共查询到20条相似文献,搜索用时 31 毫秒
1.
JW Romano KG Williams RN Shurtliff C Ginocchio M Kaplan 《Canadian Metallurgical Quarterly》1997,26(1-2):15-28
The phosphorylation state of neurofilaments plays an important role in the control of cytoskeletal integrity, axonal transport, and axon diameter. Immunocytochemical analyses of spinal cord revealed axonal localization of all protein phosphatase subunits. To determine whether protein phosphatases associate with axonal neurofilaments, neurofilament proteins were isolated from bovine spinal cord white matter by gel filtration. approximately 15% of the total phosphorylase a phosphatase activity was present in the neurofilament fraction. The catalytic subunits of PP1 and PP2A, as well as the A and B alpha regulatory subunits of PP2A, were detected in the neurofilament fraction by immunoblotting, whereas PP2B and PP2C were found exclusively in the low molecular weight soluble fractions. PP1 and PP2A subunits could be partially dissociated from neurofilaments by high salt but not by phosphatase inhibitors, indicating that the interaction does not involve the catalytic site. In both neurofilament and soluble fractions, 75% of the phosphatase activity towards exogenous phosphorylase a could be attributed to PP2A, and the remainder to PP1 as shown with specific inhibitors. Neurofilament proteins were phosphorylated in vitro by associated protein kinases which appeared to include protein kinase A, calcium/calmodulin-dependent protein kinase, and heparin-sensitive and -insensitive cofactor-independent kinases. Dephosphorylation of phosphorylated neurofilament subunits was mainly (60%) catalyzed by associated PP2A, with PP1 contributing minor activity (10-20%). These studies suggest that neurofilament-associated PP1 and PP2A play an important role in the regulation of neurofilament phosphorylation. 相似文献
2.
The protein phosphatase activity of peripheral blood T lymphocytes (PBLs) was examined to quantify the contribution of calcineurin and other members of the family of serine/threonine protein phosphatases. Using selective phosphatase inhibitors, the fractional phosphatase activities of calcineurin, protein phosphatases 1 (PP1), 2A (PP2A), and 2C (PP2C) were determined. Okadaic acid was used to inhibit the activity of both PP1 and PP2A while cyclosporin A/cyclophilin or trifluoperazine were used as a specific inhibitors of the calmodulin-dependent phosphatase calcineurin. Using a [32P]labeled 19-residue phosphopeptide substrate, RII peptide, it was found that PP1 and PP2A comprise the majority of the total phosphatase activity in PBLs with okadaic acid inhibiting 80% of the phosphatase activity. The remaining 20% of the phosphatase activity can be attributed primarily to calcineurin since it was Ca2+ dependent, sensitive to inhibition by the calmodulin antagonist trifluoperazine, and inhibited by the complex of cyclosporin A (CsA) and cyclophilin. These results indicate that PBL extracts contain little PP2C activity. In addition, PBLs treated with CsA had measurably lower calcineurin activity in cell lysates. The measurement of calcineurin activity may provide a useful means of assessing the extent of immunosuppression during drug therapy. 相似文献
3.
RE Honkanen 《Canadian Metallurgical Quarterly》1993,330(3):283-286
Cantharidin, a natural toxicant of blister beetles, is a strong inhibitor of protein phosphatases types 1 (PP1) and 2A (PP2A). Like okadaic acid, cantharidin inhibits the activity of the purified catalytic subunit of PP2A (IC50 = 0.16 microM) at a lower concentration than that of PP1 (IC50 = 1.7 microM) and only inhibits the activity of protein phosphatase type 2B (PP2B) at high concentrations. Dose-inhibition studies conducted with whole cell homogenates indicate that cantharidin also inhibits the native forms of these enzymes. Thus, cantharidin, which is economical and readily available, may be useful as an additional probe for studying the functions of serine/threonine protein phosphatases. 相似文献
4.
