The presence of a heat-stable activator of phosphoprotein phosphatase for the dephosphorylation of phosphorylated histone in rabbit liver.

A heat-stable protein activator of phosphoprotein phosphatase for the dephosphorylation of phosphorylated histone has been identified in rabbit liver. The protein activator is different than the previously observed heat-stable protein inhibitor of rabbit liver phosphoprotein phosphatase. It stimulates the dephosphorylation of histone by increasing the Vmax of the reaction as well as decreasing the Km for histone.     

The role of acid phosphatase activity during enzymic dephosphorylation of phytates byAspergillus niger phytase

Acid phosphatase present in preparations ofAspergillus niger phytase accelerated dephosphorylation of sodium phytate. Its influence on the reaction rate and distribution ofmyo-inositol phosphates was most apparent at low pH value (2.5) and when acid-hydrolysed substrate was de-esterified enzymatically. With partly purified phytase, the predominant inositol form was tetraphosphate but a preparation having acid phosphatase activity caused an even distribution of lower inositol phosphates after a few hours.     

Lysosomal acid phosphatase is not involved in the dephosphorylation of mannose 6-phosphate containing lysosomal proteins.

The mannose 6-phosphate (Man6P) residues that are necessary for the targeting of newly synthesized lysosomal proteins are dephosphorylated after delivery of lysosomal proteins to lysosomes. To examine the role of lysosomal acid phosphatase (LAP) for the dephosphorylation of Man6P residues in lysosomal proteins, the phosphorylation of endogenous lysosomal proteins and of internalized arylsulfatase A was analyzed in mouse L-cells that overexpress human LAP. Non-transfected L-cells dephosphorylate endogenous lysosomal proteins slowly (half time approximately 13 h) as well as internalized arylsulfatase A. A more than 100-fold overexpression of LAP in these cells did not affect the dephosphorylation rate. Control experiments showed that the internalized arylsulfatase A and overexpressed LAP partially colocalize and that under in vitro conditions purified LAP does not dephosphorylate arylsulfatase A. Taken together, these results indicate that LAP is not the mannose 6-phosphatase that dephosphorylates lysosomal proteins after their delivery to lysosomes.     

Factors controlling the Pb(II) promoted dephosphorylation of nucleotides

The conditions under which Pb(II) promotes dephosphorylation of nucleotides have been studied with the Pb(II) complexes of several isomers of AMP, dAMP, GMP, and dGMP. A number of factors which together control the dephosphorylation reaction have been identified. These include the tendency of Pb(II)-induced nucleotide base stacking, as evidenced by large enhancement in ultraviolet circular dichroism, to occur in the complexes and limit the reaction; hydroxylation of the metal, either with weakening of the lead-nucleotide binding, or eventually with displacement of the nucleotide; and the solubility of the complexes, which limits the reaction, but is increased by raising the temperature and by hydroxylation of the complexes. The pH range in which both base stacking and metal hydrolysis are minimized can define a "reaction window" for the complexes.     

Effect of ethanol on the activity and conformation of Penaeus penicillatus acid phosphatase.

The effect of ethanol on the activity of Penaeus penicillatus acid phosphatase has been studied. The results show that ethanol significantly inhibits enzyme activity as a non-competitive inhibitor, with Ki 8.75%. The conformational changes of the enzyme molecule induced by ethanol were followed using fluorescence emission, ultraviolet difference and circular dichroism (CD) spectra. Increasing the ethanol concentration caused the fluorescence emission intensity of the enzyme to increase. The ultraviolet difference spectra of the enzyme denatured with ethanol had two negative peaks at 220 and 278 nm, and a positive peak at 240 nm. Increasing the ethanol concentration produced a small shoulder peak at 287 nm in addition to the increases in the negative magnitudes of the 220 and 278 nm peaks. The changes of the fluorescence and ultraviolet difference spectra reflected the changes of the microenvironments of the tryptophan and tyrosine residues of the enzyme. The CD spectrum changes of the enzyme show that the secondary structure of the enzyme also changed. The results suggest that ethanol is a non-competitive inhibitor and the conformational integrity of the enzyme is essential for its activity.     

