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S Y Takahashi - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation by Ca2+/calmodulin-dependent Protein Kinase II and Protein Kinase C of sepiapterin reductase, the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin.
1994Co-Authors: S Katoh, Tsutomu Sueoka, Y Yamamoto, S Y TakahashiAbstract:Sepiapterin reductase, the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin, was stoichiometrically phosphorylated by Ca2+/calmodulin-dependent Protein Kinase II and Protein Kinase C (Ca2+/phospholipid-dependent Protein Kinase) in vitro. Maximal incorporation of phosphate into the enzyme subunit by these was 3.05 +/- 0.05 (n = 4) and 0.74 +/- 0.03 (n = 5) 32P mol per mol enzyme subunit, respectively. The enzyme was not phosphorylated by cyclic nucleotide-dependent Protein Kinase of either the cAMP-dependent or cGMP-dependent type in this study. Dihydropteridine reductase, another enzyme working in direct supply of tetrahydrobiopterin, was also a good substrate for Ca2+/calmodulin-dependent Protein Kinase II. Phosphorylation of sepiapterin reductase by these Protein Kinases modified the kinetic properties of the enzyme. It is likely that these multifunctional Ca(2+)-activated Protein Kinases may play a role in the regulation of the physiological function of the BH4-generating enzymes in vivo, as was previously found in the case of BH4-requiring enzymes.
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phosphorylation by ca2 calmodulin dependent Protein Kinase ii and Protein Kinase c of sepiapterin reductase the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin
1994Co-Authors: S Katoh, Y Yamamoto, Terumi Sueoka, S Y TakahashiAbstract:Sepiapterin reductase, the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin, was stoichiometrically phosphorylated by Ca2+/calmodulin-dependent Protein Kinase II and Protein Kinase C (Ca2+/phospholipid-dependent Protein Kinase) in vitro. Maximal incorporation of phosphate into the enzyme subunit by these was 3.05 ± 0.05 (n = 4) and 0.74 ± 0.03 (n = 5) 32P mol per mol enzyme subunit, respectively. The enzyme was not phosphorylated by cyclic nucleotide-dependent Protein Kinase of either the cAMP-dependent or cGMP-dependent type in this study. Dihydropteridine reductase, another enzyme working in direct supply of tetrahydrobiopterin, was also a good substrate for Ca2+/calmodulin-dependent Protein Kinase II. Phosphorylation of sepiapterin reductase by these Protein Kinases modified the kinetic properties of the enzyme. It is likely that these multifunctional Ca2+-activated Protein Kinases may play a role in the regulation of the physiological function of the BH4-generating enzymes in vivo, as was previously found in the case of BH4-requiring enzymes.
Enrique Rozengurt - One of the best experts on this subject based on the ideXlab platform.
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the c terminal tail of Protein Kinase d2 and Protein Kinase d3 regulates their intracellular distribution
2006Co-Authors: Romeo Papazyan, Enrique Rozengurt, Osvaldo ReyAbstract:We generated a set of GFP-tagged chimeras between Protein Kinase D2 (PKD2) and Protein Kinase D3 (PKD3) to examine in live cells the contribution of their C-terminal region to their intracellular localization. We found that the catalytic domain of PKD2 and PKD3 can localize to the nucleus when expressed without other Kinase domains. However, when the C-terminal tail of PKD2 was added to its catalytic domain, the nuclear localization of the resulting Protein was inhibited. In contrast, the nuclear localization of the CD of PKD3 was not inhibited by its C-terminal tail. Furthermore, the exchange of the C-terminal tail of PKD2 and PKD3 in the full-length Proteins was sufficient to exchange their intracellular localization. Collectively, these data demonstrate that the short C-terminal tail of these Kinases plays a critical role in determining their cytoplasmic/nuclear localization.