LM Leoni HC Shih L Deng C Tuey G Walter DA Carson HB Cottam 《Canadian Metallurgical Quarterly》1998,55(7):1105-1111
Protein phosphatase 2A (PP2A) is one of the most important and abundant serine/threonine phosphatases in mammalian tissues and plays a role in gene expression, cell division, and signal transduction. PP2A is activated by ceramide, which is produced by the hydrolysis of membrane sphingomyelin in response to a variety of stress-related stimuli. To further study the role of ceramide-mediated signal transduction in cellular processes such as senescence and apoptosis, we designed and synthesized a series of low molecular weight aromatic compounds, mainly of the isoquinolone and tetralone classes, and evaluated their ability to inhibit enzymes known to be activated by ceramide. Those enzymes studied were ceramide-activated protein kinase, protein kinase C zeta and PP2A. Of these, only PP2A was found to be inhibited. A few of the compounds inhibited both ceramide-activated as well as basal PP2A activity. In addition, several of the compounds activated PP2A by up to 300% above basal enzyme activity, but only in the presence of ceramide. Thus, modulation (both inhibition and activation) of the catatylic activity of ceramide-activated PP2A is demonstrated by certain low molecular weight aromatic compounds. 相似文献
5.
Alkaline phosphatase activity is regulated by various hormones and growth factors at least in part through the phosphorylation of target proteins during the bone cell differentiation. To investigate the role of protein phosphorylation in alkaline phosphatase activity in MC3T3-E1 osteoblast, we used okadaic acid which is a potent specific inhibitor of serine/threonine protein phosphatases to type 1 and 2A. Alkaline phosphatase activity in cellular layer was measured by spectrophotometer using p-nitrophenyl phosphate as substrate and data were expressed as p-nitrophenyl of nmol/min/mg of protein. Okadaic acid (1-50 ng/ml) caused the inhibition of alkaline phosphatase activity in MC3TC-E1 cells. At 50 ng/ml of okadaic acid showed the maximal inhibitory effect on alkaline phosphatase activity. Okadaic acid (50 ng/ml) also inhibited alkaline phosphatase activity in all differentiation stages. These results indicate that okadaic acid inhibits alkaline phosphatase activity in MC3T3-E1 cells. 相似文献
6.
Signals mediated by G-protein-linked receptors display agonist-induced attenuation and recovery involving both protein kinases and phosphatases. The role of protein kinases and phosphatases in agonist-induced attenuation and recovery of beta-adrenergic receptors was explored by two complementary approaches, antisense RNA suppression and co-immunoprecipitation of target elements. Protein phosphatases 2A and 2B are associated with the unstimulated receptor, the latter displaying a transient decrease followed by a 2-fold increase in the levels of association at 30 min following challenge with agonist. Protein kinase A displays a robust, agonist-induced association with beta-adrenergic receptors over the same period. Suppression of phosphatases 2A and 2B with antisense RNA or inhibition of their activity with calyculin A and FK506, respectively, blocks resensitization following agonist removal. Recycling of receptors to the plasma membrane following agonist-promoted sequestration is severely impaired by loss of either phosphatase 2B or protein kinase C. In addition, loss of protein kinase C diminishes association of phosphatase 2B with beta-adrenergic receptors. Overlay assays performed with the RII subunit of protein kinase A and co-immunoprecipitations reveal proteins of the A kinase-anchoring proteins (AKAP) family, including AKAP250 also known as gravin, associated with the beta-adrenergic receptor. Suppression of gravin expression disrupts recovery from agonist-induced desensitization, confirming the role of gravin in organization of G-protein-linked signaling complexes. The Ht31 peptide, which blocks AKAP protein-protein interactions, blocks association of beta-adrenergic receptors with protein kinase A. These data are the first to reveal dynamic complexes of beta-adrenergic receptors with protein kinases and phosphatases acting via an anchoring protein, gravin. 相似文献
7.
A phosphatase which exhibits strong activity toward phosphorylated atrial natriuretic peptide (ANP) was identified in the soluble fraction of rat brain homogenate. This ANP phosphatase has a neutral pH optimum, does not require divalent cations for activity, is inhibited by low concentrations of okadaic acid (50% inhibition at 1 nM) and preferentially dephosphorylates the alpha subunit of phosphorylase kinase. These properties are characteristic of serine/threonine protein phosphatase type 2A (PP2A). The apparent molecular mass of the ANP phosphatase (160 kDa), as estimated by gel filtration, is similar to that of the native heterotrimeric form of PP2A. In addition, phosphorylated ANP is an excellent substrate for the purified catalytic subunit of PP2A (Km = 42 microM, Vmax = 10.3 mumol x min-1 x mg-1). In contrast, protein phosphatase 2B (PP2B) has only very low ANP phosphatase activity (Km = 2.5 microM, Vmax = 0.008 mumol x min-1 x mg-1), and the catalytic subunit of protein phosphatase type 1 (PP1) as well as purified protein phosphatase type 2C (PP2C) are essentially inactive on ANP. These findings are consistent with the observation that PP2A-like activity accounts for virtually all ANP dephosphorylation in brain homogenate. While the phosphorylation of ANP in vitro by cAMP-dependent protein kinase is well documented, this is a first report on a phosphatase that efficiently can reverse this modification. 相似文献
8.
