1. Volume 42, Issue 5, Pages 569-583 (June 2011)
CARD-Mediated Autoinhibition of cIAP1's E3 Ligase Activity Suppresses Cell Proliferation and Migration 
Juanita Lopez, Sidonie Wicky John, Tencho Tenev, Gilles J.P. Rautureau, Mark G. Hinds, Floriana Francalanci, Rebecca Wilson, Meike Broemer, Massimo M. Santoro, Catherine L. Day, Pascal Meier 
Molecular Cell 
Volume 42, Issue 5, Pages (June 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2011 42, 569-583DOI: (10.1016/j.molcel.2011.04.008)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1 Sequence and Structure of cIAP1's CARD
(A) Schematic diagram of the modular domain architecture of cIAP1 protein.
(B) Sequence alignment and secondary structure of the CARD from vertebrate cIAP1. Limits of the α helices for human cIAP1 CARD by colored bars and indicated α1–α6. Protein sequences were aligned in Clustal. Asterisks indicate identical residues, colons highly conserved, and periods weakly conserved. Hs, Homo sapiens; Mm, Mus musculus, Xl, Xenopus laevis; Gg, Gallus gallus; Dr, Danio rerio. Sequence numbering above the sequences is for the human sequence.
(C) Ribbon representation of the human cIAP1 CARD structure (residues 454–543). The colors of the helices match those shown in (B).
(D) Molecular surfaces of the same residues as shown in (C), with identical and highly conserved residues as indicated in (B) colored cyan and pink, respectively. Identical residues are labeled. The surface represented in the left panel has the identical orientation as the ribbon representation and the right panel represents a 180° rotation.
See also Figures S1 and S4.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
The CARD Inhibits Spontaneous Activation of cIAP1's E3 Ligase Activity
(A) Schematic diagram of the Flp-In™T-REx™-HEK293shcIAP1 cell system in which endogenous cIAP1 can be knocked down via inducible expression of mir30-based short hairpin RNA targeting cIAP1's 3′ UTR. These cells also carry a single FRT site that allows Flp-mediated integration of transgenes into the same transcriptionally regulatable genomic locus. Expression of the transgene and shcIAP1 is induced after treatment with Doxocycline. TRE, tetracyclin response element; UBC, ubiquitin promoter; FRT, flippase recognition target; Tet Op, tetracycline operon; Tet-R, tet repressor protein; rtTA3, reverse Tet transactivator (rtTA3).
(B) Expression analysis of the indicated isogenic Flp-In™T-REx™-HEK293shcIAP1 cell lines with the indicated antibodies.
(C) In vivo ubiquitylation assay. Cells were transfected with His-tagged Ub, and ubiquitylated proteins were purified under denaturing conditions and analyzed by immunoblotting.
(D) CARD mutant cIAP1 possesses a significant shorter protein half-life. CHX chase experiment. Expression of the indicated transgenes was induced for 72 hr, after which cells were treated with CHX and analyzed by immunoblot analysis. The graph depicts the levels of cIAP1 protein expressed in % of their levels at the 0 time point. All values are normalized to Actin. Note, CHX treatment is toxic at 24 hr, hence the loss of Actin at this time point.
(E) Dox washout experiment. Cells were incubated in Dox for 72 hr after which cells were washed and further incubated in the absence of Dox for the indicated time points.
See also Figures S2 and S3.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
The CARD Mediates Autoinhibition through an Electrostatic Intramolecular Interaction that Prevents E2 Binding
(A) Schematic diagram of the constructs used. Below is the sequence of the CARD of human cIAP1; mutated basic and acidic residues are depicted in blue and red, respectively.
(B) Ribbon and surface representation of cIAP1 CARD depicting the position of the mutated basic and acidic residues.
(C and D) Yeast two-hybrid analysis studying the interaction between cIAP1 and UbcH5b. TRAF2 was used as a positive control, while pACT2 and pGBT9 were used as negative controls. Three single colonies for each cotransformation were patched out on fresh SD-Leu-Trp plates and subsequently replica plated onto nonselective (SD-Leu-Trp) or selective medium (SD-Leu-Trp-His, containing the indicated 3AT concentration).
See also Figures S3 and S4.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
The CARD Autoinhibits cIAP1's E3 Ligase Activity by Suppressing RING Dimerization and E2 Activation
(A) Schematic diagram of the cIAP1 fragments used in the in vitro assays.
(B) ΔCARD mutants are significantly more active in promoting autoubiquitylation than cIAP1B3/U/C/R. Samples of cIAP1B3/U/C/R (residues 255–618 of human cIAP1) and cIAP1B3/U/R (ΔCARD) were mixed with E1, UbcH5b, and Ub for the indicated time points and analyzed with SDS-PAGE to resolve the ubiquitylated species. The monoubiquitylated species are indicated by asterisks.
(C) E2∼Ub discharge assay. Wild-type cIAP1B3/U/C/R is less effective than cIAP1B3/U/R (ΔCARD) at promoting Ub hydrolysis.
