We also show that pericentrosomal CD133 captures GABARAP at centrosomes in order to inhibit GABARAP-mediated ULK1 activation, and the subsequent initiation of autophagy. Results CD133 is transported from the plasma membrane to the pericentrosomal region CD133 is a pentaspan transmembrane protein. system. Pericentrosomal CD133 is then recycled to the plasma membrane via recycling endosomes. At the pericentrosomal region, endosomal CD133 captures GABARAP, an initiator of autophagy, and inhibits GABARAP-mediated ULK1 activation and the subsequent initiation of autophagy. Furthermore, pericentrosomal CD133 suppresses cell differentiation, such as primary cilium formation and neurite outgrowth, by inhibiting autophagy. Thus, the present results provide evidence to suggest that pericentrosomal CD133 has the unique property of maintaining the undifferentiated status of cells by inhibiting autophagy. Introduction CD133, also called prominin 1, was originally identified as a cell surface marker of human haematopoietic stem cells and mouse neuroepithelial cells1C3. It was subsequently reported to function as a marker of cancer stem cells in solid tumours, such as brain tumours4, colon cancer5,6, and hepatocellular carcinoma (HCC)7. The CD133-positive cell population has a greater self-renewal ability and chemoresistance phenotype than the CD133-negative cell population. The expression of CD133 correlates with malignant characteristics and a poor prognosis in many tumours8. CD133 is a pentaspan transmembranous protein that not only undergoes glycosylation at high levels, but also binds to cholesterol9. CD133 is phosphorylated in its intracellular C-terminal domain by Src family tyrosine kinases10. As a result, it activates the p85 subunit of phosphoinositide 3-kinase (PI-3K) by binding, and PI-3K, in turn, MEK162 (ARRY-438162, Binimetinib) activates downstream targets such as Akt, thereby promoting cell proliferation in glioma stem cells11. CD133 is stabilized by binding with histone deacetylase 6 (HDAC6), and enhances the transcriptional activity of -catenin, resulting in the acceleration of cell growth and suppression of cell differentiation12. CD133 is also known to function as a cancer stem cell marker in many cancers including neuroblastoma. When the expression of CD133 is down-regulated in neuroblastoma cells, neural differentiation frequently occurs13. Thus, CD133 is not only associated with tumour cell growth, but also regulates cell differentiation. Recent studies reported that CD133 is directly involved in the cell survival of glioma and HCC through its role in the regulation of autophagy14,15. Autophagy is a highly conserved protein/organelle degradation system that is responsible for MEK162 (ARRY-438162, Binimetinib) the turnover of long-lived proteins and disposal of excess or damaged organelles in order to maintain cell homeostasis16,17. Severe growth conditions, such as low nutrient levels, activate the autophagy pathway. ULK1 is at the top of this cascade and activates the autophagy initiation complex, and elongation of the isolation membrane also occurs17,18. The isolation membrane subsequently closes and engulfs cytoplasmic constituents, forming an autophagosome. The autophagosome fuses with a lysosome, resulting in the complete degradation of the sequestered cytoplasmic components by lysosomal enzymes16,19. Although RFWD1 the underlying mechanisms currently remain unknown, CD133 appears to be preferentially processed in endosomes9,20, and it has been reported to directly participate in the autophagosome membrane fusion process, and ultimately undergoes lysosomal degradation in the cytoplasm in some nutrient-deprived microenvironments14,15,21. Autophagy also appears to serve as a critical mechanism for stem cell properties22. Autophagic activity is necessary for cell differentiation in neural stem cells (NSCs). In NSCs, autophagic activity is up-regulated during cell differentiation22,23. When autophagic activities are blocked by inhibitor(s), neurogenesis markedly decreases. Ambra1 is an autophagy component, and neuronal differentiation was shown to be impaired in or resulted in defective embryoid bodies in mouse ESCs25, suggesting a pivotal role for autophagy in early embryonic development23. Autophagic activity is also involved in primary ciliogenesis26C28. Primary cilia are sensory organelles and the key coordinators of signalling pathways during development and tissue homeostasis. Cilia typically form in the growth-resting phase of the cell cycle29. Therefore, primary cilia form in many normal cells, but not in malignant tumour cells29. In order to clarify the functions of CD133, we herein examined the cell localisation of CD133 in various cancer and normal cell lines under nutrient and nutrient-starved conditions, and found that CD133 has a unique property for autophagic processes. Mechanistically, we demonstrate that when Src family tyrosine kinase activity is weak, non-phosphorylated CD133 combined with HDAC6 is transported to endosomes, and is preferentially recruited to the MEK162 (ARRY-438162, Binimetinib) pericentrosomal region via the dynein-based traffic system. We also show that pericentrosomal CD133 captures GABARAP at centrosomes in order to inhibit GABARAP-mediated ULK1 activation, and the subsequent initiation of autophagy. Results CD133 is transported from the plasma membrane to the pericentrosomal region CD133 is a pentaspan transmembrane protein. However, a recent study showed that CD133 localises around the cytoplasm in many tumours14,30,31. Therefore, we investigated the localisation status of CD133 in CD133-positive cancer cell lines using immunostaining (Fig.?1A). While CD133 localised to the plasma membrane in Caco-2 cells, it mainly localised around the cytoplasm and partly to the perinuclear region in Huh-7 cells (Fig.?1A) as a dot-like structure. Moreover, CD133 also MEK162 (ARRY-438162, Binimetinib) specifically localised to the perinuclear region as a dot-like framework in SK-N-DZ cells (Fig.?1A). We also looked into the localisation position of Compact disc133 in these cancers cell lines using various other anti-CD133 antibodies,.