In the insulin producing cell line RINm5F distribution of serine/threonine specific protein phosphatases type 1 (PP1) and 2A (PP2A) was studied. Using different agents which inhibit or stimulate PP1 and PP2A we found that in membrane and nuclear fractions phosphatase activity was inhibited by okadaic acid (OA), protamine, heparin, and inhibitor-2 in a concentration-dependent manner. C2-ceramide had no effect. In the cytosolic fraction the inhibitory effect of okadaic acid was tenfold higher. Protamine stimulated phosphatase activity at low concentrations and became inhibitory at higher concentrations. Inhibitor-2 and heparin caused a decrease in phosphatase activity whereas C2-ceramide led to a slight activation. The data suggest that in membrane and nuclear fractions of RINmSF cells predominantly PP1 is present, whereas in the cytosol PP1 as well as PP2A can be detected. 相似文献
9.
An alkaline phosphatase was purified from conidia of a Neurospora crassa wild type strain. The M(r) of the purified native enzyme was estimated as ca 145,000 and 110,000 by gel filtration, in the presence and absence of magnesium ions, respectively. A single polypeptide band of M(r) 36,000 was detected by SDS-PAGE, suggesting that the native enzyme was a tetramer of apparently identical subunits. Conidial alkaline phosphatase was an acidic protein (pl = 4.0 +/- 0.1), with 40% carbohydrate content. Optimal pH was affected by substrate concentration and magnesium ions. Low concentrations of calcium ions (0.1 mM) had slight stimulatory effects, but in excess (5 mM) caused protein aggregates with decreased activity. The enzyme specificity against different substrates was compared with those reported for constitutive or Pi-repressible alkaline phosphatases produced by N. crassa. The results suggested that the conidial alkaline phosphatase represented a different class among other such enzymes synthesized by this organism. 相似文献
10.
The Saccharomyces cerevisiae open reading frame YFR003c encodes a small (155-amino acid) hydrophilic protein that we identified as a novel, heat-stable inhibitor of type 1 protein phosphatase (Ypi1). Ypi1 interacts physically in vitro with both Glc7 and Ppz1 phosphatase catalytic subunits, as shown by pull-down assays. Ypi1 inhibits Glc7 but appears to be less effective toward Ppz1 phosphatase activity under the conditions tested. Ypi1 contains a 48RHNVRW53 sequence, which resembles the characteristic consensus PP1 phosphatase binding motif. A W53A mutation within this motif abolishes both binding to and inhibition of Glc7 and Ppz1 phosphatases. Deletion of YPI1 is lethal, suggesting a relevant role of the inhibitor in yeast physiology. Cells overexpressing Ypi1 display a number of phenotypes consistent with an inhibitory role of this protein on Glc7, such as decreased glycogen content and an increased growth defect in a slt2/mpk1 mitogen-activated protein kinase-deficient background. Taking together, these results define Ypi1 as the first inhibitory subunit of Glc7 identified in budding yeast. 相似文献
11.
Calyculin A and okadaic acid, potent and cell permeable inhibitors of type 1 and type 2A protein phosphatases, inhibit platelet aggregation and secretion. However, the relationship between phosphatase inhibition and inhibition of platelet function is not well understood. We found that in unstimulated platelets, talin (P235) was phosphorylated at threonine residues by calyculin A. Furthermore, the extent of talin phosphorylation by calyculin A was closely correlated with its inhibition of thrombin-induced platelet aggregation. Since the binding of talin to platelet glycoprotein IIb/IIIa complex has been shown to be affected by its phosphorylation, these results suggest that type 1 and/or type 2A protein phosphatases may play a role in the regulation of membrane-cytoskeleton interaction through dephosphorylation of talin. 相似文献
12.