(D and E) cIAP1B3/U/R (ΔCARD) but not cIAP1B3/U/C/R forms a more stable dimer. MALLS analysis of samples of purified cIAP1B3/U/C/R and cIAP1B3/U/R at 10 μM (pink), 25 μM (black), and 100 μM (blue). Samples were analyzed as described in the Experimental Procedures. The calculated mass (squares in corresponding colors) and refractive index trace are shown. The dashed lines indicate the expected mass of monomeric and dimeric species.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
z-cIAP1ΔCARD Is Less Efficient than z-cIAP1WT in Rescuing tom Mutants Zebrafish
(A) Embryos from tom heterozygote intercrosses were injected at the one-cell stage with the indicated amount of mRNA encoding wild-type or z-cIAP1ΔCARD. Histograms show the pe arercentage of rescued z-cIAP1-deficient animals identified at 72 and 96 hpf. Results are the average of five independent experiments. Error bars indicate the standard deviation (SD). p < 0.01 (72h pf) and p < 0.05 (96 hpf), respectively.
(B) z-cIAP1ΔCARD is less expressed than z-cIAP1WT. Equal amount of the indicated constructs were transfected into 293 cells, and their expression levels assessed by immunoblotting with the indicated antibodies. Similar results were obtained from experiments using zebrafish embryos (data not shown).
(C) Stereomicroscope images of the vascular network of tom (a) and wild-type (e) animals, and animals rescued by z-cIAP1WT (i) and z-cIAP1ΔCARD (m) are shown. For this experiment, Tg(kdrl:GFP)s843tom mutants were used that express GFP (green) in endothelial cells. Lateral view images of the caudal portion of tom (a) animals at 72 hpf showing vascular regression. In contrast, normal blood vessel architecture of wild-type (e) and rescued z-cIAP1WT (i) and z-cIAP1ΔCARD (m) animals. The scale bar represents 250 μm. Confocal transverse sections of Tg(kdrl:GFP)s843 tom (b), wild-type (f), rescued z-cIAP1WT (j), and rescued z-cIAP1ΔCARD (n) animals at 72 hpf, stained for TUNEL (red) are also shown. Endothelial cells in tom mutants are positive for TUNEL (b and zoomed images in c and d), while WT (f and zoomed images in g and h), rescued z-cIAP1WT (j and zoomed images in k and l), and z-cIAP1ΔCARD (n and zoomed images in o and p) rescued animals display no TUNEL staining. Scale bars represent 50 μm.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
cIAP1ΔCARD Is More Effective in Restoring Expression of the Myc-Luciferase Reporter Gene and Promoting Cell Proliferation
(A and B) SM-mediated inhibition of cIAPs suppresses expression of a Myc-Luciferase reporter gene. 293T cells were left untreated or treated with the indicated SM or TNF (B) and assayed for reporter activity. Error bars indicate the SD.
(C and D) Knockdown of cIAP1 (C) reduces Myc-Luciferase reporter activity, while expression of cIAP1WT and cIAP1ΔCARD (D) promotes reporter gene expression. Notably, cIAP1ΔCARD is more potent in driving Myc-Luciferase reporter activity than wild-type cIAP1, even though it is less expressed than cIAP1WT (right panel). The expression of the indicated proteins was evaluated by immunoblotting with the indicated antibodies.
(E) cIAP1ΔCARD is more efficient in targeting Mad1 for degradation in a RING-finger dependent manner.
(F) Equal numbers of stably reconstituted cIAP1/cIAP2-DKO MEFs were seeded, and cell proliferation was measured after the indicated time points by ATP assay using CelltiterGlo. Error bars indicate the standard error (SE).
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
CARD-Mediated Autoinhibition of cIAP1 Suppresses Cell Migration
(A) Knockdown of cIAP1, like knockdown of caspase-8 (Barbero et al., 2009), suppresses cell migration. Stereomicroscope images of MCF-7 cells incubated with the indicated siRNA oligos and assayed for their ability to close a defined 500 μm gap in a cell migration assay. Note that for scratch assays used in this experiment, we used defined culture inserts (IBIDI) the removal of which generates a 500 μm gap between cells.
(B and C) SM-mediated depletion of cIAPs suppresses cell migration in MCF-7 cells (B) and wild-type MEFs (C). Cells were incubated in the presence and absence of the indicated SM and the gap width measured over time, and depicted in the graph to the right. Results are the average of triplicate measures of a representative experiment. Error bars represent the SE. Asterisks indicate p < ns, not significant.
(D) cIAP1/cIAP2 DKO MEFs reconstituted with cIAP1ΔCARD close the gap faster than the ones reconstituted with cIAP1WT. Of note, for this experiment we used pools of cells expressing comparable levels of cIAP1.
(E) Expression of the caspase-8 inhibitor CrmA suppresses cell migration.
(F) Treatment with the RIP1-kinase inhibitor Necrostatin-1 (40 μM) rescues the migration defect of DKO MEFs.
(G) cIAP1ΔCARD is more efficient in targeting RIP1 for degradation in a RING-finger dependent manner.
(H) Model depicting the role of the CARD in keeping cIAP1 in a closed, inactive conformation, see the main text for details.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions