果蠅酪胺酸去磷酸酶dPTP61F與磷酸激酶dAbl透過對蛋白質Kette的調節而影響細胞骨架蛋白actin之組合

Drosophila protein tyrosine phosphatase dPTP61F and kinase dAbl coordinate to control Kette for regulation of actin organization

10.6342/NTU.2008.01903

辜雪硯

Kette ； dPTP61F ； dAbl ； 細胞骨架 ； actin ； 果蠅 ； 酪胺酸去磷酸酶 ； Kette ； dPTP61F ； dAbl ； actin ； drosophila ； phosphatase

臺灣大學生化科學研究所學位論文

2008年

碩士

孟子青

英文

藉由干擾性核醣核酸(RNA Interference; RNAi)技術，在果蠅不同的組織中，專一性地抑制酪胺酸去磷酸酵素dPTP61F的表現，顯示dPTP61F負責調控果蠅多種組織器官的發育。為了進一步了解dPTP61F所調控的分子機制，我們利用質譜儀鑑定的方式找到dPTP61F可能的調控的受質為蛋白質Kette；另一方面，我們證明Kette受到磷酸激酶dAbl的調控。有趣的是，我們發現dAbl同時也是dPTP61F的受質，表示dPTP61F與dAbl共同調控著Kette的磷酸化程度，而dPTP61F更同時會對於Kette與其上游的dAbl進行去磷酸化作用，以更快速有效地抑制Kette的訊息傳遞。此外，我們在果蠅中驗證了Kette、dPTP61F與dAbl之間的交互作用確實會造成果蠅眼睛發育的缺陷，顯示在生物體中，此三種蛋白質之間的確參與相同的訊息傳遞途徑。以定點突變的方式，我們找到蛋白質Kette上受到dPTP61F與dAbl調控的特定的酪胺酸，當此酪胺酸突變之後，Kette的磷酸化程度不再受到dPTP61F與dAbl的影響。利用免疫螢光染色法，我們觀察到Kette的磷酸化對細胞骨架蛋白actin的影響，當Kette的磷酸化程度上升，會造成Kette由原本散佈在細胞質中變成分布在細胞膜附近，而此時細胞骨架蛋白actin也明顯出現在細胞膜附近聚合的現象；而當我們將Kette上受到dPTP61F與dAbl調控的酪胺酸突變之後，Kette則失去往細胞膜分布與調控細胞骨架蛋白actin聚合的能力。這些實驗結果顯示dPTP61F會藉由調控Kette與dAbl，進而影響細胞骨架蛋白actin，最後造成細胞形狀與附著能力的改變。

To date, the functional role of Drosophila protein tyrosine phosphatases (PTPs) remains largely elusive. We have characterized dPTP61F, which is the smallest member in the Drosophila PTP family and the ortholog of human PTP1B and T-Cell PTP, to delineate its involvement in controlling signal transduction pathways and developmental processes. Employing the RNAi-based, tissue-specific knockdown technique in flies, we found that dPTP61F plays an essential role in the formation of the central nervous system, eyes, and bristles. The ubiquitous suppression of dPTP61F expression led to the lethality that occurred at the third instar larval stage. We used the substrate trapping approach in combination with the mass spectrometry-based analysis to identify that Kette, the Drosophila ortholog of human NAP-1 and a key component of WASP and SCAR/WAVE complexes for controlling actin polymerization, is a potential substrate of dPTP61F. When ectopically expressed in Drosophila S2 cells, the tyrosine phosphorylation level of Kette was down-regulated by dPTP61F. Conversely, the tyrosine phosphorylation level of Kette was up-regulated by dAbl, the Drosophila ortholog of human tyrosine kinase c-Abl. We also showed that dAbl itself is a substrate of dPTP61F. Interestingly, Kette, dPTP61F, and dAbl genetically interact with each other, suggesting that tyrosine phosphorylation-dependent signaling may be important in regulating the activity of Kette in vivo. To test this hypothesis further, the subcellular localization of Kette was investigated in S2 cells. Importantly, only the WT form but not the YF mutant form of Kette was relocated to plasma membrane in response to suppressed expression of endogenous dPTP61F or overexpression of ectopic dAbl, consistent with the current view that Kette regulates actin dynamics at the membrane proximity.