Cell Cycle Regulation In Oogenesis

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Over the last year we have generated a series of reagents to explore the regulation of TORC1 activity in vivo. While the precise control of TORC1 activity is critical to the growth and development of metazoans, there are limited tools to explore the cell biology of the GATOR-Rag-GTPAse-TORC1 signaling axis in vivo in multicellular animals. To address this deficiency, we developed tools to examine the regulation of the GATOR-Rag GTPase-TORC1 signaling axis in vivo, at the single cell and subcellular level, in the model organism Drosophila. The advantage of our system is three-fold (1) It allows for the study of a wide variety of cell types in different developmental and physiological contexts. (2) It uses CRSPR/Cas9 to tag genes at their chromosomal localization, or transgenes that are expressed from their endogenous promoters, to avoid artifacts from over or under expression. (3) It can be used in combination with the powerful molecular genetic tools available in Drosophila to examine gene function and complex genetic interactions. We have used these new reagents to define novel roles for the GATOR2 complex in the regulation of TORC1 activity and the regulation of the endo lysosomal membrane system in multiple cell types. Additionally, we determined that the GATOR2 complex has independent roles in TORC1 regulation and MiT/TFE protein protection and thus is central to coordinating cellular metabolism with the control of the lysosomal-autophagic system. We found that in cells with deletions of the GATOR2 subunits WDR24, MIOS or SEH1, but not WDR59, members of the MiT/TFEs family of transcription factors, TFEB, TFE3 and MITF, are ubiquitylated and degraded by the proteasome. Low levels of MiT/TFEs result in a decrease in the transcription of a wide-range of lysosomal-autophagic genes, including V-ATPase subunits, lysosomal enzymes (Cathepsins), structure proteins (LAMPs and LAMTORs) and several ATG genes (ATG9), causing a significantly increased lysosomal pH and a systematic failure in digestion of lysosomal cargoes. We identified a trio of E3 liagases, HERC2, UBE3A and STUB1, that target Mi/TFEs for ubiquitin-mediated degradation in GATOR2 KO cells. Finally, we determined that in autophagy-addict pancreatic ductal adenocarcinoma (PDAC) cells the GATOR2 complex is essential for maintaining PDAC cell proliferation, invasion, and malignancy by fulfilling its dual roles in cell metabolism: activation of TORC1 and protection of MiT/TFEs. To complement these studies, we knocked down the GATOR2 component WDR24 in two kidney cancer-cell models, UOK257 and UOK124. The UOK 257 cell line was derived from a human renal carcinoma of an individual with Birt-Hogg-Dubé (BHD) syndrome resulting from a mutation in the FLCN gene. FLCN regulates lysosome function by promoting the TORC1-dependent phosphorylation and cytoplasmic sequestration of TFEB. In the absence of FLCN, TFEB is not recruited to the lysosome but instead enters the nucleus and is active. The UOK124 cell line was derived from a Xp11.2 translocation renal cell carcinoma (tRCC). Xp11.2 tRCCs represent an aggressive type of kidney cancer resulting from genomic rearrangements of TFE3, that produce oncogenic TFE3 fusion proteins 17. We determined that that WDR24 KD result in markedly decreased levels of TFEB and TFE3 in UOK257 and UOK124 tRCC cells. We will continue to analyze how GATOR2 impacts these two cancer cell lines using strategies similar to those we employed to follow MiT/TFE levels and function in HeLa and PDAC cells. Our data indicate that the GATOR2 complex acts upstream of the TORC1 regulation of MiT/TFE activity. Thus, the GATOR2 complex may make an attractive target for chemotherapeutic intervention. In summary, we demonstrated that the GATOR2 complex has independent roles in TORC1 regulation and MiT/TFE protein protection and thus is central to coordinating cellular metabolism with control of the lysosomal-autophagic system.