Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Ribosomal S6 kinase 2 (S6K2) is a serine/threonine kinase identified as a homologue of p70 ribosomal S6 kinase 1 (S6K1). S6K1 and S6K2 show different cellular localization as well as divergent amino acid sequences in non-catalytic domains, suggesting that their cellular functions and/or regulation may not be identical. Many of the serine/threonine residues that become phosphorylated and contribute to S6K1 activation are conserved in S6K2. In this study we carry out mutational analyses of these serine/threonine residues on S6K2 in order to elucidate the mechanism of S6K2 regulation. We find that Thr-228 and Ser-370 are crucial for S6K2 activity, and the three proline-directed serines in the autoinhibitory domain, Ser-410, Ser-417 and Ser-423, play a role in S6K2 activity regulation in a mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase (MEK)-dependent manner. However, unlike S6K1, changing Thr-388 to glutamic acid in S6K2 renders the kinase fully active. This activity was resistant to the effects of rapamycin or wortmannin, indicating that mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) regulate S6K2 activity via Thr-388. MEK-dependent phosphorylation of the autoinhibitory serines in S6K2 occurs prior to Thr-388 activation. Combining T388E and T228A mutations inhibited S6K2 activation, and a kinase-inactive phosphoinositide-dependent protein kinase (PDK1) diminished T388E activity, suggesting that the role of Thr-388 is to allow further phosphorylation of Thr-228 by PDK1. Thr-388 fails to become phosphorylated in Ser-370 mutants, suggesting that the role of Ser-370 phosphorylation may be to allow Thr-388 phosphorylation. Finally, using the rapamycin-resistant T388E mutant, we provide evidence that S6K2 can phosphorylate S6 in vivo.
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PMID:Mutational analysis of ribosomal S6 kinase 2 shows differential regulation of its kinase activity from that of ribosomal S6 kinase 1. 1271 46

Stimulation of intestinal fructose absorption by phorbol 12-myristate 13-acetate (PMA) results from rapid insertion of GLUT2 into the brush-border membrane and correlates with protein kinase C (PKC) betaII activation. We have therefore investigated the role of phosphatidylinositol 3 (PI3)-kinase and mammalian target of rapamycin in the regulation of fructose absorption by PKC betaII phosphorylation. In isolated jejunal loops, stimulation of fructose absorption by PMA was inhibited by preperfusion with wortmannin or rapamycin, which blocked GLUT2 activation and insertion into the brush-border membrane. Antibodies to the last 18 and last 10 residues of the C-terminal region of PKC betaII recognized several species differentially in Western blots. Extensive cleavage of native enzyme (80/78 kDa) to a catalytic domain product of 49 kDa occurred. PMA and sugars provoked turnover and degradation of PKC betaII by dephosphorylation to a 42-kDa species, which was converted to polyubiquitylated species detected at 180 and 250+ kDa. PMA increased the level of the PKC betaII 49-kDa species, which correlates with the GLUT2 level; wortmannin and rapamycin blocked these effects of PMA. Rapamycin and wortmannin inhibited PKC betaII turnover. PI3-kinase, PDK-1, and protein kinase B were present in the brush-border membrane, where their levels were increased by PMA and blocked by the inhibitors. We conclude that GLUT2-mediated fructose absorption is regulated through PI3-kinase and mammalian target of rapamycin-dependent pathways, which control phosphorylation of PKC betaII and its substrate-induced turnover and ubiquitin-dependent degradation. These findings suggest possible mechanisms for short term control of intestinal sugar absorption by insulin and amino acids.
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PMID:Intestinal sugar absorption is regulated by phosphorylation and turnover of protein kinase C betaII mediated by phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent pathways. 1276 74

The TOR protein is a phosphoinositide kinase-related kinase widely conserved among eukaryotes. Fission yeast tor1 encodes an ortholog of TOR, which is required for sexual development and growth under stressed conditions. We isolated gad8, which encodes a Ser/Thr kinase of the AGC family, as a high-copy suppressor of the sterility of a tor1 mutant. Disruption of gad8 caused phenotypes similar to those of tor1 disruption. Gad8p was less phosphorylated and its kinase activity was undetectable in tor1Delta cells. Three amino acid residues corresponding to conserved phosphorylation sites in the AGC family kinases, namely Thr387 in the activation loop, Ser527 in the turn motif and Ser546 in the hydrophobic motif, were important for the kinase activity of Gad8p. Tor1p was responsible for the phosphorylation of Ser527 and Ser546, whereas Ksg1p, a PDK1-like kinase, appeared to phosphorylate Thr387 directly. Altogether, Tor1p, Ksg1p and Gad8p appear to constitute a signaling module for sexual development and growth under stressed conditions in fission yeast, which resembles the mTOR-PDK1-S6K1 system in mammals and may represent a basic signaling module ubiquitous in eukaryotes.
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PMID:Schizosaccharomyces pombe AGC family kinase Gad8p forms a conserved signaling module with TOR and PDK1-like kinases. 1280 21

