Introduction:
The kinase complex mTORC1 serves as a nexus of metabolic control, which becomes activated in response to nutrient availability. mTORC1 signalling and metabolic activity become upregulated during cardiac hypertrophic to induce protein synthesis required for cellular growth. We previously showed that mTORC1 inhibition attenuates cardiac protein synthesis and hypertrophic growth in response to pressure overload, however full mTORC1 inhibition may be associated with undesired side effects which limits its clinical usage in the context of cardiac hypertrophy. Here, we screened for novel downstream targets of mTORC1 that mediate its control over protein synthesis and metabolic activity.
Methods:
Dynamic mTOR-dependent cap-binding proteins were identified using an APEX2 proximity labeling system together with pharmacological mTOR inhibition using Torin1. Hela, HEK239 and primary cardiomyocytes were used in vitro. Cardiomyocyte hypertrophy was induced with 50µM phenylephrine. Dependence of eIF4G1 phosphorylation on mTORC1 was tested using rapamycin, an mTORC1 inhibitor. Dynamics of eIF4G1 phosphorylation was investigated by time course experiments. Knockdown of eIF4G1 was achieved using siRNA. Translation was assessed through a puromycin incorporation assay. Metabolic activity of living cells was examined using an MTT assay at different timepoints to evaluate metabolic activity over time.
Results:
In order to identify specific translation-regulating downstream effectors of mTORC1, we performed a proximity-labeling screen for mTOR-dependent mRNA-cap-binding proteins, identifying the translation initiation factor eIF4G1, with currently unknown functions in cardiac hypertrophy. mTORC1-dependent phosphorylation of eIF4G1 occurred at evolutionary conserved sites of its interdomain linker during growth conditions, including Ser1147, which was confirmed by western blotting. Testing the dependence of the phosphorylation of eIF4G1 on nutrient availability and mTORC1 activity revealed that eIF4G1S1147 was unphosphorylated during starvation and also upon rapamycin administration. While knockdown of eIF4G1 did not show any impact on bulk protein synthesis in Hela cells at baseline, it significantly reduced metabolic activity within cells over time. Similar to HEK and Hela cells, eIF4G1S1147 became phosphorylated by mTORC1 in cardiomyocyte. Contrary to HEK/Hela cells, eIF4G1 knockdown resulted in a suppression of protein synthesis during a1-adrenergic receptor stimulation, indicating that eIF4G1 regulation is essential for cardiomyocyte growth.
Conclusions:
We show that eIF4G1 is an mTORC1 phospho-target, which is essential for the upregulation of protein synthesis in cardiomyocyte hypertrophy, but not at baseline. In addition, early experiments reveal an involvement of eIF4G1 in cellular metabolism. Future experiments will determine whether eIF4G1 may be targeted to counteract cardiomyocyte hypertrophy.
