The Novel ALG-2 Target Protein CDIP1 Promotes Cell Death by Interacting with ESCRT-I and VAPA/B.

Ryuta Inukai,Kanako Mori,Keiko Kuwata,Masatoshi Maki,Chihiro Suzuki,Terunao Takahara,Hideki Shibata

doi:10.3390/ijms22031175

Abstract

Apoptosis-linked gene 2 (ALG-2, also known as PDCD6) is a member of the penta-EF-hand (PEF) family of Ca2+-binding proteins. The murine gene encoding ALG-2 was originally reported to be an essential gene for apoptosis. However, the role of ALG-2 in cell death pathways has remained elusive. In the present study, we found that cell death-inducing p53 target protein 1 (CDIP1), a pro-apoptotic protein, interacts with ALG-2 in a Ca2+-dependent manner. Co-immunoprecipitation analysis of GFP-fused CDIP1 (GFP-CDIP1) revealed that GFP-CDIP1 associates with tumor susceptibility gene 101 (TSG101), a known target of ALG-2 and a subunit of endosomal sorting complex required for transport-I (ESCRT-I). ESCRT-I is a heterotetrameric complex composed of TSG101, VPS28, VPS37 and MVB12/UBAP1. Of diverse ESCRT-I species originating from four VPS37 isoforms (A, B, C, and D), CDIP1 preferentially associates with ESCRT-I containing VPS37B or VPS37C in part through the adaptor function of ALG-2. Overexpression of GFP-CDIP1 in HEK293 cells caused caspase-3/7-mediated cell death. In addition, the cell death was enhanced by co-expression of ALG-2 and ESCRT-I, indicating that ALG-2 likely promotes CDIP1-induced cell death by promoting the association between CDIP1 and ESCRT-I. We also found that CDIP1 binds to vesicle-associated membrane protein-associated protein (VAP)A and VAPB through the two phenylalanines in an acidic tract (FFAT)-like motif in the C-terminal region of CDIP1, mutations of which resulted in reduction of CDIP1-induced cell death. Therefore, our findings suggest that different expression levels of ALG-2, ESCRT-I subunits, VAPA and VAPB may have an impact on sensitivity of anticancer drugs associated with CDIP1 expression.

Highlights

Apoptosis-linked gene 2 (ALG-2, gene symbol PDCD6) is a 22-kDa protein having five repetitive EF-hand motifs called the penta-EF-hand (PEF) domain [1]
We demonstrated that apoptosis-linked gene gene 22 (ALG-2) interacts with CDIP1 in a Ca2+ -dependent manner and that ALG-2 functions as an adaptor bridging CDIP1 and endosomal sorting complex required for transport-I (ESCRT-I)
On the molecular basis for the involvement of ALG-2 in CDIP1-induced cell death, we considered the possibility that ALG-2 may mediate the physical association of CDIP1 with a specific binding protein by the adaptor function of ALG-2 [2,3]

Summary

Introduction

Apoptosis-linked gene 2 (ALG-2, gene symbol PDCD6) is a 22-kDa protein having five repetitive EF-hand motifs called the penta-EF-hand (PEF) domain [1]. It forms a homodimer or a heterodimer with its paralogous protein Peflin and interacts Ca2+ -dependently with a variety of target proteins that function in multifaceted cellular processes including apoptosis, cancer development, signal transduction, membrane trafficking, and posttranscriptional control (see [2] for a review). ALG-2 bridges two target proteins, ALG2-interacting protein X (ALIX, known as AIP1 and HP95) and endosomal sorting complex required for transport (ESCRT)-I [11,12]. Among the growing list of functions of ESCRT in cellular processes related to membrane remodeling, ALG-2 functions as an activator of ALIX in the multivesicular body sorting pathway [13] and as an ESCRT-0 in plasma membrane repair [14,15,16]

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Conclusion