3.2
Sequestration at non-mitochondrial sitesNon-mitochondrial localization of proapoptotic BH proteins has become the rule
rather than the exception. Bid, one of the first described family members possessing
only a BH3 domain, lacks a signal/anchor sequence and is predominantly cytoplasmic
(Wang, K. et al., 1996). Caspase-8—or several other proteases, including granzyme
B (Barry et al., 2000) and lysosomal proteases (Stoka et al., 2001)- cleaves Bid to
yield a COOH-terminal, 15-kDa, truncated Bid (tBid), which then is translocated
to mitochondrial membranes (Li, H. et al., 1998; Luo et al., 1998). Phosphorylation
of Bid by casein kinases I and II at serine/threonine residues flanking the caspase-8
cleavage site inhibits caspase-mediated proteolysis (Desagher et al., 2001).
McDonnell et al. (1999) proposed that alterations in surface charge and
hydrophobicity of the truncated Bid are responsible for intracellular relocalization to
mitochondrial membranes. Mutations in the tBid BH3 domain, which disrupt
heterodimerization with Bcl-2 and Bcl-xL, did not interfere with trafficking of tBid
to mitochondria (Luo et al., 1998). Two studies suggest that mitochondrial targeting
of tBid does not require ‘receptor’ protein at the mitochondrial surface. Lutter et al.
(2000) examined the affinity of tBid for liposomes with different lipid compositions.
Maximal tBid binding was obtained only with liposomes containing >20%
cardiolipin, a mitochondria-specific phospholipid normally restricted to the inner
mitochondrial membrane. Intracellular targeting of tBid was also impaired in the
cardiolipin-deficient CHO mutant cell line PGS. Zha et al. (2000) reported post-
translational modification of tBid by myristoylation at an NH 2 -terminal glycine
generated after cleavage by caspase-8. Furthermore, the tight association of the two
tBid fragments (p7 and p15) after caspase-8-mediated cleavage prevented efficient
binding of non-myristoylated p15 tBid to mitochondrial membranes (Chou et al.,
1999; Zha et al., 2000). Thus, post-translational myristoylation appears to be required
for relocalization of the p7-p15 tBid complex to mitochondria. Since myristoylation
is also employed for trafficking to other membrane compartments, the specificity of
myristoylated tBid may reside in conformational effects on tBid (Zha et al., 2000).
The proapoptotic Bad protein also lacks a COOH-terminal signal/anchor
sequence but has two consensus 14–3-3 binding sites. Phosphorylation at serines 112
and 136 within the 14–3-3 binding site results in cytoplasmic sequestration of Bad
bound to 14–3-3 proteins. Bad phosphorylation occurs downstream of growth factor
signals and has been attributed to several kinases: protein kinase B/Akt (Ser^136 ) (Datta
et al., 1997; del Paso et al., 1997), mitochondrial-anchored protein kinase A (Ser^112 )
(Zha,J. et al., 1996; Harada et al., 1999), p70S6 kinase (Ser^136 ) (Harada et al., 2001),
and PAKl kinase (Schurmann et al., 2000). Ser^155 in the Bad BH3 domain is also
phosphorylated by protein kinase A (Datta et al., 2000; Virdee et al., 2000; Zhou et
al., 2000). Phosphorylation at this site inhibits BH3-dependent interactions of Bad
with antiapoptotic BH proteins. A fourth phosphorylation site, Ser^170 , also regulates
the proapoptotic activity of Bad through an unknown mechanism (Dramsi et al.,
2002). Complementary phosphatase activity has been alternatively ascribed to PPIA
MAKING SENSE OF THE BCL-2 FAMILY OF APOPTOSIS REGULATORS 65