Magnesium-dependent Phosphatase Research Articles

A comprehensive overview of PPM1A: From structure to disease.

PPM1A (magnesium-dependent phosphatase 1 A, also known as PP2Cα) is a member of the Ser/Thr protein phosphatase family. Protein phosphatases catalyze the removal of phosphate groups from proteins via hydrolysis, thus opposing the role of protein kinases. The PP2C family is generally considered a negative regulator in the eukaryotic stress response pathway. PPM1A can bind and dephosphorylate various proteins and is therefore involved in the regulation of a wide range of physiological processes. It plays a crucial role in transcriptional regulation, cell proliferation, and apoptosis and has been suggested to be closely related to the occurrence and development of cancers of the lung, bladder, and breast, amongst others. Moreover, it is closely related to certain autoimmune diseases and neurodegenerative diseases. In this review, we provide an insight into currently available knowledge of PPM1A, including its structure, biological function, involvement in signaling pathways, and association with diseases. Lastly, we discuss whether PPM1A could be targeted for therapy of certain human conditions.

Magnesium-dependent Phosphatase (MDP) 1 is a Potential Suppressor of Gastric Cancer.

Recurrence is the leading cause of treatment failure and death in patients with gastric cancer (GC). However, the mechanism underlying GC recurrence remains unclear, and prognostic markers are still lacking. We analyzed DNA methylation profiles in gastric cancer cases with shorter survival (<1 year) or longer survival (> 3 years), and identified candidate genes associated with GC recurrence. Then, the biological effects of these genes on gastric cancer were studied. A novel gene, magnesium-dependent phosphatase 1 (mdp1), was identified as a candidate gene whose DNA methylation was higher in GC samples from patients with shorter survival and lower in patients with longer survival. MDP1 protein was highly expressed in GC tissues with longer survival time, and also had a tendency to be expressed in highly differentiated GC samples. Forced expression of MDP1 in GC cell line BGC-823 inhibited cell proliferation, whereas the knockdown of MDP1 protein promoted cell growth. Overexpression of MDP1 in BGC-823 cells also enhanced cell senescence and apoptosis. Cytoplasmic kinase protein c-Jun N-terminal kinase (JNK) and signal transducer and activator of transcription 3 (Stat3) were found to mediate the biological function of MDP1. These results suggest that MDP1 protein suppresses the survival of gastric cancer cells and loss of MDP expression may benefit the recurrence of gastric cancer.

Genome-wide transcriptomic analysis of a desert willow, Salix psammophila, reveals the function of hub genes SpMDP1 and SpWRKY33 in drought tolerance

BackgroundDrought is a major environmental constraint to plant growth, development and productivity. Compared with most willows that are generally susceptible to drought, the desert willow Salix psammophila has extraordinary adaptation to drought stress. However, its molecular basis of drought tolerance is still largely unknown.ResultsDuring polyethylene glycol 6000 (PEG 6000)-simulated drought stress, we found that the osmotic adjustment substances were accumulated and the antioxidant enzyme activities were enhanced in S. psammophila roots. A total of 8172 differentially expressed genes were identified in roots of S. psammophila through RNA-Sequencing. Based on K-means clustering, their expression patterns were classified into nine clusters, which were enriched in several stress-related processes including transcriptional regulation, response to various stresses, cell death, etc. Moreover, 672 transcription factors from 45 gene families were differentially expressed under drought stress. Furthermore, a weighted gene co-expression network was constructed, and eight genes were identified as hub genes. We demonstrated the function of two hub genes, magnesium-dependent phosphatase 1 (SpMDP1) and SpWRKY33, through overexpression in Arabidopsis thaliana. Overexpression of the two hub genes enhanced the drought tolerance in transgenic plants, suggesting that the identification of candidate drought tolerance genes in this study was highly efficient and credible.ConclusionsOur study analyzed the physiological and molecular responses to drought stress in S. psammophila, and these results contribute to dissect the mechanism of drought tolerance of S. psammophila and facilitate identification of critical genes involved in drought tolerance for willow breeding.

