for the deletion that removes all three of these genes (Grether et al., 1995; Chen et
al., 1996; White et al., 1996). This demonstrates that each gene is sufficient to induce
apoptosis in the absence of the others. Expression of SkI appears to be a less potent
inducer of cell death, although it can enhance death induced by Rpr or Grim
(Christich et al., 2002; Srinivasula et al., 2002; Wing et al., 2002).
Analysis of mutant embryos lacking rpr, hid, and grim suggests that these proteins
are likely to act as initiators rather than effectors of apoptosis. When these mutant
embryos are exposed to high doses of x-rays, some apoptosis is induced (White et al.,
1994). Thus, at least some of the effector machinery is present in these embryos.
Caspase activation is essential for the proapoptotic activity of Rpr, Hid, Grim, and
Skl (Grether et al., 1995; Chen et al., 1996; White et al., 1996; Srinivasula et al.,
2002; Wing et al., 2002). Coexpression of the broad-spectrum caspase inhibitor p35
can block cell death induced by ectopic expression of all four genes.
How do Rpr, Hid, Grim, and SkI activate caspases to initiate apoptosis? The first
clue came from experiments in the laboratory of Lois Miller, demonstrating that Rpr,
Hid, and Grim were able to bind to IAPs (inhibitor of apoptosis proteins), a family
of antiapoptotic proteins (Vucic et al., 1997 ; 1998). Originally identified in
baculovirus, antiapoptotic IAPs are found in insects and mammals (reviewed in
Chapter 3). IAPs are characterized by the presence of one to three copies of a conserved
Zn binding motif called the baculovirus IAP repeat or BIR domain. In addition,
many IAPs also contain a C-terminal ring-finger domain. Both of these motifs appear
to be essential for IAP function. IAPs exert their antiapoptotic activity by inhibiting
caspases.
Three IAPs DIAP1, DIAP2, and Deterin, have been characterized in Drosophila
(Hay et al., 1995; Jones et al., 2000). Overexpression of DIAP1 or DIAP2 has been
shown to suppress apoptosis induced by overexpression of Rpr, Hid, or Grim in flies
and in cultured cells (Hay et al., 1995; Vucic et al., 1997, 1998). In cultured cells,
apoptosis induced by overexpressed Rpr can be suppressed by overexpression of
Deterin as well (Jones et al., 2000). DIAP1 can also partially inhibit apoptosis
resulting from SkI expression in insect cells (Srinivasula et al., 2002; Wing et al., 2002).
A role for DIAP2 or Deterin in inhibiting developmental apoptosis has not been
demonstrated. However, decreased levels of DIAP1 enhance killing by ectopically
expressed Rpr, Grim, and Hid (Hay et al., 1995; Lisi et al., 2000). This suggests that
DIAP1 is necessary to prevent apoptosis. Strong support for this hypothesis comes
from the phenotype of embryos homozygous for DIAP1 mutations. Loss of DIAP1
function results in massive apoptosis early in embryogenesis (Wang, S.L. et al., 1999;
Goyal et al., 2000; Lisi et al., 2000). Importantly, the apoptosis resulting from the
absence of DIAP1 is not dependent on Rpr, Grim, or Hid function. DIAP1 is
therefore required to inhibit cell death, and Rpr, Hid, and Grim inhibit DIAP1
function.
Direct physical interaction between Rpr, Grim, Hid, or Skl and DIAP1 is likely
to be required for inhibition of DIAP1 antiapoptotic activity. Mutant alleles of
DIAP1 have been identified that block apoptosis induced by ectopic expression of
Rpr and Hid (Goyal et al., 2000). These mutations result in DIAP1 proteins that can
APOPTOSIS IN DROSOPHILA 187