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.
Kato
Masahiko
M
Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639.
Fukuda
Hiroyuki
H
Nonaka
Takashi
T
Imajoh-Ohmi
Shinobu
S
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D017209:Apoptosis; D002135:Calcium-Binding Proteins / Q000276:immunology; D002154:Calpain / Q000037:antagonists & inhibitors; D006801:Humans; D019169:Jurkat Cells; D018613:Microscopy, Confocal; D020409:Molecular Motor Proteins / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D018995:Myosin Heavy Chains / Q000276:immunology / Q000378:metabolism*; D010455:Peptides / Q000032:analysis; D061988:Proteasome Inhibitors
D000906:Antibodies; D002135:Calcium-Binding Proteins; C103814:MYH9 protein, human; D020409:Molecular Motor Proteins; D010455:Peptides; D061988:Proteasome Inhibitors; C033668:calpastatin; D002154:Calpain; D018995:Myosin Heavy Chains