Ischaemic preconditioning (IPC) protects the heart against myocardial infarction acutely as well as several hours later (e.g. 24-48 h). The mechanism of the profound cardioprotection is not completely explored. We hypothesized that PI3K/PDK1/Akt/mTOR/p70S6K-mediated pro-survival pathway is involved in delayed cardioprotection induced by IPC. Under Hypnorm-Diazepam anaesthesia, male New Zealand White rabbits were either sham-operated (SC) or preconditioned by four cycles of 5-min ischaemia and 10-min reperfusion on day 1. Twenty-four hours after recovery, the animals were anaesthetized with sodium pentobarbitone and subjected to 30-min ischaemia followed by 180-min reperfusion. Wortmannin (0.6 mg/kg, i.v.), an irreversible PI3 kinase (PI3K) inhibitor, rapamycin (0.25 mg/kg, i.v.), which prevents the phosphorylation of p70S6 kinase (p70S6K), or DMSO (control vehicle) was given 15 min prior to IPC. IPC significantly reduced infarct size compared to the control group (SC) (31.9 +/- 5.8% (n = 7) vs. 54.9 +/- 2.9% (n = 6), P < 0.05). Wortmannin and rapamycin alone had no effect on infarct size (56.3 +/- 1.6% (n = 6) and 54.7 +/- 3.8% (n = 6), respectively). However, when wortmannin or rapamycin were given prior to IPC the protection was completely abolished (49.9 +/- 2.8% (n = 6), 45.1 +/- 4.6% (n = 7), P < 0.05 vs. IPC). Western blot analysis showed that wortmannin, at a dose of 0.6 mg/kg, and rapamycin, at a dose of 0.25 mg/kg, were sufficient to prevent phosphorylation of Akt and p70S6K, respectively, when the inhibitors were given prior to IPC. We conclude that PI3K/PDK1/Akt/mTOR/p70S6K-signalling pathway plays an essential role in the development of the cardioprotection against infarction in rabbits.
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PMID:Second window of protection following myocardial preconditioning: an essential role for PI3 kinase and p70S6 kinase. 1296 24

The p70 S6 kinase (p70 S6K) was the first signaling element in mammalian cells shown to be inhibited by rapamycin. The activity of the p70 S6K in mammalian cell is upregulated by extracellular amino acids (especially leucine) and by signals from receptor tyrosine kinases (RTKs), primarily through activation of the type 1A PI-3 kinase. The amino acid-/rapamycin-sensitive input and the PI-3 kinase input are co-dominant but largely independent, in that deletion of the amino-terminal and carboxy-terminal noncatalytic sequences flanking the p70 S6K catalytic domain renders the kinase insensitive to inhibition by both rapamycin and by withdrawal of amino acids, whereas this p70 S6K mutant remains responsive to activation by RTKs and to inhibition by wortmannin. At a molecular level, this dual control of p70 S6K activity is attributable to phosphorylation of the two p70 S6K sites: The Ptd Ins 3,4,5P3-dependent kinasel (PDK1) phosphorylates p70 S6K at a Thr on the activation loop, whereas mTOR phosphorylates a Thr located in a hydrophobic motif carboxyterminal to the catalytic domain. Together these two phosphorylations engender a strong, positively cooperative activation of p70 S6K, so that each is indispensable for physiologic regulation. Like RTKs, the p70 S6K appears early in metazoan evolution and comes to represent an important site at which the more ancient, nutrient-responsive TOR pathway converges with the RTK/PI-3 kinase pathway in the control of cell growth. Dual regulation of p70 S6K is seen in Drosophila; however, this convergence is not yet evident in Caenorhabditis elegans, wherein nutrient activation of the insulin receptor (InsR) pathway negatively regulates dauer development and longevity, whereas the TOR pathway regulates overall mRNA translation through effectors distinct from p70 S6K, as in yeast. The C. elegans TOR and InsR pathways show none of the cross- or convergent regulation seen in mammalian cells. The nature of the elements that couple nutrient sufficiency to TOR activity remain to be discovered, and the mechanisms by which RTKs influence TOR activity in mammalian cells require further study. One pathway for RTK control involves the tuberous sclerosis complex, which is absent in C. elegans, but of major importance in Drosophila and higher metazoans.
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PMID:TOR action in mammalian cells and in Caenorhabditis elegans. 1456 Sep 55

