Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.2 (PDK1)
 2,238 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The activity of pig heart pyruvate dehydrogenase kinase was assayed by the incorporation of [(32)P]phosphate from [gamma-(32)P]ATP into the dehydrogenase complex. There was a very close correlation between this incorporation and the loss of pyruvate dehydrogenase activity with all preparations studied. 2. Nucleoside triphosphates other than ATP (at 100mum) and cyclic 3':5'-nucleotides (at 10mum) had no significant effect on kinase activity. 3. The K(m) for thiamin pyrophosphate in the pyruvate dehydrogenase reaction was 0.76mum. Sodium pyrophosphate, adenylyl imidodiphosphate, ADP and GTP were competitive inhibitors against thiamin pyrophosphate in the dehydrogenase reaction. 4. The K(m) for ATP of the intrinsic kinase assayed in three preparations of pig heart pyruvate dehydrogenase was in the range 13.9-25.4mum. Inhibition by ADP and adenylyl imidodiphosphate was predominantly competitive, but there was nevertheless a definite non-competitive element. Thiamin pyrophosphate and sodium pyrophosphate were uncompetitive inhibitors against ATP. It is suggested that ADP and adenylyl imidodiphosphate inhibit the kinase mainly by binding to the ATP site and that the adenosine moiety may be involved in this binding. It is suggested that thiamin pyrophosphate, sodium pyrophosphate, adenylyl imidodiphosphate and ADP may inhibit the kinase by binding through pyrophosphate or imidodiphosphate moieties at some site other than the ATP site. It is not known whether this is the coenzyme-binding site in the pyruvate dehydrogenase reaction. 5. The K(m) for pyruvate in the pyruvate dehydrogenase reaction was 35.5mum. 2-Oxobutyrate and 3-hydroxypyruvate but not glyoxylate were also substrates; all three compounds inhibited pyruvate oxidation. 6. In preparations of pig heart pyruvate dehydrogenase free of thiamin pyrophosphate, pyruvate inhibited the kinase reaction at all concentrations in the range 25-500mum. The inhibition was uncompetitive. In the presence of thiamin pyrophosphate (endogenous or added at 2 or 10mum) the kinase activity was enhanced by low concentrations of pyruvate (25-100mum) and inhibited by a high concentration (500mum). Activation of the kinase reaction was not seen when sodium pyrophosphate was substituted for thiamin pyrophosphate. 7. Under the conditions of the kinase assay, pig heart pyruvate dehydrogenase forms (14)CO(2) from [1-(14)C]pyruvate in the presence of thiamin pyrophosphate. Previous work suggests that the products may include acetoin. Acetoin activated the kinase reaction in the presence of thiamin pyrophosphate but not with sodium pyrophosphate. It is suggested that acetoin formation may contribute to activation of the kinase reaction by low pyruvate concentrations in the presence of thiamin pyrophosphate. 8. Pyruvate effected the conversion of pyruvate dehydrogenase phosphate into pyruvate dehydrogenase in rat heart mitochondria incubated with 5mm-2-oxoglutarate and 0.5mm-l-malate as respiratory substrates. It is suggested that this effect of pyruvate is due to inhibition of the pyruvate dehydrogenase kinase reaction in the mitochondrion. 9. Pyruvate dehydrogenase kinase activity was inhibited by high concentrations of Mg(2+) (15mm) and by Ca(2+) (10nm-10mum) at low Mg(2+) (0.15mm) but not at high Mg(2+) (15mm).
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PMID:Regulation of heart muscle pyruvate dehydrogenase kinase. 446 46

