2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643391摘要：SIK2和SIK3屬於蛋白激酶大家族，CAMK Ser/Thr蛋白激酶家族以及AMPK次家族成員。SIK2在脂肪組織以及癌細胞株普遍存在。然而SIK3則是普遍存在，且在癌細胞中高度表現！現已知SIK2可經由磷酸化CRTC2（又叫TORC2）co-activator 而達到抑制CREB所調控之基因表現。當PKA經由GPCR所活化之cAMP刺激CREB時，PKA同時對SIK2之Ser587磷酸化，達到抑制其活性；在肝臟，若SIK2之活性遭抑制，TORC2之活性增加，同時PGC-1α之活性也跟著上升，達到葡萄糖產生的效果。若SIK2/Ser358受到胰島素的刺激，經由AKT磷酸化後則其活性增加，可加速葡萄糖之代謝！SIK2亦可經由磷酸化IRS1/Ser789達到減緩胰島素訊息傳遞！肥胖小鼠的脂肪組織中，SIK2之量及活性均增加，這也是造成胰島素抗性及第二型糖尿病的原因之一！肥胖症造成ER逆境並活化JNK所導致的IRS-1磷酸化，它與SIK2之共同對IRS-1之影響導致胰島素訊息傳遞受阻。除了脂肪組織外，免疫系統B細胞之分化成漿細胞(Plasma cell)以產生免疫球蛋白之機轉中，ER逆境反應以及維持ER之高度功能非常重要！由我們的研究中，SIK3在Plasma細胞含量高，而SIK2則少，因此SIK3可能在免疫球蛋白的製造及輸出細胞外較重要！如同SIK2，SIK3亦可受到PKA之負向調控，它在胰島素在islet β細胞中之合成及輸出是否也和SIK2一樣或各有功能，也是一重要且尚待解答的問題！我們的初步結果顯示一旦SIK2或SIK3之量受到降低，細胞株立即產生UPR反應，但eIF2α/S51之磷酸化則減少，其中機轉如何？是本研究探討的中心課題。SIK2可與p97/VCP以及PP2A共同存在於蛋白質複合體中，PP2A則與BCL-2共同作用，若BCL-2之磷酸化程度受到PP2A作用而減少時，ER之功能方能維持，若BCL-2因PP2A活性降低，使得BCL-2磷酸化繼而經由proteasome降解，ER stress便增加。因此，SIK2與PP2A及BCL-2之間的調控為本研究之主題之一。此外，SIK2如何維持ER蛋白質如HMG-CoA reductase之恆定(經由ERAD)也是探討的方向。<br> Abstract: SIK1, SIK2 and SIK3 belong to the protein kinase superfamily, CAMK Ser/Thr protein kinase family, AMPK subfamily. SIK2 is expressed abundantly in adipose tissues and ubiquitously in cancer cell lines. SIK2 represses CREB-mediated gene expression by phosphorylating the CRTC2/TORC2. When PKA activates CREB, it simultaneously inactivates SIK2 by means of phosphorylation at Ser587, which reduces its kinase activity toward TORC2/Ser171. Impairment of SIK2 signaling in the liver, e.g., by knockdown with siRNA and via hyperphosphorylation at Ser587 by PKA, enhances dephosphorylation of TORC2 followed by upregulation of PGC-1α gene expression, which in turn results in enhanced gluconeogenesis. In contrast, phosphorylation of SIK2 at Ser358 by AKT is found to upregulate TORC2 phosphorylation activity when insulin signaling was activated during refeeding. Furthermore, SIK2 could attenuate insulin signal transduction by phosphorylating IRS1/Ser789. The functions of SIK2 in insulin resistance of diabetic animals remain unclear. Obesity causes ER stress and leads to suppression of insulin signal transduction by activation of JNK and subsequent phosphorylation and inactivation of IRS-1. Thus, obesity caused ER stress is a central feature of insulin resistance and type-2 diabetes and SIK2 is likely to have important roles. In addition to adipose tissues from obese animals, terminal B cell differentiation requires a massive increase in the synthesis of antibodies in response to infection. Components of the UPR are essential in the differentiation of quiescent B cells to plasma cells, allowing them to become secretory factories dedicated to antibody production. The signal transduction pathways that control this feat have not been clearly delineated. Since SIK2 or SIK3 may participate in the differentiation of B cells and antibody secretion of plasma cells. Like SIK2, SIK3 interacts with TORC2 and may be regulated by cAMP-dependent protein kinase. In both HEK293 and pancreatic islet β cells, SIK3 is down regulated by forskolin treatment. SIK2 or SIK3 could function in regulating insulin secretion in pancreas and antibody and inflammatory cytokine secretion in plasma cells by modulating the functions of ER. When the endogenous level of SIK2 or SIK3 was reduced by siRNA-mediated knockdown, the cells respond with UPR as shown by the activation of ATF6, Xbp-1S, NF-κB and Grp78 transcription together with the decreased ER-associated protein degradation (ERAD). Paradoxically, the phosphorylation of eIF2α/Ser51 is decreased when SIK2 is knockdown. These results may suggest that SIK2 is important for the regulating the protein phosphatase 1 activity and the phosphorylated eIF2α/Ser51 level. SIK2, p97/VCP, PP2A, and Hsp70 could be isolated as a complex by tandem affinity purification (TAP). P97/VCP is a key component of ERAD. PP2A plays manyregulatory functions for various signaling pathways, e.g., the activities of KATP channel and Na+/K+-ATPase and BCL-2, linked to ER homeostasis. Functional interaction among SIK2, p97/VCP and PP2A will be investigated.There are three goals of this proposal: (1) To study the regulation of insulin signaling by SIK2 and SIK3, (2) To study the regulation of UPR by SIK2 and SIK3, especially how these kinases maintain the homeostasis of ER as related to ERAD and protein secretion, and (3) To study the differential modulation of the kinase activity of SIK2 and SIK3.