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expression and activity of Protein Kinase d Protein Kinase c μ in myocardium evidence forα1 adrenergic receptor and Protein Kinase c mediated regulation
2000Co-Authors: Enrique Rozengurt, Robert S Haworth, Martin W Goss, Metin AvkiranAbstract:Abstract R. S. Haworth, M. W. Goss, E. Rozengurt and M. Avkiran. Expression and Activity of Protein Kinase D/Protein Kinase C μ in Myocardium: Evidence for α 1 -Adrenergic Receptor- and Protein Kinase C-Mediated Regulation. Journal of Molecular and Cellular Cardiology (2000) 32 , 1013–1023. Protein Kinase D (PKD), which is also known as Protein Kinase C (PKC) μ , is a novel serine/threonine Kinase that can be activated in parallel with or downstream of PKC in various cell types, but its expression and regulation in myocardium have not been characterized. In the present study, two Proteins of 110 and 115 kDa were detected in rat ventricular myocardium using antibodies directed at the extreme N- or C-terminus of PKD. Both Proteins were highly expressed in the fetal heart but showed a developmental decline in abundance. Fractionation studies showed that PKD was distributed between myocyte and non-myocyte fractions in the neonatal heart, but was found predominantly in the non-myocyte fraction in the adult heart. In cultured neonatal rat ventricular myocytes, an in vitro Kinase assay revealed increased autophosphorylation of PKD (EC 50 2.8 n m ) in response to phorbol-12-myristate-13-acetate (PMA). Exposure to norepinephrine also induced a dose-dependent increase in PKD autophosphorylation (EC 50 0.6 μ m ). Pretreatment with the α 1 -adrenergic receptor (AR) antagonist prazosin blocked norepinephrine-induced PKD autophosphorylation, while the β 1 -AR antagonist atenolol had no effect, indicating that activation of PKD by norepinephrine occurred via the α 1 -AR. Involvement of the α 1 -AR was confirmed by exposure of myocytes to the α 1 -AR agonist phenylephrine, which induced a similar profile of PKD autophosphorylation to norepinephrine (EC 50 0.6 μ m ). The effects of both α 1 -AR stimulation and PMA on PKD autophosphorylation were mediated by PKC, since these effects could be attenuated by pretreatment of myocytes with the PKC inhibitor bisindolylmaleimide. These data show that PKD is expressed in rat ventricular myocardium, where its expression is subject to developmental control, and that PKD activity in ventricular myocytes is regulated through α 1 -AR- and PKC-mediated pathways.
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bombesin vasopressin endothelin bradykinin and platelet derived growth factor rapidly activate Protein Kinase d through a Protein Kinase c dependent signal transduction pathway
1997Co-Authors: Jose L Zugaza, James Sinnettsmith, Richard T Waldron, Enrique RozengurtAbstract:Protein Kinase D (PKD) is a serine/threonine Protein Kinase that is activated by phorbol esters via Protein Kinase C in intact cells. To assess the physiological significance of this putative pathway, we examined the regulation of PKD in living cells by mitogenic regulatory peptides and by platelet-derived growth factors (PDGF). Our results demonstrate that bombesin rapidly induces PKD activation in Swiss 3T3 cells, as shown by autophosphorylation and syntide-2 phosphorylation assays. Maximum PKD activation (14-fold above base-line levels) was obtained 90 s after bombesin stimulation. Bombesin also induced PKD activation in Rat-1 cells stably transfected with the bombesin/gastrin releasing peptide (GRP) receptor and in COS-7 cells transiently co-transfected with PKD and bombesin/GRP receptor expression constructs. No inducible Kinase activity was demonstrated when COS-7 cells were transfected with a Kinase-deficient PKD mutant. Bombesin-mediated PKD activation was prevented by treatment of Swiss 3T3 cells with the Protein Kinase C inhibitors GF 1092030X and Ro 31-8220. In contrast, these compounds did not inhibit PKD activity when added directly in vitro. Vasopressin, endothelin, and bradykinin also activated PKD in Swiss 3T3 cells through a PKC-dependent pathway. Platelet-derived growth factor-stimulated PKD activation in Swiss 3T3 cells and in porcine aortic endothelial cells stably transfected with PDGF-β receptors. Treatment with GF 1092030X or Ro 31-8220 inhibited PKD activation induced by PDGF. Thus, our results indicate that PKD is activated by multiple signaling peptides through a Protein Kinase C-dependent signal transduction pathway in a variety of cell types.
