インスリンによる3-Phosphoinositide-dependent protein kinase 1(PDK1)のリン酸化機構  [in Japanese] Insulin-induced phosphorylation of 3-Phosphoinositide-dependent protein kinase 1(PDK1) in CHO cells  [in Japanese]

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Author(s)

    • 日野 泰久 Hino Yasuhisa
    • 神戸大学医学部内科学第二講座 Second Department of Internal Medicine, Kobe University School of Medicine

Abstract

3-phosphoinositede-dependent protein kinase 1 (PDK1) は phosphatidyl-inositol (PI) 3-キナーゼの下流で機能する多くの蛋白キナーゼをリン酸化し, 活性化する蛋白キナーゼである。インスリン受容体とPDK1をともに安定的に発現したCHO細胞をインスリンで処理すると, SDS-PAGE上のPDK1の移動度の低下が生じた。また, 熱刺激や過酸化水素刺激でも同様の移動度の低下が生じた。放射性リン酸標識実験により, インスリンによるPDK1の移動度の低下はPDK1のリン酸化の増強を反映することが明らかとなった。PI3-キナーゼの薬理学的阻害剤である wortmannin は, インスリンによるPDK1の移動度の低下及びPDK1のリン酸化を抑制し, 恒常的活性型PI3-キナーゼは刺激非存在下にもPDK1の移動度の低下を促進した。キナーゼ欠損型のPDK1も野性型PDK1と同様にインスリン刺激により移動度が低下した。優位抑制型Akt, 優位抑制型 protein kinase C (PKC) λ, p70S6キナーゼの活性化を阻害する薬理学的阻害剤である rapamycin はインスリン依存性のPDK1の移動度の低下を阻害しなかった。以上の結果により, インスリン刺激によりPDK1はPI3-キナーゼ依存性にリン酸化され, このPDK1のリン酸化にはAkt, p70S6キナーゼ及びPKCλは関与しないことが示唆された。 / PDK1 is a serine-threonine kinase that contributes to the phosphorylation and activation of various other protein kinases, including several that act downstream of phosphoinositide (PI) 3-kinase. In CHO cells expressing both recombinant PDK1 and insulin receptors, as well as heat shock and hydrogen peroxide each induced a shift in the electrophoretic mobility of PDK1. Labeling of cells with ^<32>P revealed that this mobility shift reflected an increase in PDK1 phosphorylation. The PI 3-kinase inhibitor wortmannin abolished the insulin-induced mobility shift of and incorporation of ^<32>P into PDK1, and a constitutively active mutant of PI 3-kinase induced the mobility shift in the absence of insulin. Insulin also induced similar shifts in the mobilities of two kinase-deficient mutants of PDK1, and dominant negative or constitutively active mutants of Akt or of the λ isoform of protein kinase C did not affect PDK1 mobility. Rapamycin, which inhibited p70 S6 kinase activation, did not affect the insulin-induced shift in PDK1 mobility. These results suggest that insulin induces phosphorylation of PDK1 by a PI 3-kinase-dependent mechanism, and that the kinase activity of PDK1 or signals mediated through Akt, p70 S6 kinase, or protein kinase Cλ do not contribute to this effect.

PDK1 is a serine-threonine kinase that contributes to the phosphorylation and activation of various other protein kinases, including several that act downstream of phosphoinositide (PI) 3-kinase. In CHO cells expressing both recombinant PDK1 and insulin receptors, as well as heat shock and hydrogen peroxide each induced a shift in the electrophoretic mobility of PDK1. Labeling of cells with ^<32>P revealed that this mobility shift reflected an increase in PDK1 phosphorylation. The PI 3-kinase inhibitor wortmannin abolished the insulin-induced mobility shift of and incorporation of ^<32>P into PDK1, and a constitutively active mutant of PI 3-kinase induced the mobility shift in the absence of insulin. Insulin also induced similar shifts in the mobilities of two kinase-deficient mutants of PDK1, and dominant negative or constitutively active mutants of Akt or of the λ isoform of protein kinase C did not affect PDK1 mobility. Rapamycin, which inhibited p70 S6 kinase activation, did not affect the insulin-induced shift in PDK1 mobility. These results suggest that insulin induces phosphorylation of PDK1 by a PI 3-kinase-dependent mechanism, and that the kinase activity of PDK1 or signals mediated through Akt, p70 S6 kinase, or protein kinase Cλ do not contribute to this effect.

Journal

  • Medical journal of Kobe University

    Medical journal of Kobe University 61(4), 131-138, 2001-03

    Kobe University

Keywords

Codes

  • NII Article ID (NAID)
    110004652904
  • NII NACSIS-CAT ID (NCID)
    AN00085973
  • Text Lang
    JPN
  • Article Type
    departmental bulletin paper
  • ISSN
    0075-6431
  • Data Source
    NII-ELS  IR 
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