SIK蛋白異構體在分泌腺體上的功能特異性

Abstract

AMPK被認為參與在人體的恆定，而AMPK family其中的SIKs一開始在高鹽餵食的大鼠腎上腺被發現，同時也藉由磷酸化輔因子TORC2使其將其留在細胞質而無法進入細胞核內，CREB ( cAMP-response element-binding protein )這個轉錄因子就可以被SIKs調控，藉由CREB與TORC2的結合以調控醣類、蛋白質、脂質等的合成及細胞存活。SIKs同時也藉由調控動植物細胞的離子進出進行離子的恆定。而在2012年Aguilera實驗室所發表的研究中指出，SIKs可能會在相同器官相同刺激下表現量都會上升只是時間點不同，並且扮演類似的角色。因此我們合理懷疑在高鹽餵食下的大鼠腎上腺，表現量上升的並不只是SIK1。免疫組織染色可以確定SIK在腎上腺上的蛋白表現量和位置。我們的研究結果顯示，SIK2在腎上腺的髓質和皮質的束狀帶細胞有大量表現，此外SIK2在腎上腺皮質束狀帶分泌細胞上具有不同的酵素活性表現。由以上我們可以確定SIK2確實參與腎上腺皮質束狀帶細胞的分泌作用。接下來我選用小鼠腎上腺皮質束狀帶細胞株Y1進行定量PCR和西方墨點法來確定SIKs在Y1中確實有穩定表現，接著我利用免疫螢光染色發現了SIK2與緻密核心的囊泡的標記蛋白Rab27A位置重疊，確定了SIK2在Y1中存在於緻密核心的囊泡上，並且藉由ACTH的刺激證明了我們在Y1發現的緻密核心的囊泡有參與生理上的調節，但是囊泡中攜帶的是什麼尚無研究，這也是下一步我們實驗室想了解的。

AMPK is involved in the maintenance of energy balance in human body. Salt-inducible kinases (SIK1/2/3 isoforms) belong to the AMP-activated protein kinase (AMPK) family, and were first cloned from the adrenal glands of rats fed with high salt diet. Through phosphorylation and subsequent cytoplasmic sequestration of the coactivator TORC2, SIKs can down-regulate transcriptional activation of the cAMP-response element-binding protein CREB. This SIK-dependent transcriptional regulation has been shown to be an important determinant for survival and protein, lipid and carbohydrate produce. SIKs also control iron homeostasis via regulate intracellular iron balance. in Aguilera’s lab, the paper published in 2012 showed that there is cooperation and sequential induction of SIK1 and SIK2 in neuron. Under stimulated, SIK1 and SIK2 will be induced in different time at the same organ. Base on the reference, was SIK2 also induced from the adrenal glands of rats fed with high salt diet? Immunohistochemistry approaches were used to determine the protein expression profile of all three SIK isoforms. In additional, SIK2 kinase activity is involved in its DCV function. Next, the western blotting and Quantitative PCR in Y1 mouse adrenocortical zona fasciculata tumor cell demonstrated that Y1 cell is a suitable cell line model for studying SIK2 function. Then, the Immunofluorescence staining showed that punctate distribution of SIK2 in cytoplasm resembling vesicles and SIK2 colocalization with DCV marker Rab27A.Thus, we suggest that SIK2 might play a dominant role in DCV secretion of adrenal cortex zone fasciculata. ACTH treatment induced SIKs express level and kinase activity changed. Further studies on the vesicle contain is the next step in our lab research.