果蠅酪氨酸去磷酸酶PTP61F功能之探討：由細胞內分佈位置的不同決定其受質專一性

Characterization of Drosophila Protein Tyrosine Phosphatase PTP61F: Determination of Substrate Specificity by Subcellular Localization of PTP Isoforms

李函(Han Lee)

指導教授 ： 孟子青

國立臺灣大學/生命科學院/生化科學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.10029

摘要

果蠅酪氨酸去磷酸酶dPTP61F是人類PTP1B及TCPTP的異物種同源基因，研究已知這兩種人類PTP參與了許多訊息路徑的調控，因此，對dPTP61F功能的探討將使我們對PTP在訊息傳遞及發育過程所扮演的角色有更深入的了解。dPTP61F基因會在3’-端發生選擇性剪切而產生兩種在羧基端擁有不同胺基酸序列的PTP，分別為表現在細胞內膜狀構造上的dPTP61Fm，和位於細胞核內的 dPTP61Fn。這兩種isoforms擁有完全相同的PTP domain以及一致的酵素活性，暗示著兩者對受質的專一性取決於它們在細胞內空間分佈的不同。
本篇論文中，我們分別對這兩種PTP isoforms進行了功能上的探討。首先，利用RNAi的方式抑制dPTP61F基因的表現，可發現dIR受胰島素刺激而產生的磷酸化程度明顯增強，顯示dPTP61F是果蠅的胰島素訊息傳遞中的負向調控者。此外，當在S2細胞內大量表現dPTP61Fm或dPTP61Fn蛋白質，發現僅有表現在細胞質的dPTP61Fm可對dIR造成去磷酸化，而dPTP61Fn則不能，說明了PTP對受質的專一性與其空間上的分佈一致。接著我們利用果蠅與哺乳類在演化上保守的性質對dPTP61Fn的功能加以研究，已知dPTP61F的哺乳類同源蛋白TCPTP具有一個表現在細胞核內的isoform，能夠將轉錄因子STAT去磷酸化而抑制其活性，因此我們推測在果蠅JAK/STAT路徑中，dPTP61Fn亦可透過將STAT92E去磷酸化而抑制其轉錄活性；另一方面，dPTP61Fm則可能具有與PTP1B類似的功能，可辨認JAK (Hop)為受質。利用短暫轉染的方式，我們發現dPTP61Fm能對Hop造成明顯的去磷酸化，當表現的是不具去磷酸酶活性的C/S 突變蛋白，則會出現顯性抑制（dominant-negative）的現象，顯示Hop的磷酸化持續受到內生性PTP的抑制。同樣的實驗若表現dPTP61Fn蛋白質，則不會對Hop的磷酸化有所影響，但此時可觀察到STAT92E的磷酸化程度下降，且表現dPTP61Fn C/S突變蛋白時也會產生顯性抑制的效果，這些結果指出STAT92E極有可能為dPTP61F的受質。有趣的是，當細胞以Upd刺激造成JAK/STAT訊息路徑的活化，隨著處理時間增加，可觀察到dPTP61F蛋白質的量逐漸上升。利用dsRNA抑制STAT92E的表現並觀察dPTP61F蛋白質及mRNA表現量的變化，我們確認了dPTP61F基因的表現受到STAT92E的轉錄活性所調控，且dPTP61Fm與dPTP61Fn的mRNA都會因為Upd訊息路徑的活化而增加，暗示著dPTP61F的兩個isoforms皆參與在JAK/STAT路徑的負向調控迴路中。
目前，我們已發現dPTP61F在胰島素訊息傳遞以及JAK/STAT路徑中扮演著負向的調控角色，且dPTP61F的兩種isoforms會依其細胞內分佈的區域而展現不同的受質專一性。由於胰島素與JAK/STAT的訊息傳遞路徑與許多重要的生理現象緊密相關，代表著dPTP61F在維持果蠅的正常生長發育上扮演著重要的角色。

關鍵字

果蠅酪氨酸去磷酸酶dPTP61F ； 受質專一性

並列摘要

The Drosophila protein tyrosine phosphatase dPTP61F is an ortholog of human PTP1B and TCPTP, both of which are involved in the regulation of various signaling pathways. Thus, it is essential to characterize the function of dPTP61F for revealing the potential role of PTPs in the control of signal transcution and development. The dPTP61F gene undergoes alternative splicing at 3’-end of the transcript, resulting in an internal membrane-associated form dPTP61Fm and a nucleus-localized form dPTP61Fn. These two PTP isoforms share the identical catalytic domain, suggesting that the substrate specificity may be modulated by the subcellular location of the phosphatase. In the current study, we have explored the functional roles of both two isoforms of dPTP61F.
Applying RNA interference (RNAi) approach to knock down both isoforms of dPTP61F, we found that the tyrosine phosphorylation of dIR was enhanced in S2 cells stimulated with insulin, suggesting that dPTP61F may function as a negative regulator in insulin-mediated signal transduction. Moreover, when overexpressed in S2 cells, only the dPTP61Fm, but not the dPTP61Fn, was capable of dephosphorylating dIR. Furthermore, we showed that the access of dIR was determined by the distinct cytosolic/plasma membrane localization of dPTP61Fm, as confirmed by results from immunofluorescence staining in S2 cells.
We further explored the potential role of the nuclear form of dPTP61F. It has been shown that the evolutionarily conserved ortholog of dPTP61Fn, the mammalian TCPTP, which dephosphorylates and inactivates transcription factor STAT, is present in nucleus. Therefore, we proposed that dPTP61Fn may antagonize JAK/STAT signaling by facilitating dephosphorylation of Drosophila STAT92E. In contrast, dPTP61Fm may exert a similar role as mammalian PTP1B, which recognizes JAK (Hop) as a potential substrate.
We observed that, when dPTP61Fm was overexpressed, Hop was significantly dephosphorylated. Forced expression of phosphatase-dead (C/S) mutant form of dPTP61Fm showed dominant negative effect. These results suggest that dPTP61Fm is indeed a Hop phosphatase and that the phosphorylation level of Hop is constitutively suppressed by the endogenous dPTP61Fm. Furthermore, the dephosphorylation event is executed only by the membrane-bound PTP isoform. In addition, we also found that the phosphorylation level of STAT92E was decreased when dPTP61Fn was overexpressed, suggesting that STAT92E is a potential substrate of the nuclear form of PTP. Interestingly, the protein expression of dPTP61F was increased in response to activation of Upd signaling following a time-dependent manner. Using the RNAi approach, we demonstrated that the up-regulation of dPTP61F gene expression was modulated by the transcription activity of STAT92E. Performing a RT-PCR experiment, we also confirmed that both the amount of dPTP61Fm and dPTP61Fn tarnscripts were affected by JAK/STAT activity. These results implicated that dPTP61F is a putative target gene of STAT and that both isoforms of this phosphatase may participate in the negative feedback regulation of JAK/STAT signaling pathway.
In summary, our studies have revealed the negative regulatory role of dPTP61F in Drosophila insulin and JAK/STAT signaling pathways. We showed that each of the dPTP61F isoform has specific functions in different compartments inside a single cell. Both pathways control important signaling events, suggesting that dPTP61F may play an essential role for the maintanance of proper cellular processes during Drosophila development.

並列關鍵字

Drosophila protein tyrosine phosphatase ； dPTP61F ； substrate specificity

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