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Evidence that catalysis by yeast inorganic pyrophosphatase proceeds by direct phosphoryl transfer to water and not via a phosphoryl enzyme intermediate.

In this work, we show that adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) is a substrate for yeast inorganic pyrophosphatase (PPase) (EC 3.6.1.1) and further, using chirally labeled [gamma-17O,18O]ATP gamma S, that enzyme-catalyzed hydrolysis to produce chiral inorganic thio[17O,18O]phosphate proceeds with inversion of configuration. Both the synthesis of chiral ATP gamma S and the determination of inorganic thiophosphate configuration were carried out as described by Webb [Webb, M. R. (1982) Methods Enzymol. 87, 301-316]. We also show in a single turnover experiment performed in H2(18)O that 1 mol each of 18O16O3P and 16O4P is produced per mol of inorganic pyrophosphate hydrolyzed, a strong indication that oxygen uptake to form inorganic phosphate on PPase catalysis of inorganic pyrophosphate hydrolysis comes directly from H2O. These two results provide strong evidence for the conclusion that PPase catalyzes inorganic pyrophosphate hydrolysis via a single-step direct phosphoryl transfer to water and does not involve formation of a phosphorylated enzyme intermediate.

Adenosine Triphosphate↗

Phosphorylation of the inhibitory subunit of troponin in perfused hearts of mice deficient in phosphorylase kinase. Evidence for the phosphorylation of troponin by adenosine 3':5'-phosphate-dependent protein kinase in vivo.

When hearts from control and phosphorylase kinase-deficient (I strain) mice were perfused with 0.1 micrometer-DL-isoprenaline, there was a parallel increase in contraction, cyclic AMP concentration and troponin I phosphorylation. However, there was no increase in phosphorylase a in the I-strain hearts, whereas the control hearts showed a large increase. Assays of I-strain heart extracts showed a normal cyclic AMP-dependent protein kinase activity but no phosphorylase kinase activity. It is concluded that troponin I is phosphorylated in intact hearts by protein kinase and not phosphorylase kinase.

Animals↗

Glycolysis and oxidative phosphorylation as a function of cytosolic phosphorylation state and power output of the muscle cell.

A mathematical description of the regulation of ATP production in muscle cells is presented whereby the activity of OxP can be calculated as a function of (1) free [ADP] as the substrate and (2) a second driving force PhiDelta G (kilojoules per mole) resulting from the difference of free energy Delta G(ox,ap) (kilojoules per mole)-Delta G(ATP,cyt) (kilojoules per mole). In turn, the term Delta G(ox,ap) results from the proton motive force and the generation of ATP in the matrix space including the ATP-ADP exchange, whereas the phosphorylation state of the CHEP-sytem is described by Delta G(ATP,cyt). Regulation of glycolysis is calculated as a function of free [ADP] and [AMP] at the level of PFK. The PFK is inhibited by a decreasing pH resulting from lactate accumulation. The ATP/PCr equilibrium of the CHEP-system is calculated by algebraic equations. The dynamic behaviour of the metabolic control of ATP production as a function of ATP consumption is calculated by a system of two 1st-order non-linear differential equations, including a time delay considering oxygen transport. Lactate distribution and elimination is calculated using a two-compartment model with an active lactate producing, and a passive, space including lactate elimination by combustion. The simulation of the dynamics of energy metabolism of muscle cells is performed by the stepwise solution of the differential equations with a 5th-order Runge-Kutta-Fehlberg-routine. Examples of various applications of the simulation of the dynamics of energy supply demonstrate the qualitative and quantitative congruence to the behaviour of metabolic processes in experiments during rest, exercise and recovery.

Animals↗

Axonal regeneration in old multiple sclerosis plaques. Immunohistochemical study with monoclonal antibodies to phosphorylated and non-phosphorylated neurofilament proteins.

