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

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 ( e ndoplasmic reticulum-associated a poptosis-involved protein containing P H and F YVE 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 Ca 2+ influx triggered by TNF-α and Ca 2+ 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.