The Identification And Functional Study Of MTORC1-regulated Proteins In Mouse Embryonic Fibroblast

Mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that exists as two protein complexes in mammalian cells, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Only mTORC1 activity can be inhibited by rapamycin. mTORC1 plays important roles in regulating cell growth and proliferation. Although the aberrant activation of mTORC1 has been observed in many human diseases, such as cancer, diabetes, obesity and neurodegeneration, the molecular mechanism of mTORCl signaling related to disease development has yet to be explored. One important function of mTORC1 is regulating gene expression at both transcriptional and translational levels. However, the proteins regulated by mTORC1 activation and their roles in mTORC1 downstream functions are still poorly understood. Therefore, targeting on the mTORCl-regulated proteins could help us uncover the downstream functions of mTORC1.In this study, a quantitative proteomic analysis was performed in WT MEFs (wild type mouse embryonic fibroblasts), TSC2-/- MEFs and rapamycin-treated TSC2-/- MEFs. A total of 58 mTORCl-regulated proteins were identified, most of which could be categorized into eight functional groups including cytoskeleton organization, mitochondrial function, ER function, transcription regulation, protein nuclear import process, glycolysis, lipid metabolism and cell cycle. This result first provided evidences to support that mTORC1 activation positively regulated the expressions of several key proteins in protein nuclear import pathway including KPNA2, RanGAP1, RanBP2, NUP93 and NUP107.Three proteins, KPNA2, RanGAP1 and RanBP2, were selected for further validation by Western blot analysis, and the results suggested that mTORC 1 activation positively regulated their abundances in MEFs. The regulation of KPNA2 by mTORC1 could also be validated in other mouse and human cells. Then we further explored how mTORC1 regulated KPNA2. KPNA2 protein abundance was not changed by inhibiting S6K1 or 4E-BP1, two well-characterized mTORC1 substrates that involved in translational regulation. However, KPNA2 mRNA abundance was positively regulated by mTORC1. These results suggested that mTORCl regulated KPNA2 at the transcriptional level, rather than at the S6K1- and 4E-BP1-dependent translational level.In our proteomic data, nine key enzymes in glycolysis pathway were identified and all of their abundances were significantly increased (p<0.05) in response to mTORC 1 activation and significantly decreased (p<0.05) due to mTORC1 inhibition. We further confirmed that mTORC1 activation positively regulated the expressions of glycolytic genes using real-time PCR. Previous reports have revealed that KPNA2 could regulate the expression of glycolytic genes. We therefore hypothesized that KPNA2 might play important roles in regulating glycolytic genes downstream of mTORC 1. We knocked down the expression of KPNA2 in TSC2-/- MEFs and found that the mRNA abundance of glycolytic genes was significantly decreased. The result proved that KPNA2 really involved in regulating glycolytic genes downstream of mTORC 1.Since KPNA2 could recognize and bind the nuclear localization sequence (NLS) of HIF1α, a transcriptional factor in regulating glycolytic genes downstream of mTORC1, we examined whether KPNA2 regulates glycolytic genes through affecting the expression or subcellular localization of HIF1α. Although mTORC1 activation did increase HIF1α abundance and HIF1α knockdown significantly decreased the expressions of glycolytic genes induced by mTORC1, KPNA2 knockdown had no effects on the abundance and subcellular localization of HIF1α. Therefore, KPNA2 participates in the expression of glycolytic genes by mTORC1 in a HIF1α-independent manner. Furthermore, HIF1α also had no influence on KPNA2 expression, because HIF1α knockdown did not change KPNA2 abundance. The above results indicate that mTORC 1 regulates the expression of glycolytic genes through two parallel pathways, by controlling HIF1α and KPNA2 expression, respectively.Taken together, this study profiled the mTORC1-regulated proteins in MEFs, and found several proteins/enzymes in protein nuclear import pathway and glycolysis pathway were positively regulated by mTORC1 activation. Combining multiple molecular biology methods, we first discovered that mTORC 1 positively regulated the expression of the nuclear transport protein KPNA2, and further proved that KPNA2 played important roles in regulating glycolytic genes by mTORC1 with a HIF1α-independent manner. Therefore, the study provides additional angle for understanding mTORC1 functions and its regulation towards glycolytic genes.