高滲透壓甘油反應磷酸化激酶調控鳥糞嘌呤核苷酸交換因子以活化第一腺嘌呤核苷二磷酸核醣化相似因子之探討

Abstract

腺嘌呤核苷二磷酸核醣化相似因子 (ARF-like protein 1, ARL1) 屬於腺嘌呤核苷二磷酸核醣化因子家族的成員之一 (ADP-ribosylation factors, ARFs)。在真核生物中，這類蛋白被報導參與許多重要的細胞功能，包含調控囊泡運輸、影響細胞骨架的組裝以及維持細胞內的離子恆定等等，但是作用機制的細節仍不甚清楚。在酵母菌的研究當中，已知鳥糞嘌呤核苷酸交換因子 (Guanine nucleotide exchange factor) Syt1p 會催化 Arl1p 的活性並使其前往反式高基氏體網路 (trans-Golgi network)。活化態的 Arl1p 將吸引高基氏體蛋白 (Imh1p) 至反式高基氏體網路並作用於下游的囊泡運輸。在我們先前的發表成果中，我們發現內質網壓力 (ER stress) 的誘發將促使內質網上的磷酸化激酶 Ire1p 活性上升，Ire1p將透過 Syt1p 的第416號絲氨酸磷酸化轉譯後修飾進一步大量活化 Arl1p。此外，我們也證實這條路徑的啟動有助於細胞抵擋內質網壓力。而在本篇研究中，我們發現在細胞經歷內質網壓力的過程中， Hog1p 是另一個調控 Syt1p的磷酸化激酶。不同於 Ire1p 的是，Hog1p 作用於 Syt1p 的第297號絲氨酸磷酸化轉譯後修飾以活化 Arl1p。同時，我們亦發現不受壓力激活的 Hog1p 也可以調控 Arl1p 的活化，說明在壓力環境以及正常生理狀態下，Hog1p 皆扮演活化 Arl1p 的重要角色。總結本篇除了再次說明 Syt1p 的磷酸化轉譯後修飾對於 Arl1p 活化的重要性，我們亦提出 Hog1p 在正常生理狀態中所執行的新功能。

ARF-like (ARL) proteins play important roles in regulating vesicular trafficking at the Golgi compartments, modulating cytoskeleton dynamics and maintaining ion homeostasis. In Saccharomyces cerevisiae, guanine-nucleotide exchange factor (GEF) Syt1p facilitates Arl1p activation to recruit golgin protein Imh1p to trans-Golgi network (TGN). Our previous findings have elucidated the induction of unfolded protein response (UPR) highly promotes activation of Arl1p and that ER-resident kinase Ire1p is responsible for the up-regulation through transcription alteration (Proc. Natl. Acad. Sci. U S A. 113:E1683-E1690). Moreover, the SYT1-ARL1-IMH1 signaling is required for ER stress resistance. In this study, we identified MAP kinase Hog1p as a new regulator of the ARL1 pathway for UPR. We found that MAP kinase controls Arl1p activation and non-stressed Hog1p mutant can rescue Imh1p Golgi localization in MAP kinase-deleted cells. We further showed that Hog1p might directly phosphorylate Syt1p at serine 297 to promote the activation of Arl1p, which performs a distinct route from Ire1p-mediated Syt1p phosphorylation at serine 416. Collectively, we demonstrate that Syt1p phosphorylation is regulated by multi-regulators, which is important for the activation and function of Arl1p under stress as well as normal growth conditions.