細胞凋零死亡過程中，Caspase-3對酪氨酸去磷酸酶PTP-PEST的降解機制及活性調控之探討

Abstract

細胞凋零死亡是生物體中的重要機制，有助於生物體正常生長以組織發育的平衡。過去研究指出，細胞凋亡是由許多複雜的訊息傳遞因子所調控，其中，酪氨酸磷酸化這項已知普遍參與在許多訊息傳遞路徑的蛋白質修飾作用，也被觀察到可能與細胞凋亡的調控有關，此作用本身是透過酪氨酸激酶 (PTK) 與酪氨酸去磷酸酶 (PTP) 所調控，本篇論文的研究主題，便是探討凋零死亡調控過程，究竟是否有PTP參與在其中。以dATP處理293T cell lysates，使lysates中模擬caspases活化的環境，我們利用膠內酪氨酸去磷酸酶活性分析法觀察，發現一個分子量大約115 kDa PTP的活性隨著dATP處理而下降，伴隨著幾個較小PTPs的出現，可能是115 kDa PTP發生降解所產生。我們隨後利用免疫移除法實驗，證實115 kDa PTP是PTP-PEST，而新出現兩個短片段PTP (78 kDa與58 kDa) 則是PTP-PEST的降解產物。根據觀察，我們認為PTP-PEST很可能是在caspases活化的狀態當中，最主要會發生降解的PTP之一。在細胞培養的系統當中，以Staurosporine刺激細胞凋零死亡，同樣可以觀察到類似的現象；而此降解作用會因caspases抑制物，Z-VAD的處理而被抑制，顯示PTP-PEST之降解是由caspases所引發。為了找出究竟是否有caspase會專一性地執行這項降解作用，我們在293T lysates中各別對幾個重要的caspase做免疫移除處理，結果顯示，移除caspase-3、caspase-9會抑制PTP-PEST降解的發生，而移除caspase-7則不會造成影響；透過in vitro環境下caspase-3與GST fusion PTP-PEST直接反應的結果，顯示PTP-PEST屬於caspase-3的受質。在比較全長PTP-PEST及降解PTP-PEST之間差異的探討上，我們觀察到降解產物反而具有較高的酵素活性。根據目前的文獻，這篇論文的研究結果，不但是第一個發現PTP做為caspase的受質，同時也揭露PTPs的降解產物可能透過其較高的酵素活性，在細胞凋亡的調控中扮演重要的角色。

Apoptosis is a fundamental biological process which is crucial for development and tissue homeostasis. Previous studies have shown that, the protein tyrosine phosphorylation, which is controlled by PTKs and PTPs, participates in the regulation of apoptosis. In the current study, we investigated the possible role of PTPs. Our initial experiments demonstrated that the addition of dATP into 293T lysates imitated the apoptosis circumstance. We then applied the technique of “in gel” phosphatase activity assay for analyzing those samples. Our data clearly showed that a PTP with molecule weight about 115 kDa had an obvious decrease of activity in response to the caspase activation, while a few smaller PTPs emerged, presumedly being the cleaved products of the ~115kDa PTP. The immunodepletion experiments identified the 115 kDa PTP as the PTP-PEST, a widely expressed cytosolic tyrosine phophatase. Our data further demonstrated that the smaller forms of PTPs (78 kDa and 58 kDa), which appeared in response to caspase activation, were the cleaved PTP-PEST. According to these observations, we proposed that the PTP-PEST might be the predominant PTP being cleaved by caspases during apoptosis. To further test this hypothesis, we applied a similar approach of in-gel phosphatase assay in the cell culture models. Our results showed the cleavage of PTP-PEST occurred in the Staurosporine treated HeLa and Rat-1 cells. Furthermore, such an event was indeed caspases-dependent manifested by the fact that Z-VAD, a caspases inhibitor, attenuated the cleavage of PTP-PEST. We then decided to identify the caspase that is responsible for the hypothesis of PTP-PEST. For this purpose, immunodepletion of individual caspases was carried out prior to the addition of dATP into 293T lysates. Our results showed that the removal of caspase-3 and -9 prevented this cleavage, but the depletion of caspase-7 did not, suggesting that caspase-3 is the primary protease in this signaling event. To further provide direct evidence, GST fusion PTP-PEST was incubated with the purified caspase-3. Our data demonstrated that three cleaved products with various lengths of deletions in the C-terminus of PTP-PEST were generated. In search of the biological significance of caspase-3-mediated hypothesis of PTP-PEST, we also found that the cleaved forms exhibited about 2-fold higher phosphatase activity than the full-length form. To the best of our knowledge, this is not only the first report to demonstrate that a tyrosine-specific phosphatase is indeed the substrate of activated caspases, but also for the first time shows that the cleaved forms of PTP may play an important role in the regulation of apoptotic signaling pathways.