S known about the regulation of its receptors and signaling in these cells. We demonstrate here a novel, autophagy-independent role for beclin 1 in regulating TGF- signaling in both primary mouse neurons and fibroblasts. Our data show that beclin 1 facilitates localization of ALK5 with both the retromer complex and Rab11 in neurons, and provides direct evidence that beclin 1 regulates ALK5 recycling in COS7 cells. The conservation of a beclin 1 function in ALK5 sorting and TGF- signaling inaP-Smad / total Smad*P-Smad Smad 2 Smad 3 Beclin 1 NSEtotal Smad/NSEctrl shRNAbec shRNAb1.c2.0 1.5 1.0 0.5 0.1.0.****0.ctrl bec shRNA shRNActrl shRNAbec shRNAdBeclin 1 Vps15 Atg14 Vps34 Ambra Beclin 1 Vps38/ Vps15 Vps34 UVRAG AmbraeRelative SEAP Activity1.1.****0.********0.Ash R N AroAANNNntRRRshcoshshinVP S3GVRbeTGFig. 7 Beclin 1 regulates TGF- signaling. a Representative western blot from infected primary forebrain neurons. Starred band is non-specific. Quantification of (b) P-Smad/total Smad levels, and (c) Smad relative to NSE from three independent experiments (n = 3/group). All bars are mean ?SEM, **p 0.01, ****p 0.0001. d Mammalian homologs of beclin 1 complexes. Beclin 1 interacts mutually exclusively with Atg14 and UVRAG. In yeast, the ATG14 complex functions specifically in autophagy, while the Vps38 (homologous to UVRAG) complex functions specifically in the vacuolar protein sorting pathway. e) SEAP activity in media collected from F11 cells expressing ctrl shRNA or shRNA targeting beclin 1, VPS34, UVRAG, ATG14, or ATG7. Data are combined results from 2? independent experiments (n = 3/group). Values were normalized to ctrl shRNA levels for each experiment. Data were analyzed by one-way ANOVA with Dunnet's post-test. All bar graphs are mean ?SEM.UAATGclAshRNAlO'Brien et al. Molecular Neurodegeneration (2015) 10:Page 10 ofmultiple cell types suggests beclin 1 is fundamental to the regulation of this pathway. Our results are consistent with a recent report Capecitabine that identified ALK5 as one of over a hundred receptors down-regulated at the surface of cells depleted for either SNX27 or VPS35 [35]. Additionally, an independent report from Yin et al. recently demonstrated a role for the retromer in polarized distribution of TBRII in MDCK cells [36]. TBRII is endocytosed from the apical membrane and recycled through a Rab11-positive recycling endosome to the basolateral membrane in a retromerdependent manner. While this study did not find a direct interaction between ALK5 and the retromer component VPS26, the observations on the effect of retromer knockdown on ALK5 levels is consistent with our data. These PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/6833145 authors found that although ALK5 does not lose its polarized localization upon retromer knockdown (in contrast to TBRII), levels of ALK5 at the basolateral membrane appear to decrease. This is consistent with a role of the retromer in ALK5 recycling, though not polarized localization of this receptor. The fact that we see only a partial decrease in the colocalization of ALK5 with VPS35 and Rab11 upon beclin 1 knockdown may be due either to incomplete knockdown or to the presence of additional, beclin 1independent retromer recruitment mechanisms. Indeed, Rojas et al. have previously proposed a model where both Rab5 and Rab7 are required for retromer recruitment to endosomes [37]. Rab5 recruits the PI3K complex to generate PI3P on endosomes, which in turn recruits the sorting nexin subcomplex of the retromer. These authors suggest that the interaction.