Thermostability of the PYL–PP2C Heterodimer Is Dependent on Magnesium: In Silico Insights into the Link between Heat Stress Response and Magnesium Deficiency in Plants

Magnesium deficiency increases the susceptibility of plants toward heat stress. The correlation between magnesium levels and stress response has been studied at the physiological level; albeit, the molecular explanation to this relationship remains elusive. Among diverse pathways implicated in the heat stress, the abscisic acid (ABA) signal modulates the heat stress response by magnesium dependent phosphatases (PP2Cs). Exclusively, sequestration of PP2Cs by ABA receptors (PYLs) in the heterodimer form activates the stress response through ABA responsive transcription factors. In this study, the molecular interplay between magnesium levels and ABA related heat stress response was investigated. Molecular dynamics simulations have been applied to two different PYL-PP2C heterodimer systems representing normal and magnesium deficient conditions. The heterodimer conformation and stability were delineated at high temperatures mimicking heat stress. Results showed that the thermostability of the heat stress response heterodimer was significantly dependent on the magnesium. Furthermore, a conserved aromatic cluster at the dimer interface acted synergistically with the metal to confer thermostability to the heterodimer structure. These structural insights into one of the possible links between magnesium levels and stress highlight the importance of metal micronutrients for tuning the stability of the stress-related proteins and optimizing tolerance.

Regulation of the hydrolytic activity of Mg²⁺-dependent phosphatase models by intramolecular NH···O hydrogen bonds.

Magnesium-dependent phosphatase models containing intramolecular NH···O hydrogen bonds were synthesized and structurally characterized by X-ray analysis. The Mg-O bond distances varied with the mode of the hydrogen bonds. (1)H NMR spectra in nonpolar solvents revealed that the acidity of the coordinated water molecule was regulated by the hydrogen bonds. Further, stoichiometric hydrolysis of phosphoric ester significantly depended on the hydrogen bonds. Zinc analogues showed similar but smaller dependencies, which suggest the indispensable role of Mg(2+) ion in the activation of the enzymes.

Structure of a haloacid dehalogenase superfamily phosphatase PH1421 fromPyrococcus horikoshiiOT3: oligomeric state and thermoadaptation mechanism

PH1421 from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 is a hypothetical protein belonging to the haloacid dehalogenase (HAD) superfamily. To gain insight into its biological function and thermostabilization mechanism, the crystal structure of PH1421 has been determined at 1.6 A resolution. The crystallographic asymmetric unit contains a homodimer. The monomeric protomer is composed of two distinct domains, a small cap domain and a large core domain, which agrees well with the typical domain organization of HAD subfamily II. Based on structure-based amino-acid sequence alignment and enzymatic analysis, PH1421 is suggested to be a magnesium-dependent phosphatase that is similar to the dimeric HAD phosphatase TA0175 from the mesothermophilic archaeon Thermoplasma acidophilum. Further comparison between the crystal structures of PH1421 and TA0175 revealed a marked structural similarity in the interprotomer dimer association. The common dimer interface with interprotomer twofold symmetry is characterized by a well conserved hydrophobic core consisting of the beta1-alpha1 loop and helices alpha1 and alpha2 of the core domain and additional contacts including the beta7-beta8 loop of the cap domain, which constitutes part of the putative active site of the enzyme. Several factors that potentially contribute to the higher thermal stability of PH1421 were identified: (i) an increase in intraprotomer hydrophobic interactions, (ii) a decrease in denaturation entropy from amino-acid composition and (iii) an increased number of intraprotomer ion pairs. These results suggest that the PH1421 protomer itself has an intrinsically higher thermal stability when compared with the mesothermophilic orthologue TA0175.

Progress in deciphering the biological role of the human soluble epoxide hydrolase phosphatase activity

The soluble epoxide hydrolase (sEH) is a unique enzyme in that it has two enzymatic activities on two separate domains: a C‐terminal epoxide hydrolase (Cterm‐EH), and a N‐terminal phosphatase (Nterm‐phos). The Cterm‐EH has been reported to hydrolyze epoxy‐fatty acids, especially epoxy‐eicosatrienoic acids (EETs) that are endogenous regulators of blood pressure and inflammation. Thus, Cterm‐EH inhibition is novel pharmacological approach to treat hypertension, pain and inflammation. The N‐terminal domain function is still not well known. It was thought to only help stabilize the C‐terminal domain. However, we recently described its magnesium dependent phosphatase activity on some lipid phosphates. We will report our progress on determining the substrate selectivity of the Nterm‐phos, as well as novel as specific inhibitors for this activity. The results will be discussed in light of possible interaction between the two domains, and biological roles of both sEH activities. This work was supported in part by NIEHS Grant R37 ES02710, NIEHS SBRP Grant P42 ES04699, and NIEHS CEHS Grant P30 ES05707.

