Browsing by Subject "Mechanistic Target of Rapamycin Complex 1"
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Item Crosstalk Signaling Between cAMP and mTORC1(August 2021) Melick, Chase Hunter; DeBerardinis, Ralph J.; Cobb, Melanie H.; Pan, Duojia; Jewell, Jenna L.The mammalian target of rapamycin complex 1 (mTORC1) senses multiple stimuli to regulate anabolic and catabolic processes. G-protein-coupled receptors (GPCRs) paired to Gs proteins increase cyclic adenosine 3'5' monophosphate (cAMP) to activate protein kinase A (PKA), which phosphorylates Raptor at Ser 791 resulting in potent mTORC1 inhibition. We identified a novel mTORC1-interacting protein called A-kinase anchoring protein 8L (AKAP8L). Using biochemical assays, we found that the N-terminal region of AKAP8L binds to mTORC1 in the cytoplasm. Importantly, loss of AKAP8L decreased mTORC1-mediated processes such as translation, cell growth and cell proliferation. AKAPs anchor protein kinase A (PKA) through PKA regulatory subunits, and we show that AKAP8L can anchor PKA through regulatory subunit I (RI). Full-length AKAP8L restored mTORC1-regulated biology, whereas AKAP8L missing the N-terminal region that confers interaction with mTORC1 did not. Additionally, we have shown that H89 (N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide), a well-characterized ATP-mimetic kinase inhibitor, renders the phosphorylation of S6K1 and AKT resistant to mTOR inhibitors across multiple cell lines. Moreover, H89 prevented the dephosphorylation of AKT and S6K1 under nutrient depleted conditions. PKA and other known H89-targeted kinases do not alter the phosphorylation status of S6K1 and AKT. Pharmacological inhibition of some phosphatases also enhanced S6K1 and AKT phosphorylation. These findings suggest a new unknown target for H89 by which it sustains the phosphorylation status of S6K1 and AKT, resulting in mTOR signaling. Lastly, we identified A-kinase anchoring protein 13 (AKAP13) as a crucial scaffold involved in GPCR-Gs signaling to mTORC1. AKAP13 potently enhances Raptor Ser 791 phosphorylation and inhibits mTORC1 activity. Consistently, in cells where Raptor Ser 791 is mutated to Ala, AKAP13 is unable to supress mTORC1 activity. AKAP13 mediates mTORC1-induced cell proliferation, cell size and colony formation. Interestingly, AKAP13 expression inversely correlates with mTORC1 activation and positively correlates with overall lung adenocarcinoma patient survival. Our results place the GPCR-Gas signaling pathway to mTORC1 as a potential target that may be beneficial for human diseases with hyperactivated mTORC1.Item Neural Mechanisms and Behaviors in Models of Conditional Nprl2 Loss(2020-08-01T05:00:00.000Z) Dentel, Brianne Marie; Johnson, Jane E.; Tu, Benjamin; Pascual, Juan M.; Tsai, PeterThe amino acid sensitive arm of mTORC1 regulation signals through the GATOR1 complex. Loss of function of GATOR1 contributes to several neurodevelopmental disorders and medically refractive epilepsy. Mutations to one of the essential subunits of GATOR1, NPRL2, are sufficient to cause focal epilepsy and schizophrenia; yet, little is known about its role in the nervous system. Here we demonstrate the loss of Nprl2 in excitatory cells in the neocortex and hippocampus is sufficient to cause mTOR-related pathology, decreased survival and spontaneous seizures. By inhibiting mTOR activity with rapamycin we were able to rescue brain size, seizures and survival. We also show that loss of Nprl2 results in a down-regulation of synaptic proteins, and several metabolic disruptions. Furthermore, we demonstrated that the significantly increased glycine was a primary mechanism which increased synaptic excitability. This suggests that targeting the glycine binding site on the NMDA receptor may be a targeted therapy for future study. We also demonstrated, in three different cell-specific conditional knockout models, distinct behavioral profiles which points to the importance of Nprl2 in various neurodevelopmental disorders. These findings demonstrate the multifaceted effects of Nprl2 loss in excitatory cells- which demonstrate seizures and early mortality; excitatory and inhibitory neurons- which had seizures, hyperactivity, social and learning deficits; inhibitory cells- which demonstrated severe hyperactivity, social and learning deficits; and Purkinje cell-specific loss- which had seizure susceptibility, reversal learning deficits and delayed-onset social deficits and altered PPI. These findings