Tyrosine dephosphorylation and ethanol inhibition of N-Methyl-D-aspartate receptor function

The inhibitory effect of ethanol on N-methyl-d-aspartate receptors (NMDARs) is well documented in several brain regions. However, the molecular mechanisms by which ethanol affects NMDARs are not well understood. In contrast to the inhibitory effect of ethanol, phosphorylation of the NMDAR potentiates channel currents (Lu, W. Y., Xiong, Z. G., Lei, S., Orser, B. A., Dudek, E., Browning, M. D., and MacDonald, J. F. (1999) Nat. Neurosci. 2, 331-338). We have previously shown that protein kinase C activators induce tyrosine phosphorylation and potentiation of the NMDAR (Grosshans, D. R., Clayton, D. R., Coultrap, S. J., and Browning, M. D. (2002) Nat. Neurosci. 5, 27-33). We therefore hypothesized that the ethanol inhibition of NMDARs might be due to changes in tyrosine phosphorylation of NMDAR subunits. In support of this hypothesis, we found that tyrosine phosphorylation of both NR2A and NR2B subunits was significantly reduced following in situ exposure of hippocampal slices to 100 mm ethanol. Specifically, phosphorylation of tyrosine 1472 on NR2B was reduced 23.5%. These data suggest a possible mechanism by which ethanol may inhibit the NMDAR via activation of a tyrosine phosphatase. Electrophysiological studies demonstrated that ethanol inhibited NMDAR field excitatory postsynaptic potential slope and amplitude to a similar degree as previously reported by our laboratory and others (Schummers, J., Bentz, S., and Browning, M. D. (1997) Alcohol Clin. Exp. Res. 21, 404-408). Inclusion of bpV(phen), a potent phosphotyrosine phosphatase inhibitor, in the recording chamber prior to and during ethanol exposure significantly reduced the inhibitory effect of ethanol on NMDAR field excitatory postsynaptic potentials. Taken together, these data suggest that phosphatase-mediated dephosphorylation of NMDAR subunits may play an important role in mediating the inhibitory effects of ethanol on the N-methyl-D-aspartate receptor.     

Hydrolytic breakdown of lactoferricin by lactic acid bacteria

Lactoferricin is a 25-amino acid antimicrobial peptide fragment that is liberated by pepsin digestion of lactoferrin present in bovine milk. Along with its antibacterial properties, lactoferricin has also been reported to have immunostimulatory, antiviral, and anticarcinogenic effects. These attributes provide lactoferricin and other natural bioactive peptides with the potential to be functional food ingredients that can be used by the food industry in a variety of applications. At present, commercial uses of these types of compounds are limited by the scarcity of information on their ability to survive food processing environments. We have monitored the degradation of lactoferricin during its incubation with two types of lactic acid bacteria used in the yogurt-making industry, Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, with the aim of assessing the stability of this milk protein-derived peptide under simulated yogurt-making conditions. Analysis of the hydrolysis products isolated from these experiments indicates degradation of this peptide near neutral pH by lactic acid bacteria-associated peptidases, the extent of which was influenced by the bacterial strain used. However, the data also showed that compared to other milk-derived bioactive peptides that undergo complete degradation under these conditions, the 25-amino acid lactoferricin is apparently more resistant, with approximately 50% of the starting material remaining after 4 h of incubation. These findings imply that lactoferricin, as a natural milk protein-derived peptide, has potential applications in the commercial production of yogurt-like fermented dairy products as a multi-functional food ingredient.     

Site-selective dephosphorylation of the platelet-derived growth factor beta-receptor by the receptor-like protein-tyrosine phosphatase DEP-1

Ligand stimulation of PDGF beta-receptors leads to autophosphorylation of the regulatory tyrosine 857 and of tyrosine residues that in their phosphorylated form serve as docking sites for Src homology 2 domain-containing proteins. Regulation of the PDGF beta-receptor by protein-tyrosine phosphatases is poorly understood. We have investigated PDGF beta-receptor dephosphorylation by receptor-like protein-tyrosine phosphatase DEP-1 using a cell line with inducible DEP-1 expression and by characterizing in vitro dephosphorylation of the PDGF beta-receptor and of receptor-derived phosphopeptides by DEP-1. After DEP-1 induction PDGF beta-receptor.DEP-1 complexes and reduced receptor tyrosine phosphorylation were observed. Phosphopeptide analysis of the PDGF beta-receptors from DEP-1-expressing cells and of the receptors dephosphorylated in vitro by DEP-1 demonstrated that dephosphorylation of autophosphorylation sites of the receptor differed and revealed that the regulatory Tyr(P)(857) was not a preferred site for DEP-1 dephosphorylation. When dephosphorylation of synthetic receptor-derived peptides was analyzed, the selectivity was reproduced, indicating that amino acid sequence surrounding the phosphorylation sites is the major determinant of selectivity. This notion is supported by the observation that the poorly dephosphorylated Tyr(P)(562) and Tyr(P)(857), in contrast to other analyzed phosphorylation sites, are surrounded by basic amino acid residues at positions -4 and +3 relative to the tyrosine residue. Our study demonstrates that DEP-1 dephosphorylation of the PDGF beta-receptor is site-selective and may lead to modulation, rather than general attenuation, of signaling.     