The effects of tautomycin and its derivatives on protein phosphatases PP1 and PP2A and their apoptosis-inducing activity toward human leukemia Jurkat cells were examined, and the relationship between chemical structure and function was discussed. Among the compounds we examined, tautomycin was the most potent inhibitor and the most effective inducer of apoptosis. It inhibited PP1 and PP2A enzymatic activity concentration-dependently with IC50 values of 20 and 75 pM, respectively, in the presence of 0.01% Brij-35, and an LC50 value of 1 microM. Esterification of the anhydride moiety of tautomycin markedly increased the IC50 for the protein phosphatases. The C1'-C7' fragment of tautomycin had no inhibitory effect, but the fragment containing the C22-C26 moiety was inhibitory. These results suggest that the C22-C26 moiety is essential for inhibition of protein phosphatase activity and that the anhydride moiety enhances the inhibition. However, the esterification of the anhydride did not decrease, nor did the inclusion of the C22-C26 moiety increase the apoptosis-inducing activity. On the other hand, the C1-C18 moiety of tautomycin was essential for induction of apoptosis, and the conformation and the arrangement of functionalities of the C18-C26 carbon chain affected the apoptosis activity. However, modification of C1-C18, C1-C21, or C1-C26 compounds had little effect on phosphatase inhibitory activity. Our results strongly suggest that different moieties of tautomycin are involved in protein phosphatase inhibition and induction of apoptosis. 相似文献
13.
MA Nesmeianova VI Krupianko AE Kalinin LIu Kadyrova 《Canadian Metallurgical Quarterly》1996,61(1):89-99
Natural and mutant alkaline phosphatases with amino acid substitutions in the processing site and N-terminal domain of the mature polypeptide chain Val for Ala(-1), Gln for Glu (+4) and simultaneously Gln for Glu (+4) and Ala for Arg (+1) have been isolated from the periplasm and cultural fluid of E. coli. It has been found that these substitutions have little effect on the dependence of the enzyme activity on pH, ionic strength and temperature but influence its isoenzymic spectrum and decrease (almost twofold) the maximal rate of the enzyme-catalyzed reaction. Extracellular enzymes display, in contrast with periplasmic ones, other catalytic properties (Vmax) and binding activity (Km). After translocation through the outer membrane all the enzymes display decreased Vmax and increased Km. These changes are especially well-pronounced in case of the mutant protein PhoA46 which contains an uncleaved signal peptide due to the impossibility of processing resulting from the substitution of Val for Ala(-1). The Vmax for this protein is decreased 20 times, while the Km is increased 4-fold. The protein also shows a higher (in comparison with other proteins) sensitivity towards proteolytic enzymes and is less resistant upon storage. The experimental data suggest that the changes in the N-end of alkaline phosphatase located at a long distance from its active center influence the enzyme function. 相似文献
14.
O Larsson CJ Barker A Sj?holm H Carlqvist RH Michell A Bertorello T Nilsson RE Honkanen GW Mayr J Zwiller PO Berggren 《Canadian Metallurgical Quarterly》1997,278(5337):471-474
Inositol hexakisphosphate (InsP6), the dominant inositol phosphate in insulin-secreting pancreatic beta cells, inhibited the serine-threonine protein phosphatases type 1, type 2A, and type 3 in a concentration-dependent manner. The activity of voltage-gated L-type calcium channels is increased in cells treated with inhibitors of serine-threonine protein phosphatases. Thus, the increased calcium channel activity obtained in the presence of InsP6 might result from the inhibition of phosphatase activity. Glucose elicited a transient increase in InsP6 concentration, which indicates that this inositol polyphosphate may modulate calcium influx over the plasma membrane and serve as a signal in the pancreatic beta cell stimulus-secretion coupling. 相似文献
15.