Recent evidence indicates that mutations in the gene encoding the WNK1 [with no K (lysine) protein kinase-1] results in an inherited hypertension syndrome called pseudohypoaldosteronism type II. The mechanisms by which WNK1 is regulated or the substrates it phosphorylates are currently unknown. We noticed that Thr-60 of WNK1, which lies N-terminal to the catalytic domain, is located within a PKB (protein kinase B) phosphorylation consensus sequence. We found that PKB phosphorylated WNK1 efficiently compared with known substrates, and both peptide map and mutational analysis revealed that the major PKB site of phosphorylation was Thr-60. Employing a phosphospecific Thr-60 WNK1 antibody, we demonstrated that IGF1 (insulin-like growth factor) stimulation of HEK-293 cells induced phosphorylation of endogenously expressed WNK1 at Thr-60. Consistent with PKB mediating this phosphorylation, inhibitors of PI 3-kinase (phosphoinositide 3-kinase; wortmannin and LY294002) but not inhibitors of mammalian target of rapamycin (rapamycin) or MEK1 (mitogen-activated protein kinase kinase-1) activation (PD184352), inhibited IGF1-induced phosphorylation of endogenous WNK1 at Thr-60. Moreover, IGF1-induced phosphorylation of endogenous WNK1 did not occur in PDK1-/- ES (embryonic stem) cells, in which PKB is not activated. In contrast, IGF1 still induced normal phosphorylation of WNK1 in PDK1(L155E/L155E) knock-in ES cells in which PKB, but not S6K (p70 ribosomal S6 kinase) or SGK1 (serum- and glucocorticoid-induced protein kinase 1), is activated. Our study provides strong pharmacological and genetic evidence that PKB mediates the phosphorylation of WNK1 at Thr-60 in vivo. We also performed experiments which suggest that the phosphorylation of WNK1 by PKB is not regulating its kinase activity or cellular localization directly. These results provide the first connection between the PI 3-kinase/PKB pathway and WNK1, suggesting a mechanism by which this pathway may influence blood pressure.
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PMID:WNK1, the kinase mutated in an inherited high-blood-pressure syndrome, is a novel PKB (protein kinase B)/Akt substrate. 1461 43

We have examined the role of endogenous 70-kDa S6 kinase (p70(S6K)) in actin cytoskeletal organization and cell migration in Swiss 3T3 fibroblasts. Association of p70(S6K) with the actin cytoskeleton was demonstrated by cosedimentation of p70(S6K) with F-actin and by subcellular fractionation in which p70(S6K) activity was measured in the F-actin cytoskeletal fraction. Immunocytochemical studies showed that p70(S6K), Akt1, PDK1, and p85 phosphoinositide 3-kinase (PI 3-kinase) were localized to the actin arc, a caveolin-enriched cytoskeletal structure located at the leading edge of migrating cells. Using a phospho-specific antibody to mammalian target of rapamycin (mTOR), we find that activated mTOR is enriched at the actin arc, suggesting that activation of the p70(S6K) signaling pathway is important to cell migration. Using the actin arc to assess migration, epidermal growth factor (EGF) stimulation was found to induce actin arc formation, an effect that was blocked by rapamycin treatment. We show further that actin stress fibers may function to down-regulate p70(S6K). Fibronectin stimulated stress fiber formation in the absence of growth factors and caused an inactivation of p70(S6K). Conversely, cytochalasin D and the Rho kinase inhibitor Y-27632, both of which cause stress fiber disruption, increased p70(S6K) activity. These studies provide evidence that the p70(S6K) pathway is important for signaling at two F-actin microdomains in cells and regulates cell migration.
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PMID:Role of the p70(S6K) pathway in regulating the actin cytoskeleton and cell migration. 1514 49