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Protein Kinase d pkd activation in intact cells through a Protein Kinase c dependent signal transduction pathway
1996Co-Authors: Jose L Zugaza, James Sinnettsmith, J Van Lint, Enrique RozengurtAbstract:Protein Kinase D (PKD) is a serine/threonine Protein Kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids. Here, we examine the regulation of PKD in living cells. Our results demonstrate that tumour-promoting phorbol esters, membrane-permeant diacylglycerol and serum growth factors rapidly induced PKD activation in immortalized cell lines (e.g. Swiss 3T3 and Rat-1 cells), in secondary cultures of mouse embryo fibroblasts and in COS-7 cells transiently transfected with a PKD expression construct. PKD activation was maintained during cell disruption and immunopurification and was associated with an electrophoretic mobility shift and enhanced 32P incorporation into the enzyme, but was reversed by treatment with alkaline phosphatase. PKD was activated, deactivated and reactivated in response to consecutive cycles of addition and removal of PDB. PKD activation was completely abrogated by exposure of the cells to the Protein Kinase C inhibitors GF I and Ro 31-8220. In contrast, these compounds did not inhibit PKD activity when added directly in vitro. Co-transfection of PKD with constitutively activated mutants of PKCs showed that PKCepsilon and eta but not PKCzeta strongly induced PKD activation in COS-7 cells. Thus, our results indicate that PKD is activated in living cells through a PKC-dependent signal transduction pathway.
S Katoh - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation by Ca2+/calmodulin-dependent Protein Kinase II and Protein Kinase C of sepiapterin reductase, the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin.
1994Co-Authors: S Katoh, Tsutomu Sueoka, Y Yamamoto, S Y TakahashiAbstract:Sepiapterin reductase, the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin, was stoichiometrically phosphorylated by Ca2+/calmodulin-dependent Protein Kinase II and Protein Kinase C (Ca2+/phospholipid-dependent Protein Kinase) in vitro. Maximal incorporation of phosphate into the enzyme subunit by these was 3.05 +/- 0.05 (n = 4) and 0.74 +/- 0.03 (n = 5) 32P mol per mol enzyme subunit, respectively. The enzyme was not phosphorylated by cyclic nucleotide-dependent Protein Kinase of either the cAMP-dependent or cGMP-dependent type in this study. Dihydropteridine reductase, another enzyme working in direct supply of tetrahydrobiopterin, was also a good substrate for Ca2+/calmodulin-dependent Protein Kinase II. Phosphorylation of sepiapterin reductase by these Protein Kinases modified the kinetic properties of the enzyme. It is likely that these multifunctional Ca(2+)-activated Protein Kinases may play a role in the regulation of the physiological function of the BH4-generating enzymes in vivo, as was previously found in the case of BH4-requiring enzymes.
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phosphorylation by ca2 calmodulin dependent Protein Kinase ii and Protein Kinase c of sepiapterin reductase the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin
1994Co-Authors: S Katoh, Y Yamamoto, Terumi Sueoka, S Y TakahashiAbstract:Sepiapterin reductase, the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin, was stoichiometrically phosphorylated by Ca2+/calmodulin-dependent Protein Kinase II and Protein Kinase C (Ca2+/phospholipid-dependent Protein Kinase) in vitro. Maximal incorporation of phosphate into the enzyme subunit by these was 3.05 ± 0.05 (n = 4) and 0.74 ± 0.03 (n = 5) 32P mol per mol enzyme subunit, respectively. The enzyme was not phosphorylated by cyclic nucleotide-dependent Protein Kinase of either the cAMP-dependent or cGMP-dependent type in this study. Dihydropteridine reductase, another enzyme working in direct supply of tetrahydrobiopterin, was also a good substrate for Ca2+/calmodulin-dependent Protein Kinase II. Phosphorylation of sepiapterin reductase by these Protein Kinases modified the kinetic properties of the enzyme. It is likely that these multifunctional Ca2+-activated Protein Kinases may play a role in the regulation of the physiological function of the BH4-generating enzymes in vivo, as was previously found in the case of BH4-requiring enzymes.
