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)

Rapamycin is an immunosuppressant which antagonizes cellular proliferation by inhibiting the function of mTOR. The mTOR:FKBP12: rapamycin complex blocks G1/S transition by inhibiting downstream targets essential for cell cycle progression. One such target is p70S6k1 (S6K1), a serine/threonine kinase which is inactivated by the mTOR : FKBP12 : rapamycin complex, and which has been linked to translational control by virtue of its ability to phosphorylate the ribosomal protein S6. In the current work, we describe cloning and characterization of a novel S6K1 homolog, p54 S6 kinase 2 (p54S6k2/S6K2). Similar to S6K1, S6K2 is activated by mitogens and by constitutively active PI3K, and is inhibited by rapamycin as well as wortmannin. Differences between activation of S6K1 and S6K2 by PDK1 were observed, suggesting potential differences in the regulation of these homologs. Strikingly, S6K2 activity and S6 phosphorylation were both intact in S6K1-/-ES cell, indicating a possible role for S6K2 in in vivo S6 phosphorylation. Interestingly, we found two isoforms of S6K2 which are localized to distinct cellular compartments; the smaller form resides in the detergent-soluble fraction, whereas the larger form is found in the particulate fraction. Our findings demonstrate the existence of a family of rapamycin-sensitive protein kinases potentially involved in S6 phosphorylation, translational control, and transduction of mTOR signals.
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PMID:Characterization of S6K2, a novel kinase homologous to S6K1. 1049 Aug 47

Translation of terminal oligopyrimidine tract (TOP) mRNAs, which encode multiple components of the protein synthesis machinery, is known to be controlled by mitogenic stimuli. We now show that the ability of cells to progress through the cell cycle is not a prerequisite for this mode of regulation. TOP mRNAs can be translationally activated when PC12 or embryonic stem (ES) cells are induced to grow (increase their size) by nerve growth factor and retinoic acid, respectively, while remaining mitotically arrested. However, both growth and mitogenic signals converge via the phosphatidylinositol 3-kinase (PI3-kinase)-mediated pathway and are transduced to efficiently translate TOP mRNAs. Translational activation of TOP mRNAs can be abolished by LY294002, a PI3-kinase inhibitor, or by overexpression of PTEN as well as by dominant-negative mutants of PI3-kinase or its effectors, PDK1 and protein kinase Balpha (PKBalpha). Likewise, overexpression of constitutively active PI3-kinase or PKBalpha can relieve the translational repression of TOP mRNAs in quiescent cells. Both mitogenic and growth signals lead to phosphorylation of ribosomal protein S6 (rpS6), which precedes the translational activation of TOP mRNAs. Nevertheless, neither rpS6 phosphorylation nor its kinase, S6K1, is essential for the translational response of these mRNAs. Thus, TOP mRNAs can be translationally activated by growth or mitogenic stimuli of ES cells, whose rpS6 is constitutively unphosphorylated due to the disruption of both alleles of S6K1. Similarly, complete inhibition of mammalian target of rapamycin (mTOR) and its effector S6K by rapamycin in various cell lines has only a mild repressive effect on the translation of TOP mRNAs. It therefore appears that translation of TOP mRNAs is primarily regulated by growth and mitogenic cues through the PI3-kinase pathway, with a minor role, if any, for the mTOR pathway.
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PMID:Transduction of growth or mitogenic signals into translational activation of TOP mRNAs is fully reliant on the phosphatidylinositol 3-kinase-mediated pathway but requires neither S6K1 nor rpS6 phosphorylation. 1241 14