Cryostat sections of two old plaques removed at autopsy from the spinal cord of a 62-year-old man with multiple sclerosis of 24-year duration were studied by indirect immunofluorescence with antibodies to neurofilament proteins, glial fibrillary acidic protein (GFAP), glial hyaluronate-binding protein (GHAP), vimentin and laminin. The neurofilament monoclonal antibodies used in this study reacted with phosphorylated epitopes of the two large polypeptides of the neurofilament triplet (NF 150K, NF 200K). As previously reported [Dahl D, Labkovsky B, Bignami A (1989) Brain Res Bull 22:225-232], the neurofilament antibodies either stained axons in the distal stump of transected sciatic nerve in the early stages of regeneration or late in the process, i.e., after regenerating axons had reached the distal stump of the transected sciatic nerve. Both multiple sclerosis plaques were positive for GFAP and vimentin, but negative for GHAP, while astrocytes in myelinated spinal cord white matter stained with both GFAP and GHAP antibodies. Laminin immunoreactivity in the plaques and normal spinal cord was confined to blood vessels. One plaque was almost devoid of axons as evidenced by indirect immunofluorescence with neurofilament antibodies. Another plaque was packed with bundles of thin axons running an irregular course in the densely gliosed tissue. Axons in the plaque only stained with neurofilament antibodies reacting with sciatic nerve in the early stages of regeneration while axons in the surrounding myelinated white matter were decorated by all neurofilament antibodies, regardless of the time of appearance of immunoreactivity in crushed sciatic nerve. It is concluded that reactive astrocytes forming glial scars do not constitute a non-permissible substrate for axonal growth.

Antibodies, Monoclonal↗

Phosphorylation site mutants of the mannitol transport protein enzyme IImtl of Escherichia coli: studies on the interaction between the mannitol translocating C-domain and the phosphorylation site on the energy-coupling B-domain.

Mannitol binding and translocation catalyzed by the C domain of the Escherichia coli mannitol transport protein enzyme IImtl is influenced by domain B. This interaction was studied by monitoring the effects of mutating the B domain phosphorylation site, C384, on the kinetics of mannitol binding to the C domain. The dissociation constants for mannitol to the C384 mutants in inside-out membrane vesicles varied from 45 nM for the wild-type enzyme to 306 nM for the mutants. The rate constants pertinent to the binding equilibrium were also altered by the mutations. The association rate of mannitol to the cytoplasmic binding site in the mutants was accelerated for all mutants. The exchange rate of bound mannitol on the wild-type enzyme was shown to be pH dependent with a pKa of approximately 8 and increasing rates at higher pH. This rate was increased for all the mutants, but the pKas differed for the various mutants. The exchange rate for binding to the isolated IICmtl, however, was not pH dependent and exhibited a low rate. Exchange measured at 4 degrees C showed that, of the two steps, binding and occlusion, involved in binding to wild-type EIImtl in inside-out vesicles, only one could be detected for the C384E and C384L mutants. This suggests that the mutations increased the rate of the occlusion step so that it was no longer separable from the initial binding step or that the mutations eliminated the occlusion step altogether. The change in the mannitol binding kinetics of the C domain indicates that the B and C domains of EIImtl influence each other's conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

Dissociation of calcium from the phosphorylated calcium-transporting adenosine triphosphatase of sarcoplasmic reticulum: kinetic equivalence of the calcium ions bound to the phosphorylated enzyme.