Peripherin/rds and rom-1 are homologous integral membrane protein subunits found as an oligomeric complex at the rim regions of rod and cone photoreceptor outer segment disks. These proteins are essential for the morphogenesis of normal outer segments and have been linked to a variety of human retinal degenerative diseases. Previous studies have suggested that disulfide-linked homodimers of peripherin/rds and rom-1 can associate noncovalently to form higher order structures. We have characterized the hydrodynamic properties of Triton X-100 solubilized peripherin/rds-rom-1 complexes from bovine ROS membranes by gel exclusion chromatography on Sepharose C1-6B and velocity sedimentation through H2O- and D2O-based sucrose gradients. A single hydrodynamic species is observed which has a Stokes radius of 6.2 nm, a sedimentation coefficient (S20,w) of 5.8 S, and a partial specific volume of 0.83 mL/g. From these data the molecular mass of the detergent-peripherin/rds-rom-1 complex is calculated to be 240 kDa. The protein component of this complex is estimated to be 135 kDa, providing direct evidence that the solubilized peripherin/rds-rom-1 complex is a tetramer. The abundance of this complex as measured by competitive ELISA and immunoaffinity purification is approximately 4% of total bovine ROS membrane protein and indicates that peripherin/rds-rom-1 tetramers are present at a relatively high average surface density (ca. 4100/ microns m2) at the rim surfaces of rod outer segment disks. 相似文献
16.
JJ Hens M De Wit WE Ghijsen AG Leenders HW Boddeke R Kissmehl VM Wiegant U Weller WH Gispen PN De Graan 《Canadian Metallurgical Quarterly》1998,71(5):1978-1986
Neurotransmission requires rapid docking, fusion, and recycling of neurotransmitter vesicles. Several of the proteins involved in this complex Ca2+-regulated mechanism have been identified as substrates for protein kinases and phosphatases, e.g., the synapsins, synaptotagmin, rabphilin3A, synaptobrevin, munc18, MARCKS, dynamin I, and B-50/GAP-43. So far most attention has focused on the role of kinases in the release processes, but recent evidence indicates that phosphatases may be as important. Therefore, we investigated the role of the Ca2+/calmodulin-dependent protein phosphatase calcineurin in exocytosis and subsequent vesicle recycling. Calcineurin-neutralizing antibodies, which blocked dynamin I dephosphorylation by endogenous synaptosomal calcineurin activity, but had no effect on the activity of protein phosphatases 1 or 2A, were introduced into rat permeabilized nerve terminals and inhibited Ca2+-induced release of [3H]noradrenaline and neuropeptide cholecystokinin-8 in a specific and concentration-dependent manner. Our data show that the Ca2+/calmodulin-dependent phosphatase calcineurin plays an essential role in exocytosis and/or vesicle recycling of noradrenaline and cholecystokinin-8, transmitters stored in large dense-cored vesicles. 相似文献
17.
The regulation of glycogen synthase (GS) and glycogen phosphorylase (GP) activity by phosphorylation/ dephosphorylation has been proposed to be via changes in activities of several different protein (serine/threonine) phosphatases and kinases, including protein phosphatase (PP) 1/2A, PP2C, and cAMP-dependent protein kinase (PKA). In order to determine whether PP1/2A, PP2C, and/or PKA activities are related to GS and/or GP activities, these enzymes were measured in freeze-clamped liver biopsies obtained under basal fasting conditions from 16 obese monkeys. Four monkeys were normoglycemic and normoinsulinemic, five were hyperinsulinemic, and seven had type 2 diabetes (NIDDM). Liver glycogen and glucose 6-phosphate (G6P) contents were also determine. Basal enzyme activities and basal substrate concentrations were not significantly different between the three group of obese monkeys; however, there were several significant linear relationships observed when the monkeys were treated as one group. Therefore, multiple regression was used to determine the correlation between key variables. GS fractional activity was correlated to GP fractional activity (p < 0.05) and to PP2C activity (p = 0.005) (adjusted R2, 53%). GP independent activity was correlated to GS independent activity (p < 0.07) and to PKA fractional activity (p = 0.005) (adjusted R2, 64%). PP2C activity was correlated to GS fractional activity (p < 0.0005) and to PP1/2A activity (p < 0.0001) (adjusted R2, 83%). PKA fractional activity was correlated to GP total activity (p < 0.0005) and to age (p = 0.001) (adjusted R2, 82%). G6P content was correlated to glycogen content (p < 0.05) and to PP2C activity (p = 0.0005) (adjusted R2, 73%). In conclusion, PP2C and PKA are involved in the regulation of GS and GP activity in the basal state in liver of obese monkeys with a wide range of glucose tolerance. 相似文献
18.
Glucose metabolism in the photoreceptor rod outer segment produces both ATP (GTP) and NADPH to support phototransduction and NADPH-requiring processes in this organelle. Glycolysis in isolated bovine rod outer segments produces 44.0 +/- 6.4 nmol of ATP/min/mg of protein or 5.7 mM ATP/min. This rate of ATP production is more than sufficient to maintain the basal rate of cGMP synthesis (0.86 mM cGMP/min) in the dark requiring 1.7 mM ATP/min. Following photoexcitation, the 4.5-fold increase in the turnover of cGMP requires an ATP synthesis rate of up to 7.7 mM ATP/min (Ames, A., Walseth, T. F., Heyman, R. A., Barad, M., Graeff, R. M., and Goldberg, N. D. (1986) J. Biol. Chem. 261, 13034-13042). Under these conditions the rate of ATP production by glycolysis as measured in isolated rod outer segments is not sufficient for the regeneration of cGMP. Additional energy is most likely provided by the phosphocreatine shuttle which transports high energy phosphate groups in the form of creatine phosphate from the rod inner segment to the rod outer segment for conversion to ATP. The hexose monophosphate pathway in bovine rod outer segments can produce up to 39.8 +/- 2.2 nmol of NADPH/min/mg of protein. This rate of NADPH production is sufficient to support both the reduction of retinal to retinol (1.2 +/- 0.2 nmol of NADPH/min/mg of protein) following the photobleaching of rhodopsin and glutathione reduction (1.1 +/- 0.1 nmol of NADPH/min/mg of protein) for the protection of rod outer segments from oxidative damage. These studies provide insight into the contribution of anaerobic glycolysis and the hexose monophosphate pathway in providing energy and nucleotides for phototransduction and other outer segment processes. 相似文献
19.
In eukaryotic cells a number of different proteins with important regulatory functions are reversibly methyl-esterified at carboxyl-terminal prenylcysteine residues. These proteins include the low molecular weight GTP-binding proteins, the gamma-subunit of the heterotrimeric G-proteins, and the nuclear lamins. The methylating enzymes that catalyze this type of carboxyl methylation reaction are integral membrane proteins, and the methylated protein products tend to be membrane-associated. Analyses of protein carboxyl methylation in a wide range of vertebrate tissues revealed a major carboxyl-methylated protein that was clearly distinct from those that are modified at prenylcysteine groups (Volker, C., Miller, R.A., McCleary, W.R., Rao, A., Poenie, M., Backer, J.M., and Stock, J.B. (1991) J. Biol. Chem. 266, 21515-21522). This M(r) = 36,000 protein is localized to the cytosol. Unlike the prenylcysteine methyltransferases, the enzyme that catalyzes the methylation of the 36-kDa protein is found in the cytosol. The 36-kDa methylated protein has been purified from bovine brain. Sequence analysis of several peptides clearly shows that the protein is the catalytic subunit of protein phosphatase 2A. A soluble 40-kDa methyltransferase that catalyzes the reaction has also been purified. 相似文献
20.
CB Mamoun DJ Sullivan R Banerjee DE Goldberg 《Canadian Metallurgical Quarterly》1998,273(18):11241-11247
We have cloned a gene from Plasmodium falciparum with homology to the Mg2+-dependent serine/threonine protein phosphatase 2C (PP2C) family. The predicted coding region is 920 amino acids long, twice the size of other members of this family. We show that this protein can be divided into two halves (Pf2C-1 and Pf2C-2), each a complete phosphatase unit with homology to other phosphatases of this class. To study the function of this PP2C, we have tested the ability of different constructs to complement conditional null mutants of yeast. Our results show that expression of the full-length protein, the first half alone, the second half alone, or a hybrid with the N terminus of the first half and the C terminus of the second half was able to complement the heat shock response defect of a Schizosaccharomyces pombe strain with a PP2C (PTC1) deletion. Recombinant P. falciparum PP2C expressed in Escherichia coli was active in dephosphorylating 32P-labeled casein in an Mg2+- or Mn2+-dependent reaction. Each half alone was also active in recombinant form. Using the two-hybrid system, we have shown that the two halves can interact. Gel filtration assay of P. falciparum protein extracts suggests that full-length PfPP2C is a dimer, and phosphatase activity competition experiments indicate that dimerization of PfPP2C is required for its optimal activity. This unusual phosphatase molecule appears to be composed of four catalytic units on two polypeptide chains. 相似文献