Identification of Barkor as a mammalian autophagy-specific factor for Beclin 1 and class III phosphatidylinositol 3-kinase. Xanya Sofra Weiss

Identification of Barkor as a mammalian autophagy-specific factor for Beclin 1 and class III phosphatidylinositol 3-kinase Qiming Suna, Weiliang Fana, Keling Chena, Xiaojun Dingb, She Chenb, and Qing Zhonga,1 aDivision of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; and bNational Institute of Biological Sciences, Beijing 102206, China Communicated by Xiaodong Wang, University of Texas Southwestern Medical Center, Dallas, TX, October 17, 2008 (received for review October 10, 2008) Autophagy mediates the cellular response to nutrient deprivation, protein aggregation, and pathogen invasion in human. Dysfunction of autophagy has been implicated in multiple human diseases including cancer. The identification of novel autophagy factors in mammalian cells will provide critical mechanistic insights into how this complicated cellular pathway responds to a broad range of challenges. Here, we report the cloning of an autophagy-specific protein that we called Barkor (Beclin 1-associated autophagy-related key regulator) through direct interaction with Beclin 1 in the human phosphatidylinositol 3-kinase class III complex. Barkor shares 18% sequence identity and 32% sequence similarity with yeast Atg14. Elimination of Barkor expression by RNA interference compromises starvation- and rapamycin-induced LC3 lipidation and autophagosome formation. Overexpression of Barkor leads to autophagy activation and increased number and enlarged volume of autophagosomes. Tellingly, Barkor is also required for suppression of the autophagy-mediated intracellular survival of Salmonella typhimurium in mammalian cells. Mechanistically, Barkor competes with UV radiation resistance associated gene product (UVRAG) for interaction with Beclin 1, and the complex formation of Barkor and Beclin1 is required for their localizations to autophagosomes. Therefore, we define a regulatory signaling pathway mediated by Barkor that positively controls autophagy through Beclin 1 and represents a potential target for drug development in the treatment of human diseases implicated in autophagic dysfunction. Atg14 autophagosome LC3 Salmonella UVRAG One of the central regulators of autophagy in mammalian cells is Beclin 1 (1–3). Beclin 1 is a component of the class III phosphatidylinositol 3-kinase (PI3KC3) complex, which also contains a PI3K catalytic subunit and a regulatory subunit (p150) (4). Beclin 1 was identified as a haploid insufficient tumor suppressor gene (3). It is monoallelically deleted in ovarian, breast, and prostate cancers. Heterozygous Beclin 1 / mice have reduced autophagy activity and increased incidence of spontaneous tumors (5, 6). Allelic loss of Beclin 1 leads to genome instability upon metabolic stress (7, 8). All of this evidence illustrates a role for Beclin 1 and autophagy in cancer development. Notably, Beclin 1 and PI3KC3 have pleiotropic functions in multiple cellular processes. PI3KC3 is not only required for autophagy, but also has broad functions in endocytic protein sorting (9). Functional equivalents of Beclin 1/PI3KC3/p150 in yeast, Vps30/ Atg6-Vps15-Vps34, are known to play a critical role in autophagy and in vacuolar protein sorting (VPS) (1, 10). The specificity of PI3KC3 in yeast is determined by different complex compositions. Two regulatory proteins, Atg14 and Vps38, direct the core PI3K complex to either the phagophore assembly site (PAS) for autophagy or the endosome for VPS (10, 11), respectively, to execute their functions in autophagy or VPS. Atg14 is required for mediating the localization of the core PI3KC3 complex to PAS and is also important in recruiting downstream Atg proteins such as Atg2, Atg8, Atg16, and the Atg12-Atg5 conjugate to the PAS for membrane elongation and vesicle completion (12, 13). In contrast, Vps38 is responsible for the endosomal localization of the PI3K complex (11). Surprisingly, such regulatory mechanisms directing PI3KC3 specificity have not been identified in mammals. How the function of Beclin 1 is specifically directed toward autophagosomes in mammalian cells has remained elusive. We speculate that there are autophagy-specific factors mediating Beclin 1 activity in autophagy. We used a biochemical approach to purify and proteomic methods to characterize the Beclin 1 complex. Here, we report the identification of a Beclin 1- associated protein that promotes autophagy specifically through the interaction with Beclin 1. Results Identification of Barkor as a Beclin 1-Interacting Protein. To search for Beclin 1 regulatory proteins, we generated a cell line from human osteosarcoma U2OS cells that is stably transfected with ZZ-Beclin 1-FLAG under the control of doxycycline [supporting information (SI) Fig. S1A]. The expression of Beclin 1 was adjusted by the titration of doxycycline, and a dose (20 ng/mL) that induces expression of tagged Beclin 1 close to the endogenous level was selected (Fig. S1B). The tagged Beclin 1 was purified from cell extracts by sequential affinity chromatography steps, and the final FLAGpeptide eluate was subjected to 4–12% gradient SDS/PAGE and visualized by silver staining (Fig. 1A). The indicated bands were excised and analyzed by mass spectrometry. In addition to the known components of the Beclin 1 complex, namely the PI3K catalytic subunit, p150 regulatory subunit, and UVRAG, we also identified a 68-kDa protein by mass spectrometry, KIAA0831 (Fig. 1A), which we called Barkor (Beclin 1-associated autophagy related key regulator). We were able to purify the same complex from human embryonic kidney 293T cells expressing tagged Beclin 1, indicating that the formation of this complex is not cell type-specific (Fig. 1B). Bioinformatic analysis revealed that Barkor contains an N-terminal zinc finger motif and a central coiled-coil domain (CCD) (Fig. S2) and a domain organization similar to Atg14 in yeast. Barkor also shares 18% sequence identity and 32% sequence similarity with yeast Atg14 (Fig. S3). The identities of these interacting proteins were further confirmed by immunoblotting analysis (Fig. S4). Although another Beclin 1-interacting protein, Bcl-2 (14), could not be visualized by silver staining, its presence in the final eluate was validated by immunoblotting (Fig. S4). The interaction of Barkor and Beclin 1 was further confirmed by the Author contributions: Q.Z. designed research; Q.S., W.F., and K.C. performed research; X.D. and S.C. contributed new reagents/analytic tools; Q.S., W.F., and Q.Z. analyzed data; and Q.S. and Q.Z. wrote the paper. The authors declare no conflict of interest. Freely available online through the PNAS open access option. 1Towhomcorrespondence should be addressed at: Department of Molecular and Cell Biology, University of California, 316 Barker Hall, Berkeley, CA.

Xanya Sofra Weiss

Xanya Sofra Weiss