a coiled coil domain with sequence similarity to the death effector domains (DEDs)
of procaspase-8 and -10, could weakly associate with procaspase-8 in cotransfected
cells (Ng and Shore, 1998). We have recently observed that in a physiologic setting
BAP31 can recruit a novel procaspase-8 isoform, procaspase-8L (Breckenridge et al.,
2002). Procaspase-8L is characterized by the Nex domain, a 59-residue domain that
extends procaspase-8/a at the N-terminus and facilitates a specific interaction with
BAP31. Procaspase-8L is localized at the cytosolic face of the ER membrane in resting
cells, and after apoptotic signaling by the E1A oncogene, endogenous procaspase-8L,
but not procaspase-8, is recruited to BAP31. Concomitant with this recruitment,
procaspase-8L undergoes processing by a mechanism inhibited by Bcl-2 but
independent of FADD, an adapter molecule required for recruitment and activation
of procaspase-8 at death receptors. Cells deficient in Bap31 and its cellular homolog,
Bap29, do not process procaspase-8L in response to E1A and show reduced caspase-8
and -3 activity, and apoptosis. Oncogene-induced apoptosis is also known to proceed
though the mitochondrial pathway (Soengas et al., 1999); therefore, BAP31-induced
caspase activation at the ER may cooperate with the apoptosome to bring about full
activation of downstream executioner caspases. Activation of Procaspase-8L by
BAP31 at the ER membrane may also place the caspase in a strategic location to cleave
BAP31 itself, an event that can mediate cross-talk between the ER and mitochondria
(below).
In addition to being a regulator of procaspase-8L, BAP31 is also a caspase-8
substrate (Ng et al., 1997). The cytosolic tail of human BAP31 encodes two identical
caspase recognition sequences that are rapidly cleaved by caspase-8 after activation of
the Fas death receptor (Nguyen, 2000; our unpublished data). Fas initiates apoptosis
by directly recruiting and activating procaspase-8 at the plasma membrane.
Caspase-8, in turn, cleaves the BH3-only molecule, BID, generating tBID, which
induces Cyt.c release from mitochondria and subsequent activation of downstream
caspases through the formation of the apoptosome (Korsmeyer et al., 2000). In some
cell types, Fas signaling does not require the participation of the mitochondria, and
caspase-8 can directly cleave and activate downstream caspases (Scaffidi et al., 1998).
We have found that stable expression of a caspase-resistant BAP31 (crBAP31) mutant
strongly inhibits Fas-induced apoptosis in KB cells (Nguyen et al., 2000), a cell line
that requires amplification through the mitochondria. While crBAP31 has a very
weak influence on Fas-induced caspase activation or cleavage of caspase substrates, it
strongly inhibits Cyt.c release, mitochondrial depolarization, cytoskeletal
reorganization, and membrane blebbing. The fact that crBAP31 inhibits these events,
in the face of caspase activation and BID cleavage, suggests that in intact cells the ER
exerts a restraint on apoptotic mitochondrial transition and cytoplasmic restructuring
that is overcome by BAP31 cleavage in the Fas pathway. Interestingly, caspase-cleaved
BAP31 (p20BAP31) autonomously induces apoptosis by a mitochondria-dependent
pathway (Breckenridge and Shore, unpublished), suggesting that caspase cleavage of
BAP31 converts it from an inhibitor to an activator of mitochondrial dysfunction.
This phenomenon has been observed with other antiapoptotic proteins such as Bcl-2
and Bcl-xL (Cheng et al., 1997; Clem et al., 1998).
THE ROLE OF THE ENDOPLASMIC RETICULUM IN APOPTOSIS 115