The anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Bcl-XL, play a central role in preventing the induction of apoptosis via the intrinsic apoptotic pathway. It has been previously shown that induction of apoptosis by the pro-apoptotic Bcl-2 family member Bok is not antagonized by either Bcl-2 or Bcl-xL, suggesting that Bok might have a unique role in the apoptotic cascade. We showed here that human Bok is the only member of the Bcl-2 family to have a leucine-rich sequence indicative of a nuclear export signal within its BH3 domain. Western blot analysis of nuclear and cytoplasmic fractions identified Bok in both the nucleus and the cytoplasm of HEK 293T cells, HeLa cells, and breast cancer cells, and its nuclear concentration increased after treatment of those cells with leptomycin B, an inhibitor of the exportin Crm1. Immunocytochemistry of flag-tagged Bok confirmed its nuclear localization. Mutating the nuclear export signal of Bok by site-directed mutagenesis resulted in an increase in its nuclear localization and apoptotic activity. We also found that Crm1 interacted with wild-type Bok but not with the mutated form. These results suggest that nuclear export of Bok is a regulated process mediated by Crm1, and constitutes the first report of a link between the apoptotic activity and nuclear localization of a pro-apoptotic member of the Bcl-2 family.
Copyright 2005 Wiley-Liss, Inc.