| Abstract: | Inhibitor of apoptosis (IAP) proteins are widely expressed throughout
nature and suppress cell death under a variety of circumstances. X-linked IAP,
the prototypical IAP in mammals, inhibits apoptosis largely through direct
inhibition of the initiator caspase-9 and the effector caspase-3 and -7. Two
additional IAP family members, cellular IAP1 (cIAP1) and cIAP2, were once
thought to also inhibit caspases, but more recent studies have suggested
otherwise. Here we demonstrate that cIAP1 does not significantly inhibit the
proteolytic activities of effector caspases on fluorogenic or endogenous
substrates. However, cIAP1 does bind to caspase-3 and -7 and does so,
remarkably, at distinct steps prior to or following the removal of their
prodomains, respectively. Indeed, cIAP1 bound to an exposed IAP-binding motif,
AKPD, on the N terminus of the large subunit of fully mature caspase-7,
whereas cIAP1 bound to partially processed caspase-3 in a manner that required
its prodomain and cleavage between its large and small subunits but did not
involve a classical IAP-binding motif. As a ubiquitin-protein isopeptide
ligase, cIAP1 ubiquitinated caspase-3 and -7, concomitant with binding, in a
reaction catalyzed by members of the UbcH5 subfamily (ubiquitin carrier
protein/ubiquitin-conjugating enzymes), and in the case of caspase-3,
differentially by UbcH8. Moreover, wild-type caspase-7 and a chimeric
caspase-3 (bearing the AKPD motif) were degraded in vivo in a
proteasome-dependent manner. Thus, cIAPs likely suppress apoptosis, at least
in part, by facilitating the ubiquitination and turnover of active effector
caspases in cells.Apoptosis is a programmed form of cell death that is generally executed
through the activation of
caspases,2 cysteine
proteases that exhibit an almost absolute preference for cleavage after
aspartate residues. Caspases are synthesized as single-chain zymogens,
containing a prodomain, as well as large and small subunits that include
residues required for substrate recognition and cleavage
(1). During death receptor or
mitochondria-dependent apoptosis, the long prodomain-containing initiator
caspase-8/10 and -9 are recruited via their adapter proteins, Fas-associated
death domain and apoptotic protease-activating factor-1 (Apaf-1), to
multimeric caspase-activating complexes known as the death-inducing signaling
complex and the apoptosome, respectively
(1,
2). In the latter case,
mitochondrial outer membrane permeabilization (MOMP) is required to mediate
the release of cytochrome c from the intermembrane space into the
cytosol, where it stimulates dATP/ATP-dependent oligomerization of Apaf-1 into
the apoptosome (2). Once
recruited, all initiator caspases are concentrated within their respective
complexes and are thought to be activated as a result of dimerization, with
concomitant autocatalytic cleavage of the activation loops that separate their
large and small subunits (1).
However, unlike caspase-8 and -10, caspase-9 must remain bound to the
apoptosome to exhibit significant catalytic activity, so that in addition to
promoting dimerization, the apoptosome may also induce conformational changes
in caspase-9 that are necessary for its activation
(3–6).In contrast to initiator caspases, effector caspases, such as caspase-3 and
-7, contain short prodomains and exist normally as latent dimers, wherein
their activation loops sterically hinder substrate access and hold the
substrate binding pocket in an inactive conformation
(1). Effector caspases are
directly activated by caspase-8, -9, and -10, and following cleavage of
caspase-3 between its large and small subunits, the two-chain p20/p12 form
becomes a catalytically active heterotetramer and undergoes subsequent
autocatalytic processing between its prodomain and large subunits to generate
the fully mature p17/p12 form of the enzyme
(7). Similarly, procaspase-7 is
also activated following cleavage of its activation loop to generate its
two-chain p22/p12 form; however, it remains unclear whether removal of its
prodomain in cells (to generate its p19/p12 form) is accomplished primarily
via autocatalysis, active caspase-3, or perhaps by serine proteases at a
non-aspartate residue (8,
9). Caspase-3 and -7 exhibit
significant sequence and structural homology, differing primarily in their
short prodomains. Despite this fact, caspase-3 processes a wider array of
protein substrates during apoptosis and is largely responsible for dismantling
the cell (10). Thus,
interesting questions remain regarding the physiological roles of caspase-7,
whether caspase-7 activity is differentially regulated compared with
caspase-3, and what structural features determine (and in some cases limit)
its substrate specificity.Given the devastating consequences of unfettered caspase activation, cells
have evolved mechanisms to regulate caspase activity. For example, IAPs,
originally identified in baculoviruses, possess one or more baculovirus IAP
repeat (BIR) domains, and at least one of the eight family members, XIAP,
selectively inhibits the activities of caspase-9, -3, and -7
(1,
11). Mechanistically, the BIR3
domain in XIAP binds to an exposed IBM on the N terminus of the small subunit
of processed caspase-9, situated directly above the active site, and limits
the access of substrates (12,
13). By contrast, the linker
region (located between the BIR1 and BIR2 domains in XIAP) lies across the
active sites of caspase-3 and -7 and binds in a reverse orientation to
substrates, thereby preventing cleavage of the linker while simultaneously
preventing the access of substrates
(14,
15). The BIR2 domain then
stabilizes the linker-caspase-3 (and linker-caspase-7) interactions further by
binding to an exposed IBM on the N terminus of the small subunit in the
adjacent caspase dimer (14,
16). Importantly, IAP
antagonists, such as Smac/DIABLO and Omi/HtrA2, are normally sequestered to
the intermembrane space of mitochondria and are released (along with
cytochrome c) into the cytoplasm during apoptosis. As IAP antagonists
also possess IBMs, they bind to BIR domains and prevent or relieve the
inhibition of caspases by IAPs
(1).Previously, two additional IAP family members, cIAP1 and cIAP2, were also
thought to inhibit caspases, but more recent studies suggest that these IAPs
bind but do not inhibit caspases
(17–19).
Nevertheless, various studies have shown that cIAPs can protect cells from
apoptosis, are overexpressed or mutated in some cancers, and can promote
tumorigenesis
(20–25),
raising questions as to how these IAPs inhibit cell death or whether they have
additional functions (26).
XIAP, cIAP1, and cIAP2 possess C-terminal RING zinc finger domains with E3
ubiquitin (Ub) ligase activities capable of catalyzing the ubiquitination and
subsequent proteasomal degradation of cellular targets, including themselves
(27,
28). Moreover, cIAPs have been
shown to ubiquitinate several factors, including TNF receptor-associated
factor 2, the serine/threonine kinase NIK, receptor-interacting protein 1, and
the IAP antagonist Smac
(29–34).
However, although there is some evidence to support a direct role for
ubiquitination in the regulation of effector caspases by XIAP
(35,
36), the role of cIAPs in this
process remains unclear, particularly in vivo. We demonstrate herein
that cIAP1 binds to caspase-3 and -7 at unique steps in their processing,
prior to or following the removal of their prodomains, respectively. Moreover,
rather than directly inhibiting these effector caspases, cIAP1 ubiquitinates
them and targets them for proteasome-dependent degradation, thereby
suppressing apoptosis. |
