Identification of Myo18A as a caspase-3 substrate and its role in apoptosis

• Main Authors: Shu Wan Chang, 張書菀
• Other Authors: J. S. Yu
• Format: Others
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/18891427016499096361

碩士 === 長庚大學 === 生物醫學研究所 === 97 === 英文摘要 Previously, our lab has employed a proteomic approach to identify Myo18A as a novel PAK2-binding partner. To further investigate the biological functions of Myo18A, I performed sequence analysis of Myo18A, which revealed that several consensus caspase-3 cleavage sites are present in the Myo18A’s C-terminal part. We first monitored the fate of Myo18A protein in apoptosis. I used UV irradiation or staurosporine (STS) to induce apoptosis in human A431 cells, and found that Myo18A could be cleaved in response to the two apoptotic stimuli. To determine whether Myo18A is cleaved by caspase3, I performed an in vitro protease assay using recombinant caspase-3 to process Myo18A-GFP fusion protein. I further confirmed the presence of two fragments during apoptosis using a C-terminally tagged Myo18A-GFP construct. As the consensus caspase-3 cleavage sequence is DXXD, and Myo18A was cleaved into two fragments (222 kDa and 8 kDa), we deduced the putative cleavage site and generated two point mutants. Among the two mutants, the D1981N was resistant to cleavage during apoptosis. Taken together, these results demonstrated that the DRVD1981 sequence in Myo18A underwent caspase3-mediated cleavage during apoptosis. Finally, I generated NF-Myo18A(1-1981) and CF-Myo18A(1982-2054) truncation mutants that mimic the cleavage products of Myo18A. I found that NF-Myo18A could induce apoptosis when transfected into A431 cells, and that CF-Myo18A could be localized to the nucleus.