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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This study examines whether the serine/threonine protein kinase, Akt, is involved in the cross-talk between epidermal growth factor (EGF) and insulin-related growth factor I (IGF-I) receptors and ER-alpha. Treatment of MCF-7 cells with either EGF or IGF-I resulted in a rapid phosphorylation of Akt and a 14- to 16-fold increase in Akt activity, respectively. Akt activation was blocked by inhibitors of phosphatidylinositol 3-kinase, but not by an inhibitor of the ribosomal protein kinase p70S6K. Stable transfection of cells with a dominant negative Akt mutant blocked the effects of EGF and IGF-I on ER-alpha expression and activity, whereas stable transfection of cells with a constitutively active Akt mutant mimicked the effects of EGF and IGF-I. In the latter cells, there was a decrease in the amount of ER-alpha protein and messenger RNA (70-80%) and an increase in the amount of progesterone receptor protein, messenger RNA (4- to 9- and by 3.5- to 7-fold, respectively) and pS2 (3- to 5-fold). Coexpression of wild-type ER-alpha and the dominant negative Akt mutant in COS-1 cells also blocked the growth factor-stimulated activation of ER-alpha, but coexpression of the wild-type receptor with the constitutively active Akt mutant increased ER-alpha activity. Receptor activation was blocked by an antiestrogen. Studies using mutants of ER-alpha demonstrated that Akt increased estrogen receptor activity through the amino-terminal activation function-1 (AF-1). Serines S104 S106, S118, and S167 appear to play a role in the activation of ER-alpha by Akt.
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PMID:A role for Akt in mediating the estrogenic functions of epidermal growth factor and insulin-like growth factor I. 1110 61

Protein metabolism in eukaryotic organisms is defined by a synthesis-degradation equilibrium that is subject to regulation by hormonal and nutritional signals. In mammalian tissues such as skeletal muscle, glucocorticoid hormones specify a catabolic response that influences both protein synthetic and protein degradative pathways. With regard to the former, glucocorticoids attenuate mRNA translation at two levels: translational efficiency, i.e. translation initiation, and translational capacity, i.e. ribosome biogenesis. Glucocorticoids may impair translational capacity through the ribosomal S6 protein kinase (p70 S6K), a recognized glucocorticoid target and an effector of ribosomal protein synthesis. We demonstrate here that the reduction in growth factor-activated p70 S6K activity by glucocorticoids depends upon a functional glucocorticoid receptor (GR) and that the GR is both necessary and sufficient to render p70 S6K subject to glucocorticoid regulation. Furthermore, the DNA binding and transcriptional activation but not repression properties of the GR are indispensable for p70 S6K regulation. Finally, a mutational analysis of the p70 S6K carboxyl terminus indicates that this region confers glucocorticoid sensitivity, and thus glucocorticoids may facilitate autoinhibition of the enzyme ultimately reducing the efficiency with which T389 is phosphorylated.
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PMID:The activated glucocorticoid receptor modulates presumptive autoregulation of ribosomal protein S6 protein kinase, p70 S6K. 1170 93

The substrates of most protein kinases remain unknown because of the difficulty tracing signaling pathways and identifying sites of protein phosphorylation. Here we describe a method useful in detecting subclasses of protein kinase substrates. Although the method is broadly applicable to any protein kinase for which a substrate consensus motif has been identified, we illustrate here the use of antibodies broadly reactive against phosphorylated Ser/Thr-motifs typical of AGC kinase substrates. Phosphopeptide libraries with fixed residues corresponding to consensus motifs RXRXXT*/S* (Akt motif) and S*XR (protein kinase C motif) were used as antigens to generate antibodies that recognize many different phosphoproteins containing the fixed motif. Because most AGC kinase members are phosphorylated and activated by phosphoinositide-dependent protein kinase-1 (PDK1), we used PDK1-/- ES cells to profile potential AGC kinase substrates downstream of PDK1. To identify phosphoproteins detected using the Akt substrate antibody, we characterized the antibody binding specificity to generate a specificity matrix useful in predicting antibody reactivity. Using this approach we predicted and then identified a 30-kDa phosphoprotein detected by both Akt and protein kinase C substrate antibodies as S6 ribosomal protein. Phosphospecific motif antibodies offer a new approach to protein kinase substrate identification that combines immunoreactivity data with protein data base searches based upon antibody specificity.
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PMID:Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs. 1215 8

