Endocytosis and Endolysosomal Trafficking of Receptor Tyrosine Kinases in the Regulation of Chaperone-Mediated Autophagy

Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal proteolysis, where proteins bearing consensus motifs are individually selected for lysosomal degradation. By selectively targeting a subset of the proteome, CMA downregulates fundamental anabolic processes, including glycolysis, fatty acid synthesis, and translation at the cytoplasmic ribosome. CMA regulates the abundance of proteins whose overaccumulation contributes to age-related diseases, including Parkinson’s disease, Alzheimer’s disease, fatty-liver disease, oncogenesis, and atherosclerosis. Conversely, we have found that CMA is constitutively active in three long-lived mouse stocks with perturbed insulin signaling, which also results in very low rates of spontaneous cancers. These findings support a widely-held view that CMA promotes resistance to age-related diseases. Thus, we have undertaken the long-term goal of identifying and refining interventions for activating CMA therapeutically. This study will contribute to our long-term vision by characterizing novel mechanisms of CMA regulation, downstream of two druggable RTKS34, the insulin (INS) receptor (INSR) and the plateletderived growth factor (PDGF) receptor (PDGFR). We recently showed that CMA is negatively regulated by the PI3K/PDPK1/AKT signaling axis, and that two inhibitors of class I PI3K activate CMA in mice. However, the serious side effects of these drugs limit their translational potential. The PI3K/PDPK1/AKT signaling axis is activated downstream of ligand binding to receptor tyrosine kinases (RTKs). Both the endocytosis and the endolysosomal trafficking of activated RTKs are required for activation of AKT in the endolysosomal system. AKT, localized to the lysosomal membrane, is a critical negative regulator of CMA. These findings suggest the hypothesis that the endocytosis and endolysosomal trafficking of RTKs is required for the activation of AKT at the lysosomal membrane, which suppresses CMA. Consistent with this hypothesis, our unpublished preliminary data show that inhibition of dynamin-mediated endocytosis, or MAP4-based endosomal trafficking, activate CMA. CMA is activated in cell culture by serum starvation. Both PDGF and INS are sufficient to block the activation of CMA by serum starvation, suggesting that signaling downstream of PDGFR and INSR inhibit CMA. Specific Aim 1 will evaluate the role of two RTKs, the insulin receptor (INSR) and PDGF receptor (PDGFR), and their clathrin-mediated endocytosis, in CMA regulation. We expect that this aim will provide a more sophisticated view of CMA regulation and lay the groundwork for future studies concerned with the relationship between RTKs, CMA, and oncogenesis. Specific Aim 2 will evaluate microtubule-based trafficking (controlled by MAP4) of endocytic vesicles and endosomes containing active RTKs complexed to PIK3CA, for its role in regulating lysosomal AKT and CMA. We expect that the findings of this aim will resolve important unanswered questions about how trafficking in the endolysosomal system regulates CMA, and provide insights into new therapeutic targets for activating CMA, without the serious side effects of class I PI3K inhibitors.