The SGK1 protein belongs to the AGC gene family of kinases that are regulated by phosphorylation mediated by PDK1. SGK1 regulation is accomplished by several pathways including growth-factor and stress-mediated signaling. We have expanded the analysis of SGK1 regulation in epithelial cells. We used HA-tagged SGK1 to transiently transfect MDCK cells and study the regulation of SGK1 upon stimulation with HGF, cAMP or upon adhesion of the cells to immobilized fibronectin. In addition, we studied the regulation of SGK1 activity by small GTP-binding proteins of the Rho family. Treatment of MDCK cells with HGF leads to a time-dependent activation of SGK1 that is blocked by wortmanin. This activation requires the conserved phosphorylation site present in the activation loop of the kinase (T256 in SGK1) and the phosphorylation site present in a hydrophobic domain at its C-terminus (S422 in SGK1), which are targets for PDK1/PDK2-mediated regulation of SGK1. We tested whether SGK1 could be activated by cAMP as it contains a putative PKA site. We were unable to demonstrate a significant activation of HA-SGK1 by cAMP stimulation under conditions where we detect cAMP-mediated phosphorylation of the transcription factor CREB. Cotransfection of SGK1 with activated small GTP-binding proteins revealed that Rac1, but not Rho or Rap1, induces activation of SGK1. However, this activation was wortmanin insensitive and dominant-negative Rac1 did not inhibit the HGF-mediated activation of SGK1. Adhesion of MDCK cells to immobilized fibronectin also leads to activation of SGK1. However, it appears that the integrin-mediated activation of HA-SGK1 differs from AKT activation in the fact that AKT phosphorylation was blocked by wortmanin (or LY294002) whereas HA-SGK1 was not. The adhesion-dependent activation, however, requires the intact phosphorylation sites of SGK1. Co-transfection of HA-SGK1 with RacV12 results in increased activity in adherent cells compared with HA-SGK1 alone. Since RacN17 failed to inhibit adhesion dependent-activation of SGK1, it suggests that integrin activation is achieved by a parallel Rac-independent pathway. The activation of SGK1 by HGF and integrin provides a link between HGF-mediated protection of MDCK from de-attachment induced apoptosis (anoikis). We demonstrate that dephosphorylation of the transcription factor FKRHL1 induced by cell de-attachment is prevented by activated SGK1, suggesting that SGK1 regulates cell survival pathways. In summary, we demonstrate that SGK1 activation could be achieved through signaling pathways involved in the regulation of cell survival, cell-cell and cell-matrix interactions. SGK1 activation can be accomplished via HGF, PI-3K-dependent pathways and by integrin-mediated, PI-3K independent pathways. In addition, activation of SGK1 by the small GTP-binding protein Rac1 has been observed.
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PMID:Activation of SGK1 by HGF, Rac1 and integrin-mediated cell adhesion in MDCK cells: PI-3K-dependent and -independent pathways. 1195 29

cAMP regulates a wide range of processes through its downstream effectors including PKA, and the family of guanine nucleotide exchange factors. Depending on the cell type, cAMP inhibits or stimulates growth and proliferation in a PKA-dependent or independent manner. PKA-independent effects are mediated by PI 3-kinases-Akt signaling and EPAC1 (exchange protein directly activated by cAMP) activation. Recently, we reported PKA-independent activation of the protein kinase Akt as well co-immunoprecipitation of Epac1 with Rap1, p-Akt(Thr-308), and p-Akt(Ser-473) in forskolin-stimulated macrophages. To further probe the role of Epac1 in Akt protein kinase activation and cellular proliferation, we employed the cAMP analog 8-CPT-2-O-Me-cAMP, which selectively binds to Epac1 and triggers Epac1 signaling. We show the association of Epac1 with activated Akt kinases by co-immunoprecipitation and GST-pulldown assays. Silencing Epac1 gene expression by RNA interference significantly reduced levels of Epac1 mRNA, Epac protein, Rap1 GTP, p-ERK1/2, p-B-Raf, p110alpha catalytic subunit of PI 3-kinase, p-PDK, and p-p(70s6k). Silencing Epac1 gene expression by RNA interference also suppressed 8-CPT-2-O-Me-cAMP-upregulated protein and DNA synthesis. Concomitantly, 8-CPT-2-O-Me-cAMP-mediated upregulation of Akt(Thr-308) protein kinase activity and p-Akt(Thr-308) levels was prevented in plasma membranes and nuclei of the cells. In contrast, silencing Epac1 gene expression reduced Akt(Ser-473) kinase activity and p-Akt(Ser-473) levels in plasma membranes, but showed negligible effects on nuclear activity. In conclusion, we show that cAMP-induced Akt kinase activation and cellular proliferation is mediated by Epac1 which appears to function as an accessory protein for Akt activation.
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PMID:The cAMP-activated GTP exchange factor, Epac1 upregulates plasma membrane and nuclear Akt kinase activities in 8-CPT-2-O-Me-cAMP-stimulated macrophages: Gene silencing of the cAMP-activated GTP exchange Epac1 prevents 8-CPT-2-O-Me-cAMP activation of Akt activity in macrophages. 1849 29

