A role for human WIPI3 in lysosomal phosphoinositide signaling

• Applicant: Professorin Dr. Tassula Proikas-Cezanne
• Subject Area: Cell Biology
• Term: since 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 323732846

Lysosomal function and autophagy critically contribute to secure the maintenance of cellular homeostasis and many human diseases are characterized by autophagy/lysosome dysfunction. Inaccurate phosphoinositide signaling, in particular pathways involving phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), impact both autophagy and lysosomal function. Underlying molecular mechanisms involved, eminently in the context of human pathologies caused by mutations in phosphoinositide metabolizing enzymes, such as mutations in the phosphoinositide 5-phosphatase FIG4 causing Charcot-Marie-Tooth neuropathy type 4J (CMT4J), are insufficiently understood. In order to contribute to the understanding of the roles of PI3P and PI(3,5)P2 in the control of autophagy and lysosome function we addressed the function of the human WIPI (WD-repeat protein interacting with phosphoinositide) proteins (WIPI1-WIPI4), PI3P- and PI(3,5)P2-binding proteins that we identified earlier, in the process of autophagy. We showed that all four WIPI proteins fulfill distinct scaffold functions in the signal control of autophagy and at the nascent autophagosome. Whereas WIPI1 and WIPI2 function as PI3P effectors at the nascent autophagosome, WIPI3 and WIPI4 function in the signal control of autophagy upstream of PtdIns3P production. In response to AMPK activation, WIPI4 is released from a WIPI4-ATG2/AMPK-ULK1 complex and translocates, together with ATG2, to the nascent autophagosome for size controlling membrane expansion, to which WIPI3 also contributes. Upstream and on lysosomes, WIPI3 associates the TSC complex, which controls lysosomal TORC1 activity. Addressing the function of WIPI3 in lysosomal phosphoinositide signaling in the first funding period we detailed the lysosomal PI3P- and PI(3,5)P2-dependent localization of WIPI3 at lysosomes. Furthermore, using image-based high-throughput shRNA screening we identified DDR1 and ABL1/2 to control membrane localization of WIPI3, and using SILAC-based qPhospho-proteomics and qPCR-based screening of 84 mTOR targets, we found that WIPI3 controls TORC1 via LAMTOR1 and DEPTOR. We propose to dissect the underlying molecular details of WIPI3-mediated TORC1 regulation via DEPTOR and LAMTOR1, as well as the control of WIPI3 by DDR1 and ABL protein kinases in the second function period. In addition, we will continue to study how FIG4 mutations in CMT4J impact autophagy and lysosome function as we demonstrated that selective autophagic-lysosomal degradation is impaired in CMT4J human patient-derived primary fibroblasts.

• DFG Programme: Research Units
• Subproject of: FOR 2625: Mechanisms of Lysosomal Homeostasis