Phosphoinositide-3 kinases (PI3Ks) are a family of evolutionary conserved lipid kinases that mediate many cellular responses in both physiologic and pathophysiologic states. Class I PI3K can be activated by either receptor tyrosine kinase (RTK)/cytokine receptor activation (class I(A)) or G-protein-coupled receptors (GPCR) (class I(B)). Once activated PI3Ks generate phosphatidylinositols (PtdIns) (3,4,5)P(3) leading to the recruitment and activation of Akt/protein kinase B (PKB), PDK1 and monomeric G-proteins (e.g. Rac-GTPases), which then activate a range of downstream targets including glycogen synthase kinase-3beta (GSK-3beta), mammalian target of rapamycin (mTOR), p70S6 kinase, endothelial nitric oxide synthase (eNOS) and several anti-apoptotic effectors. Class I(A) (PI3Kalpha, beta and delta) and class I(B) (PI3Kgamma) PI3Ks mediate distinct phenotypes in the heart and under negative control by the 3'-lipid phosphatase, phosphatase and tensin homolog on chromosome ten (PTEN) which dephosphorylate PtdIns(3,4,5)P(3) into PtdIns(4,5)P(2). PI3Kalpha, gamma and PTEN are expressed in cardiomyocytes, fibroblasts, endothelial cells and vascular smooth muscle cells where they modulate cell survival/apoptosis, hypertrophy, contractility, metabolism and mechanotransduction. Several transgenic and knockout models support a fundamental role of PI3K/PTEN signaling in the regulation of myocardial contractility and hypertrophy. Consequently the PI3K/PTEN signaling pathways are involved in a wide variety of diseases including cardiac hypertrophy, heart failure, preconditioning and hypertension. In this review, we discuss the biochemistry and molecular biology of PI3K (class I isoforms) and PTEN and their critical role in cardiovascular physiology and diseases.
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PMID:The role of phosphoinositide-3 kinase and PTEN in cardiovascular physiology and disease. 1527 15

We detected a protein in rabbit skeletal muscle extracts that was phosphorylated rapidly by PKBa (protein kinase Ba), but not by SGK1 (serum- and glucocorticoid-induced kinase 1), and identified it as the cytoskeletal protein FLNc (filamin C). PKBa phosphorylated FLNc at Ser2213 in vitro, which lies in an insert not present in the FLNa and FLNb isoforms. Ser2213 became phosphorylated when C2C12 myoblasts were stimulated with insulin or epidermal growth factor, and phosphorylation was prevented by low concentrations of wortmannin, at which it is a relatively specific inhibitor of phosphoinositide 3-kinase. PD 184352 [an inhibitor of the classical MAPK (mitogen-activated protein kinase) cascade] and/or rapamycin [an inhibitor of mTOR (mammalian target of rapamycin)] had no effect. Insulin also induced the phosphorylation of FLNc at Ser2213 in cardiac muscle in vivo, but not in cardiac muscle that does not express PDK1 (3-phosphoinositide-dependent kinase 1), the upstream activator of PKB. These results identify the muscle-specific isoform FLNc as a new physiological substrate for PKB.
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PMID:Identification of filamin C as a new physiological substrate of PKBalpha using KESTREL. 1546 88

Difficulties in achieving long-term survival of lung cancer patients treated with conventional therapies suggest that novel approaches are required. Although several genes have been investigated for antitumor activities using gene delivery, problems surrounding the methods used such as efficiency, specificity, and toxicity hinder its application as an effective therapy. This has lead to the re-emergence of aerosol gene delivery as a noninvasive approach to lung cancer therapy. In this study, glucosylated conjugated polyethylenimine (glucosylated PEI) was used as carrier. After confirming the efficiency of glucosylated PEI carriers in lungs, the potential effects of the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) tumor suppressor gene on Akt downstream pathways were investigated. Aerosol containing glucosylated PEI and recombinant plasmid pcDNA3.0-PTEN complex was delivered into K-ras null lung cancer model mice through a nose-only inhalation system. Investigation of proteins in the phosphatidylinositol 3'-kinase/Akt signaling pathway in PTEN-delivered mouse lung revealed that the PTEN protein was highly expressed, whereas the protein levels of PDK1, total Akt1, phospho-(Thr-308)-Akt, phospho-(Ser-2448)-mTOR, p70S6K, and 4E-BP1 were decreased to varying degrees. Additionally, the kinase activities of both Akt and mTOR were suppressed. Finally, apoptosis was detected in PTEN-delivered mouse lung by terminal deoxynucleotidyltransferase-mediated nick end labeling assay, suggesting that our aerosol PTEN delivery is capable of functionally altering cell phenotype in vivo. In summary, Western blot analysis, kinase assays, immunohistochemistry, and terminal deoxynucleotidyltransferase-mediated nick end labeling assays suggest that our aerosol gene delivery technique is compatible with in vivo gene delivery and can be applied as a noninvasive gene therapy.
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PMID:Aerosol delivery of glucosylated polyethylenimine/phosphatase and tensin homologue deleted on chromosome 10 complex suppresses Akt downstream pathways in the lung of K-ras null mice. 1552 Feb 4


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