C by d-chiro-Inositol−Galactosamine, a Putative Mediator Mimetic of Insulin Action

Insulin-stimulated glucose disposal in skeletal muscle proceeds predominantly through a nonoxidative pathway with glycogen synthase as a rate-limiting enzyme, yet the mechanisms for insulin activation of glycogen synthase are not understood despite years of investigation. Isolation of putative insulin second messengers from beef liver yielded a pseudo-disaccharide consisting of pinitol (3-O-methyl-d-chiro-inositol) beta-1,4 linked to galactosamine chelated with Mn(2+) (called INS2). Here we show that chemically synthesized INS2 has biological activity that significantly enhances insulin reduction of hyperglycemia in streptozotocin diabetic rats. We used computer modeling to dock INS2 onto the known three-dimensional crystal structure of protein phosphatase 2C (PP2C). Modeling and FlexX/CScore energy minimization predicted a unique favorable site on PP2C for INS2 in a surface cleft adjacent to the catalytic center. Binding of INS2 is predicted to involve formation of multiple H-bonds, including one with residue Asp163. Wild-type PP2C activity assayed with a phosphopeptide substrate was potently stimulated in a dose-dependent manner by INS2. In contrast, the D163A mutant of PP2C was not activated by INS2. The D163A mutant and wild-type PP2C in the absence of INS2 had the same Mn(2+)-dependent phosphatase activity with p-nitrophenyl phosphate as a substrate, showing that this mutation did not disrupt the catalytic site. We propose that INS2 allosterically activates PP2C, fulfilling the role of a putative mediator mimetic of insulin signaling to promote protein dephosphorylation and metabolic responses.

Modulation of integrin signal transduction by ILKAP, a protein phosphatase 2C associating with the integrin-linked kinase, ILK1.

ILKAP, a protein serine/threonine (S/T) phosphatase of the PP2C family, was isolated in a yeast two-hybrid screen baited with integrin-linked kinase, ILK1. Association of ILK1 and ILKAP was independent of the catalytic activity of either partner, as assayed in co-precipitation and two-hybrid experiments. Condi tional expression of ILKAP in HEK 293 cells resulted in selective inhibition of ECM- and growth factor-stimulated ILK1 activity, but did not inhibit Raf-1 kinase activity. A catalytic mutant of ILKAP, H154D, did not inhibit ILK1 kinase activity. Two cellular targets of ILK1, glycogen synthase kinase 3 beta (GSK3beta) and protein kinase B (PKB)/AKT, were differentially affected by ILKAP-mediated inhibition of ILK1. Catalytically active, but not mutant ILKAP, strongly inhibited insulin-like growth factor-1-stimulated GSK3beta phosphorylation on Ser9, but did not affect phosphorylation of PKB on Ser473, suggesting that ILKAP selectively affects ILK-mediated GSK3beta signalling. Consistent with this, active, but not H154D mutant or the related PP2Calpha, selectively inhibited transactivation of a Tcf/Lef reporter gene, TOPFlash, in 293 cells. We propose that ILKAP regulates ILK1 activity, targeting ILK1 signalling of Wnt pathway components via modulation of GSK3beta phosphorylation.

Mutational analysis of protein phosphatase 2C involved in abscisic acid signal transduction in higher plants.

Protein phosphatase 2C (PP2C) is a class of ubiquitous and evolutionarily conserved serine/threonine PP involved in stress responses in yeasts, mammals, and plants. Here, I present mutational analysis of two Arabidopsis thaliana PP2Cs, encoded by ABI1 and AtPP2C, involved in the plant stress hormone abscisic acid (ABA) signaling in maize mesophyll protoplasts. Consistent with the crystal structure of the human PP2C, the mutation of two conserved motifs in ABI1, predicted to be involved in metal binding and catalysis, abolished PP2C activity. Surprisingly, although the DGH177-179KLN mutant lost the ability to be a negative regulator in ABA signaling, the MED141-143IGH mutant still inhibited ABA-inducible transcription, perhaps through a dominant interfering effect. Moreover, two G to D mutations near the DGH motif eliminated PP2C activity but displayed opposite effects on ABA signaling. The G174D mutant had no effect but the G180D mutant showed strong inhibitory effect on ABA-inducible transcription. Based on the results that a constitutive PP2C blocks but constitutive Ca2+-dependent protein kinases (CDPKs) activate ABA responses, the MED141-143IGH and G180D dominant mutants are unlikely to impede the wild-type PP2C and cause hyperphosphorylation of substrates. In contrast, these dominant mutants could trap cellular targets and prevent phosphorylation by PKs required for ABA signaling. The equivalent mutations in AtPP2C showed similar effects on ABA responses. This study suggests a mechanism for the action of dominant PP2C mutants that could serve as valuable tools to understand protein-protein interactions mediating ABA signal transduction in higher plants.

