Apoptosis factor EI24/PIG8 is a novel endoplasmic reticulum-localized Bcl-2-binding protein which is associated with suppression of breast cancer invasiveness.
p53 is a critical tumor suppressor which removes cells with DNA damage by regulating expression and activity of a select group of p53-induced genes (PIG) that subsequently induce apoptosis. PIG8 was also identified as a gene induced by etoposide and named etoposide-induced gene 24 (EI24). Later experiments established EI24/PIG8 as a proapoptotic factor and suggested that it may function as a tumor suppressor. Indeed, EI24/PIG8 is relatively highly mutated in aggressive breast cancers and is located in a region which expresses frequent loss of heterozygosity. However, despite these important observations, the activity and role of EI24/PIG8 remain largely unknown. We used (immmuno)fluorescence microscopy and subcellular fractionation techniques to show that EI24/PIG8 is localized in the endoplasmic reticulum (ER). Pull-down experiments showed that it specifically binds with Bcl-2, a death regulator known to reside in mitochondria, ER, and the nuclear envelope. EI24/PIG8-Bcl-2 binding was corroborated by coimmunoprecipitation and other in vitro and in vivo protein-protein binding assays. Further analysis showed that EI24/PIG8 uses its N-terminal region to bind the BH3 domain in Bcl-2. Finally, we used immunohistochemical techniques to analyze expression of EI24/PIG8 in breast cancer tissue progression arrays and showed that loss of EI24/PIG8 is associated with tumor invasiveness but not with the development of the primary tumor. These results suggest that EI24/PIG8 is a novel, ER-localized Bcl-2-binding protein which may contribute to apoptosis by modulating the activity and/or function of Bcl-2 in this organelle. EI24/PIG8 may serve to prevent tumor spreading, consistent with its suspected role as a tumor suppressor.
Zhao
Xiansi
X
Department of Otolaryngology and Biochemistry, Cancer Research Center, Boston University School of Medicine, Boston, MA 02118, USA.
Ayer
Robert E
RE
Davis
Shannon L
SL
Ames
Sarah J
SJ
Florence
Brian
B
Torchinsky
Cyrus
C
Liou
James S
JS
Shen
Ling
L
Spanjaard
Remco A
RA
D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D009361:Neoplasm Invasiveness; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; C402592:EI24 protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; D019253:Proto-Oncogene Proteins c-bcl-2