Yeast cells starved for inorganic phosphate on a glucose-containing medium arrest growth and enter the resting phase G0. We show that re-addition of phosphate rapidly affects well known protein kinase A targets: trehalase activation, trehalose mobilization, loss of heat resistance, repression of STRE-controlled genes and induction of ribosomal protein genes. Phosphate-induced activation of trehalase is independent of protein synthesis and of an increase in ATP. It is dependent on the presence of glucose, which can be detected independently by the G-protein coupled receptor Gpr1 and by the glucose-phosphorylation dependent system. Addition of phosphate does not trigger a cAMP signal. Despite this, lowering of protein kinase A activity by mutations in the TPK genes strongly reduces trehalase activation. Inactivation of phosphate transport by deletion of PHO84 abolishes phosphate signalling at standard concentrations, arguing against the existence of a transport-independent receptor. The non-metabolizable phosphate analogue arsenate also triggered signalling. Constitutive expression of the Pho84, Pho87, Pho89, Pho90 and Pho91 phosphate carriers indicated pronounced differences in their transport and signalling capacities in phosphate-starved cells. Pho90 and Pho91 sustained highest phosphate transport but did not sustain trehalase activation. Pho84 sustained both transport and rapid signalling, whereas Pho87 was poor in transport but positive for signalling. Pho89 displayed very low phosphate transport and was negative for signalling. Although the results confirmed that rapid signalling is independent of growth recovery, long-term mobilization of trehalose was much better correlated with growth recovery than with trehalase activation. These results demonstrate that phosphate acts as a nutrient signal for activation of the protein kinase A pathway in yeast in a glucose-dependent way and they indicate that the Pho84 and Pho87 carriers act as specific phosphate sensors for rapid phosphate signalling.
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PMID:Inorganic phosphate is sensed by specific phosphate carriers and acts in concert with glucose as a nutrient signal for activation of the protein kinase A pathway in the yeast Saccharomyces cerevisiae. 1258 67

During mitosis, the cyclin-dependent kinase, Cdc2, signals the inactivation of major anabolic processes such as transcription, mRNA processing, translation, and ribosome biogenesis, thereby providing energy needed for the radical and energetically costly structural reorganization of the cell. This is accomplished by phosphorylation and inactivation of several key anabolic elements, including TFIIIB, TFIID, RNA polymerase II, poly(A) polymerase, and translation elongation factor 1gamma. We report here that ribosomal S6 kinase 1 (S6K1), a protein kinase linked to the translation of ribosomal protein mRNAs, is also subject to regulation by Cdc2 in mitosis. In mitotic HeLa cells, when the activity of Cdc2 is high, S6K1 is phosphorylated at multiple Ser/Thr, Pro (S/TP) sites, including Ser(371), Ser(411), Thr(421), and Ser(424). Concomitant with this, the phosphorylation of the hydrophobic motif site, Thr(389), is reduced resulting in a decrease in the specific activity of S6K1. The mitotic S/TP phosphorylation sites are readily phosphorylated by Cdc2.cyclin B in vitro. These proline-directed phosphorylations are sensitive to chemical inhibitors of Cdc2 but not to inhibitors of mammalian target of rapamycin, phosphatidylinositol 3-kinase, MEK1/2, or p38. In murine FT210 cells arrested in mitosis, conditional inactivation of Cdc2 reduces phosphorylation of S6K1 at S/TP sites while simultaneously increasing phosphorylation of Thr(389) and of the S6K1 substrate, RPS6. A physical interaction exists between Cdc2 and S6K1, and this interaction is enhanced in mitotic cells. These results suggest that Cdc2 provides a signal that triggers inactivation of S6K1 in mitosis, presumably serving to spare energy for costly mitotic processes at the expense of ribosomal protein synthesis.
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PMID:Mitotic regulation of ribosomal S6 kinase 1 involves Ser/Thr, Pro phosphorylation of consensus and non-consensus sites by Cdc2. 1258 35

Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) is a powerful method for measurement of gene expression for diagnostic and prognostic studies of non-Hodgkin's lymphomas (NHL). In order for this technique to gain wide applicability, it is critically important to establish a uniform method for normalization of RNA input. In this study, we have determined the best method to quantify the RNA/cDNA input per reaction and searched for the most useful endogenous control genes for normalization of the measurements, based on their abundance and lowest variability between different types of lymphoid cells. To accomplish these aims, we have analyzed the RNA expression of 11 potential endogenous control genes (glyceraldehyde-3-phosphate dehydrogenase, beta-actin, peptidylprolyl isomerase A, beta 2 microglobulin, protein kinase cGMP-dependent, type I, hypoxanthine phosphoribosyltransferase 1, TATA box binding protein, transferrin receptor, large ribosomal protein, beta-glucoronidase and 18S ribosomal RNA). In all, 12 different B- and T-cell lymphoma/leukemia cell lines, 80 B- and T-cell NHL specimens, and resting and activated normal B and T lymphocytes were screened. Normalization of the nucleic acid input by spectrophotometric OD(260) measurement of RNA proved more reliable than spectrophotometric or fluorometric measurements of cDNA or than electrophoretic estimation of the ribosomal and mRNA fractions. The protein kinase cGMP-dependent, type I (PRKG1) and the TBP genes were expressed at common abundance and exhibited the lowest variability among the cell specimens. We suggest that for further lymphoma studies based on the real-time RT-PCR quantification of gene expression, that RNA input in each reaction be equalized between the specimens by spectrophotometric OD(260) measurements. The expression of the gene of interest in different samples should be normalized by concomitant measurement of the PRKG1 and/or the TBP gene products.
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PMID:Optimization of quantitative real-time RT-PCR parameters for the study of lymphoid malignancies. 1268 39

Addition of a nitrogen source to yeast (Saccharomyces cerevisiae) cells starved for nitrogen on a glucose-containing medium triggers activation of protein kinase A (PKA) targets through a pathway that requires for sustained activation both a fermentable carbon source and a complete growth medium (fermentable growth medium induced or FGM pathway). Trehalase is activated, trehalose and glycogen content as well as heat resistance drop rapidly, STRE-controlled genes are repressed, and ribosomal protein genes are induced. We show that the rapid effect of amino acids on these targets specifically requires the general amino acid permease Gap1. In the gap1Delta strain, transport of high concentrations of l-citrulline occurs at a high rate but without activation of trehalase. Metabolism of the amino acids is not required. Point mutants in Gap1 with reduced or deficient transport also showed reduced or deficient signalling. However, two mutations, S391A and S397A, were identified with a differential effect on transport and signalling for l-glutamate and l-citrulline. Specific truncations of the C-terminus of Gap1 (e.g. last 14 or 26 amino acids) did not reduce transport activity but caused the same phenotype as in strains with constitutively high PKA activity also during growth with ammonium as sole nitrogen source. The overactive PKA phenotype was abolished by mutations in the Tpk1 or Tpk2 catalytic subunits. We conclude that Gap1 acts as an amino acid sensor for rapid activation of the FGM signalling pathway which controls the PKA targets, that transport through Gap1 is connected to signalling and that specific truncations of the C-terminus result in permanently activating Gap1 alleles.
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PMID:The Gap1 general amino acid permease acts as an amino acid sensor for activation of protein kinase A targets in the yeast Saccharomyces cerevisiae. 1461 51

