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)

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

A novel thermodynamically-balanced inside-out (TBIO) method of primer design was developed and compared with a thermodynamically-balanced conventional (TBC) method of primer design for PCR-based gene synthesis of codon-optimized gene sequences for the human protein kinase B-2 (PKB2; 1494 bp), p70 ribosomal S6 subunit protein kinase-1 (S6K1; 1622 bp) and phosphoinositide-dependent protein kinase-1 (PDK1; 1712 bp). Each of the 60mer TBIO primers coded for identical nucleotide regions that the 60mer TBC primers covered, except that half of the TBIO primers were reverse complement sequences. In addition, the TBIO and TBC primers contained identical regions of temperature- optimized primer overlaps. The TBC method was optimized to generate sequential overlapping fragments (approximately 0.4-0.5 kb) for each of the gene sequences, and simultaneous and sequential combinations of overlapping fragments were tested for their ability to be assembled under an array of PCR conditions. However, no fully synthesized gene sequences could be obtained by this approach. In contrast, the TBIO method generated an initial central fragment (approximately 0.4-0.5 kb), which could be gel purified and used for further inside-out bidirectional elongation by additional increments of 0.4-0.5 kb. By using the newly developed TBIO method of PCR-based gene synthesis, error-free synthetic genes for the human protein kinases PKB2, S6K1 and PDK1 were obtained with little or no corrective mutagenesis.
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PMID:Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences. 1460 36

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

The conformation of a bisindolylmaleimide may be controlled by the size of a macrocyclic ring in which it is constrained. A range of techniques were used to demonstrate that the tether controls both the ratio of the two limiting conformers (syn and anti) in solution and the extent of conjugation between the maleimide and indole rings. Screening the conformationally diverse bisindolylmaleimides against a panel of protein kinases allowed their ATP binding sites to be compared using a chemical approach which, like sequence alignment, does not require detailed structural information. This approach lead to the conclusion that several AGC group protein kinases (including PKCalpha, PKCbeta, MSK1, p70 S6K, PDK-1, and MAPKAP-K1alpha) may be best inhibited by bisindolylmaleimides which adopt a compressed approximately C2-symmetric anti conformation; in constrast, GSK3beta may be best inhibited by bisindolylmaleimides whose ground state is a distorted syn conformation. It is concluded that PDK-1, whose structure has been determined by X-ray crystallography, and its mutants, may serve as particularly useful surrogates for the study of PKC inhibitors.
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PMID:Comparison of the ATP binding sites of protein kinases using conformationally diverse bisindolylmaleimides. 1610 47

Many cancers possess elevated levels of PtdIns(3,4,5)P(3), the second messenger that induces activation of the protein kinases PKB/Akt and S6K and thereby stimulates cell proliferation, growth, and survival. The importance of this pathway in tumorigenesis has been highlighted by the finding that PTEN, the lipid phosphatase that breaks down PtdIns(3,4,5)P(3) to PtdIns(4,5)P(2), is frequently mutated in human cancer. Cells lacking PTEN possess elevated levels of PtdIns(3,4,5)P(3), PKB, and S6K activity and heterozygous PTEN(+/-) mice develop a variety of tumors. Knockout of PKBalpha in PTEN-deficient cells reduces aggressive growth and promotes apoptosis, whereas treatment of PTEN(+/-) mice with rapamycin, an inhibitor of the activation of S6K, reduces neoplasia. We explored the importance of PDK1, the protein kinase that activates PKB and S6K, in mediating tumorigenesis caused by the deletion of PTEN. We demonstrate that reducing the expression of PDK1 in PTEN(+/-) mice, markedly protects these animals from developing a wide range of tumors. Our findings provide genetic evidence that PDK1 is a key effector in mediating neoplasia resulting from loss of PTEN and also validate PDK1 as a promising anticancer target for the prevention of tumors that possess elevated PKB and S6K activity.
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PMID:Hypomorphic mutation of PDK1 suppresses tumorigenesis in PTEN(+/-) mice. 1624 31

The eukaryotic translation initiation factor 4B (eIF4B) plays a critical role in recruiting the 40S ribosomal subunit to the mRNA. In response to insulin, eIF4B is phosphorylated on Ser422 by S6K in a rapamycin-sensitive manner. Here we demonstrate that the p90 ribosomal protein S6 kinase (RSK) phosphorylates eIF4B on the same residue. The relative contribution of the RSK and S6K modules to the phosphorylation of eIF4B is growth factor-dependent, and the two phosphorylation events exhibit very different kinetics. The S6K and RSK proteins are members of the AGC protein kinase family, and require PDK1 phosphorylation for activation. Consistent with this requirement, phosphorylation of eIF4B Ser422 is abrogated in PDK1 null embryonic stem cells. Phosphorylation of eIF4B on Ser422 by RSK and S6K is physiologically significant, as it increases the interaction of eIF4B with the eukaryotic translation initiation factor 3.
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PMID:The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity. 1676 66

Although trastuzumab has been successfully used in patients with HER2-overexpressing metastatic breast cancer, resistance is a common problem that ultimately culminates in treatment failure. In light of the importance of Akt signaling in trastuzumab's antitumor action, we hypothesized that concurrent inhibition of Akt could enhance trastuzumab sensitivity and moreover reverse the resistant phenotype in HER2-positive breast cancer cells. Based on our finding that celecoxib mediates antitumor effects through the inhibition of phosphoinositide-dependent kinase-1 (PDK-1)/Akt signaling independently of cyclooxygenase-2 (COX-2), we used celecoxib as a scaffold to develop a COX-2-inactive PDK-1 inhibitor, 2-amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide (OSU-03012). Here, we investigated the effect of OSU-03012 on trastuzumab-mediated apoptosis in four breast cancer cell lines with different HER2 expression and trastuzumab-resistance status, including MDA-MB-231, BT474, SKBR3, and insulin-like growth factor-I receptor-overexpressing SKBR3 (SKBR3/IGF-IR). Effects of trastuzumab and OSU-03012, individually or in combination, on cell viability and changes in pertinent biomarkers including HER2 expression, phosphorylation of Akt, p27(kip1), and the PDK-1 substrate p70(S6K) were assessed. OSU-03012 alone was able to trigger apoptosis in all cell lines with equal potency (IC(50) = 3-4 microM), suggesting no cross-resistance with trastuzumab. Medium dose-effect analysis indicates that OSU-03012 potentiated trastuzumab's antiproliferative effect in HER2-positive cells, especially in SKBR3/IGF-IR cells, through the down-regulation of PDK-1/Akt signaling. This synergy, however, was not observed in HER2-negative MDA-MB-231 cells. This combination treatment represents a novel strategy to increase the efficacy of trastuzumab and to overcome trastuzumab resistance in the treatment of HER2-positive breast cancer.
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PMID:Overcoming trastuzumab resistance in HER2-overexpressing breast cancer cells by using a novel celecoxib-derived phosphoinositide-dependent kinase-1 inhibitor. 1688 35

Mammalian target of rapamycin (mTOR) controls cell growth and proliferation via the raptor-mTOR (TORC1) and rictor-mTOR (TORC2) protein complexes. Recent biochemical studies suggested that TORC2 is the elusive PDK2 for Akt/PKB Ser473 phosphorylation in the hydrophobic motif. Phosphorylation at Ser473, along with Thr308 of its activation loop, is deemed necessary for Akt function, although the regulatory mechanisms and physiological importance of each phosphorylation site remain to be fully understood. Here, we report that SIN1/MIP1 is an essential TORC2/PDK2 subunit. Genetic ablation of sin1 abolished Akt-Ser473 phosphorylation and disrupted rictor-mTOR interaction but maintained Thr308 phosphorylation. Surprisingly, defective Ser473 phosphorylation affected only a subset of Akt targets in vivo, including FoxO1/3a, while other Akt targets, TSC2 and GSK3, and the TORC1 effectors, S6K and 4E-BP1, were unaffected. Our findings reveal that the SIN1-rictor-mTOR function in Akt-Ser473 phosphorylation is required for TORC2 function in cell survival but is dispensable for TORC1 function.
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PMID:SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. 1696 53

Balloon cells of distinct focal cortical dysplasias type IIb (FCD(IIb)) and giant cells of cortical tubers in tuberous sclerosis (TSC) constitute neuropathological hallmarks and cytological similarities. In TSC, frequent mutations in the TSC1 or TSC2 genes result in mTOR-signaling activity. Here, we addressed whether Pi3K-pathway activation differentiates balloon cells from giant cells. We used immunohistochemistry with antibodies against p-PDK1 (S241), p-Akt (S473), p-tuberin (T1462), p-p70(S6K) (T389), p-p70(S6K) (T229) and phalloidin-staining to analyze stress fiber formation in balloon cells of FCD(IIb) (n = 23) compared with cortical tuber giant cells (n = 5) and adjacent normal CNS tissue as control. We have further established an in vitro assay to assess potential phosphorylation between Akt and S6. We observed phosphorylated (p-)PDK1, p-Akt, p-tuberin, and p-p70-kDa S6-kinase (p-p70(S6K); residue T229) in balloon cells, whereas giant cells showed only equivalent levels of p-tuberin, p-p70(S6K) and stress fibers. Furthermore, Pi3K-cascade activity in balloon cells may reflect pathway "cross-talk". An in vitro assay revealed S6, a major target of p70(S6K), to increase phosphorylation of Akt. Our data suggest recruitment of different Pi3K-cascade factors in the molecular pathogenesis of giant cells in cortical tubers vs. balloon cells in FCD(IIb) and provides new implications for the development of treatment strategies for these cortical malformations.
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PMID:Differential Pi3K-pathway activation in cortical tubers and focal cortical dysplasias with balloon cells. 1738 47


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