CIAP1 Cooperatively Inhibits Procaspase-3 Activation by the Caspase-9 Apoptosome

Stephen P Burke,Lucinda Smith,Jeffrey B Smith

doi:10.1074/jbc.m110.125955

Stephen P Burke, Lucinda Smith + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m110.125955

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Abstract

Although early studies of inhibitor of apoptosis proteins (IAPs) suggested that cIAP1 directly binds and inhibits caspases similarly to X-linked IAP (XIAP), a recent one found that micromolar concentrations of cIAP1 only weakly inhibit caspase-3, -7, or -9. Here, we show that cIAP1 specifically and cooperatively blocks the cytochrome c-dependent apoptosome in vitro. Hence, cIAP1 prevented the activation of procaspase-3 but had no effect on the processing of procaspase-9 or the activity of prior activated caspase-3. Like cIAP1, XIAP had no effect on procaspase-9 processing and was a more potent inhibitor of procaspase-3 activation than of already activated caspase-3 activity. Inhibition of procaspase-3 activation depended on BIR2 and BIR3 of cIAP1 and was independent of BIR1, RING, CARD, and UBA domains. Smac prevented cIAP1 from inhibiting procaspase-3 activation and reversed the inhibition by prior addition of cIAP1. A procaspase-9 mutant (D315A) that cannot produce the p12 subunit was resistant to inhibition by cIAP1. Therefore, the N-terminal Ala-Thr-Pro-Phe motif of the p12 subunit of the caspase-9 apoptosome facilitates apoptosome blockade. Consequently, cIAP1 cooperatively interacts with oligomerized processed caspase-9 in the apoptosome and blocks procaspase-3 activation.

Highlights

The baculovirus IAP repeat (BIR) domains of cIAP1 were sufficient for the inhibition of procaspase-3 activation, which was independent of the RING and CARD domains of cIAP1
Micromolar concentrations of GST itself (Յ7 ␮M) or of GSTSurvivin (Յ6 ␮M) had no effect on cytochrome c-dependent apoptosome activation. It appears that X-linked IAP (XIAP) and cIAP1 may be sufficiently potent to regulate apoptosome activation intracellularly
Smac antagonized the inhibitory action of cIAP1 with an EC50 of 30 nM (Fig. 4A)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Transfection—911 cells were grown in Dulbecco’s modified Eagle’s medium/Ham’s F-12 (50:50) containing 10% FBS [25]. Apoptosome Assay of Caspase-3-like Activity and Western Blot Analysis—S100 or S64 (1–1.5 ␮g/␮l) was incubated for the indicated interval (15– 60 min) in buffer containing 25 mM Tris-HCl (pH 7.5), 50 mM KCl, 0.2 mM EGTA (pH 7.5), 1 mM DTT, and 5 ␮M PS-341 (bortezomib), a proteasome inhibitor, in the presence and absence of the indicated concentration of bovine cytochrome c (Sigma) and 1 mM Na2ATP (Sigma) unless indicated otherwise. Following incubation with or without cytochrome c and ATP, a 4-␮l sample was diluted with 36 ␮l of caspase assay buffer, which contained 25 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.2 mM EGTA, and 0.25 mM Ac-DEVD-AMC (a fluorogenic tetrapeptide substrate) [25]. Following a 30-min incubation at 37 °C, the caspase reaction was stopped by dilution with ice-cold buffer containing 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA. His10-cIAP1 and Smac56-His were purchased from R&D Systems

RESULTS

No of experiments nM

DISCUSSION