DISSECTION OF FUNCTIONAL DOMAINS IN BCL-2-ALPHA BY SITE-DIRECTED MUTAGENESIS

Bcl-2 alpha is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. How Bcl-2 confers cell survival is unknown, although antioxidant and antiprotease functions have been proposed. In addition, protein structures of Bcl-2 that are crucial for its survival activity are still ill-defined. Bcl-2 can occur as Bcl-2 alpha or Bcl-2 beta, two alternatively spliced forms which solely differ in their carboxyl termini. The finding that Bcl-2 alpha is active and membrane bound, but Bcl-2 beta is inactive and cytosolic, indicates that the carboxyl terminus contributes to the survival activity of Bcl-2. This region contains two subdomains, a domain X with unknown function and a hydrophobic stretch reported to mediate membrane assocation of Bcl-2 alpha Recently Bcl-2-related proteins have been identified. These include Bar that heterodimerizes with Bcl-2 and, when overxpressed, counteracts Bcl-2. Bar contains two highly conserved regions of sequence homology with Bcl-2, referred to as Bcl-2 homology 1 and 2 (BH1 and BH2) domains. Site-directed mutagenesis studies have revealed that both domains are not only novel dimerization motifs for the interaction of Bar with Bcl-2 but also crucial for the survival activity of Bcl-2. Interestingly, the C-terminal end of BH2 encompasses the Bcl-2 alpha/beta splice site, as well as part of domain X in Bcl-2 alpha. To better define the role of domain X and the hydrophobic C-terminal stretch of Bcl-2 alpha for its survival activity, we created various deletion and truncation mutations in these regions by site-directed mutagenesis. We show here that membrane attachment and therefore the hydrophobic stretch is not required for the survival activity of Bcl-2, but part of domain X appears to be indispensable.