Philip Cohen - One of the best experts on this subject based on the ideXlab platform.
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the activation of Protein Kinase b by h2o2 or heat shock is mediated by phosphoinositide 3 Kinase and not by mitogen activated Protein Kinase activated Protein Kinase 2
1998Co-Authors: Morag Shaw, Philip Cohen, Dario R AlessiAbstract:Protein Kinase B (PKB) isoforms became activated [and glycogen synthase Kinase-3 (GSK3) became inhibited] when mouse Swiss 3T3 fibroblasts were exposed to oxidative stress (H2O2) or heat shock, but not when they were exposed to osmotic shock (0.5 M sorbitol or 0. 7 M NaCl), chemical stress (sodium arsenite), the Protein-synthesis inhibitor anisomycin, or UV radiation. In contrast, all seven stimuli activated mitogen-activated Protein Kinase-activated Protein Kinase-2 (MAPKAP-K2). The activation of MAPKAP-K2 was suppressed by the drug SB 203580, but not by inhibitors of phosphoinositide (phosphatidylinositide, PI) 3-Kinase. In contrast, the activation of PKB isoforms and the inhibition of GSK3 by oxidative stress or heat shock were prevented by inhibitors of PI 3-Kinase, but not by SB 203580. Thus the activation of PKB by oxidative stress or heat shock is mediated by PI 3-Kinase and not by MAPKAP-K2. PKBalpha and PKBgamma were also activated by heat shock and oxidative stress in human embryonic kidney 293 cells and PKBgamma was activated by heat shock in NIH 3T3 cells; in each case activation was suppressed by inhibitors of PI 3-Kinase. The activation of PKB isoforms by H2O2 may underlie some of the insulin-mimetic effects of this compound.
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identification of regulatory phosphorylation sites in mitogen activated Protein Kinase mapk activated Protein Kinase 1a p90 rsk that are inducible by mapk
1998Co-Authors: Kevin N Dalby, Nick Morrice, Barry F Caudwell, Joseph Avruch, Philip CohenAbstract:Abstract Mitogen-activated Protein Kinase-activated Protein Kinase-1 (MAPKAP-K1; also known as p90rsk) contains two Protein Kinase domains in a single polypeptide. The N-terminal Kinase domain is necessary for the phosphorylation of peptide substrates, whereas the C-terminal Kinase domain is required for full activation of the N-terminal domain. Here we identify six sites in MAPKAP-K1a that become phosphorylated in transfected COS-1 cells. The inactive form of MAPKAP-K1a in unstimulated cells is partially phosphorylated at Ser222 and Ser733. Stimulation with phorbol 12-myristate 13-acetate induces the phosphorylation of Thr360, Ser364, Thr574, and Ser381 and increases the phosphorylation of Ser222 and Ser733. Our data indicate that mitogen-activated Protein Kinase activates the C-terminal Kinase domain by phosphorylating Thr574 and contributes to the activation of the N-terminal Kinase domain by phosphorylating Ser364. The activated C-terminal domain phosphorylates Ser381, which, together with phosphorylation of Ser364, activates the N-terminal Kinase domain. The phosphorylation of Ser222and Ser733, which can be catalyzed by the N-terminal domain, does not activate MAPKAP-K1a per se, but Ser222 phosphorylation appears to be required for activation. Ser222, Ser364, and Ser381 are situated in analogous positions to phosphorylation sites in Protein Kinase B, Protein Kinase C, and p70S6K, suggesting a common mechanism of activation for these growth factor-stimulated Protein Kinases.
