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James L. Maller - One of the best experts on this subject based on the ideXlab platform.
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A purified S6 Kinase Kinase from Xenopus eggs activates S6 Kinase II and autophosphorylates on serine, threonine, and tyrosine residues.
The Journal of biological chemistry, 1992Co-Authors: C B Barrett, E Erikson, James L. MallerAbstract:S6 Kinases I and II have been purified previously from Xenopus eggs and shown to be activated by phosphorylation on serine and threonine residues. An S6 Kinase clone, closely related to S6 Kinase II, was subsequently identified and the protein product was expressed in a baculovirus system. Using this protein, termed "rsk" for Ribosomal Protein S6 Kinase, as a substrate, we have purified to homogeneity from unfertilized Xenopus eggs a 41-kDa serine/threonine Kinase termed rsk Kinase. Both microtubule-associated protein-2 and myelin basic protein are good substrates for rsk Kinase, whereas alpha-casein, histone H1, protamine, and phosvitin are not. rsk Kinase is inhibited by low concentrations of heparin as well as by beta-glycerophosphate and calcium. Activation of rsk Kinase during Xenopus oocyte maturation is correlated with phosphorylation on threonine and tyrosine residues. However, in vitro, rsk Kinase undergoes autophosphorylation on serine, threonine, and tyrosine residues, identifying it as a "dual specificity" enzyme. Purified rsk Kinase can be inactivated in vitro by either a 37-kDa T-cell protein-tyrosine phosphatase or the serine/threonine protein phosphatase 2A. Phosphatase-treated S6KII can be reactivated by rsk Kinase, and S6 Kinase activity in resting oocyte extracts increases significantly when purified rsk Kinase is added. The availability of purified rsk Kinase will enhance study of the signal transduction pathway(s) regulating phosphorylation of ribosomal protein S6 in Xenopus oocytes.
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purification and characterisation of the insulin stimulated protein Kinase from rabbit skeletal muscle close similarity to S6 Kinase ii
FEBS Journal, 1991Co-Authors: Alain Lavoinne, James L. Maller, Eleanor Erikson, Joseph Avruch, Daniel J Price, Philip CohenAbstract:The insulin-stimulated protein Kinase (ISPK) was purified over 50,000-fold from extracts of rabbit skeletal muscle by a procedure involving chromatography on phosphocellulose, fractionation with ammonium sulphate, and further chromatography on DEAE-cellulose, phenyl-Superose, Mono S and Mono Q. About 10 micrograms enzyme was isolated from 800 g muscle (one rabbit) in four days with an overall recovery of 5%. The purified enzyme showed a single protein-staining band of apparent molecular mass 91 kDa when analysed by SDS/polyacrylamide gel electrophoresis. The ISPK comigrated during SDS/polyacrylamide gel electrophoresis with the enzyme S6 Kinase II from Xenopus eggs, and was recognised in immunoblotting and immunoprecipitation experiments by antibodies raised against S6 Kinase II. The substrate specificities of ISPK and S6 Kinase II were also very similar and like S6 Kinase II, ISPK that had been inactivated by protein phosphatase 2A could be reactivated by incubation with mitogen-activated protein Kinase and MgATP. ISPK was distinct from an insulin-stimulated 70-kDa S6 Kinase from rat liver in both substrate specificity and immunological cross reactivity. It is concluded that ISPK is closely related in structure to S6 Kinase II and may be a mammalian equivalent of this enzyme. The possibility that ISPK is involved in mediating a number of the actions of insulin is discussed.
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Purification and characterization of ribosomal protein S6 Kinase I from Xenopus eggs.
The Journal of biological chemistry, 1991Co-Authors: Eleanor Erikson, James L. MallerAbstract:Abstract Ribosomal protein S6 Kinase I has been purified from unfertilized Xenopus eggs to near homogeneity as a Mr = 90,000 protein. S6 Kinase I is phosphorylated when activated in vivo and can be phosphorylated by mitogen-activated protein Kinase in vitro. The purified enzyme is inactivated upon treatment with protein phosphatase 2A. Immunological data and analysis of substrate specificity demonstrate that S6 Kinase I is related to, but distinct from, the previously characterized S6 Kinase II. Both enzymes are members of the ribosomal protein S6 Kinase (rsk) gene family.