In Kluyveromyces lactis, the casein kinase I (Rag8p) regulates the transcription of glycolytic genes and the expression of the low-affinity glucose transporter gene RAG1. This control involves the transcription factor Sck1p, a homologue of Sgc1p of Saccharomyces cerevisiae. SGC1 is known to interact genetically with ScGCR1 and ScGCR2, which code for regulators of glycolytic gene expression. Therefore, we studied the role of KlGCR1 and KlGCR2 genes in K. lactis. The Klgcr1 null mutant could not grow on glucose when respiration was blocked by antimycin A (Rag(- )phenotype). In contrast, the Klgcr2 null mutant could grow under the same conditions, although at a reduced rate. In both mutants, the transcription of glycolytic genes was affected, while that of ribosomal protein genes was not modified. Furthermore, the transcription of the glucose permease genes was also found to be affected in the two mutants, although dissimilarly. While RAG1 transcription decreased at high glucose concentrations, the expression of the high-affinity glucose permease gene HGT1 was unexpectedly impaired under gluconeogenic conditions, in the absence of glucose. Gel mobility shift assays performed with purified maltose-binding protein-KlGcr1p showed that KlGcr1p could interact directly with the promoters of the glycolytic genes, but not with the promoters of the glucose permease genes. Thus, the control exerted by KlGcr1p and KlGcr2p upon glucose transporter genes is probably indirect.
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PMID:Regulation of glycolysis in Kluyveromyces lactis: role of KlGCR1 and KlGCR2 in glucose uptake and catabolism. 1468 65

Transport of lipids and proteins is a highly regulated process, which is required to maintain the integrity of various intracellular organelles in eukaryotic cells. Mutations along the yeast secretory pathway repress transcription of rRNA, tRNA, and ribosomal protein genes. Here, we show that these mutations also lead to a rapid and specific attenuation of translation initiation that occurs prior to the transcriptional inhibition of ribosomal components. Using distinct vesicular transport mutants and chlorpromazine, we have identified the eIF2alpha kinase Gcn2p and the eIF4E binding protein Eap1p as major mediators of the translation attenuation response. Finally, in chlorpromazine-treated cells, this response does not require Wsc1p or the protein kinase Pkc1p, both of which are upstream of the transcriptional repression of ribosomal components. Altogether, our results suggest that yeast cells not only evolved a transcriptional but also a translational control to assure efficient attenuation of protein synthesis when membranes are stressed.
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PMID:A membrane transport defect leads to a rapid attenuation of translation initiation in Saccharomyces cerevisiae. 1496 43

Previous studies have shown that oral administration of leucine to fasted rats results in a preferential increase in liver in the translation of mRNAs containing an oligopyrimidine sequence at the 5'-end of the message (i.e. a TOP sequence). TOP mRNAs include those encoding the ribosomal proteins (rp) and translation elongation factors. In cells in culture, the preponderance of evidence suggests that translation of TOP mRNAs is regulated by the mammalian target of rapamycin (mTOR), a protein kinase that signals through ribosomal protein S6 kinase (S6K1) to rpS6. However, the results of previous studies were recently challenged by several reports suggesting that translation of TOP mRNAs is independent of mTOR, S6K1, and S6 phosphorylation. The purpose of the present study was to evaluate the role of mTOR in the stimulation of TOP mRNA translation by leucine in vivo. Fasted rats were treated with the mTOR inhibitor, rapamycin, prior to oral administration of leucine. It was found that rapamycin severely attenuated leucine-induced signaling through mTOR in liver. In addition, rapamycin prevented the enhanced translation of TOP mRNAs in rats administered leucine, as assessed by a decrease in the proportion of TOP mRNAs associated with polysomes (i.e. those mRNAs being actively translated). Instead, in rapamycin-treated rats, ribosomal protein mRNAs accumulated in the fraction containing monosomes (mRNA bound to one ribosome). The results suggest that in liver in vivo, mTOR-dependent signaling is critical for maximal stimulation of TOP mRNA translation.
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PMID:The mTOR signaling pathway mediates control of ribosomal protein mRNA translation in rat liver. 1531 64


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