© 2005 The Japanese Biochemical Society
BIOCHEMISTRY |
Cleavage of Nonmuscle Myosin Heavy Chain-A during Apoptosis in Human Jurkat T Cells
Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639
1 To whom correspondence should be addressed. Tel: +81-3-5449-5311, Fax: +81-3-5449-5491, E-mail: ohmi{at}ims.u-tokyo.ac.jp
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.
* Present address: Department of Biochemistry, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666.
Present address: Department of Molecular Neurobiology, Tokyo Institute of Psychiatry, 2-1-8, Kamikitazawa, Setagaya-ku, Tokyo 156-8585.
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