Characterization of interior cleavage of retinoblastoma protein in apoptosis

Previously we reported that at the onset of apoptotic execution, retinoblastoma protein (RB) was cleaved in its interior region, resulting in production of two major fragments, p48 and p68, and that the RB interior cleavage was mediated by a caspase-like activity. Here, we further characterized the RB interior cleavage process in human leukemia cells treated with the anticancer agent etoposide. We found that the RB interior cleavage activity was much more sensitive to two specific tetrapeptide caspase inhibitors, YVAD-CMK and DEVD-FMK, than the poly(ADP-ribose) polymerase cleavage activity, suggesting that two distinct caspases are involved in these processes. Several Asp residues are located in amino acids 341-421 of RB protein, and cleavage of any one of these sites by a caspase would generate a p48, which contains the amino terminus, and a p68 fragment, which contains the A/B pocket and the carboxyl terminus. This hypothesis was supported by the fact that the p48 and p68 fragments had selective binding affinity to different RB antibodies and that the p48 was found only in the low-salt-extracted cytoplasmic fraction, while the p68 was only in the nuclear fraction, of the apoptotic cells. However, the nuclear binding partner of the p68 RB fragment is not the transcription factor E2F-1 since a specific E2F-1 antibody coimmunoprecipitated only the unphosphorylated form of RB, but not the p68 fragment. Lastly, we confirmed that RB also underwent dephosphorylation and carboxyl terminal cleavage during apoptosis, as we and others reported previously.