Dephosphorylation of PKCdelta by protein phosphatase 2Ac and its inhibition by nucleotides

The protein phosphatases PP1(c), PP2A(c) and PP2Calpha are shown to dephosphorylate protein kinase Cdelta (PKCdelta) in vitro; of these PP2A(c) displayed the highest specific activity towards PKCdelta. The role of PP2A(c) in the dephosphorylation of PKCdelta in cells was supported by the demonstration that these proteins could be co-immunoprecipitated from NIH3T3 cells. However the observation that binding of Mg-ATP to PKCdelta could protect the enzyme from dephosphorylation by PP2A(c) in vitro indicates that an additional input/factor is required for dephosphorylation in vivo.     

Insulin mediator causes dephosphorylation of the alpha subunit of pyruvate dehydrogenase by stimulating phosphatase activity

Insulin treatment of rats results in an increased amount or activity of insulin mediators in liver and skeletal muscle. These mediators stimulated pyruvate dehydrogenase and inhibited adenylate cyclase. The insulin-generated mediators caused dephosphorylation of the alpha subunit of pyruvate dehydrogenase in mitochondria prelabeled with [gamma-32P]ATP. An assay was developed which quantitatively measured mediator activity by determining the rate of alpha-subunit dephosphorylation. The dephosphorylation was directly proportional to the amount of mediator added and was directly related to activation of pyruvate dehydrogenase. The decrease of alpha-subunit phosphorylation resulted from stimulation of pyruvate dehydrogenase phosphatase, since it occurred in the absence of ATP and was inhibited by NaF. These data further delineate the mechanism of insulin mediator activation of pyruvate dehydrogenase.     

Molecular mechanism of ERK dephosphorylation by striatal‐enriched protein tyrosine phosphatase

Striatal‐enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal‐regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho‐ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho‐ERK by STEP is not known. Therefore, we examined STEP activity toward para‐nitrophenyl phosphate, phospho‐tyrosine‐containing peptides, and the full‐length phospho‐ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N‐terminal regulatory region and key residues in its active site. Specifically, both kinase interaction motif (KIM) and kinase‐specific sequence of STEP were required for ERK interaction. In addition to the N‐terminal kinase‐specific sequence region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho‐ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho‐ERK peptide sequence through its active site, and the contact of STEP F311 with phospho‐ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP‐ERK recognition, which could serve as a potential therapy for neurological disorders.

     

Mechanism of substrate dephosphorylation in low Mr protein tyrosine phosphatase.

Substrate dephosphorylation by the low molecular weight protein tyrosine phosphatases proceeds via nucleophilic substitution at the phosphorous atom yielding a cysteinyl phosphate intermediate. However, several questions regarding the exact reaction mechanism remain unanswered. Starting from the crystal structure of the enzyme we study the energetics of this reaction, using the empirical valence bond method in combination with molecular dynamics and free energy perturbation simulations. The free energy profiles of two mechanisms corresponding to different protonation states of the reacting groups are examined along stepwise and concerted pathways. The activation barriers calculated relative to the enzyme-substrate complex are very similar for both monoanionic and dianionic substrates, but taking the substrate binding step into account shows that hydrolysis of monoanionic substrates is strongly favored by the enzyme, because a dianionic substrate will not bind when the reacting cysteine is ionized. The calculated activation barrier for dephosphorylation of monoanionic phenyl phosphate according to this novel mechanism is 14 kcal mol(-1), which is in good agreement with experimental data. Proteins 1999;36:370-379.