cIAP1 binds to and induces loss of endogenous XIAP. (a) Stable isogenic cell lines expressing cIAP1 or EGFP were established using the Flp-In T-REx system, and were induced for 30 h with doxycyclin. Lysates were immunoprecipitated with anti-FLAG beads and blots probed with anti-XIAP, anti-Flag, and anti-TRAF2. The experiment was repeated with three independently generated cIAP1 stable lines. (b) Degradation of endogenous XIAP induced by the minimal RING domain. Stable isogenic cell lines were cultured for 16 h with or without doxycyclin to induce expression of the relevant construct (second blot). Lysates alone were run on 4–20% gradient SDS/PAGE gels and blotted with anti-XIAP, anti-HSP70, anti-Bcl-w (mbw), anti-c-cbl, anti-TRAF2, and anti-TRAF4. (c) Stable cell lines expressing mbw or mbw cIAP1 RING were induced for the indicated times, and lysates were probed as before. (d) The more highly expressed cIAP1 RING fusion is more effective at promoting degradation of XIAP than full length cIAP1. Note that the lysates were probed with an antibody raised to ML-IAP RING that crossreacts with cIAP1 RING. *, Nonspecific cross-reactive.

The C-terminal six residues of cIAP1 are essential for binding to and degradation of XIAP. (a) Inducible isogenic stables stable cell lines were generated with TRAF2, EGFP cIAP1 RING, or EGFP cIAP1 RING ΔC mutant constructs and treated for 16 h with or without doxycyclin. *, Nonspecific cross-reactive band. (b) EGFP cIAP1 RING ΔC mutants are unable to interact with full length XIAP. Cells were transiently transfected with equal amounts of Flag-tagged XIAP and EGFP cIAP1 RING constructs, lysates used for Flag bead immunoprecipitation (IP) were run on SDS/PAGE gels, and membranes were blotted with anti-GFP and then subsequently with anti-Flag. *, anti-Flag nonspecific cross-reactive band. (c) XIAP proteins lacking either the RING or XIAP BIR1 are unable to interact with cIAP1 RING. 293T cells were transiently transfected with 0.6 μg of XIAP domain constructs and 0.4 μg of EGFP cIAP1 RING constructs and lysates and IPs were run on SDS/PAGE gels and transferred to poly(vinylidene difluoride) membranes, and blotted with anti-GFP, and then subsequently with anti-Flag. *, The residual EGFP cIAP1 RING signal from previous anti-GFP blot. (d) Recombinant RING proteins fused to GST were purified and incubated with recombinant XIAP RING in PBS buffer and 0.2% Tween. XIAP ring was detected with anti-His.

Lineup of RING fingers and a model for XIAP or cIAP1 RING–RING interaction. A subset of 13 (from >50 RING finger proteins) was selected to emphasize the conservation across large phylogenetic differences of the C-terminal extension of certain RING fingers, exactly 13 amino acids after the final zinc-coordinating cysteine. For comparison, three RING finger proteins whose levels were not decreased with cIAP1 RING (c-cbl, TRAF2, and TRAF4), and which have different flanking sequences, are shown. Lowercase, RING consensus residues; uppercase, flanking residues of RING; yellow, similar charged residues; pink, similar polar residues; gray, similar hydrophobic; white on black, conserved zinc-coordinating residues. Structures from 1JM7 (24) showing the BARD1 BRCA1 RING–RING interaction, produced with rasmol (16). A schematic showing a speculative model for the C-terminal 13 amino acids of cIAP1 RING interacting with the N-terminal helix preceding the XIAP RING domain.

XIAP regulation by cIAP1 is proteasome-dependent and does not occur at the level of transcription. (a) Inducible isogenic stable cell lines mbw, mbw cIAP1 short RING, and mbw XIAP RING constructs were treated for 16 h with or without doxycyclin to induce expression of the relevant construct. RNA was isolated and Northern blot performed with the XIAP RING probe. (b) Proteasomal inhibitors impair cIAP1-induced degradation of endogenous XIAP. Stable cell lines expressing EGFP cIAP1 RING or EYFP cIAP1 RING were induced with or without doxycyclin for 8 h. LLnL (50 μM), PS341 (1 μM), or zVAD-fmk (50 μM) were added at the same time as the doxycyclin for the period of the induction. (c) Stable cell lines expressing EYFP cIAP1 RING or EYFP cIAP1 RING ΔC were either induced or not and then lysed directly in 8-M urea lysis buffer. The lysates were run on an SDS/PAGE gel and probed with antibodies to XIAP or cIAP1.

cIAP1 RING sensitizes melanoma cell lines to cisplatin. (a) Melanoma cell lines were transiently transfected with pcDNA5 FRT TO EGFP, EGFP cIAP1 RING, or EGFP cIAP1 RING ΔC mutants together with pcDNA6 Tet repressor at a ratio of 2:3. After ≈16 h, doxycyclin was used to induce expression of the relevant construct. Where indicated, 24 h after transfection, the induced cells were treated for a further 16 h with 8 μg/ml (ColoF, WW, SKMel28, and RM) or 4 μg/ml (A2058) cisplatin. Cells were then harvested, stained for annexin V/propidium iodide uptake, and run on a FACS scanner. The means of the results of three (ColoF, A2058), four (WW, SKMel28), and five (RM) independent experiments are represented as mean ± SD. (b) Stable inducible WW, RM, and Sk Mel28 cells were induced to express EGFP cIAP1 RING or EGFP cIAP1 RING ΔC for 24 h. Western blots indicate depletion of XIAP by the expressed constructs.