GSKIP透過不同的GSK3和PKA binding domains提供粒線體fission protein Drp1磷酸化所需, 在H2O2誘導的氧化壓力下能夠賦予神經保護作用.

GSKIP is required for mitochondrial fission protein Drp1 phosphorylation via distinct GSK3 and PKA binding domains, conferring neuroprotection under H2O2 induced-oxidative stress.

黃韋勝(Wei-sheng Huang)

指導教授 ： 洪義人

高雄醫學大學/醫學院/生物化學研究所/碩士(2013年)

摘要

GSKIP已被確定為最小AKAP，並保有PKA和GSK3β binding domains。GSKIP binding GSK3β與神經軸突生長(neurite outgrowth)有關，其與PKA binding的還是沒有找到生理意義。在這項研究中，我們首先證明GSKIP，GSK3β，PKA和DRP1，形成一個複合體。為了探討GSKIP binding PKA有何功能上的意義，應用了不同的刺激物來界定出所參與的訊號通路。使用異位過度表達 (ectopic overexpressed) GSKIP wt，V41/L45P (loss of PKA binding) 和L130P (loss of GSK3β binding) 缺失的突變體(defective mutant)，我們觀察到V41/L45P缺失的突變體與autophagy是不相關的，但顯示出H2O2的處理下不管有無活化cAMP信號會增加細胞凋亡標誌物的表達量，證明GSKIP的PKA binding domains可能有助於對抗細胞凋亡作用。PKA介導 DRP1 S637磷酸化已被證實與粒腺體形態延長(elongated mitochondrial morphology)有關。過度表達GSKIP及其他的突變體，在一個spatial-temporal manner GSKIP wt與兩個缺失的突變體相比DRP1 S637磷酸化提高7∼8倍，證明L130和V41/L45對於GSKIP保護作用與DRP1的關聯性是同等重要的。值得注意的是，GSK3β也能作為支架蛋白(scaffold protein)抓住DRP1讓 PKA 能直接磷酸化其S637。此外，GSKIP wt，但不是缺失的突變體，經由過氧化氫（H2O2）和forskolin的處理協調後表現出粒腺體形態延長。此外，異位表達DRP1 S637D，一個phosphomimetic mutant,，在這個細胞表達的缺失的突變體成功恢復粒腺體形態延長，而S637A則沒有此現象。總而言之，我們研究結果說明了GSKIP能夠去抵抗H2O2誘導的細胞凋亡可能的生理角色，經由協調活化PKA 介導 DRP1磷酸化S637還有GSK3β anchoring Drp1而非GSK3β 激酶活性來影響線粒體形態。

關鍵字

蛋白質激酶A ； 肝糖合成激酶3β ； GSK3β相互作用蛋白 ； 氧化壓力

並列摘要

GSKIP has been identified as smallest A-kinase anchor proteins (AKAP), and retains both PKA and GSK3beta binding domains. The GSK3β binding of GSKIP is linked to neurite outgrowth, but no physiological significance was found for its PKA binding. In this study, we firstly demonstrated that GSKIP, GSK3β , PKA and Drp1 form a local working complex. To explore the functional consequences of PKA binding of GSKIP, different stimuli were applied to delineate the signaling pathways involved. Using ectopic overexpressed GSKIP wt, V41/L45P (loss of PKA binding) and L130P (loss of GSK3β binding) defective mutants, we observed that the V41/L45P defective mutant was not associated with autophagy but showed increased expression levels of apoptotic markers under H2O2 insult regardless of activated cAMP signaling, suggesting that the GSKIP PKA binding domain may contribute to an anti-apoptotic effect. PKA-mediated Drp1 S637 phosphorylation has been linked to elongated mitochondrial morphology. Overexpressed GSKIP and its mutants, Drp1 S637 phosphorylation was enhanced 7~8-fold in the GSKIP wt group compared to both defective mutant groups in a spatial-temporal manner, indicating that both L130 and V41/L45 are important to Drp1-associated protective action of GSKIP. Notably, GSK3β also functions as a scaffold protein that recruits Drp1 for PKA directly phosphorylating at S637. Silencing GSKIP resulted in a decrease of Drp1 S637 and GSK3β S9 phosphorylation. Furthermore, GSKIP wt, but not the two defective mutants, exhibited elongated mitochondrial morphology after compromising the effects attributed from H2O2 and forskolin. Moreover, ectopic expressed Drp1 S637D, a phosphomimetic mutant, but not S637A, restored the elongated mitochondrial morphology of the cells expressing the two defective mutants. Altogether, our data illustrated a possible physiological role of GSKIP associated with acquired resistance to H2O2-induced apoptosis, influencing mitochondrial morphology by coordinating both the activation of PKA-mediated Drp1 phosphorylation at S637 and tethering GSK3β-mediated Drp1 recruitment by anchoring rather than kinase activity.

並列關鍵字

PKA ； GSK3β ； GSKIP ； AKAP ； mitochondrial morphology ； oxidative stress

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