The internalization of 45Ca by the calcium-transporting ATPase into sarcoplasmic reticulum vesicles from rabbit muscle was measured during a single turnover of the enzyme by using a quench of 7 mM ADP and EGTA (25 degrees C, 5 mM MgCl2, 100 mM KCl, 40 mM MOPS.Tris, pH 7.0). Intact vesicles containing either 10-20 microM or 20 mM Ca2+ were preincubated with 45Ca for approximately 20 s and then mixed with 0.20-0.25 mM ATP and excess EGTA to give 70% phosphorylation of Etot with the rate constant k = 300 s-1. The two 45Ca ions bound to the phosphoenzyme (EP) become insensitive to the quench with ADP as they are internalized in a first-order reaction with a rate constant of k = approximately 30 s-1. The first and second Ca2+ ions that bind to the free enzyme were selectively labeled by mixing the enzyme and 45Ca with excess 40Ca, or by mixing the enzyme and 40Ca with 45Ca, for 50 ms prior to the addition of ATP and EGTA. The internalization of each ion into loaded or empty vesicles follows first-order kinetics with k = approximately 30 s-1; there is no indication of biphasic kinetics or an induction period for the internalization of either Ca2+ ion. The presence of 20 mM Ca2+ inside the vesicles has no effect on the kinetics or the extent of internalization of either or both of the individual ions. The Ca2+ ions bound to the phosphoenzyme are kinetically equivalent. A first-order reaction for the internalization of the individual Ca2+ ions is consistent with a rate-limiting conformational change of the phosphoenzyme with kc = 30 s-1, followed by rapid dissociation of the Ca2+ ions from separate independent binding sites on E approximately P.Ca2; lumenal calcium does not inhibit the dissociation of calcium from these sites. Alternatively, the Ca2+ ions may dissociate sequentially from E approximately P.Ca2 following a rate-limiting conformational change. However, the order of dissociation of the individual ions can not be distinguished. An ordered-sequential mechanism for dissociation requires that the ions dissociate much faster (k greater than or equal to 10(5) s-1) than the forward and reverse reactions for the conformational change (k-c = approximately 3000 s-1). Finally, the Ca2+ ions may exchange their positions rapidly on the phosphoenzyme (kmix greater than or equal to 10(5) s-1) before dissociating. A Hill slope of nH = 1.0-1.2, with K0.5 = 0.8-0.9 mM, for the inhibition of turnover by binding of Ca2+ to the low-affinity transport sites of the phosphoenzyme was obtained from rate measurements at six different concentrations of Mg2+.

Adenosine Diphosphate↗

A cationic lanthanide complex binds selectively to phosphorylated tyrosine sites, aiding NMR analysis of the phosphorylated insulin receptor peptide fragment.

The binding of two cationic europium complexes to a differentially phosphorylated insulin receptor peptide has been studied by emission spectroscopy and (31)P NMR and (1)H NMR TOCSY methods. Analysis of the europium emission and NMR spectral data was consistent with the presence of species in slow exchange on the NMR and emission timescales, in agreement with selective binding of the lanthanide ion to the phospho-tyrosine site, allowing such complexes to be considered as prototypical chemoselective paramagnetic derivatising agents.

Cations↗

Regulation of pig heart pyruvate dehydrogenase by phosphorylation. Studies on the subunit and phosphorylation stoicheiometries.

1. The molecular weights of the subunits of purified pig heart pyruvate dehydrogenase complex were determined by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis and were: pyruvate decarboxylase, alpha-subunit 40600, beta-subunit 35100; dihydrolipoyl acetyltransferase 76100; dihydrolipoyl dehydrogenase 58200. 2. Inactivation of the pyruvate dehydrogenase complex by its integral kinase corresponded to the incorporation of 0.46nmol of P/unit of complex activity inactivated. 3. Further incorporation of phosphate into the complex occurred to a limit of 1.27nmol of P/unit of complex inactivated (approx. 3 times that required for inactivation). 4. Phosphate was incorporated only into the alpha-subunit of the decarboxylase. 5. The molar ratio of phosphate to alpha-subunits of the decarboxylase was estimated by radioamidination of amino groups of pyruvate dehydrogenase [(32)P]phosphate complex by using methyl [1-(14)C]acetimidate, followed by separation of alpha-subunits by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis. Inactivation of the complex (0.46nmol of P/unit of complex inactivated) corresponded to a molar ratio of one phosphate group per two alpha-chains (i.e. one phosphate group/alpha(2)beta(2) tetramer). Complete phosphorylation corresponded to three phosphate groups per alpha(2)beta(2) tetramer. 6. Subunit molar ratios in the complex were also estimated by the radioamidination technique. Results corresponded most closely to molar ratios of 4 alpha-subunits:4 beta-subunits:2 dihydrolipoyl acetyltransferase subunits:1 dihydrolipoyl dehydrogenase subunit.

Chemical Phenomena↗

A decade of site- and phosphorylation state-specific antibodies: recent advances in studies of spatiotemporal protein phosphorylation.

From 1990 to 2001, numerous site- and phosphorylation state-specific antibodies have been developed and many are now commercially available. These antibodies have facilitated understanding of the cytoskeletal organization, signal transduction and transcriptional mechanisms as well as clinical diseases. This review is an attempt to cover all these aspects.

Animals↗

alpha-Thrombin induces rapid and sustained Akt phosphorylation by beta-arrestin1-dependent and -independent mechanisms, and only the sustained Akt phosphorylation is essential for G1 phase progression.