Interaction of Fcp1 Phosphatase with Elongating RNA Polymerase II Holoenzyme, Enzymatic Mechanism of Action, and Genetic Interaction with Elongator

Fcp1 de-phosphorylates the RNA polymerase II (RNAPII) C-terminal domain (CTD) in vitro, and mutation of the yeast FCP1 gene results in global transcription defects and increased CTD phosphorylation levels in vivo. Here we show that the Fcp1 protein associates with elongating RNAPII holoenzyme in vitro. Our data suggest that the association of Fcp1 with elongating polymerase results in CTD de-phosphorylation when the native ternary RNAPII0-DNA-RNA complex is disrupted. Surprisingly, highly purified yeast Fcp1 dephosphorylates serine 5 but not serine 2 of the RNAPII CTD repeat. Only free RNAPII0(Ser-5) and not RNAPII0-DNA-RNA ternary complexes act as a good substrate in the Fcp1 CTD de-phosphorylation reaction. In contrast, TFIIH CTD kinase has a pronounced preference for RNAPII incorporated into a ternary complex. Interestingly, the Fcp1 reaction mechanism appears to entail phosphoryl transfer from RNAPII0 directly to Fcp1. Elongator fails to affect the phosphatase activity of Fcp1 in vitro, but genetic evidence points to a functional overlap between Elongator and Fcp1 in vivo. Genetic interactions between Elongator and a number of other transcription factors are also reported. Together, these results shed new light on mechanisms that drive the transcription cycle and point to a role for Fcp1 in the recycling of RNAPII after dissociation from active genes.

Predominant phosphatase in the ocular lens regulated by physiological concentrations of magnesium and calcium

A magnesium-dependent phosphatase with a molecular weight of about 55 kDa was found in the lens of chicken embryo, mouse, rabbit and bovine. It appears to be unique to the lens and, when activated by magnesium, accounts for the majority of the phosphatase activity in the lens. Phosphatases in the lens were separated by using high-resolution gel permeation column chromatography, and phosphatase activity was determined with p-nitrophenyl phosphate ( pNPP) as a substrate. The 55-kDa phosphatase showed very low basal activity, but the activity was increased concentration-dependently by magnesium ion (Mg 2+), and at 1 mM Mg 2+, this enzyme accounted for over 50% of the total phosphatase activity in the lens. Calcium potently inhibited the magnesium-activated phosphatase activity in a dose-dependent (IC 50; about 50 μM), uncompetitive manner. The phosphatase activity was high in the acidic pH range, with an optimum pH value of 5.5. The characteristics of the lens Mg 2+-dependent phosphatase, such as substrate specificity and sensitivity to various phosphatase inhibitors, showed little similarity to those of any reported phosphatase. The Mg 2+ and Ca 2+ concentrations that were found to be effective in this study are similar to physiological concentrations in the lens; therefore it is likely that these ions physiologically regulate the 55-kDa phosphatase activity in the lens.

A phosphatase of undefined function is common to the photoreceptive microvilli of several arthropod species

It has previously been demonstrated, using an ultracytochemical technique, that the photoreceptive microvilli of crab retinae contain a magnesium-dependent phosphatase that hydrolyses the artificial substrate 4-nitrophenylphosphate. Whilst many phosphatases hydrolyse 4-nitrophenylphosphate, the properties of the microvillar enzyme indicated that it is not a conventional acid or alkaline phosphatase. Using the same technique, it is now shown that a similar activity resides in the rhabdomeric microvilli of both the lateral compound eye and the ventral photoreceptors of Limulus polyphemus as well as in the compound eyes of the freshwater crayfish Cherax destructor and the fly Lucilia cuprina. Control cytochemical procedures performed on crayfish and fly showed that in these species too the activity is magnesium-dependent and is not due to a Na+/K+ ATPase.

