"果蠅高基氏體蛋白GOLPH3,Rotini,調控Wingless,及Tor訊息傳導路徑中Sin1的表現"

Abstract

在果蠅中的人類高基氏體蛋白GOLPH3同源物Rti 是一個在高基氏體中負責調控EXT蛋白群的回送運輸的蛋白質。EXT蛋白群是生合成HSPG支鏈所需要的酵素群。當Rti增量或缺失時皆會導致HSPG 表現量減少以及Hh訊息傳導路徑的失常。 在果蠅中Wnt/Wingless (Wg) 調控翅脈以及翅膀周圍剛毛的形成。在翅碟上，EXT缺失的細胞會造成Wg的表現量減少，但此現象並未在Rti異常表現的細胞中觀察到。相反地在Rti增量表現時會導致Wg累積在原本合成Wg的細胞內，而Rti缺失時則不會影響Wg的表現量。因此Rti影響Wg的分佈表現並非源自於HSPG的缺失。此外，當Rti增量表現時，Wg會被限制在細胞內而無法送至細胞膜上，因此 Wg訊息傳導的下游的目標無法被誘發，最後造成翅膀周圍的剛毛缺失。 Wntless (Wls) 是一個膜蛋白，負責將Wg由高基氏體送往細胞膜，而自身由retromer回收至高機氏體以利進行下一循環。文獻指出當retromer缺失時，會造成Wg累積在細胞內以及細胞外的Wg量減少，這些現象皆與Rti增量表現時類似。考量Rti與retromer在免疫沈澱中有交互作用，且在細胞中坐落在一起，推測Rti增量表現時會阻斷retromer回收Wls，細胞內Wls不足而導致Wg無法正常被運輸至細胞膜上。 在另一方面，分別將同源rti突變以及野生型果蠅的卵巢萃取蛋白純化後以二維電泳分析。結果顯示有些許的蛋白質表現量異常，經質譜分析鑑定這些蛋白質身分，其中包含一個Tor complex2 (TORC2)的成員Sin1。 文獻中GOLPH3已知會透過未明機制參與Tor訊息傳導路徑，此外在 Rti增量表現時所引起的果蠅眼睛皺縮性狀與Tor突變的果蠅眼睛性狀類似。推測Rti 是否透過透過調控Sin1參與Tor訊息傳導路徑。因此我們在S2細胞中證實Rti的表現會負調控Sin1的表現。 此外，TORC2的下游訊息傳導目標Akt及其磷酸化 (pAktS505) 也受Rti所調控。Rti的表現量正調控Akt及pAktS505的量，而因此我們推測Rti正向調控Akt及其磷酸化，引起Tor下游訊息傳導活化而最終負回饋抑制Sin1的表現量。

Drosophila homologue of human Golgi protein GOLPH3, Rotini (Rti), regulates the retrograde trafficking of EXT proteins, which are required for the polymerization of Heparan Sulfate Proteoglycans (HSPGs) GAG chains. Both loss- and gain-of-function rti causes the reduction of HSPGs expression and Hedgehog signaling. In other words, both loss- and gain-of-function rti represent the loss-of-function of EXTs. Drosophila Wnt/Wingless (Wg) is required in a wide range of patterning events, including defining the wing blade and specifying the wing margin. In wing margin, the expression of Wg is reduced in EXT clone but not in rti mutant cells. Instead, Wg accumulates inside the Wg producing cells in rti gain-of-function condition, and expressed normally in rti loss-of-function condition. For instance the way Rti affects the expression pattern of Wg is HSPG independent. In addition, when Rti is overexpressed, Wg was restricted inside the producing cells but not secreted to the cell surface. Wg downstream signaling targets are not induced consequently. For example, the bristles on the wing margin are lost because of the defect of Wg signaling pathway. Wntless (Wls), a multipass transmembrane protein, is required for sending Wg from Golgi onto the cell surface. Wls are recycled back to trans-Golgi by an essential regulator called retromer. The failure of retromer will cause the accumulation of Wg inside the cell and the reduction of extracellular Wg secreted. These phenotypes are similar to those found in Rti gain-of-function. Considering that Rti interacts and colocalizes with the cargo recognition complex of the retromer, and Wg accumulation occurs in Rti gain-of-function condition, we postulated that overexpressed Rti may interrupt the retromer dependent retrograde recycling of Wls resulting in the restriction of Wg inside the Wg producing cells. On the other hand, we purified total proteins of rti GLC and OR ovaries to perform two-dimensional gel electrophoresis. We observed that some proteins with abnormal expression level between ovaries of rti GLC and OR. After in gel trypsin digestion and LC-MS/MS analysis, we identified these proteins including a protein named Sin1, which is a component of Tor complex 2 (TORC2). Since GOLPH3 was reported involving in the Tor signaling pathway with unknown mechanism, and the reduced eye phenotype is similar between rti and tor mutant condition, we were interested in whether Rti involved in Tor pathway through modulating Sin1. In S2 cells, we demonstrated that Rti negatively regulated Sin1, and the expression of Sin1 was dosage dependence on the expression of Rti. In addition, Akt and pAktS505, which are the downstream signal targets of TORC2, were also modulated by the expression of Rti. Rti positively regulates the expression of Akt and pAktS505 in vivo. In conclusion we postulate that Rti may facilitate the phosphorylation of Akt on S505, and the down-stream signaling pathway triggers the negative feedback to negatively regulate Sin1.