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characterization of a 3 phosphoinositide dependent Protein Kinase which phosphorylates and activates Protein Kinase bα
1997Co-Authors: Dario R Alessi, S R James, Peter C Downes, Andrew B Holmes, Piers R J Gaffney, Colin B Reese, Philip CohenAbstract:Abstract Background: Protein Kinase B (PKB), also known as c-Akt, is activated rapidly when mammalian cells are stimulated with insulin and growth factors, and much of the current interest in this enzyme stems from the observation that it lies ‘downstream' of phosphoinositide 3-Kinase on intracellular signalling pathways. We recently showed that insulin or insulin-like growth factor 1 induce the phosphorylation of PKB at two residues, Thr308 and Ser473. The phosphorylation of both residues is required for maximal activation of PKB. The Kinases that phosphorylate PKB are, however, unknown. Results: We have purified 500 000-fold from rabbit skeletal muscle extracts a Protein Kinase which phosphorylates PKB α at Thr308 and increases its activity over 30-fold. We tested the Kinase in the presence of several inositol phospholipids and found that only low micromolar concentrations of the D enantiomers of either phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P 3 ) or PtdIns(3,4)P 2 were effective in potently activating the Kinase, which has been named PtdIns(3,4,5)P 3 -dependent Protein Kinase-1 (PDK1). None of the inositol phospholipids tested activated or inhibited PKB α or induced its phosphorylation under the conditions used. PDK1 activity was not affected by wortmannin, indicating that it is not likely to be a member of the phosphoinositide 3-Kinase family. Conclusions: PDK1 is likely to be one of the Protein Kinases that mediate the activation of PKB by insulin and growth factors. PDK1 may, therefore, play a key role in mediating many of the actions of the second messenger(s) PtdIns(3,4,5)P 3 and/or PtdIns(3,4)P 2 .
Avihu Boneh - One of the best experts on this subject based on the ideXlab platform.
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inhibition of calcium activated phospholipid dependent Protein Kinase Protein Kinase c activity in sphingolipidoses
1991Co-Authors: Avihu BonehAbstract:Sphingolipidoses arise as a consequence of a deficiency of specific lysosomal enzymes involved in the catabolism of particular sphingolipids. As a result lipid compounds, specific for each disease, accumulate progressively in tissues in which synthesis and/or degradation are most active, e.g. brain, liver and spleen. Additional changes found in the brain tissue of patients with a neurodegenerative phenotype include neuronal degeneration, brain atrophy, gliosis and demyelination. In these patients, symptoms and signs appear during the first year of life, with loss of developmental qualifications which had been obtained at an earlier age. To date the underlying mechanism linking the sphingolipid accumulation, the histopathology and the functional derangement of cellular processes leading to neurological dysfunction has not been elucidated. Sphingolipids are complex lipids which contain sphingosine (or a related base) as their backbone. Lysosphingolipids differ from their parental sphingolipids by not having the amide-linked fatty acid at the 2-amino position of the sphingoid base. Sphingolipids and lysosphingolipids are degraded by the same catabolic enzymes (Eto et al., 1974), thus a genetic deficiency in one such enzyme results in the accumulation of the two classes of compounds. Lysosphingolipids are toxic to cells, but the mechanism of this cytotoxicity is not known. Results of several recent studies indicate that sphingosine (Hannun et al., 1986), lysosphingotipids (Hannun and Bell, 1987) and gangliosides (Kreutter et al., 1987) inhibit the activity of calcium-activated, phospholipid-dependent Protein Kinase (Protein Kinase C). Protein Kinase C is a ubiquitous serine and threonine Kinase which is tightly linked to the phosphatidylinositol/calcium signal transduction pathway, and has been implicated in the regulation of a large variety of cellular processes (Nishizuka, 1986). In view of the observation that lysosphingolipids modulate Protein Kinase C activity, by which they could affect cell regulation and cell function, Hannun and Bell (1987) suggested that this finding represents the missing functional link between the accumulation of sphingolipids and the pathogenesis of the sphingolipidoses. This paper provides preliminary evidence in support of this hypothesis.
