Apoptosis-linked gene 2 binds to the death domain of Fas and dissociates from Fas during Fas-mediated apoptosis in Jurkat cells

Apoptosis-linked gene 2 (ALG-2) is a member of the family of Ca2+-binding p roteins with penta-EF-hand and is essential for the execution of apoptosis by various signals including Fas activation. We studied the regulation of A LG-2 during Fas-mediated apoptosis in Jurkat cells. The 22-kDa ALG-2 protei n is cleaved and becomes a 19-kDa protein after Fas activation. The appeara nce of 19-kDa ALG-2 protein increases for 4 h after treatment with 200 ng/m l of anti-Fas Ab treatment and gradually degrades afterward. Confocal micro scopic analysis showed that ALG-2 translocated from the plasma membrane to the cytosol during Fas-mediated apoptosis. Therefore, we examined if ALG-2 interacts with Fas. The protein-protein interaction of ALG-2 with Fas was d emonstrated using yeast two-hybrid assays as well as in vitro GST pull-down assay. Endogenous ALG-2 was immunoprecipitated with anti-Fas Ab in Jurkat cells without Fas activation. However, the endogenous ALG-2 was no longer i mmunoprecipitated with anti-Fas Ab 2 h after anti-Fas Ab treatment. This st udy, for the first time, presents a direct molecular connection of ALG-2 to apoptosis by its direct interaction with Fas, and enlists ALG-2 as a new m ember of posttranslationally modified proteins during Fas-mediated apoptoti c process. (C) 2001 Academic Press.