EAPF/Phafin-2, a novel endoplasmic reticulum-associated protein, facilitates TNF-alpha-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 (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-alpha-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-alpha-induced apoptosis, respectively. We demonstrate that EAPF/Phafin-2 induces a much sharper and more rapid Ca2+ influx triggered by TNF-alpha 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-alpha-induced activity of caspase 3 (but not caspase 8 or 9), and promotes TNF-alpha-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 eIF2alpha. Therefore, our results demonstrate that EAPF/Phafin-2 facilitates TNF-alpha-induced cellular apoptosis through an ER-mitochondrial apoptotic pathway, which may improve our understanding of drug-induced cancer cell death and cancer chemotherapy.
Li
Changfei
C
Institute of Immunology, Zhejiang University, Hangzhou 310031, People's Republic of China.
Liu
Qiuyan
Q
Li
Nan
N
Chen
Wei
W
Wang
Li
L
Wang
Yuzhen
Y
Yu
Yizhi
Y
Cao
Xuetao
X
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014018:Tissue Distribution; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D020169:Caspases; D002118:Calcium