Sister chromatid cohesion during DNA replication plays a pivotal role in accurate chromosomal segregation in the eukaryotic cell cycle. Rad21 is one of the major cohesin subunits that keeps sister chromatids together until anaphase when proteolytic cleavage by separase allows the chromosomes to separate. Mitotic cleavage sites in Rad21 in yeast as well as in humans have been mapped (12
). Here we show that hRad21 cleavage occurs during apoptosis and is induced by various agents, including DNA-damaging (ionizing radiation and topoisomerase inhibitors) and non-DNA-damaging agents (cycloheximide treatment, cytokine withdrawal, and treatment with proteasome inhibitors). We have biochemically mapped the apoptotic cleavage site in human Rad21 (PDSPD279
/S), which is distinct from the mitotic cleavage sites (DREIMR172
/E and IEEPSR450
/L) previously described (12
). The apoptotic cleavage site is conserved among vertebrate species, and it is likely that cleavage is mediated by a nuclear caspase or caspase-like molecule, as this cleavage site bears the characteristic caspase-3 subfamily recognition motif (DXXD) and hRad21 is cleaved in vitro by the two major apoptosis executioner caspases, caspase-3 and caspase-7. hRad21 cleavage is not restricted to transformed cancer cells, as induction of apoptosis resulted in hRad21 cleavage in the primary cell line IMR90 (data not shown) as well as the nontransformed immortal cell line EL-12.
Cleavage of hRad21 appears to be an early event in the apoptotic pathway. The immunofluorescence experiments and Western blot analysis of nuclear and cytoplasmic fractions of cells undergoing apoptosis demonstrate the translocation of the hRad21 C-terminal cleavage products to the cytoplasm early (3 to 4 h after insult) in apoptosis. Our results clearly show that hRad21 proteolysis by a caspase family protease at D279/S leads to the production of a proapoptotic C-terminal cleavage product. The specific protease that cleaves hRad21 in vivo and promotes hRad21-induced apoptosis is yet to be identified. Nuclear changes determined by Annexin V staining and examination of the morphology of DAPI-stained nuclei indicate a strong temporal relationship between hRad21 cleavage and apoptosis. As determined by Annexin V staining, hRad21 cleavage correlates well with the early events of apoptosis when the cell membrane remains intact. Furthermore, the progressive increase in the cleavage of hRad21 correlates well with the level of caspase activation, as determined by assaying of by caspase-3 activity. Translocation of the 64-kDa hRad21 cleavage product to the cytoplasm early in apoptosis may act as a nuclear signal that promotes and accelerates subsequent events of apoptosis. The specificity of this product was determined further, as the N-terminal hRad21 cleavage product neither translocates nor has the ability to induce apoptosis. We have not explored the role of the 60-kDa hRad21 product generated at a cleavage site other than D279/S in the apoptotic process.
The physiological significance of cohesin hRad21 cleavage in apoptosis is intriguing. The nuclear signal(s) that detects subsequent events of apoptosis in the cytoplasm and mitochondria has remained elusive. It is possible that cleavage of hRad21 at the onset of apoptosis and the translocation of the C-terminal cleavage product to the cytoplasm act as cues to accelerate the apoptotic process. Supporting evidence in favor of this possibility include the following: (i) hRad21 is not normally cytoplasmic; (ii) early in apoptosis, hRad21 is found in the cytoplasm; and (iii) directed expression of either the C-terminal or full-length hRad21 to the cytoplasm induces apoptosis. It is not clear whether localization of C-terminal hRad21 to the cytoplasm is due to an active or a passive transport process following cleavage. The carboxy-terminal fragment contains a putative nucleolar localization signal sequence, which argues against a passive transport process. These findings further strengthen the notion that the translocation of the C-terminal hRad21 protein to the cytoplasm may play a functional role in apoptosis.
We have firmly established the proapoptotic activity of the C-terminal hRad21 cleavage product by several apoptotic assays, including Annexin V staining, TUNEL methods, quantitative measurement of DAPI-stained nuclear morphology, and assaying of caspase-3 activity. However, the exact mechanism by which cleaved hRad21 induces apoptosis requires further investigation. It is interesting that a BLAST search of the apoptosis database (www.apoptosis-db.org
) indicated that C-terminal hRad21 possesses a stretch of 80 aa (aa 282 to 362) that has homology to the tumor necrosis factor receptor superfamily and other apoptosis-inducing proteins, including TRAIL-R2 and death receptor 5. However, the functional significance of this domain in apoptosis-inducing proteins is not known.
The caspase-mediated proteolysis of hRad21 and the partial removal of hRad21 from the nucleus may also expose the chromosomal DNA to DNase and other proteins responsible for chromatin condensation and apoptotic DNA fragmentation. hRad21 was originally isolated in fission yeast as an essential protein with a role in the repair of DNA double-strand breaks induced by ionizing radiation (2
). It is therefore logical to think that disruption of the DNA repair function of hRad21 may be necessary during the execution of apoptosis. This notion has been strengthened by recent findings that a number of DNA repair enzymes such as Rad51 (15
), ATM (13
), DNA-PK (4
), and PARP (22
) and cell cycle regulators such as retinoblastoma protein (9
) are cleaved by caspases. Coordinated destruction of the DNA repair machinery and cell cycle regulators by the caspase family of proteases therefore constitutes a physiologically relevant process that promotes and accelerates chromosomal condensation and DNA fragmentation without interference by the cell cycle and DNA repair machinery. Unlike hRad21, however, cleavage products of these other DNA repair proteins have not been reported to play a direct role in promoting apoptosis. In this case, cleavage of hRad21 by caspases may play a unique role in amplifying the apoptotic signal by elevating the level of caspase activity. A similar mechanism for amplifying the apoptotic signal for the caspase substrate vimentin has recently been described (3
The p53 tumor suppressor protein plays a central role in the regulation of the cell cycle and apoptosis after DNA damage (17
). In the event that DNA damage is more severe and not repairable, p53 directs the cells into apoptosis through the Bax/Bcl-2 pathway. p53 status does not appear to have any effect on the apoptotic cleavage of hRad21 after DNA damage (i.e., UV and ionizing radiation), indicating the lack of involvement of the p53 pathway in hRad21 cleavage. It is possible that a parallel p53-independent pathway may regulate the genotoxic-damage-induced cleavage of hRad21.
Finally, it is interesting that cleavage of cohesin hRad21 is carried out by a separase in mitosis and by a caspase in apoptosis at different sites in the protein. Both of these proteases belong to the distantly related CD clan protease family (38
), suggesting an evolutionarily conserved mechanism shared by the mitotic and apoptotic machinery. hRad21 may serve as the link between the two key cellular processes of mitosis and apoptosis. In summary, in contrast to the previously described functions of Rad21, i.e., in chromosome segregation and DNA repair, cleavage of the cohesion protein and translocation of the C-terminal cleavage product to the cytoplasm are early events in the apoptotic pathway that amplify the apoptotic signal in a positive-feedback manner, possibly by activating more caspases. These results provide the framework for identification of the physiologic role of hRad21 in the apoptotic response in normal and malignant cells.