Src homology 2-containing inositol 5'-phosphatase 2 (SHIP2) possesses 5'-phosphatase activity to specifically hydrolyze the phosphatidylinositol 3-kinase product PI(3,4,5)P3 in the regulation of insulin signaling. In the present study, we examined the impact of SHIP2 on the regulation of insulin signaling leading to protein synthesis in 3T3-L1 adipocytes cultured with standard and excess concentrations of amino acids. Insulin-induced translocation of PDK1 to the plasma membrane, phosphorylation of Akt and p70S6-kinase and ribosomal protein S6, increase in the amount of 4E-BP1 gamma-form, association of eIF4E with eIF4G, and protein synthesis were decreased by overexpression of wild-type SHIP2 by adenovirus-mediated gene transfer. The effect of SHIP2 overexpression on the regulation of insulin-induced phosphorylation of Akt and p70S6-kinase was somewhat augmented by the incubation with 5-fold excess concentrations of amino acids for 30 min. In contrast, the impact of SHIP2 expression was diminished in insulin-induced phosphorylation of p70S6-kinase and S6, but not of Akt, after the incubation for 16 h. Interestingly, incubation with the excess concentrations of amino acids for 30 min induced activation of phosphatidylinositol 3-kinase and phosphorylation of Akt, whereas phosphorylation of p70S6-kinase and S6 was decreased. Furthermore, although the exposure for longer time periods up to 24 h did not elicit phosphorylation of Akt, it markedly induced phosphorylation of p70S6-kinase and S6. These results indicate that SHIP2 plays an important role in the negative regulation of insulin signaling for the protein synthesis and that the impact of SHIP2 is altered, dependent on the acute or chronic exposure of excess concentrations of amino acids in culture.
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PMID:Impact of Src homology 2-containing inositol 5'-phosphatase 2 on the regulation of insulin signaling leading to protein synthesis in 3T3-L1 adipocytes cultured with excess amino acids. 1504 64

A series of 30 N10-substituted phenoxazines were synthesized and screened as potential inhibitors of Akt. In cellular assays at 5 mum, 17 compounds inhibited insulin-like growth factor 1 (IGF-I)-stimulated phosphorylation of Akt (Ser-473) by at least 50% but did not inhibit IGF-I-stimulated phosphorylation of Erk-1/2 (Thr-202/Tyr-204). Substitutions at the 2-position (Cl or CF3) did not alter inhibitory activity, whereas N10-substitutions with derivatives having acetyl (20B) or morpholino (12B) side chain lost activity compared with propyl or butyl substituents (7B and 14B). Inhibition of Akt phosphorylation was associated with the inhibition of IGF-I stimulation of the mammalian target of rapamycin phosphorylation (Ser-2448 and Ser-2481), phosphorylation of p70 S6 kinase (Thr-389), and ribosomal protein S6 (Ser-235/236) in Rh1, Rh18, and Rh30 cell lines. The two most potent compounds 10-[4'-(N-diethylamino)butyl]-2-chlorophenoxazine (10B) and 10-[4'-[(beta-hydroxyethyl)piperazino]butyl]-2-chlorophenoxazine (15B) (in vitro, IC50 approximately 1-2 microM) were studied further. Inhibition of Akt phosphorylation correlated with inhibition of its kinase activity as determined in vitro after immunoprecipitation. Akt inhibitory phenoxazines did not inhibit the activity of recombinant phosphatidylinositol 3'-kinase, PDK1, or SGK1 but potently inhibited the kinase activity of recombinant Akt and Akt deltaPH, a mutant lacking the pleckstrin homology domain. Akt inhibitory phenoxazines blocked IGF-I-stimulated nuclear translocation of Akt in Rh1 cells and suppressed growth of Rh1, Rh18, and Rh30 cells (IC50 2-5 microM), whereas "inactive" derivatives were > or = 10-fold less potent inhibitors of cell growth. In contrast to rapamycin analogs, Akt inhibitory phenoxazines induced significant levels of apoptosis under serum-containing culture conditions at concentrations of agent consistent with Akt inhibition. Thus, the cellular responses to phenoxazine inhibitors of Akt appear qualitatively different from the rapamycin analogs. Modeling studies suggest inhibitory phenoxazines may bind in the ATP-binding site, although ATP competition studies were unable to distinguish between competitive and noncompetitive inhibition.
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PMID:Identification of N10-substituted phenoxazines as potent and specific inhibitors of Akt signaling. 1600 6