Pyruvate kinase phosphatase

1. Introduction Liver pyruvate kinase can be phosphorylated and simultaneously inactivated by CAMP-dependent pro- tein kinase; the inactivated enzyme can be reactivated by a multifunctional protein phosphatase (review [ 13). The inactivation of pyruvate kinase has been observed in isolated hepatocytes incubated in the presence of glucagon and in this case the decrease in pyruvate kinase activity was correlated with the stimulation of gluconeogenesis [2]. The glucagon induced inactiva- tion of pyruvate kinase is however transient and reac- tivation occurred within 20 min when sub-optimal doses of glucagon were added. This reactivation was more rapid in the presence of insulin [2,3]. The purpose of this work was to investigate the nature of the enzyme that reactivates pyruvate kinase and its relationship with the well-known protein plios- phatases that act on glycogen phosphorylase a. 2. Methods The inactive form of pyruvate kinase was partially purified from livers of anesthetized rats given glucagon (i.v., 1 mg/kg) 5 min prior to sacrifice. Livers were homogenized at 0°C in 4vol. 0.1 M KF, 15 mM EGTA and 50 mM glycyl glycine, at pH 7.4. Following cen- trifugation at 18 800 X g for 10 min, pyruvate kinase was purified essentially as in [4] by treatment at pH 5.2, ammonium sulfate precipitation and a single chromatography on DEAE-cellulose. The enzyme was eluted from the DEAE-column (1.5 cm X 10 cm) with 100 mM potassium phosphate (pH 7.2) at a rate of 5-6 ml/min. The peak fraction was dialyzed over- night against a buffer containing 30% glycerol, 20 mM mercaptoethanol, and 100 mM Tris at pH 7.2 and stored at 5°C. When the final pyruvate kinase prepara- tion was assayed as described below at 0.15 mM PEP (1.‘6.1s), the activity was consistently <IO% of the maximal velocity measured at 5 mM PEP (V,). This value of the v,.,s/V’s ratio is characteristic of the inac- tive form of pyruvate kinase [2,5]. When purified pyruvate kinase was incubated in the presence of 10 mM MgC12, no reactivation occurred indicating that no magnesium-dependent phosphatase activity was present in this preparation. The addition of exog genous pyruvate kinase phosphatase caused a complete reactivation of pyruvate kinase as evidenced by an increase in the v,.,,/Vs ratio from 0.1-0.45. The liver extract, used as a source of pyruvate kinase phosphatase was prepared as follows. Livers were homogenized in 2 vol. 50 mM imidazole (pH 7.4), 250 mM sucrose and 0.5 dithiothreitol the homogenate was centrifuged at 18 800 X g for 10 min at 0°C. The high-speed supernatant was prepared by centrifuging the extract at 106 500 X g for 30 min. Additional treatment is noted in the legend of table 1. The reactivation of pyruvate kinase was measured at 25°C in the presence of 50 mM Tris (pH 7.2), 5% glycerol, bovine serum albumin (1 mgiml), 25 mM KCI, 10 mM MgCI,, 20 mM mercaptoethanol and non- saturating levels of pyruvate kinase (8 units/ml), unless noted otherwise. The reaction was initiated by the addition of the pyruvate kinase phosphatase prep-

Regulation of Hepatic Acetyl Coenzyme A Carboxylase by Phosphorylation and Dephosphorylation

Partially purified acetyl-CoA carboxylase (EC 6.4.1.2) was activated several-fold by incubation with magnesium ions. The activation was inhibited by fluoride. Partially purified but not highly purified enzyme was inactivated by ATP in a reaction which was both time- and temperature-dependent. Inactivation by ATP was greatly enhanced by a protein fraction devoid of carboxylase activity, and the inactivation was independent of adenosine 3′,5′-monophosphate. Whereas inactivation of the enzyme in the presence of [γ-32P] ATP resulted in significant amounts of protein-bound 32P, incubation with [U-14C]ATP led to little incorporation of isotope into protein. The 32P label accompanied the carboxylase activity through ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The 32P-labeled carboxylase was precipitated by the antibody to homogeneous acetyl-CoA carboxylase. The evidence is consistent with a mechanism for controlling acetyl-CoA carboxylase activity by an interconversion involving phosphorylation and dephosphorylation reactions. It appears that the carboxylase protein is phosphorylated, and the activity decreased, by an ATP-dependent kinase. It is reactivated by dephosphorylation that is mediated by a magnesium-dependent phosphatase.