Cleavage of nonmuscle myosin heavy chain-A during apoptosis in human Jurkat T cells.
We have previously reported that calpastatin, an endogenous inhibitory protein of calpain, is cleaved by a caspase-3-like protease during apoptosis in human Jurkat T cells [Kato, M. et al. (2000) J. Biochem. 127, 297-305]. In this study, we found that nonmuscle myosin heavy chain-A (NMHC-A) is cleaved during apoptosis in Jurkat cells by using a cleavage-site-directed antibody for calpastatin. The cleavage-site-directed antibody was raised against the amino-terminal fragment of calpastatin, and this antibody detected the in vitro cleaved calpastatin fragment. Although cleaved calpastatin was not detected, a 95-kDa polypeptide (p95) was detected in apoptotic cells by this antibody. This p95 was identified as the carboxyl-terminal fragment of NMHC-A based on the results of peptide mass spectrometry fingerprinting and amino-terminal sequencing. Furthermore, two cleavage sites on NMHC-A, Asp-1153 and Asp-1948, were determined, and three cleaved fragments of NMHC-A, one cleaved at Asp-1153 and the other two cleaved at Asp-1948, were detected by cleavage-site-directed antibodies against each cleavage site. The results of confocal immunofluorescence microscopic analysis show that the cleavage at Asp-1948 occurs faster than that at Asp-1153 during apoptosis. In addition, the Asp-1153 cleaved fragment was distributed diffusely in the cytoplasm of apoptotic cells, whereas the Asp-1948 cleaved fragments were detected as condensed dots. In conclusion, our findings can be summarized as follows: (i) NMHC-A is cleaved at two sites during apoptosis, (ii) the timing of cleavage is different between these two cleavage sites, and (iii) the distribution of cleaved fragments is different in apoptotic cells.[1]References
- Cleavage of nonmuscle myosin heavy chain-A during apoptosis in human Jurkat T cells. Kato, M., Fukuda, H., Nonaka, T., Imajoh-Ohmi, S. J. Biochem. (2005) [Pubmed]
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