Apaf-1XL is an inactive isoform compared with Apaf-1L

Apaf-1 plays a crucial role in the cytochrome c/dATP-dependent activation o f caspase-9 and -3. We found that the human myeloid leukemic K562 cells wer e more resistant to cytochrome c-induced activation of caspase-9 and -3 in a cell-free system compared with the human T-lymphoblastic subclone CEM/VLB 100 cells. Apaf-1 cDNA sequencing revealed an additional insert of 11 aa be tween the CARD and CED-4 (ATPase) domains in K562 cells, which was identica l to the sequence of Apaf-1XL. Immunoprecipitation of Apaf-1 with caspase-9 after a cell-free reaction demonstrated that Apaf-1XL in the K562 cell lin e showed a lower binding ability to caspase-9 compared with Apaf-1L protein . The resistance of K562 cells to cytochrome c-dependent apoptosis may be p artly due to this Apaf-1XL form. These results suggest that the additional insert between CARD and CED-4 domains might affect Apaf-1 recruitment of ca spase-9 during apoptosis. (C) 2001 Academic Press.