highlight the significant role NPRL2 has in the nervous system and future studies in these models will aid in understanding and potentially developing targeted therapies to address the molecular and cellular mechanisms underlying NDDs and seizures in NPRL2 loss.Item TRPML1 Promotes Protein Homeostasis in Melanoma Cells by Negatively Regulating MAPK and mTORC1 Signaling(2019-07-08) Kasitinon, Stacy Yuan; DeBerardinis, Ralph J.; Morrison, Sean J.; Mendell, Joshua T.; Vernino, StevenA major goal of studying melanoma is to identify therapeutic vulnerabilities that can be exploited to improve patient treatment. Melanoma cells are particularly sensitive to perturbations in ion homeostasis, especially when ion gradients are perturbed in combination with MAP kinase inhibition. I hypothesized that melanoma cells preferentially require certain ion channels and transporters for growth and survival. I thus screened ion channels and transporters throughout the genome to identify those required by human melanoma cells but not by normal human melanocytes. I discovered that Mucolipin-1 (MCOLN1), which encodes the lysosomal cation channel TRPML1, is preferentially required for the survival and proliferation of melanoma cells. Loss of MCOLN1/TRPML1 function impaired the growth of patient-derived melanomas in culture and in xenografts but did not affect the growth of human melanocytes. TRPML1 expression was elevated in melanoma cells relative to melanocytes and was required in melanoma cells to negatively regulate MAPK pathway and mTORC1 signaling. TRPML1-deficient melanoma cells exhibited decreased survival, proliferation, tumor growth, and macropinocytosis as well as serine depletion and proteotoxic stress. All of these phenotypes were partially or completely rescued by mTORC1 inhibition. Melanoma cells thus increase TRPML1 expression relative to melanocytes to attenuate MAPK and mTORC1 signaling. This helps melanoma cells prevent overactivation of these oncogenic signaling pathways, sustain macropinocytosis and avoid proteotoxic stress. Further investigation of the role of TRPML1 in melanoma may ultimately guide future patient therapies and contribute to our understanding of ion channels and transporters in cancer.Item Yeast Ataxin-2 (Pbp1) Condensates Regulate TORC1 Activity and Autophagy in Response to Cellular Redox State(2018-11-26) Yang, Yu-San; O'Donnell, Kathryn A.; Tu, Benjamin; DeBerardinis, Ralph J.; Potts, Patrick RyanYeast ataxin-2, also known as Pbp1 (Poly(A) binding protein-binding protein 1), is an intrinsically disordered protein that has earlier been implicated in stress granule formation, RNA biology, and neurodegenerative disease. However, the normal endogenous function of Pbp1 and ataxin-2 remains poorly understood. In this dissertation, I identified Pbp1 as a dedicated regulator of TORC1 signaling and autophagy under conditions that require mitochondrial respiration. Unlike the autophagy-deficient atg mutants that harbor severe growth defects, pbp1 null mutants exhibited significantly increased cell growth despite lack of autophagy. I discovered that Pbp1 binds to TORC1 specifically during respiratory growth, but utilizes an additional methionine-rich, low complexity (LC) region to inhibit TORC1. This LC region of Pbp1 forms reversible cross-β fibrils that facilitate phase transition of the protein into either liquid-like or gel-like states in vitro and enables self-association of full-length Pbp1 into pelletable assemblies in vivo. Sequence analysis revealed that Pbp1 LC region contains an unusually high frequency of methionine residues (24 methionines in 150 a.a.) compared to the rest of the yeast proteome. I showed that the phase separation of Pbp1 is mediated by these methionine residues, which are sensitive to H2O2-mediated oxidation and mitochondrial toxins in living cells. I also observed that the phase separation of Pbp1 mediated by its C-terminal LC region is responsive to the activity state of mitochondria and required for TORC1 inhibition. Mutants that weaken phase separation in vitro exhibit reduced capacity to inhibit TORC1 and induce autophagy in vivo. Loss of Pbp1 leads to mitochondrial dysfunction and reduced fitness during nutritional stress. Thus, Pbp1 forms a condensate in response to respiratory status to regulate TORC1 signaling. These observations offer a mechanistic explanation describing how reversible formation of condensates formed from the LC region of Pbp1 has evolved as a sensor of cellular redox state.