Functional discontinuities in prothymosin alpha caused by caspase cleavagein apoptotic cells

Our study examines the effect of apoptosis on prothymosin alpha, an abundan t, nuclear protein intimately involved with proliferation of all mammalian cells. When HeLa cells were treated with actinomycin D, with etoposide, or with staurosporine following synchronization with hydroxyurea, they underwe nt apoptosis based on several specific criteria, including fragmentation of DNA and activation of specific caspases. Similarly treated NIH3T3 cells ar rested and displayed no indicators of apoptosis. In HeLa, but not in NIH3T3 cells, prothymosin or levels declined precipitously and a truncated versio n of the protein was formed. The following observations implicate caspase a ctivity: (1) The truncated polypeptide arose only in the treated HeLa cell cultures. (2) The appearance of the truncated polypeptide coincided with th e activation of caspase 3 and the cleavage of poly(ADP-ribose) polymerase, a known caspase substrate. (3) Carbobenzoxy-DEVD-fluoromethylketone, a cell -permeable caspase 3 inhibitor, blocked cleavage and degradation of prothym osin alpha. (4) The same inhibitor, when added to mixed extracts of apoptot ic and normal cells, prevented cleavage of intact prothymosin alpha. (5) Re combinant caspase 3 and, to a much lesser extent, caspase 7 truncated purif ied prothymosin alpha. (6) In HeLa cells, cleavage occurred at three overla pping caspase 3-like sites with the consensus sequence D-X-X-D and released 10 to 14 residues from the carboxyl terminus, including the core nuclear l ocalization signal. Two immediate consequences of the cleavage were observe d: truncated prothymosin alpha was no longer confined to the nucleus and it was deficient in phosphate. These data suggest that the disabling of proth ymosin alpha is a significant event in apoptosis. Published 2000 Wiley-Liss , Inc.(dagger)