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[21] Xenopus ribosomal protein S6 Kinase II
Methods in enzymology, 1991Co-Authors: Erikson E, James L. Maller, Raymond L. EriksonAbstract:Publisher Summary Extracts of unfertilized eggs display a 10- to 20-fold increase in S6 Kinase activity that can be resolved into two peaks by DEAE-Sephacel chromatography. This chapter describes the purification of the S6 Kinase activity from the second peak, S6 Kinase II. The purified enzyme is used to generate peptide sequence information for molecular cloning. The data on the biochemical analysis and cDNA clones suggest that the gene(s) for this enzyme(s), denoted as rsk, for ribosomal S6 Kinase, may encode a family of related proteins. Xenopus S6 Kinase II catalyzes the transfer of the γ phosphate of adenosine triphosphate (ATP) to serine residues in the S6 protein of 40S ribosomal subunits. Direct analysis of the phosphorylation sites by sequencing radiolabeled S6 peptides demonstrates that this enzyme phosphorylates the serine residues in the carboxyl region of S6: 232-RRL SS LRA S TSK S -244. The possibility that serine-247 is also phosphorylated by S6KII is still under investigation. The nomenclature and identification of these residues is based on the assumption that the sequence of Xenopus S6 is identical to that of rat liver S6.
Joseph Avruch - One of the best experts on this subject based on the ideXlab platform.
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Identification of the NIMA family Kinases NEK6/7 as regulators of the p70 ribosomal S6 Kinase.
Current biology : CB, 2001Co-Authors: Christopher Belham, Michael J Comb, Joseph AvruchAbstract:Abstract Background: The p70 S6 Kinase, like several other AGC family Kinases, requires for activation the concurrent phosphorylation of a site on its activation loop and a site carboxyterminal to the catalytic domain, situated in a hydrophobic motif site FXXFS/TF/Y, e.g.,Thr412 in p70 S6 Kinase (α1). Phosphorylation of the former site is catalyzed by PDK1, whereas the Kinase responsible for the phosphorylation of the latter site is not known. Results: The major protein Kinase that is active on the p70 S6 Kinase hydrophobic regulatory site, Thr412, was purified from rat liver and identified as the NIMA-related Kinases NEK6 and NEK7. Recombinant NEK6 phosphorylates p70 S6 Kinase at Thr412 and other sites and activates the p70 S6 Kinase in vitro and in vivo, in a manner synergistic with PDK1. Kinase-inactive NEK6 interferes with insulin activation of p70 S6 Kinase. The activity of recombinant NEK6 is dependent on its phosphorylation, but NEK6 activity is not regulated by PDK1 and is only modestly responsive to insulin and PI-3 Kinase inhibitors. Conclusion: NEK6 and probably NEK7 are novel candidate physiologic regulators of the p70 S6 Kinase.
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identification of the nima family Kinases nek6 7 as regulators of the p70 ribosomal S6 Kinase
Current Biology, 2001Co-Authors: Christopher Belham, Michael J Comb, Joseph AvruchAbstract:Abstract Background: The p70 S6 Kinase, like several other AGC family Kinases, requires for activation the concurrent phosphorylation of a site on its activation loop and a site carboxyterminal to the catalytic domain, situated in a hydrophobic motif site FXXFS/TF/Y, e.g.,Thr412 in p70 S6 Kinase (α1). Phosphorylation of the former site is catalyzed by PDK1, whereas the Kinase responsible for the phosphorylation of the latter site is not known. Results: The major protein Kinase that is active on the p70 S6 Kinase hydrophobic regulatory site, Thr412, was purified from rat liver and identified as the NIMA-related Kinases NEK6 and NEK7. Recombinant NEK6 phosphorylates p70 S6 Kinase at Thr412 and other sites and activates the p70 S6 Kinase in vitro and in vivo, in a manner synergistic with PDK1. Kinase-inactive NEK6 interferes with insulin activation of p70 S6 Kinase. The activity of recombinant NEK6 is dependent on its phosphorylation, but NEK6 activity is not regulated by PDK1 and is only modestly responsive to insulin and PI-3 Kinase inhibitors. Conclusion: NEK6 and probably NEK7 are novel candidate physiologic regulators of the p70 S6 Kinase.
