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The USP19 Deubiquitinase Regulates the Stability of c-IAP1 and c-IAP2

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The USP19 Deubiquitinase Regulates the Stability of c-IAP1 and c-IAP2

Yide Mei et al. J Biol Chem.

Abstract

The inhibitors of apoptosis (IAPs) are critical regulators of apoptosis and other fundamental cellular processes. Many IAPs are RING domain-containing ubiquitin E3 ligases that control the stability of their interacting proteins. However, how IAP stability is regulated remains unclear. Here we report that USP19, a deubiquitinating enzyme, interacts with cellular IAP 1 (c-IAP1) and c-IAP2. Knockdown of USP19 decreases levels of both c-IAPs, whereas overexpression of USP19 results in a marked increase in c-IAP levels. USP19 effectively removes ubiquitin from c-IAPs in vitro, but it stabilizes c-IAPs in vivo mainly through deubiquitinase-independent mechanisms. The deubiquitinase activity is involved in the stabilization of USP19 itself, which is facilitated by USP19 self-association. Functionally, knockdown of USP19 enhances TNFα-induced caspase activation and apoptosis in a c-IAP1 and 2-dependent manner. These results suggest that the self-ubiquitin ligase activity of c-IAPs is inhibited by USP19 and implicate deubiquitinating enzymes in the regulation of IAP stability.

Figures

FIGURE 1.
FIGURE 1.
USP19 interacts with c-IAP1, c-IAP2, and XIAP. A, interaction of USP19 with c-IAPs. FLAG-tagged c-IAP1 or c-IAP2 was transfected into HEK293T cells either alone or together with HA-USP19. Cell lysates were immunoprecipitated with anti-HA beads. The immunoprecipitates and input extracts were analyzed by Western blot. B, interaction of USP19 with c-IAP2 and XIAP. HA-USP19 or HA-USP19 CA was transfected into HEK293T cells together with vector control (V), FLAG-XIAP, or FLAG-c-IAP2. Cell lysates were subjected to immunoprecipitation with anti-FLAG M2 beads followed by Western blot analysis. C, interaction of endogenous USP19 and c-IAP2. Jurkat cell lysates were subjected to immunoprecipitation with anti-USP19 antibody. Immunoprecipitates were analyzed by Western blot using anti-c-IAP2 antibody. *, a nonspecific band. D, co-localization of exogenous USP19 and c-IAP2. HeLa cells expressing FLAG-c-IAP2 and HA-USP19 individually or in combination were immunostained with anti-FLAG and/or anti-HA antibody followed by Rhodamine-conjugated anti-mouse and/or FITC-conjugated anti-rabbit IgG.
FIGURE 2.
FIGURE 2.
Structural determinants of the USP19-c-IAP2 interaction. A, schematic representation of c-IAP2 (left), USP19 (right), and their mutants used in this work. B and C, each of the indicated c-IAP2 mutants was expressed in HEK293T cells alone or together with HA-USP19. Cell lysates were immunoprecipitated with anti-HA beads. The immunoprecipitates and extracts were analyzed by Western blot. D, FLAG-c-IAP2 N was expressed in HEK293T cells alone or together with the indicated HA-USP19 proteins. Cell lysates were analyzed as in B and C.
FIGURE 3.
FIGURE 3.
Deubiquitination of c-IAP2 by USP19. A, in vitro deubiquitination assay. Tetra-Ub peptide was treated with recombinant USP19 or USP19 CA in the presence or absence of iodoacetate (IAc). Samples were analyzed by SDS-PAGE and Coomassie Blue staining. B, ubiquitinated FLAG-c-IAP2 protein was treated with USP19 WT and USP19 CA in the presence (+) or absence (−) of iodoacetate (IAc). Reaction mixes were analyzed by Western blot. C, FLAG-c-IAP2 was co-expressed with vector control (−), HA-USP19, or HA-USP19 CA in HEK293T cells. c-IAP2 was immunoprecipitated with anti-FLAG M2 beads and detected by a polyclonal anti-FLAG antibody. Lysates were blotted for c-IAP2 with M2 and for USP19 with anti-HA antibody.
FIGURE 4.
FIGURE 4.
USP19 stabilizes c-IAPs. A, HeLa and MDA-MB231 cells were infected with lentiviruses expressing a control shRNA (−) or USP19 shRNA. Protein expression was analyzed by Western blot. Protein bands were quantified by NIH image software, and relative ratios of c-IAP1 or c-IAP2 versus actin are indicated. B, Western blot analysis of HeLa cells transfected with FLAG-c-IAP2 and increasing amounts of HA-USP19 or HA-USP19 CA. GFP was included as a control for transfection efficiency. C, HEK293T cells were transfected with FLAG-c-IAP1 (left) or FLAG-c-IAP2 (right), and vector (−), HA-USP19, or HA-USP19 CA. p53 and GFP were included as controls. Cells were treated with cycloheximide for the indicated periods of time and analyzed by Western blot. D, MDA-MB231 cells infected with the lentivirus expressing control shRNA (−) or USP19 shRNA were treated with cycloheximide (CHX) for the indicated periods of time and analyzed by Western blot.
FIGURE 5.
FIGURE 5.
Auto-deubiquitination and self-interaction of USP19. A, HEK293 cells were transfected with HA-Ub plus vector control, FLAG-USP19, or FLAG-USP19 CA. Cells were treated with or without 20 μm ALLN. FLAG-tagged proteins were immunoprecipitated and analyzed by Western blotting with anti-HA antibody (top) and Coomassie Blue staining (bottom). B, Western blot analysis of HeLa cells transfected with HA-USP19 or HA-USP19 CA and treated with cycloheximide (CHX) for the indicated periods of time. C, FLAG-USP19 was expressed in HEK293T cells alone or together with HA-USP19 or HA-USP19 CA. Cell extracts were immunoprecipitated with anti-HA antibody. Extracts and immunoprecipitates were analyzed by Western blot. Actin and GFP levels are shown as sample loading and transfection efficiency controls, respectively. D, HEK293T cells were transfected with HA-USP19 CA plus increasing amounts of FLAG-USP19 (0, 0.25, 0.5, and 1 μg). Cell extracts were analyzed by Western blot.
FIGURE 6.
FIGURE 6.
Down-regulation of USP19 enhances TNFα-induced apoptosis. A and B, MDA-MB231 cells expressing control shRNA or USP19 shRNA were treated with TNFα (20 ng/ml) for 20 h (A) or transfected with control vector (−), FLAG-c-IAP1, and FLAG-c-IAP2, and then treated with TNFα (B). Top panel: Percentages of apoptotic cells shown as the mean ± S.D. of three independent experiments. Bottom panels: expression of proteins, activation of caspase-8, and cleavage of poly(ADP-ribose) polymerase in cell extracts analyzed by Western blot.

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