Protein Tyrosine Phosphatase PTP-PEST is cleaved and activated through Caspase-3-mediated pathway Implication of a Novel Regulatory Mechanism during Apoptosis
Date Issued
2005
Date
2005
Author(s)
Liu, Ying-Chih
DOI
zh-TW
Abstract
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.
Subjects
細胞凋零
酪氨酸去磷酸酶
降解
apoptosis
caspase-3
PTP-PEST
Type
other
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