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An array of insulin-activated, proline-directed serine/threonine protein Kinases phosphorylate the p70 S6 Kinase.
The Journal of biological chemistry, 1992Co-Authors: N K Mukhopadhyay, D J Price, J M Kyriakis, J Sanghera, Steven L. Pelech, Joseph AvruchAbstract:This study characterizes the insulin-activated serine/threonine protein Kinases in H4 hepatoma cells active on a 37-residue synthetic peptide (called the SKAIPS peptide) corresponding to a putative autoinhibitory domain in the carboxyl-terminal tail of the p70 S6 Kinase as well as on recombinant p70 S6 Kinase. Three peaks of insulin-stimulated protein Kinase active on both these substrates are identified as two (possibly three) isoforms of the 40-45-kDa erk/microtubule-associated protein (MAP)-2 Kinase family and a 150-kDa form of cdc2. Although distinguishable in their substrate specificity, these protein Kinases together with the p54 MAP-2 Kinase share a major common specificity determinant reflected in the SKAIPS peptide: the requirement for a proline residue immediately carboxyl-terminal to the site of Ser/Thr phosphorylation. In addition, however, at least one peak of insulin-stimulated protein Kinase active on recombinant p70, but not on the SKAIPS peptide, is present although not yet identified. MFP/cdc2 phosphorylates both rat liver p70 S6 Kinase and recombinant p70 S6 Kinase exclusively at a set of Ser/Thr residues within the putative autoinhibitory (SKAIPS peptide) domain. erk/MAP Kinase does not phosphorylate rat liver p70 S6 Kinase, but readily phosphorylates recombinant p70 S6 Kinase at sites both within and in addition to those encompassed by the SKAIPS peptide sequences. Although the tryptic 32P-peptides bearing the cdc2 and erk/MAP Kinase phosphorylation sites co-migrate with a subset of the sites phosphorylated in situ in insulin-stimulated cells, phosphorylation of the p70 S6 Kinase by these proline-directed protein Kinases in vitro does not reproducibly activate p70 S6 Kinase activity. Thus, one or more erk/MAP Kinases and cdc2 are likely to participate in the insulin-induced phosphorylation of the p70 S6 Kinase. In addition to these Kinases, however, phosphorylation of the p70 S6 Kinase by other as yet unidentified protein Kinases is necessary to recapitulate the multisite phosphorylation required for activation of the p70 S6 Kinase.
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Cloning and expression of two human p70 S6 Kinase polypeptides differing only at their amino termini.
Molecular and cellular biology, 1991Co-Authors: J. R. Grove, D J Price, Joseph Avruch, Papia Banerjee, A. Balasubramanyam, P. J. Coffer, James R. WoodgettAbstract:Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 Kinase have been isolated; the two classes differ only in the 5' region, such that the longer polypeptide (p70 S6 Kinase alpha I; calculated Mr 58,946) consists of 525 amino acids, of which the last 502 residues are identical in sequence to the entire polypeptides encoded by the second cDNA (p70 S6 Kinase alpha II; calculated Mr 56,153). Both p70 S6 Kinase polypeptides predicted by these cDNAs are present in p70 S6 Kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both polypeptides are expressed from a single mRNA transcribed from the (longer) p70 S6 Kinase alpha I cDNA through the utilization of different translational start sites. Although the two p70 S6 Kinase polypeptides differ by only 23 amino acid residues, the slightly longer alpha I polypeptide exhibits anomalously slow mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), migrating at an apparent Mr of 90,000 probably because of the presence of six consecutive Arg residues immediately following the initiator methionine. Transient expression of p70 alpha I and alpha II S6 Kinase cDNA in COS cells results in a 2.5- to 4-fold increase in overall S6 Kinase activity. Upon immunoblotting, the recombinant p70 polypeptides appear as a closely spaced ladder of four to five bands between 65 and 70 kDa (alpha II) and 85 and 90 kDa (alpha I). Transfection with the alpha II cDNA yields only the smaller set of bands, while transfection with the alpha I cDNA generates both sets of bands. Mutation of Met-24 in the alpha I cDNA to Leu or Thr suppresses synthesis of the alpha II polypeptides. Only the p70 alpha I and alpha II polypeptides of slowest mobility on SDS-PAGE comigrate with the 70- and 90-kDa proteins observed in purified rat liver S6 Kinase. Moreover, it is the recombinant p70 polypeptides of slowest mobility that coelute with S6 Kinase activity on anion-exchange chromatography. The slower mobility and higher enzymatic activity of these p70 proteins is due to Ser/Thr phosphorylation, inasmuch as treatment with phosphatase 2A inactivates Kinase activity and increases the mobility of the bands on SDS-PAGE in an okadaic acid-sensitive manner. Thus, the recombinant p70 S6 Kinase undergoes multiple phosphorylation and partial activation in COS cells. Acquisition of S6 protein Kinase catalytic function, however, is apparently restricted to the most extensively phosphorylated recombinant polypeptides.
