鑑定SIK2蛋白激酶調控LDCV分泌的下游因子

Abstract

內分泌及外分泌腺體參與體內各種不同器官的相互調控進而影響身體的發育、生殖、代謝及動態恆定等等，對於生命體來說扮演非常重要的角色。 分泌系統的異常或缺陷會導致代謝紊亂，例如乾口症及糖尿病等代謝症候群疾病，嚴重者則會危及生命。 許多消化酵素、神經生肽或荷爾蒙等分泌型蛋白會被包裹在大型緻密核心囊泡 (large dense core vesicle, LDCV)中並進行運輸及釋放。 新生成的LDCV會被運送到細胞質中暫時儲存，稱為儲存型囊泡庫 (reserve pool, RP)或黏附在細胞膜上等待釋放的立即釋放型囊泡庫 (readily releasable pool, RRP)，兩者的動態平衡是經由細胞內部和外部的訊息來嚴格控制，然而決定儲存到RP或運送到RRP的詳細分子機制，目前仍尚在研究當中。 SIK2屬於AMPK激酶家族中的一員。根據先前我們實驗室的研究顯示，SIK做為一個PKA調控LDCV分泌的下游因子。 不同激酶活性的SIK2主要存在於不同的LDCV囊泡庫中，而利用SIK2激酶活性抑制劑處理後發現會促使LDCV的釋放增加，暗示著SIK2可能藉由其激酶活性來調控LDCV的分泌。 本篇我們利用生化分餾(biochemical fractionation)的方式分離Rinm5F胰臟癌細胞的胰島素囊泡 (insulin-containing LDCVs)，確認SIK2確實位在胰島素囊泡上。 接著以SIK2已知的磷酸化共同基因序列 (phosphorylation consensus motif)進行電腦資料運算比對，鎖定CAPS2 (Calcium-dependent secretion activator 2)和Syt12 (Synaptotagmin XII)可能是SIK2調控的下游因子。 我們證明了CAPS2和Syt12與SIK2共同存在於相同的fraction，且CAPS2與SIK2確實存在於同一個複合體，但詳細的功能仍需進一步的研究。 綜合以上結果，我們藉由SIK2激酶活性與LDCV動態的關係，並確定其可能的下游分子，將有助於建立SIK2在調控LDCV分泌過程中所扮演的角色。

Endocrine and exocrine secretory glands are important controls of the organism’s homeostasis, by communicating and coordinating among the different organ systems of the body. Defects in these secretory processes can result in life threatening diseases, while minor deregulations can cause metabolic disorders such as diabetes mellitus and xerostomia. Secretion through the large dense-core vesicles (LDCVs) is the primary route for the release of neuropeptides. In the cell, newly synthesized LDCVs are transported and temporarily stored as reserve pool (RP) or readily releasable pool (RRP) vesicles, and this dynamic equilibrium is tightly controlled by both internal and external clues. However, the molecular mechanism underlying the decision for transport and storage as RP or RRP vesicles is currently unknown. SIK2 (salt-inducible kinase 2) is a family member of the AMPK serine/threonine kinases. Previous data from our lab indicate that SIK2 is a LDCV resident and serves as substrate for PKA kinase activity, which is a major trigger of LDCVs secretion. Subsets of SIK2 proteins presenting differential kinase activities localized to distinct pools of LDCVs, while treatment with specific inhibitor of SIK2 kinase activity promotes LDCV release, suggesting that SIK2 may regulate LDCV secretion through its kinase activity. Here, we perform biochemical fractionation of LDCVs from Rinm5F insulinoma cells by sucrose gradient centrifugation, and confirm the cofractionation of SIK2 with insulin vesicles. Then, we use in silico data mining to identify Calcium-dependent secretion activator 2 (CAPS2) and Synaptotagmin XII (Syt12) as candidate substrates of SIK2 kinase activity by conservation of phosphorylation motif consensus. CAPS2 and Syt12 cofractionate with SIK2, while CAPS2 present in the SIK2-containing complex. Whether CAPS2 and Syt12 display functional association with SIK2 is under study. In parallel, we assay how different post-translational modifications affect on SIK2 kinase activity in vitro, using recombinant Syntide sequence as substrate. Together, these studies will help establish the role of SIK2 in regulating LDCV secretion, by relating the status of its kinase activity with LDCV dynamics, and identifying its putative downstream effector molecules.