In Chinese hamster embryonic fibroblasts (IIC9 cells) alpha-thrombin activates the MAPK(ERK) and phosphatidylinositol 3-OH-kinase (PI 3-kinase)/Akt pathways, and both are essential for progression through the G(1) phase of the cell cycle. We investigated in IIC9 cells, the role of beta-arrestin1 in alpha-thrombin signaling to these pathways. alpha-Thrombin stimulates rapid and sustained PI 3-kinase and Akt activities. Expression of a dominant negative beta-arrestin1 (beta-arrestin1(V53D)) inhibits rapid but not sustained PI 3-kinase and Akt activities. Surprisingly, expression of beta-arrestin1(V53D) does not block activation of the MAPK(ERK) pathway. PI 3-kinase and Akt activities are also inhibited by expression of a beta-arrestin1 mutant, which impairs binding to c-Src (beta-arrestin1(P91G-P121E)), indicating the involvement of c-Src in the rapid stimulation of the PI 3-kinase/Akt pathway. Consistent with these results, PP1, a selective inhibitor of c-Src family kinases, prevents alpha-thrombin-stimulated Akt phosphorylation. Expression of beta- arrestin1(V53D) does not prevent G(1) progression, as its expression has no effect on [(3)H]thymidine incorporation into DNA. In agreement with the ineffectiveness of beta-arrestin1(V53D) to block G(1) progression, cyclin D1 protein amounts and CDK4-cyclin D1 activity is unaffected by expression of beta-arrestin1(V53D). Thus in IIC9 cells, alpha-thrombin activates rapid beta-arrestin1-dependent and sustained beta-arrestin1-independent Akt activity, suggesting that two mechanisms are involved. Furthermore, although blocking the beta-arrestin1-independent PI 3-kinase/Akt pathway prevents G(1) progression, inhibition of the beta-arrestin1-dependent pathway does not, indicating different roles for the rapid and sustained activities.

Animals↗

Lymphocyte isoforms of mouse p50 LSP1, which are phosphorylated in mitogen-activated T cells, are formed through alternative splicing and phosphorylation.

p50 is phosphorylated in mitogen-stimulated T cells, and translocated from the membrane to the cytosol after activation of protein kinase C. Sequence analysis of p50 revealed that it is identical with LSP1, a putative calcium-binding and actin-binding protein. lymphocyte form of p50 exhibits heterogeneity in the apparent molecular mass on SDS-PAGE, 50 and 52 kDa (pp50 and pp52), and each isoform exhibits heterogeneity in the isoelectric point, when examined by two-dimensional PAGE. When the two molecular mass variants of p50 were dephosphorylated with alkaline phosphatase, both isoforms showed the same apparent molecular mass of 50 kDa on SDS-PAGE, but could be distinguished by their distinct isoelectric points. Dephosphorylated pp50 (p50a) has an acidic pI compared with dephosphorylated pp52 (p50b). Comparison of the peptide maps of purified p50a and p50b on HPLC revealed that the difference was limited to one peptide peak. NH2-terminal sequence and mass spectrometric analyses of these peptides showed that the peptides derived from p50a and p50b had the same NH2-terminal amino acid sequence up to eight residues, but had distinct molecular masses, 5,533.4 and 6,318.6 Da, respectively. These data suggested that pp52 (p50b) is the product of the previously cloned cDNA and the reduction in the molecular mass of the p50a-derived peptide could be explained by deletion of six amino acid residues, EHLIRH or HLIRHQ.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

c-Cbl is inducibly tyrosine-phosphorylated by epidermal growth factor stimulation in fibroblasts, and constitutively tyrosine-phosphorylated and associated with v-Src in v-src-transformed fibroblasts.

