呂勝春臺灣大學：分子醫學研究所沈暐倫Sheng, Wei-LunWei-LunSheng2007-11-262018-07-092007-11-262018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/51380腺苷單磷酸活化蛋白質激酶（AMPK）為一由三個次體組成的蛋白質激酶，可藉由細胞中腺苷單磷酸（AMP）的量來感知細胞的能量狀態。AMPK可因AMP與ATP比例的上升而活化，進而抑制細胞中的耗能反應並促進能量分子ATP的產生。AMP可與AMPK之 gamma 調節次體結合並活化 alpha 催化次體的酵素活性。在 gamma 2 調節次體上我們觀察到一157位之絲胺酸（Ser157）坐落於一段符合AGC家族激酶所能辨認之一致性序列（consensus sequence）的胺基酸序列中，利用質譜儀分析配合西方墨點法我們確認在細胞內Ser157確實可被磷酸化。我們將此絲胺酸突變成為殘基無法被磷酸化之丙胺酸（alanine）後，發現此 gamma 2 突變體可使與其結合的 alpha 催化次體保持在活化的狀態，而這可解釋成是因為此突變體在與AMP結合後無法促使Ser157被磷酸化，進而不能對 alpha 催化次體產生抑制的作用，因為和Ser157被磷酸化之 gamma 2 調節次體結合的 alpha 催化次體並不因AMPK活化物AICAR的處理而上升。Ser157的磷酸化是受到來自生長激素的訊號所調控，而一些與生長有關之AGC激酶的家族成員似乎參與了Ser157的磷酸化。因此，我們提出了一個 gamma 2 調節次體抑制 alpha 催化次體的機制，為經由 gamma 2 調節次體上157位絲胺酸的磷酸化所達成。AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase which senses the cellular energy state by monitoring the AMP level. It is activated when the [AMP]/[ATP] ratio is elevated, and then the ATP consumption pathways are decreased together with the acceleration of ATP production. AMP can bind to the γ regulatory subunits of AMPK complex and allosterically activate the catalytic activity of α subunits. On the γ2 subunit we have observed a potential phosphorylation site, Ser157, which lies in a consensus sequence recognized by AGC family kinase. We confirmed its phosphorylation in vivo by mass spectrometry along with Western blot using phospho-specific antibody. α subunits that associated with S157A mutant are constitutively activated, and this can be explained by the lack of negative regulation induced by AMP binding and the subsequent Ser157 phosphorylation on γ2 subunits, since the α subunits associated with Ser157-phosphorylated γ2 are not activated by AICAR, a drug that is metabolized into AMP analog in vivo. Ser157 phosphorylation is mediated by growth signals, and multiple AGC family kinases relating to cell growth are involved. Therefore, we provide here a negative regulation mechanism of AMPK by the regulatory γ2 subunit when its Ser157 is phosphorylated.Master Thesis i 致謝 ii 中文摘要 iii ABSTRACT iv CONTENTS v INTRODUCTION 1 MATERIALS AND METHODS 7 DNA constructs and site-directed mutagenesis 7 Drugs and antibodies 9 Cell culture and transfection 10 Immunoprecipitation 11 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 11 Western blot 12 Bacterial expression of His-tagged recombinant AMPK γ2 N terminal region 13 In vitro kinase assay 14 In-gel digestion 14 RESULTS 16 AMPK γ2 Ser157 is a phosphorylation site in vivo 16 Phosphorylation of Ser157 negatively regulates AMPK activity 17 AICAR induces Ser157 phosphorylation. 18 Phosphorylation of Ser157 is triggered by AMP binding and requires growth signals. 20 Activities of multiple AGC family kinases are correlated with Ser157 phosphorylation. 21 Inhibition of Akt and PKC caused direct activation of AMPK activity. 22 DISCUSSION 23 REFERENCES 30 FIGURES AND TABLES 35 Fig. 1. Amino acid sequence surrounding Ser157 on the AMPK γ2 subunit. 35 Fig. 2. Mass spectrometry analysis of AMPK γ2 phosphorylation on Ser157. 36 Fig. 3. Western blot analysis of AMPK γ2 phosphorylation on Ser157. 37 Fig. 4. S157A mutant γ2 elevated catalytic activity of α subunits. 38 Fig. 5. AICAR induced Ser157 phosphorylation on γ2 subunits but not Thr172 phosphorylation on the α subunits associated with Ser157-phosphorylated γ2. 39 Fig. 6. Thr172 and Ser157 phosphorylation status of endogenous α and overexpressed γ2 subunits during the time course of AICAR treatment. 40 Fig. 7. Ser157 phosphorylation is triggered by AMP binding and requires growth signals. 41 Fig. 8. Multiple AGC family kinases are involved in Ser157 phosphorylation. 42 Fig. 9. PKCs play an essential role in AICAR-induced Ser157 phosphorylation. 43 Fig. 10. Inhibition of Akt and PKC activity resulted in AMPK activation. 44 Fig. 11. Purification of recombinant proteins of AMPK γ2 N terminal region as the substrate for in vitro kinase assay 45 Fig. 12. Akt phosphorylated AMPK γ2 N terminal region at multiple sites in vitro. 46 Fig. 13. Schematic representation of AMPK γ2 Ser157 phosphorylation during AMPK activation. 47 Table 1. Recipe to make different percentage of polyacrylamide gel for SDS-PAGE 48987380 bytesapplication/pdfen-US腺苷單磷酸活化蛋白質激酶AICAR藥物磷酸化調節次體AGC家族AMPKAICARphosphorylationregulatory subunitAGC familyotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51380/1/ntu-96-R94448001-1.pdf