ABI2, a second protein phosphatase 2C involved in abscisic acid signal transduction in Arabidopsis

The abi2-1 (abscisic acid insensitive) mutant of Arabidopsis thaliana shows abscisic acid (ABA) insensitivity with respect to seed germination and vegetative ABA responses. We identified the ABI2 gene by a combination of positional mapping and homology to ABI1. The ABI2 protein shows 80% amino acid sequence identity to ABI1, a protein phosphatase 2C (PP2C) involved in ABA signaling. The mutation that confers the abi2-1 phenotype is equivalent to the mutation previously identified in abi1-1 and the resulting Gly 168Asp abi2 protein shows a reduced PP2C activity. Thus, a pair of highly homologous PP2Cs regulate ABA signaling.

Purification and assay of the Ptc1/Tpd1 protein phosphatase 2C from the yeast Saccharomyces cerevisiae.

AbstractOne species of protein phosphatase 2C (PP2C) in the yeast S. cerevisiae is encoded by the gene PTC1/TPD1 (1,2). This gene encodes a protein that is highly conserved in all eukaryotes. It is 38% identical to the rat protein over the entire sequence, with identity reaching up to 80% in distinct regions. TPD1 was shown to encode protein phosphatase 2C activity based on two lines of evidence: first, Ptc1/Tpd1 protein expressed in E. coli exhibits readily detectable Mg2+ or Mn2+ dependent protein phosphatase activity with 32P-labeled casein as a substrate. Second, this activity does not require Ca2+ and is resistant to okadaic acid at concentrations capable of inhibiting all the other mam families of protein phosphatases in eukaryotic organisms. These are the primary distinguishing enzymatic characteristics of mammalian PP2C (3). Yeast has at least two other PP2C species since extracts made from cells deleted for PTC1/TPD1 exhibit substantial PP2C activity (1,2). Putative genes for this activity have been cloned (1).KeywordsGlass BeadProtein PhosphataseOkadaic AcidDialysis TubingFreeze ThawThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Counteractive roles of protein phosphatase 2C (PP2C) and a MAP kinase kinase homolog in the osmoregulation of fission yeast.

With the goal of discovering the cellular functions of type 2C protein phosphatases, we have cloned and analyzed two ptc (phosphatase two C) genes, ptc2+ and ptc3+, from the fission yeast Schizosaccharomyces pombe. Together with the previously identified ptc1+ gene, the enzymes encoded by these genes account for approximately 90% of the measurable PP2C activity in fission yeast cells. No obvious growth defects result from individual disruptions of ptc genes, but a delta ptc1 delta ptc3 double mutant displays aberrant cell morphology and temperature-sensitive cell lysis that is further accentuated in a delta ptc1 delta ptc2 delta ptc3 triple mutant. These phenotypes are almost completely suppressed by the presence of osmotic stabilizers, strongly indicating that PP2C has an important role in osmoregulation. Genetic suppression of delta ptc1 delta ptc3 lethality identified two loci, mutations of which render cells hypersensitive to high-osmolarity media. One locus is identical to wis1+, encoding a MAP kinase kinase (MEK) homolog. The Wis1 sequence is most closely related to the Saccharomyces cerevisiae MEK encoded by PBS2, which is required for osmoregulation. These data indicate that divergent yeasts have functionally conserved MAP kinase pathways, which are required to increase intracellular osmotic concentrations in response to osmotic stress. Moreover, our observations implicate PP2C enzymes as also having an important role in signal transduction processes involved in osmoregulation, probably acting to negatively regulate the osmosensing signal that is transmitted through Wis1 MAP kinase kinase.

Protein phosphatase 2C, encoded by ptc1+, is important in the heat shock response of Schizosaccharomyces pombe.

Protein phosphatase 2C (PP2C), an Mg(2+)-dependent enzyme that dephosphorylates serine and threonine residues, defines one of the three major families of structurally unrelated eukaryotic protein phosphatases. Members of the two other families of protein phosphatases are known to have important cellular roles, but very little is known about the biological functions of PP2C. In this report we describe a genetic investigation of a PP2C enzyme in the fission yeast Schizosaccharomyces pombe. We discovered ptc1+ (phosphatase two C) as a multicopy suppressor gene of swo1-26, a temperature-sensitive mutation of a gene encoding the heat shock protein hsp90. The ptc1+ gene product is a 40-kDa protein with approximately 24% identity to a rat PP2C protein. Purified Ptc1 has Mg(2+)-dependent casein phosphatase activity, confirming that it is a PP2C enzyme. A ptc1 deletion mutant is viable and has approximately normal levels of PP2C activity, observations consistent with the fact that ptc1+ is a member of a multigene family. Although a ptc1 deletion mutant is viable, it has a greatly reduced ability to survive brief exposure to elevated temperature. Moreover, ptc1+ mRNA levels increase 5- to 10-fold during heat shock. These data, demonstrating that Ptc1 activity is important for survival of heat shock, provide one of the first genetic clues as to the biological functions of PP2C.