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purification and characterisation of the insulin stimulated protein Kinase from rabbit skeletal muscle close similarity to S6 Kinase ii
FEBS Journal, 1991Co-Authors: Alain Lavoinne, James L. Maller, Eleanor Erikson, Joseph Avruch, Daniel J Price, Philip CohenAbstract:The insulin-stimulated protein Kinase (ISPK) was purified over 50,000-fold from extracts of rabbit skeletal muscle by a procedure involving chromatography on phosphocellulose, fractionation with ammonium sulphate, and further chromatography on DEAE-cellulose, phenyl-Superose, Mono S and Mono Q. About 10 micrograms enzyme was isolated from 800 g muscle (one rabbit) in four days with an overall recovery of 5%. The purified enzyme showed a single protein-staining band of apparent molecular mass 91 kDa when analysed by SDS/polyacrylamide gel electrophoresis. The ISPK comigrated during SDS/polyacrylamide gel electrophoresis with the enzyme S6 Kinase II from Xenopus eggs, and was recognised in immunoblotting and immunoprecipitation experiments by antibodies raised against S6 Kinase II. The substrate specificities of ISPK and S6 Kinase II were also very similar and like S6 Kinase II, ISPK that had been inactivated by protein phosphatase 2A could be reactivated by incubation with mitogen-activated protein Kinase and MgATP. ISPK was distinct from an insulin-stimulated 70-kDa S6 Kinase from rat liver in both substrate specificity and immunological cross reactivity. It is concluded that ISPK is closely related in structure to S6 Kinase II and may be a mammalian equivalent of this enzyme. The possibility that ISPK is involved in mediating a number of the actions of insulin is discussed.
Steven L. Pelech - One of the best experts on this subject based on the ideXlab platform.
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adenosine triphosphate induces proliferation of human neural stem cells role of calcium and p70 ribosomal protein S6 Kinase
Journal of Neuroscience Research, 2003Co-Authors: Steven L. Pelech, Jae K Ryu, Hyun B Choi, Kozo Hatori, Rochelle L Heisel, James G Mclarnon, Seung U KimAbstract:Human neural stem cells (NSCs) grown in culture responded to extracellularly applied adenosine triphosphate (ATP), and the rate of proliferation increased as shown by immunocytochemical and RT-PCR analysis. Activation of P2 purinoceptors by ATP is coupled to the release of intracellular calcium ([Ca(2+)](i)) from thapsigargin-sensitive intracellular stores. ATP-induced proliferation was blocked by thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase. Neither EGTA, a calcium chelator, nor caffeine had any effect on ATP-induced [Ca(2+)](i) increases. Multiblot Kinase analysis, by which activation of 24 different Kinases could be determined, showed that application of ATP to NSCs predominantly activated p70 ribosomal protein S6 Kinase (p70 S6 Kinase). As well, rapamycin, a p70 S6 Kinase inhibitor, blocked the ATP-mediated proliferative response in NSCs. After outlining a role for p70 S6 Kinase in ATP-mediated NSC proliferation, we examined the possibility that phosphatidylinositol 3-Kinase (PI3-Kinase) acts upstream of p70 S6 Kinase. The application of wortmannin, a PI3-Kinase inhibitor, decreased both ATP-mediated p70 S6 Kinase activation and NSC proliferation. From these results, we conclude that ATP application to NSCs induces release of Ca(2+) from intracellular Ca(2+) stores and that this increase in intracellular Ca(2+) in turn promotes NSC proliferation. The increase in NSC proliferation observed following ATP application can also be mediated by PI3-Kinase-dependent p70 S6 Kinase activation.
