no longer be bound by Rpr or Hid, supporting a role for direct physical interactions
between the proteins.
The Drosophila caspases Dronc, Dcp-1, and drICE are found to bind to DIAP1
(Kaiser et al., 1998; Hawkins et al., 1999; Meier et al., 2000). DIAP1 binds to the
proform of Dronc and Dcp-1, and to the cleaved, activated form of drICE. Diap1
binding inhibits the activity of these caspases. Thus, DIAP1 is probably present in
many or all cells, blocking caspase activation and activity. Supporting this idea is the
finding that the Drosophila Apaf-1 homolog, Ark, is required for apoptosis resulting
from decreased levels of DIAP1 (Rodriguez et al., 2002; Zimmermann et al., 2002).
Apaf-1 in mammalian cells promotes the activation of certain caspases (see Chapter 7).
If DIAP1 is required to block the activity of caspases activated by Ark, the absence
of Ark could lower the general level of caspase activity in the cell, decreasing the
requirement for caspase inhibition.
Rpr, Grim, Hid, and SkI bind to the second BIR domain of DIAP1 through the
conserved 14-amino-acid stretch at their N-terminus (Vucic et al., 1998; Wang et
al., 1999c; Wu, J.W. et al., 2001; Srinivasula et al., 2002). Deletion of N-terminal
sequences abrogates the proapoptotic activity of Hid, as well as binding of Hid to
DIAP1 (Vucic et al., 1998). Deletion of this N-terminal motif from Rpr and Grim
does not completely eliminate the killing activity of Rpr and Grim. Surprisingly, both
of these truncated proteins still bind to DIAP1 when the proteins are co-overexpressed
(P.Bangs and K.White, unpublished observations; Vucic et al., 1998). This suggests
that additional domains of both Rpr and Grim may act as DIAP1 inhibitory motifs.
Other differences between the mechanisms of Rpr and Grim killing and Hid killing
are suggested by the finding that specific gain of function alleles of DIAP1 can inhibit
Rpr and Grim killing, but not Hid killing, or can inhibit Hid killing but have no
appreciable impact on Rpr or Grim killing (Lisi et al., 2000). These mutant proteins
might be predicted to show differential abilities to bind Rpr and Grim in comparison
with Hid.
Obvious sequence homologs of Rpr, Grim, and Hid have not been identified in
mammalian systems. However, two mammalian proteins have recently been shown
to act as functional homologs of these proteins. SMAC/Diablo and Omi/HtrA2 are
both mitochondrial proteins that can be released when apoptosis is induced (Du et
al., 2000; Verhagen et al., 2000; Suzuki et al., 2001; Hegde et al., 2002; Martins et
al., 2002; van Loo et al., 2002; Verhagen et al., 2002). Both proteins potentiate
caspase activation, and can bind to mammalian IAPs. Importantly, the first four
amino acids of the processed forms of SMAC/Diablo and Omi/HtrA2 are
homologous to the N-terminal IAP-inhibitory domains of Rpr, Grim, Hid, and Skl,
and bind to the same structural d omai n of the IAPs. It i s li kely that this IAP i nhi bi to ry
domain will also be found in other mammalian proteins.
188 GENETICS OF APOPTOSIS