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EAPF/Phafin-2, a novel endoplasmic reticulum-associated protein, facilitates TNF-α-triggered cellular apoptosis through endoplasmic reticulum-mitochondrial apoptotic pathway

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Abstract

We recently identified the Phafin protein family, whose members all contain an N-terminal PH domain (pleckstrin homology) and a C-terminal FYVE (Fab1, YGLO23, Vps27, and EEA1) domain. LAPF (lysosome-associated apoptosis-inducing protein containing PH and FYVE domains, also known as Phafin-1), as one representative member of this new family, has been shown to be able to initiate caspase-independent apoptosis through lysosomal-mitochondrial apoptotic pathway. Here, we describe the cloning and functional characterization of another Phafin member, EAPF (endoplasmic reticulum-associated apoptosis-involved protein containing PH and FYVE domains)/Phafin-2. Overexpression of EAPF/Phafin-2 enhances the sensitivity of L929 and MCF-7 cells to TNF-α-induced apoptosis, concomitant with its partial translocation to endoplasmic reticulum (ER). Both the PH and the FYVE domains contribute to the ER translocation of EAPF/Phafin-2 as well as EAPF/Phafin-2-enhanced apoptosis. Knockdown of mouse and human EAPF/Phafin-2 expression protects L929 cells and MCF-7 cells from TNF-α-induced apoptosis, respectively. We demonstrate that EAPF/Phafin-2 induces a much sharper and more rapid Ca2+ influx triggered by TNF-α and Ca2+ release ER contributes to the enhancement of EAPF/Phafin-2 in TNF-induced apoptosis. EAPF/Phafin-2 increases the activity of caspase 12, suggesting that EAPF/Phafin-2 is involved in ER-related apoptotic pathway. Overexpression of EAPF/Phafin-2 also enhances TNF-α-induced activity of caspase 3 (but not caspase 8 or 9), and promotes TNF-α-triggered mitochondrial membrane permeabilization (MMP) in L929 cells, including dissipation of mitochondrial membrane potential and release of AIF. Besides, EAPF/Phafin-2 also suppresses the unfolded protein response by inhibiting phosphorylation of eIF2α. Therefore, our results demonstrate that EAPF/Phafin-2 facilitates TNF-α-induced cellular apoptosis through an ER-mitochondrial apoptotic pathway, which may improve our understanding of drug-induced cancer cell death and cancer chemotherapy.

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Acknowledgments

We thank Dr. Wan T. and Zhang L. for helpful discussion. We thank Dr. Zheng Y. and Ms. Zuo X. for their expert technical assistance. This work was supported by grants from the National Key Basic Research Program of China (2004CB518807, 2007CB512403), the National Natural Science Foundation of China (30121002, 30490240), the National High Biotechnology Development Program of China (2006AA02A305) and Shanghai Committee of Science and Technology (06DJ14011).

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Authors and Affiliations

  1. Institute of Immunology, Zhejiang University, Hangzhou, 310031, People’s Republic of China

    Changfei Li, Wei Chen, Li Wang, Yuzhen Wang & Xuetao Cao

  2. Institute of Immunology and National Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai, 200433, People’s Republic of China

    Changfei Li, Qiuyan Liu, Nan Li, Yizhi Yu & Xuetao Cao

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  1. Changfei Li
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Correspondence to Xuetao Cao.

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Changfei Li, Qiuyan Liu, and Nan Li contributed equally to this work.

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Li, C., Liu, Q., Li, N. et al. EAPF/Phafin-2, a novel endoplasmic reticulum-associated protein, facilitates TNF-α-triggered cellular apoptosis through endoplasmic reticulum-mitochondrial apoptotic pathway. J Mol Med 86, 471–484 (2008). https://doi.org/10.1007/s00109-007-0298-7

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