The c-cbl gene was cloned as the cellular homolog of the v-cbl oncogene that is the transforming component of a murine tumorigenic retrovirus, CAS NS-1, though the biological roles of c-Cbl remain to be elucidated. We have previously reported that c-Cbl is implicated in the signal transduction triggered by granulocyte-macrophage colony-stimulating factor or erythropoietin in hematopoietic cells. Here, we observed tyrosine phosphorylation of C-cbl in cells expressing epidermal growth factor receptor depending on EGF stimulation and in v-src transformed cells. Furthermore, c-Cbl was revealed to associate with v-Src in vivo. By means of binding experiments using glutathione S-transferase fusion proteins, we have found that the SH2 and SH3 domains of many proteins bind to c-Cbl. These findings strongly suggest that c-Cbl is implicated in a wide variety of signal transduction pathways, including those of EGF receptor and Src protein, as well as in the signaling pathways of hematopoietic cells.

3T3 Cells↗

Phosphorus-31 nuclear-magnetic-resonance study of phosphorylated metabolites compartmentation, intracellular pH and phosphorylation state during normoxia, hypoxia and ethanol perfusion, in the perfused rat liver.

A quantitative analysis of the phosphorus-31 NMR spectra of excised perfused rat liver has been carried out at 80.9 MHz using a 30-mm sample cell. The results indicate that in liver from fed rats, all intracellular ATP is detected by NMR. In contrast, only the cytosolic fractions of Pi and ADP can be observed as indicated by careful analysis of spectra obtained from perchloric acid liver extracts and intact liver under valinomycin perfusion. In well-oxygenated perfused liver the ATP concentration is 7.4 mM. Values of 5.3 mM and 0.9 mM are found respectively for Pi and ADP concentrations in the cytosolic compartment. Cytosolic pH value (pHi) is 7.25 +/- 0.05 and free magnesium concentration 0.5 mM. Addition of 70 mM (0.4%) ethanol to the perfusate of a fed rat liver induces 25% and 38% reduction of ATP and Pi levels, respectively. A large amount of sn-glycerol 3-phosphate is synthesized (up to 11 mM) in the cytosol. After ethanol withdrawal, a large overshoot in cytosolic Pi is observed, which is indicative of a net uptake of Pi across the plasma membrane that occurred during ethanol oxidation. No significant pH variation is observed during ethanol infusion. In perfused liver of rats subjected to 48-h fasts, the concentrations of cytosolic phosphorylated metabolites are 5.3 mM, 0.8 mM and 11.5 mM for ATP, ADP and Pi, respectively. The perfusion of the liver with 70 mM ethanol does not change the adenine nucleotide levels, while the Pi content is decreased by 10%. During a 4-min hypoxia, induced by reducing the perfusion flow rate from 12 ml to 3 ml min-1 (100 g body weight)-1, ATP concentration decreases to 5.8 mM in the fed rat liver. Cytosolic Pi and ADP increase to 8.7 mM and 1.6 mM, respectively. The cytosolic pH evolves to more acidic values and reaches 7.02 +/- 0.05 at the end of the 4-min hypoxic period.

Animals↗

Regulation of phosphorylation pathways by p21 GTPases. The p21 Ras-related Rho subfamily and its role in phosphorylation signalling pathways.