PRK2/PKNgamma is a Rho effector and a member of the protein kinase C superfamily of serine/threonine kinases. Here, we explore the structure-function relationship between various motifs in the C-terminal half of PRK2 and its kinase activity and regulation. We report that two threonine residues at conserved phosphoacceptor position in the activation loop and the turn motif are essential for the catalytic activity of PRK2, but the phosphomimetic Asp-978 at hydrophobic motif is dispensable for kinase catalytic competence. Moreover, the PRK2-Delta958 mutant with the turn motif truncated still interacts with 3-phosphoinositide-dependent kinase-1 (PDK-1). Thus, both the intact hydrophobic motif and the turn motif in PRK2 are dispensable for the binding of PDK-1. We also found that while the last seven amino acid residues at the C-terminus of PRK2 are not required for the activation of the kinase by RhoA in vitro, however, the extreme C-terminal segment is critical for the full activation of PRK2 by RhoA in cells in a GTP-dependent manner. Our data suggest that the extreme C-terminus of PRK2 may represent a potential drug target for effector-specific pharmacological intervention of Rho-medicated biological processes.
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PMID:The C-terminus of PRK2/PKNgamma is required for optimal activation by RhoA in a GTP-dependent manner. 1883 41

p90 ribosomal S6 kinase (RSK1) is an effector of both Ras/MEK/MAPK and PI3K/PDK1 pathways. We present evidence that RSK1 drives p27 phosphorylation at T198 to increase RhoA-p27 binding and cell motility. RSK1 activation and p27pT198 both increase in early G(1). As for many kinase-substrate pairs, cellular RSK1 coprecipitates with p27. siRNA to RSK1 and RSK1 inhibition both rapidly reduce cellular p27pT198. RSK1 overexpression increases p27pT198, p27-cyclin D1-Cdk4 complexes, and p27 stability. Moreover, RSK1 transfectants show mislocalization of p27 to cytoplasm, increased motility, and reduced RhoA-GTP, phospho-cofilin, and actin stress fibers, all of which were reversed by shRNA to p27. Phosphorylation by RSK1 increased p27pT198 binding to RhoA in vitro, whereas p27T157A/T198A bound poorly to RhoA compared with WTp27 in cells. Coprecipitation of cellular p27-RhoA was increased in cells with constitutive PI3K activation and increased in early G(1). Thus T198 phosphorylation not only stabilizes p27 and mislocalizes p27 to the cytoplasm but also promotes RhoA-p27 interaction and RhoA pathway inhibition. These data link p27 phosphorylation at T198 and cell motility. As for other PI3K effectors, RSK1 phosphorylates p27 at T198. Because RSK1 is also activated by MAPK, the increased cell motility and metastatic potential of cancer cells with PI3K and/or MAPK pathway activation may result in part from RSK1 activation, leading to accumulation of p27T198 in the cytoplasm, p27:RhoA binding, inhibition of RhoA/Rock pathway activation, and loss of actomyosin stability.
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PMID:RSK1 drives p27Kip1 phosphorylation at T198 to promote RhoA inhibition and increase cell motility. 1947 Apr 70

To grow, eukaryotic cells must expand by inserting glycerolipids, sphingolipids, sterols, and proteins into their plasma membrane, and maintain the proper levels and bilayer distribution. A fungal cell must coordinate growth with enlargement of its cell wall. In Saccharomyces cerevisiae, a plasma membrane-localized protein kinase complex, Target of Rapamicin (TOR) complex-2 (TORC2) (mammalian ortholog is mTORC2), serves as a sensor and masterregulator of these plasma membrane- and cell wall-associated events by directly phosphorylating and thereby stimulating the activity of two types of effector protein kinases: Ypk1 (mammalian ortholog is SGK1), along with a paralog (Ypk2); and, Pkc1 (mammalian ortholog is PKN2/PRK2). Ypk1 is a central regulator of pathways and processes required for plasma membrane lipid and protein homeostasis, and requires phosphorylation on its T-loop by eisosome-associated protein kinase Pkh1 (mammalian ortholog is PDK1) and a paralog (Pkh2). For cell survival under various stresses, Ypk1 function requires TORC2-mediated phosphorylation at multiple sites near its C terminus. Pkc1 controls diverse processes, especially cell wall synthesis and integrity. Pkc1 is also regulated by Pkh1- and TORC2-dependent phosphorylation, but, in addition, by interaction with Rho1-GTP and lipids phosphatidylserine (PtdSer) and diacylglycerol (DAG). We also describe here what is currently known about the downstream substrates modulated by Ypk1-mediated and Pkc1-mediated phosphorylation.
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PMID:The TORC2-Dependent Signaling Network in the Yeast Saccharomyces cerevisiae. 2887 98