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Identification of Kinase-Phosphatase Signaling Modules Composed of p70 S6 Kinase-Protein Phosphatase 2A (PP2A) and p21-activated Kinase-PP2A
The Journal of biological chemistry, 1999Co-Authors: Ryan S. Westphal, R. Lane Coffee, Anthony Marotta, Steven L. Pelech, Brian E. WadzinskiAbstract:Abstract A growing body of evidence indicates that regulation of protein-serine/threonine phosphatase 2A (PP2A) involves its association with other cellular and viral proteins in multiprotein complexes. PP2A-containing protein complexes may exist that contribute to PP2A’s important regulatory role in many cellular processes. To identify such protein complexes, PP2A was partially purified from rat brain soluble extracts following treatment with a reversible cross-linker to stabilize large molecular size forms of PP2A. Compared with native (uncross-linked) PP2A, cross-linked PP2A revealed an enrichment of p70 S6 Kinase and two p21-activated Kinases (PAK1 and PAK3) in the PP2A complex, indicating these Kinases may associate with PP2A. The existence of protein Kinase-PP2A complexes in rat brain soluble extracts was further substantiated by the following results: 1) independent immunoprecipitation of the Kinases revealed that PP2A co-precipitated with p70 S6 Kinase and the two PAK isoforms; 2) glutathione S-transferase fusion proteins of p70 S6 Kinase and PAK3 each isolated PP2A; and 3) PAK3 and p70 S6 Kinase bound to microcystin-Sepharose (an affinity resin for PP2A-PP1). Cumulatively, these findings provide evidence for association of PP2A with p70 S6 Kinase, PAK1, and PAK3 in the context of the cellular environment. Moreover, together with the recent reports describing associations of PP2A with Ca2+/calmodulin-dependent protein Kinase IV (Westphal, R. S., Anderson, K. A., Means, A. R., and Wadzinski, B. E. (1998) Science 280, 1258–1261) and casein Kinase IIα (Heriche, J. K., Lebrin, F., Rabilloud, T., Leroy, D., Chambaz, E. M., and Goldberg, Y. (1997)Science 276, 952–955), the present data provide compelling evidence for the existence of protein Kinase-PP2A signaling modules as a new paradigm for the control of various intracellular signaling cascades.
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Role of p70 S6 Kinase in cytokine-regulated hemopoietic cell survival
Biochemistry and cell biology = Biochimie et biologie cellulaire, 1996Co-Authors: Michael P. Scheid, Steven L. Pelech, Lorin Charlton, Vincent DuronioAbstract:The signalling mechanisms required for cell survival remain relatively undefined. We and others have shown that phosphatidylinositol 3-Kinase (PI 3-Kinase) is an important enzyme in the prevention of apoptosis, and this property is independent of p21ras-MAP Kinase activation. It is therefore important to define the downstream targets of this enzyme mediating the inhibition of apoptosis. We report here than p70 S6 Kinase, a protein critical for progression through the cell cycle and a downstream effector of PI 3-Kinase, is not required for the survival of cytokine-stimulated human T-cells or murine mast cells. The potent inhibitor of p70 S6 Kinase activation, rapamycin, was unable to induce apoptosis in cells stimulated with cytokines. As well, PI 3-Kinase inhibitors that also blocked the activation of p70 S6 Kinase were able to induce apoptosis. These studies, therefore, describe a bifurcation of signalling pathways from PI 3-Kinase leading to different physiological outcomes.
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Purification and characterization of a novel ribosomal S6 Kinase from skeletal muscle of insulin-treated rats.