The oncogenic Ras p21 GTPases regulate phosphorylation pathways that underlie a wealth of activities, including growth and differentiation, in organisms ranging from yeast to human. In metazoa, growth factors trigger conversion of Ras from an inactive GDP-bound form to an active GTP-bound form. This activation of Ras leads to activation of Raf. Raf is one of the initial kinases in the cytoplasmic mitogen-activated protein kinase (MAPK) cascade, involving extracellular-signal-regulated kinases (ERK), which culminates in nuclear transcription. The Ras-related subfamily of Rho p21s, including Rho, Rac and Cdc42 are similarly active in their GTP-bound forms. These p21s mediate growth-factor-induced morphological changes involving actin-based cellular structures. For example, in mammalian fibroblasts, Rho mediates the formation of cytoskeletal stress fibres induced by lysophosphatidic acid, while Rac mediates the formation of membrane ruffles induced by platelet-derived growth factor, and Cdc42 mediates the formation of peripheral filopodia by bradykinin. In some cases, factor-induced Rac activation results in Rho activation, and factor-induced Cdc42 activation leads to Rac activation, as determined by specific morphological changes. Although separate Cdc42/Rac and Rac/Rho hierarchies exist, these might not extend into a linear form (i.e. Cdc42-->Rac-->Rho) since Cdc42 and Rho activities may be competitive or even antagonistic. Thus Cdc42-mediated formation of filopodia is accompanied by loss of stress fibres (whose formation is mediated by Rho). Recently, mammalian kinases that bind to the GTP-bound forms of Rho p21s have been isolated. These kinases include the p21-activated serine/threonine kinase (PAK), which is stimulated by binding to Cdc42 and Rac, and the Rho-binding serine/threonine kinase (ROK), which is not as strongly stimulated by binding. These kinases act as effectors for their p21 partners since they can directly affect the reorganization of the relevant actin-containing structures. ROK promotes the formation of Rho-induced actin-containing stress fibres and focal-adhesion complexes, to which the ends of the stress fibres attach. PAK stimulates the disassembly of stress fibres, which has been shown to accompany formation of Cdc42-induced peripheral-actin-containing structures, including filopodia, which with Rac-induced membrane ruffles play a role in cell movement. PAK also fosters loss of focal-adhesion complexes. Thus, there is cooperation between different Rho p21s as well as antagonism, with their associated kinases having a role in the integration of the reorganization of the actin cytoskeleton. The similarity of PAK to the Saccharomyces cerevisiae kinase Ste20p, which initiates the yeast mating/pheromone MAPK cascade, led to experiments showing that Cdc42 regulates Ste20p in this MAPK pathway. This similarity has also led to the demonstration that mammalian Cdc42 and Rac can signal to the nucleus through MAPK pathways. However, c-Jun N-terminal kinase (JNK, stress-activated protein kinase) rather than ERK, is involved. PAK have been implicated in the JNK pathway, but their exact roles are uncertain. Thus members of the Rho subfamily, and kinases that bind to these p21s are intimately involved in immediate morphological processes as well as long-term transcriptional events.

Animals↗

Phosphorylation of rat liver nuclear envelopes. II. Characterization of in vitro lipid phosphorylation.

Incubation of nuclear envelopes isolated from normal rat liver with [gamma-32P]ATP resulted in the rapid labeling of chloroform-soluble products. These products were identified as phosphatidic acid (PA), phosphatidylinositol 4-phosphate (DPI), and phosphatidylinositol 4,5-bisphosphate (TPI) based on their chromatographic mobilities on Silica Gel H and cellulose thin layer plates, and by analysis of their deacylation products by high pressure liquid chromatography. The extent of phosphorylation of these products was dependent on the method used to isolate the nuclear envelopes. Relatively gentle isolation methods, such as lysis of purified nuclei with heparin or digestion with deoxyribonuclease I, produce nuclear envelopes which possess significantly higher lipid kinase activity than do envelopes isolated by sonication. Incorporation of 32P into PA, DPI, and TPI in 2 min under standard assay conditions was 13, 152, and 22 pmol/mg of membrane protein, respectively. Degradation of labeled DPI and TPI was evident after 2-5 min of incubation. Nuclear envelope-associated diacylglycerol kinase, phosphatidylinositol kinase, and DPI kinase are characterized with regard to their pH and Mg2+ requirements. The effects of metals, phospholipids, and sulfhydryl reagents on these kinase activities are also described.

1-Phosphatidylinositol 4-Kinase↗

Metabolic properties of human platelet membranes. II. Thrombin-induced phosphorylation of membrane lipids and demonstration of phosphorylating enzymes in the platelet membrane.

Washed human platelets have been labeled with either 32Pi or glycerol-1-14C and the distribution of the label in the phospholipids determined. 32Pi was introduced primarily into polyphosphoinositides, i.e. di- and triphosphoinositide, whereas the label from glycerol which indicates de novo synthesis of lipid molecules did not appear in these phospholipids. In the course of thrombin-induced aggregation and release the phosphate incorporation into phosphatic acid, di- and triphosphoinositide was rapidly stimulated in parallel to the platelet reaction. The incorporation of glycerol did not change under the same conditions. It is concluded that phosphoinositides with rapid incorporation of phosphate groups are not as rapidly synthesized de novo and presumably form a separate phospholipid pool in the platelets. Only the phosphorylating reactions are stimulated by the thrombin aggregation. The necessary enzymes for these reactions, namely diglyceride kinase, phosphatidylinositol kinase, and phosphatidylinositol-phosphate kinase all can be shown to be associated with a well characterized platelet membrane fraction.

Blood Platelets↗