Abstract
In apoptotic cells, XRCC4 is cleaved by caspase-3 or -7 to form a 35 kDa-fragment (pN35) which is composed of the N-terminal 265 residues. This fragment includes the DNA ligase IV binding domain, but not the nuclear localization signal, of XRCC4. To investigate a role of XRCC4 cleavage in apoptosis, we examined the effect of caspase-resistant XRCC4 on staurosporine (STS)-induced apoptosis. Apoptosis was assessed by immunoblotting analysis of the cleaved-form of caspase-3, and the TUNEL assay which detects the internucleosomal DNA fragmentation. We established a XRCC4-deficient cell line M10, which is derived from a murine lymphoma cell line L5178Y. Expression in M10 cells of wild type XRCC4, but not caspase-resistant XRCC4 (XRCC4D265A), was required for induction of apoptosis by STS. Furthermore, more cleaved-forms of caspase-8, and -9, the upstream caspases of caspase-3 in the caspase-activation cascade, were detected in cells expressing wild-type XRCC4 than cells expressing caspase-resistant XRCC4 after treatment of cells with STS. Expression of the pN35 in M10 cells did not enhance STS-induced apoptosis. However, expression of the pN35 fused to the nuclear localization signal (pN35-NLS) in M10 cells enhanced the STS-induce apoptosis. In both wild-type and caspase-resistant XRCC4-expressing cells, localization of XRCC4 and DNA ligase IV changed from nucleoplasm to cytoplasm during the progression of apoptosis. These results revealed that pN35 augments apoptosis when it is located in the nucleus, and suggested that pN35 activates cleavage of both caspase-8 and -9.