TPD1 of Saccharomyces cerevisiae encodes a protein phosphatase 2C-like activity implicated in tRNA splicing and cell separation.

The Saccharomyces cerevisiae TPD1 gene has been implicated in tRNA splicing because a tpd1-1 mutant strain accumulates unspliced precursor tRNAs at high temperatures (W. H. van Zyl, N. Wills, and J. R. Broach, Genetics 123:55-68, 1989). The wild-type TPD1 gene was cloned by complementation of the tpd1-1 mutation and shown to encode a protein with substantial homology to protein phosphatase 2C (PP2C) of higher eukaryotes. Expression of Tpd1p in Escherichia coli results in PP2C-like activity. Strains deleted for the TPD1 gene exhibit multiple phenotypes: temperature-sensitive growth, accumulation of unspliced precursor tRNAs, sporulation defects, and failure of cell separation during mitotic growth. On the basis of the presence of these observable phenotypes and the fact that Tpd1p accounts for a small percentage of the observed PP2C activity, we argue that Tpd1p is a unique member of the PP2C family.

Protein Phosphatase 2C, Encoded byptc1+, Is Important in the Heat Shock Response ofSchizosaccharomyces pombe

Protein phosphatase 2C (PP2C), an Mg(2+)-dependent enzyme that dephosphorylates serine and threonine residues, defines one of the three major families of structurally unrelated eukaryotic protein phosphatases. Members of the two other families of protein phosphatases are known to have important cellular roles, but very little is known about the biological functions of PP2C. In this report we describe a genetic investigation of a PP2C enzyme in the fission yeast Schizosaccharomyces pombe. We discovered ptc1+ (phosphatase two C) as a multicopy suppressor gene of swo1-26, a temperature-sensitive mutation of a gene encoding the heat shock protein hsp90. The ptc1+ gene product is a 40-kDa protein with approximately 24% identity to a rat PP2C protein. Purified Ptc1 has Mg(2+)-dependent casein phosphatase activity, confirming that it is a PP2C enzyme. A ptc1 deletion mutant is viable and has approximately normal levels of PP2C activity, observations consistent with the fact that ptc1+ is a member of a multigene family. Although a ptc1 deletion mutant is viable, it has a greatly reduced ability to survive brief exposure to elevated temperature. Moreover, ptc1+ mRNA levels increase 5- to 10-fold during heat shock. These data, demonstrating that Ptc1 activity is important for survival of heat shock, provide one of the first genetic clues as to the biological functions of PP2C.

The regulatory and basal phosphorylation sites of hormone-sensitive lipase are dephosphorylated by protein phosphatase-1, 2A and 2C but not by protein phosphatase-2B.

The activity of hormone-sensitive lipase, the rate-limiting enzyme in adipose tissue lipolysis, is controlled by cAMP-mediated phosphorylation at a specific regulatory phosphorylation site. The lipase is also phosphorylated at a site, termed basal, without any effects on its activity [Strålfors et al. (1984) Proc. Natl Acad. Sci. USA 81, 3317-3321]. The capacity of protein phosphatase-1, 2A, 2B and 2C to dephosphorylate the lipase, selectively phosphorylated by glycogen synthase kinase-4 and cAMP-dependent protein kinase at the basal and regulatory phosphorylation sites, was compared with that towards glycogen phosphorylase and phosphorylase kinase (alpha subunit). Protein phosphatase-1, 2A and 2C were found to dephosphorylate both phosphorylation sites of hormone-sensitive lipase, while protein phosphatase-2B had no measureable activity towards any of the sites. When the activities of protein phosphatase-1, 2A and 2C were normalized with respect to the reference substrates, they were found to dephosphorylate the lipase regulatory site in the approximate relations of 1:4:3 and the basal site in the approximate relations of 1:6:4. Protein phosphatase-1 showed 20% higher and protein phosphatase-2A and 2C 80% higher activity towards the basal site compared to the regulatory site. The two phosphorylation sites of the lipase were comparable to good substrates for protein phosphatase-2A and 2C, but relatively poor substrates for protein phosphatase-1. Protein phosphatase-2C activity towards the lipase was completely dependent on Mg2+ with a half-maximal effect at 3 mM. Protamine increased the lipase dephosphorylation by protein phosphatase-1 3-5-fold with half-maximal effect at 0.6 microgram/ml, and by protein phosphatase-2A about 2-fold with half-maximal effect at 3-5 micrograms/ml, thus illustrating the potential for control of these lipase phosphatase activities.