c-IAP1 Cooperates with Myc by Acting as a Ubiquitin Ligase for Mad1 Lei Xu, Jidong Zhu, Xiaofang Hu, Hong Zhu, Hyoung Tae Kim, Joshua LaBaer, Alfred Goldberg, Junying Yuan  Molecular Cell  Volume 28, Issue 5, Pages 914-922 (December 2007) DOI: 10.1016/j.molcel.2007.10.027 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Identification of Mad1 as a Substrate of c-IAP1 Ubiquitin Ligase In Vitro and In Vitro (A) The scheme of screening for ubiquitination substrates of c-IAP1. (B) 35S-labeled Mad1 was subjected to ubiquitination assay with purified GST or GST-c-IAP1 and analyzed by autoradiography. (C) GST-Mad1 was subjected to ubiquitination assay in the presence of GST-tagged c-IAP1, c-IAP2, or XIAP (each in 1 μM final concentration). The ubiquitinated Mad1 was detected by immunoblotting against Mad1. The levels of E3s were measured by western blotting using anti-GST antibody. (D) Schematic diagrams of the c-IAP1 protein domains and fragments used in the following coimmunoprecipitation experiments: FL, full-length; BIR, baculoviral IAP repeat (rectangle); CARD, caspase recruitment domain (oval); and R, RING domain (hexagon). (E) Expression vectors of Mad1 and Flag-tagged c-IAP1 or its mutated forms were transfected as indicated. Immunoprecipitated Mad1 was detected by western blotting. Western blotting of the cell lysates using anti-Mad1 and anti-Flag were controls for protein expression. (F) Endogenous Mad1 of MG132-treated 293T cells was immunoprecipitated with anti-Mad1, and the western blot was probed with anti-c-IAP1 (top) and anti-Mad1 (bottom). Normal rabbit serum (NRS) served as a negative control. (G) 293T cells were transfected with expression vectors of Flag-Mad1 and Myc-c-IAP1 or c-IAP1-H588A (E3-inactive) for 20 hr and then treated with or without MG132 (10 μM) for 4 hr before harvesting. Anti-Flag immunoprecipitates and cell lysates were analyzed by western blotting using indicated antibodies. Molecular Cell 2007 28, 914-922DOI: (10.1016/j.molcel.2007.10.027) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 c-IAP1 Promotes Mad1 Degradation in Cells (A) 293T cells were treated with or without MG132 (10 μM) for 4 hr before harvesting and cell fractionation. Cell fractions were analyzed by western blotting using indicated antibodies. (B) Cell lysates made from 293T cells transfected with expression vectors of Mad1 and Flag-tagged c-IAP1 or its mutants were resolved by SDS-PAGE and analyzed by western blotting using anti-Mad1 antibody. (C) 293T cells were transfected with Flag-tagged c-IAP1 or its RING domain mutants (E3 inactive). Cell extracts were analyzed by western blotting with indicated antibodies. Asterisk (∗) denotes nonspecific bands. (D) HL-60 cells were infected with retrovirus containing pMSCV vector or pMSCV-Flag-c-IAP1 and selected with puromycin for 4 days. The cell lysates were analyzed by western blotting using indicated antibodies. (E) 293T cells were infected with retrovirus encoding vector, c-IAP1, or Mad1 RNAi constructs and selected with puromycin for 4 days. The levels of Mad1 and c-IAP1 were analyzed by western blotting using anti-Mad1 and anti-c-IAP1 antibodies and quantified with NIH Image J and normalized to tubulin levels (left panel). The mRNA levels of mad1 and c-iap1 in the RNAi cells were analyzed by RT-PCR (right panel). (F) 293T cells were transfected with two different siRNAs against c-IAP1. Two days after the transfection, total RNA was extracted and mRNA levels of c-iap1, c-iap2, xiap, or β-actin were analyzed by semiquantitative RT-PCR (left panel). The cell lysates from the RNAi cells were immunoblotted with the indicated antibodies (right panel). (G) 293T c-IAP1 RNAi cells or pSRP vector control cells were treated with cycloheximide (CHX) (100 ng/ml) and harvested at indicated time points. The cell lysates were analyzed by immunoblotting using antibodies for indicated proteins. (H) The levels of Mad1 in (G) were quantified with NIH Image J and normalized to tubulin levels. Molecular Cell 2007 28, 914-922DOI: (10.1016/j.molcel.2007.10.027) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 c-IAP1 Antagonizes the Tumor Suppression Function of Mad1 (A) The expression vector of mad1 was transfected alone or together with that of 3Flag-c-IAP1, 3Flag-c-IAP1H588A, 3Flag-XIAP, or pRc/CMV-c-Myc into 293T cells. The total RNAs were extracted and analyzed by semiquantitative RT-PCR for the levels of ODC1 or CCND2 mRNAs. β-actin was used as an internal control. (B) Equal numbers of different 293T RNAi stable cells were seeded, and cell proliferation was measured after indicated time points by ATP assay (right panel) using CelltiterGro (Promega). The experiments were repeated three times and the error bars represent ± SD ∗p < 0.05 versus vector (pSRP) values (Day2), ∗∗p < 0.05 versus vector (pSRP) values (Day4). The levels of c-IAP1 and Mad1 were analyzed by western blotting (left panel). (C) c-IAP1 RNAi inhibits cell transformation. Equal numbers of different SAOS2 RNAi stable cells were seeded (1 × 104 cells per plate) and cultured for 3 weeks. The colonies were stained with crystal violet. (D) 293T cells were transfected with siRNAs to c-IAP1 and/or Mad1. Twenty-four hours later, equal numbers of cells were seeded and cell proliferation was measured by counting cell number. Values are the mean ± SD of three wells. (E) c-IAP1 RNAi 293T cells and control RNAi 293T cells were cultured continuously for 1 month. The Mad1 levels were analyzed by western blotting. (F) The proliferation rates of c-IAP1 RNAi and control RNAi 293T cells that had been cultured for 1 month were measured by seeding in the same numbers and counting cell numbers daily. Values are the mean ± SD of three wells. Molecular Cell 2007 28, 914-922DOI: (10.1016/j.molcel.2007.10.027) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 c-IAP1 Cooperates with c-Myc to Promote Tumorigenesis in a Ubiquitin Ligase-Dependent Manner (A) MCF7 cells were transfected with vectors encoding Flag-tagged c-IAP1 or c-IAP1 H588A (E3 inactive) with or without c-Myc. After 24 hr, equal numbers of cells were seeded and cultured in the presence of 800 μg/ml G418 for 2 weeks. The colonies were stained by crystal violet (right panel). The levels of c-IAP1 and Myc were analyzed by western blotting using anti-Flag and anti-Myc antibodies (left panel). (B) A model for the mechanism by which c-IAP1 cooperates with Myc to promote tumorigenesis. Aberrant upregulation of c-iap1 leads to a reduction in the protein levels of Mad1 by enhancing its proteasomal degradation, which in turn promotes Myc-mediated tumorigenesis by making more Max available. Molecular Cell 2007 28, 914-922DOI: (10.1016/j.molcel.2007.10.027) Copyright © 2007 Elsevier Inc. Terms and Conditions