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

IGF-II, a potent stimulator of cellular proliferation, differentiation, and development, regulates uterine function and conceptus growth in several species. In situ hybridization analyses found that IGF-II mRNA was most abundant in the caruncular endometrial stroma of both cyclical and pregnant ewes. In the intercaruncular endometrium, IGF-II mRNA transitioned from stroma to luminal epithelium between d 14 and 20 of pregnancy. IGF-II mRNA was present in all cells of the conceptus but was particularly abundant in the yolk sac. Immunohistochemical analyses revealed that phosphorylated (p)-protooncogenic protein kinase 1, p-ribosomal protein S6 kinase, p-ERK1/2, and p-P38 MAPK proteins were present at low levels in a majority of endometrial cells but were most abundant in the nuclei of endometrial luminal epithelium and conceptus trophectoderm of pregnant ewes. In mononuclear trophectoderm cells isolated from d-15 conceptuses, IGF-II increased the abundance of p-pyruvate dehydrogenase kinase 1, p-protooncogenic protein kinase 1, p-glycogen synthase kinase 3B, p-FK506 binding protein 12-rapamycin associated protein 1, and p-ribosomal protein S6 kinase protein within 15 min, and the increase was maintained for 90 min. IGF-II also elicited a rapid increase in p-ERK1/2 and p-P38 MAPK proteins that was maximal at 15 or 30 min posttreatment. Moreover, IGF-II increased migration of trophectoderm cells. Collectively, these results support the hypothesis that IGF-II coordinately activates multiple cell signaling pathways critical to survival, growth, and differentiation of the ovine conceptus during early pregnancy.
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PMID:Insulin-like growth factor II activates phosphatidylinositol 3-kinase-protooncogenic protein kinase 1 and mitogen-activated protein kinase cell Signaling pathways, and stimulates migration of ovine trophectoderm cells. 1833 15

PDK1 (3-phosphoinositide-dependent protein kinase 1) is a key mediator of signaling by phosphoinositide 3-kinase. To gain insight into the physiological importance of PDK1 in cell proliferation and cell cycle control, we established immortalized mouse embryonic fibroblasts (MEFs) from mice homozygous for a "floxed" allele of Pdk1 and from wild-type mice. Introduction of Cre recombinase by retrovirus-mediated gene transfer resulted in the depletion of PDK1 in Pdk1(lox/lox) MEFs but not in Pdk1(+/+) MEFs. The insulin-like growth factor-1-induced phosphorylation of various downstream effectors of PDK1, including Akt, glycogen synthase kinase 3, ribosomal protein S6, and p70 S6 kinase, was markedly inhibited in the PDK1-depleted (Pdk1-KO) MEFs. The rate of serum-induced cell proliferation was reduced; progression of the cell cycle from the G(0)-G(1) phase to the S phase was delayed, and cell cycle progression at G(2)-M phase was impaired in Pdk1-KO MEFs. These cells also manifested an increased level of p27(Kip1) expression and a reduced level of cyclin D1 expression during cell cycle progression. The defect in cell cycle progression from the G(0)-G(1) to the S phase in Pdk1-KO MEFs was rescued by forced expression of cyclin D1, whereas rescue of the defect in G(2)-M progression in these cells required both overexpression of cyclin D1 and depletion of p27(Kip1) by RNA interference. These data indicate that PDK1 plays an important role in cell proliferation and cell cycle progression by controlling the expression of both cyclin D1 and p27(Kip1).
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PMID:PDK1 regulates cell proliferation and cell cycle progression through control of cyclin D1 and p27Kip1 expression. 1843 Jul 22

PDK1 (phosphoinositide-dependent protein kinase-1) catalyzes phosphorylation of Thr-229 in the T-loop of S6K1 alpha II (the 70-kDa 40 S ribosomal protein S6 kinase-1 alpha II isoform), and Thr-229 phosphorylation is synergistic with C-terminal Thr-389 phosphorylation to activate S6K1 alpha II regulatory functions in protein translation preinitiation complexes. Unlike its common AGC kinase subfamily member S6K1 alpha II, PDK1 does not contain the synergistic C-terminal phosphorylation site, and it has been proposed that phosphorylated Thr-389 in S6K1 alpha II may initially serve to trans-activate PDK1-catalyzed Thr-229 phosphorylation. Herein, we report direct binding and kinetic studies that showed PDK1 to exhibit nearly equal binding affinities and steady-state kinetic turnover numbers toward native (K(d)(S6K1) = 1.2 microm and k(cat) = 1.1 s(-1)) and the phosphomimicking T389E mutant S6K1 alpha II (K(d)(S6K1) = 1.5 microm and k(cat) = 1.2 s(-1)), although approximately 2-fold enhanced specificity was displayed for the T389E mutant (k(cat)/K(m)(S6K1) = 0.08 microm(-1) s(-1) compared with 0.04 microm(-1) s(-1)). Considering that transient kinetic binding studies showed all nucleotide and S6K1 alpha II substrates and products to rapidly associate with PDK1 (k(on) = 1-6 mum(-1) s(-1)), it was concluded that positioning a negative charge at residue Thr-389 reduced approximately 2-fold the occurrence of nonproductive binding events that precede formation of a reactive ternary complex for Thr-229 phosphorylation. In addition, steady-state kinetic data were most simply accommodated by an Ordered Bi Bi mechanism with competitive substrate inhibition, where (i) the initially formed PDK1-ATP complex phosphorylates the nucleotide-free form of the S6K1 alpha II kinase and (ii) initial binding of S6K1 alpha II precludes ATP binding to PDK1.
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PMID:Mechanism of PDK1-catalyzed Thr-229 phosphorylation of the S6K1 protein kinase. 1957 Sep 88

The Akt kinase is a critical effector in growth factor signaling. Activation of Akt driven by the growth factor dependent PI3K (phosphatidylinositol-3-OH kinase) is coupled to the plasma membrane translocation and phosphorylation of Akt on two sites by PDK1 (phosphoinositide-dependent protein kinase-1) on Thr-308 and by mTORC2 (mammalian Target of Rapamycin Complex 2) on Ser-473. In our study we examined the sub-cellular localization of mTORC2 and identified that this kinase complex predominantly resides on endoplasmic reticulum (ER). Our immunostaining analysis did not show a substantial co-localization of the mTORC2 component rictor with Golgi, lysosome, clathrin-coated vesicles, early endosomes, or plasma membrane but indicated a strong co-localization of rictor with ribosomal protein S6 and ER marker. Our biochemical study also identified the mTORC2 components rictor, SIN1, and mTOR as the highly abundant proteins in the ER fraction, whereas only small amount of these proteins are detected in the plasma membrane and cytosolic fractions. We found that growth factor signaling does not alter the ER localization of mTORC2 and also does not induce its translocation to the plasma membrane. Based on our study we suggest that the mTORC2-dependent phosphorylation of Akt on Ser-473 takes place on the surface of ER.
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PMID:Endoplasmic reticulum is a main localization site of mTORC2. 2186 82

Target of rapamycin (TOR), an evolutionarily conserved serine/threonine protein kinase, plays pivotal roles in several important cellular processes in eukaryotes. In the fission yeast Schizosaccharomyces pombe, TOR complex 1 (TORC1), which includes Tor2 as a catalytic subunit, manages the switch between cell proliferation and differentiation by sensing nutrient availability. However, little is known about the direct target of TORC1 that plays key roles in nutrient-dependent TORC1 signaling in fission yeast. Here we report that in fission yeast, three AGC kinase family members, named Psk1, Sck1 and Sck2, which exhibit high homology with human S6K1, are phosphorylated under nutrient-rich conditions and are dephosphorylated by starvation conditions. Among these, Psk1 is necessary for phosphorylation of ribosomal protein S6. Furthermore, Psk1 phosphorylation is regulated by TORC1 in nutrient-dependent and rapamycin-sensitive manners in vivo. Three conserved regulatory motifs (the activation loop, the hydrophobic and the turn motifs) in Psk1 are phosphorylated and these modifications are required for Psk1 activity. In particular, phosphorylation of the hydrophobic motif is catalyzed by TORC1 in vivo and in vitro. Ksg1, a homolog of PDK1, is also important for Psk1 phosphorylation in the activation loop and for its activity. The TORC1 components Pop3, Toc1 and Tco89, are dispensable for Psk1 regulation, but disruption of pop3(+) causes an increase in the sensitivity of TORC1 to rapamycin. Taken together, these results provide convincing evidence that TORC1/Psk1/Rps6 constitutes a nutrient-dependent signaling pathway in fission yeast.
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PMID:Psk1, an AGC kinase family member in fission yeast, is directly phosphorylated and controlled by TORC1 and functions as S6 kinase. 2297 95


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