腺苷單磷酸活化蛋白質激酶調節次體之157位絲胺酸殘基磷酸化對其活性之調控

Regulation of AMPK activity by phosphorylation of Ser157 on its regulatory gamma 2 subunit

沈暐倫(Wei-Lun Sheng)

指導教授 ： 呂勝春

國立臺灣大學/醫學院/分子醫學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.02189

摘要

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。

關鍵字

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

並列摘要

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.

並列關鍵字

AMPK ； AICAR ； phosphorylation ； regulatory subunit ； AGC family

參考文獻

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