tcBid, through destabilization of the mitochondrial membrane, could facilitate
insertion of the multidomain proteins and thereby promote channel formation.
Other ‘BH3-domain-only’ proteins have been shown to activate the multidomain
proteins in an indirect way. For example, unphosphorylated Bad binds to Bcl-2 and
Bcl-XL, neutralizing their antiapoptotic activity (Zha et al., 1997). Survival factors
such as interleukin-3 or NGF have been shown to induce phosphorylation of Bad.
Phosphorylated Bad is released from the complexes with the antiapoptotic proteins
Bcl-2 and Bcl-XL, allowing the proteins to interact with the multidomain proteins,
and prevent their proapoptotic activity. Another ‘BH3-domain-only’ protein, Bik,
has also been found to be phosphorylated; in this case, phosphorylation reduced its
proapoptotic activity (Verma et al., 2001). The ‘BH3-domain-only’ protein Bim is
bound to the microtubulin-associated dynein complex through interactions with the
LC8 dynein light chain in normal cells. After induction of apoptosis by various
stimuli, including UV irradiation, staurosporine, γ-irradiation, or growth factor
deprivation, Bim was released from the microtubuli complex (Puthalakath et al.,
1999). In the cytosol, Bim is thought to bind to antiapoptotic proteins, preventing
their function.
8.
How do the multidomain Bcl-2 proteins trigger the release of
proteins from the intermembrane space of the mitochondria?The first report indicating that mitochondria are essential for apoptosis was by
Newmeyer et al. (1994), who used a cell-free model system. It is now clear that the
proapoptotic Bcl-2 proteins execute their function at the mitochondria, although
considerable controversy persists over the molecular mechanisms. Bax is primarily
localized to the cytosol in normal tissues, but, after apoptotic stimulation, the protein
specifically translocates to the mitochondria (Hsu, Y.T. et al., 1997; Gross et al.,
1998; Zhang, H. et al., 1998). In contrast, Bak is localized to the mitochondrial
membrane at all times (Griffiths et al., 1999). Endogenous Bcl-Xs was recently shown
to be localized in the cytosol in PC12 cells; however, when the protein was
overexpressed, it was found associated with the mitochondria, suggesting that this
multidomain protein also functions at the mitochondria (Lindenboim et al., 2000).
Antiapoptotic proteins such as Bcl-2 are localized in several intracellular membranes,
including the mitochondria, ER, and nuclear envelope (Krajewski et al., 1993). This
poses the question of whether the antiapoptotic proteins might have additional
functions, perhaps even unrelated to apoptosis. At the mitochondria, the
multidomain proapoptotic proteins trigger the release of proteins from the
intermitochondrial space, including cytochrome c, apoptosis-inducing factor (AIF),
adenylate kinase, endonuclease G, and Smac/Diablo (Kluck et al., 1997; Kohler et
al., 1999; Daugas et al., 2000; Du et al., 2000; Verhagen et al., 2000; Li et al., 2001).
MITOCHONDRIA IN APOPTOSIS INDUCTION 131