The Journal of biological chemistry, 1994Co-Authors: Yong-jiang Hei, Steven L. Pelech, Xunsheng Chen, Jack Diamond, John H. McneillAbstract:The predominant 40 S ribosomal protein S6 Kinase in skeletal muscle extracts from insulin-treated rats was purified over 10,000-fold to near homogeneity with approximately 4.5% recovery of starting activity. This S6 Kinase was resolved from the catalytic subunit of cAMP-dependent protein Kinase only by the seventh and final column chromatography step. The purified S6 Kinase migrated as a tight doublet of approximately 31 kDa on an SDS-polyacrylamide gel, and it was eluted from gel filtration columns with a similar apparent M(r), which indicated that the enzyme exists as a monomer. This S6 Kinase was immunologically distinct from the other known insulin-activated S6 Kinases, i.e. p70S6K and p90rsk. It was inhibited by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and beta-glycerophosphate at concentrations routinely used to stabilize p70S6K and p90rsk. In addition to S6, phosvitin was also a substrate, whereas myelin basic protein, casein, protamine, and histones were poorly phosphorylated if at all by the purified S6 Kinase. The purified enzyme was inactivated upon incubation with serine/threonine-specific protein phosphatase 2A, which indicated that it may be an intermediary component in a cascade of insulin-activated protein Kinases.
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An array of insulin-activated, proline-directed serine/threonine protein Kinases phosphorylate the p70 S6 Kinase.
The Journal of biological chemistry, 1992Co-Authors: N K Mukhopadhyay, D J Price, J M Kyriakis, J Sanghera, Steven L. Pelech, Joseph AvruchAbstract:This study characterizes the insulin-activated serine/threonine protein Kinases in H4 hepatoma cells active on a 37-residue synthetic peptide (called the SKAIPS peptide) corresponding to a putative autoinhibitory domain in the carboxyl-terminal tail of the p70 S6 Kinase as well as on recombinant p70 S6 Kinase. Three peaks of insulin-stimulated protein Kinase active on both these substrates are identified as two (possibly three) isoforms of the 40-45-kDa erk/microtubule-associated protein (MAP)-2 Kinase family and a 150-kDa form of cdc2. Although distinguishable in their substrate specificity, these protein Kinases together with the p54 MAP-2 Kinase share a major common specificity determinant reflected in the SKAIPS peptide: the requirement for a proline residue immediately carboxyl-terminal to the site of Ser/Thr phosphorylation. In addition, however, at least one peak of insulin-stimulated protein Kinase active on recombinant p70, but not on the SKAIPS peptide, is present although not yet identified. MFP/cdc2 phosphorylates both rat liver p70 S6 Kinase and recombinant p70 S6 Kinase exclusively at a set of Ser/Thr residues within the putative autoinhibitory (SKAIPS peptide) domain. erk/MAP Kinase does not phosphorylate rat liver p70 S6 Kinase, but readily phosphorylates recombinant p70 S6 Kinase at sites both within and in addition to those encompassed by the SKAIPS peptide sequences. Although the tryptic 32P-peptides bearing the cdc2 and erk/MAP Kinase phosphorylation sites co-migrate with a subset of the sites phosphorylated in situ in insulin-stimulated cells, phosphorylation of the p70 S6 Kinase by these proline-directed protein Kinases in vitro does not reproducibly activate p70 S6 Kinase activity. Thus, one or more erk/MAP Kinases and cdc2 are likely to participate in the insulin-induced phosphorylation of the p70 S6 Kinase. In addition to these Kinases, however, phosphorylation of the p70 S6 Kinase by other as yet unidentified protein Kinases is necessary to recapitulate the multisite phosphorylation required for activation of the p70 S6 Kinase.
Alan J Whitmarsh - One of the best experts on this subject based on the ideXlab platform.
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The mTOR-S6 Kinase pathway promotes stress granule assembly
Cell Death and Differentiation, 2018Co-Authors: Aristeidis P. Sfakianos, Laura E. Mellor, Yoke Fei Pang, Paraskevi Kritsiligkou, Hope Needs, Hussein Abou-hamdan, Gino B. Poulin, Mark P. Ashe, Laurent Désaubry, Alan J WhitmarshAbstract:Stress granules are cytoplasmic mRNA-protein complexes that form upon the inhibition of translation initiation and promote cell survival in response to environmental insults. However, they are often associated with pathologies, including neurodegeneration and cancer, and changes in their dynamics are implicated in ageing. Here we show that the mTOR effector Kinases S6 Kinase 1 (S6K1) and S6 Kinase 2 (S6K2) localise to stress granules in human cells and are required for their assembly and maintenance after mild oxidative stress. The roles of S6K1 and S6K2 are distinct, with S6K1 having a more significant role in the formation of stress granules via the regulation of eIF2α phosphorylation, while S6K2 is important for their persistence. In C. elegans, the S6 Kinase orthologue RSKS-1 promotes the assembly of stress granules and its loss of function sensitises the nematodes to stress-induced death. This study identifies S6 Kinases as regulators of stress granule dynamics and provides a novel link between mTOR signalling, translation inhibition and survival.
Alain Lavoinne - One of the best experts on this subject based on the ideXlab platform.
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IDENTIFICATION OF A NOVEL CA2+-STIMULATED S6-Kinase IN RAT LIVER
Biochemical and biophysical research communications, 1997Co-Authors: Jean-christophe Harlin, Mohamed Tredano, Mark H. Rider, Mathieu Bollen, Arlette Chedeville, Alain LavoinneAbstract:Abstract Extracellular calcium addition transiently stimulated two S6 peptide Kinase activities in isolated rat hepatocytes. Mono Q chromatography revealed that the activities eluting at 0.15 M NaCl and 0.18 M NaCl were stimulated 4-fold and 2-fold, respectively. The Kinase stimulated by calcium was a 40000-Mr S6 peptide Kinase, as demonstrated by partial purification from whole liver. The protein Kinase did not crossreact with antibodies directed against the N- or C-terminal part of p70 ribosomal S6 Kinase (p70S6K) and the C-terminal part of p90 ribosomal S6 Kinase (p90rsk). Following digestion of 40000-Mr S6 peptide Kinase with trypsin, six peptides were sequenced. There was no similarity with the sequences of p70S6Kand p90rsk. Moreover, the obtained sequences could not be identified in the SwissProt or EMBL-genebank databases, suggesting that 40000-Mr S6 peptide Kinase probably represents a novel protein Kinase.
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purification and characterisation of the insulin stimulated protein Kinase from rabbit skeletal muscle close similarity to S6 Kinase ii
FEBS Journal, 1991Co-Authors: Alain Lavoinne, James L. Maller, Eleanor Erikson, Joseph Avruch, Daniel J Price, Philip CohenAbstract:The insulin-stimulated protein Kinase (ISPK) was purified over 50,000-fold from extracts of rabbit skeletal muscle by a procedure involving chromatography on phosphocellulose, fractionation with ammonium sulphate, and further chromatography on DEAE-cellulose, phenyl-Superose, Mono S and Mono Q. About 10 micrograms enzyme was isolated from 800 g muscle (one rabbit) in four days with an overall recovery of 5%. The purified enzyme showed a single protein-staining band of apparent molecular mass 91 kDa when analysed by SDS/polyacrylamide gel electrophoresis. The ISPK comigrated during SDS/polyacrylamide gel electrophoresis with the enzyme S6 Kinase II from Xenopus eggs, and was recognised in immunoblotting and immunoprecipitation experiments by antibodies raised against S6 Kinase II. The substrate specificities of ISPK and S6 Kinase II were also very similar and like S6 Kinase II, ISPK that had been inactivated by protein phosphatase 2A could be reactivated by incubation with mitogen-activated protein Kinase and MgATP. ISPK was distinct from an insulin-stimulated 70-kDa S6 Kinase from rat liver in both substrate specificity and immunological cross reactivity. It is concluded that ISPK is closely related in structure to S6 Kinase II and may be a mammalian equivalent of this enzyme. The possibility that ISPK is involved in mediating a number of the actions of insulin is discussed.
