Molecular interaction between HAX-1 and XIAP inhibits apoptosis

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Abstract

Caspase-3 is an important executor caspase that plays an essential role in apoptosis. Recently, HS1-associated protein X1 (HAX-1) was found to be a substrate of caspase-3. Although HAX-1 has serve multifunctional roles in cellular functions such as cell survival and calcium homeostasis, the detailed functional mechanism of HAX-1 remains still unclear. In this study, we performed proteomic experiments to identify the HAX-1 interactome. Through immunoprecipitation and 2D gel electrophoresis, we identified X-linked inhibitor of apoptosis protein (XIAP) as a novel HAX-1-interacting protein. By performing the GST pull-down assay, we defined the interaction domains in HAX-1 and XIAP, showing that HAX-1 binds to the BIR2 and BIR3 domains of XIAP whereas XIAP binds to the C-terminal domain of HAX-1. In addition, surface plasma resonance experiments showed that both BIR2 and BIR3 domains of XIAP bind to HAX-1 with affinity similar to that of full-length XIAP, indicating that either domain is necessary and sufficient for tight binding to HAX-1. Taken together with the observation that HAX-1 suppresses the polyubiquitination of XIAP, the cell viability assay results suggest that the formation of the HAX-1–XIAP complex inhibits apoptosis by enhancing the stability of XIAP against proteosomal degradation.

Introduction

Apoptosis is a complex biological process that kills unwanted cells during animal development, normal homeostasis and immune response. Apoptosis is mediated by a family of aspartate-specific cystein protease known as caspase [1]. Caspases play a pivotal role in both the initiation and the execution of apoptosis [2]. Among the caspase family, caspase-3, an effector caspase, plays a crucial role in the execution of apoptosis. It is activated by an initiator caspases such as caspase-9 through cleavage at a specific internal aspartate residue [2]. Activated caspase-3 cleaves downstream substrates such as molecules involved in DNA repair, apoptosis inhibitors and signaling proteins. To date, a number of proteins have been identified as substrates of caspase-3. Recently, we demonstrated that HAX-1 is cleaved by caspase-3 during apoptosis [3].

HAX-1 was first identified to associate with hematopoietic cell-specific Lyn substrate 1 (HS1), which is a component of the B-cell receptor signaling pathway [4]. Subsequently, HAX-1 was shown to interact with a diverse group of proteins including phospholamban [5], polycystic kidney disease protein PKD2 [6], SERCA2 [7], Epstein–Barr virus nuclear antigen leader protein (EBNA-LP) [8], K15 protein of Kaposi’s sarcoma-associated herpes virus [9], Omi/HtrA2 serine protease [10] and caspase-9 [11]. HAX-1 is an anti-apoptotic protein that shares homology with the BH1 and BH2 domains from the Bcl-2 family proteins [12]. Although it has been reported that HAX-1 is involved in multiple cellular functions such as cell survival and calcium homeostasis in cardiac tissue [12], the detailed functional mechanism of HAX-1 remains unclear. Therefore, the identification of new binding partners is important for understanding the functional mechanism of HAX-1.

The inhibitor of apoptosis protein (IAP) family serves as critical checkpoints of apoptotic cell death. IAP family members including X-linked inhibitor of apoptosis protein (XIAP), c-IAP1, c-IAP2 and ML-IAP, have been well characterized as inhibitors of caspases [13]. Among the IAP family members, XIAP is the most potent suppressor of apoptosis. XIAP blocks the apoptotic pathway by binding and inhibiting caspase-3, -7 and -9 [11]. XIAP contains three tandem baculovirus IAP repeat (BIR) domains followed by a RING zinc finger domain. Previous biochemical studies demonstrated that a region encompassing the second BIR domain (BIR2) is involved in the suppression of caspase-3 and caspase-7, while the third BIR domain (BIR3) binds to and inhibits caspase-9 [14]. Through its RING zinc finger domain, XIAP can function as an ubiquitin ligase both to itself and other target proteins, which are then degraded in the proteasome [15]. During apoptosis, the caspase-inhibiting function of XIAP can be antagonized by Smac/Diablo [16] and Omi/HtrA2 [10].

In this study, we identified XIAP as a novel binding partner of HAX-1 by a combination of immunoprecipitation (IP) and 2D gel electrophoresis (2-DE). By performing various biochemical experiments, we characterized the molecular interaction between HAX-1 and XIAP. First, GST pull-down assays showed that HAX-1 binds to the BIR2 and BIR3 domains of XIAP whereas XIAP binds to the C-terminal domain of HAX-1. Second, surface plasma resonance experiments demonstrated that the BIR2 and BIR3 domains of XIAP bind to HAX-1 with similar affinity. Third, ubiquitination and cell viability assays showed that the interaction between HAX-1 and XIAP contributes to cell survival by blocking the polyubiquitination of XIAP, thereby stabilizing XIAP.

Section snippets

Materials and methods

Immunoprecipitation for 2-DE. For identification of novel HAX-1 interacting proteins, pFLAG-CMV-C2 and FLAG tagged HAX-1 plasmids were transfected into Bosc 23 cells. After 48 h of transfection, the cells were harvested and lysed in Nonidet P-40 lysis buffer (150 mM NaCl, 20 mM Tris–HCl, pH 7.5, 1% Nonidet P-40 and 1% glycerol). Lysates (3.5 mg) were mixed with 15 μl of anti-FLAG M2-agarose affinity gel and incubated overnight at 4 °C. The antigen–antibody complex were collected by centrifugation at

Proteomic identification for mining novel HAX-1 binding proteins

To identify the HAX-1 interactome, we performed proteomics-based screening using a combination of IP and 2-DE. Bosc 23 cells were transfected with FLAG-tagged HAX-1 expression plasmid or FLAG control vector, and the HAX-1 protein was then isolated by IP. After IP, the eluted proteins were applied to 2-DE to separate the candidate HAX-1-interacting partners. Subsequently, the 2-DE gel was visualized by silver staining and the protein spots were analyzed by PDQuest-2-DE analysis software. Protein

Acknowledgments

This study was supported by a grant from Korea Science and Engineering Foundation (Basic Research Program No. 2008-01050) (to S.G. Park) and a grant from the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family Affairs, Republic of Korea (0720130) (to S.-W. Chi). This study was also supported by a grant from KRIBB Research Initiative Program.

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These authors contributed equally to this work.

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