Abstract 346: Tuberin S1365 Phosphorylation Regulates Mechanistic Target of Rapamycin Complex 1 (mTORC1) Pathological Signaling While Sustaining Metabolic Sensor Function

Rationale: The Mechanistic Target of Rapamycin complex 1 (mTORC1) integrates signaling and sensory inputs to maintain cardiomyocyte homeostasis, and itself is negatively regulated by the signaling nexus tuberin (TSC2). We identified a novel TSC2 phosphorylation site S1365 (pS1365) targeted by protein kinase G (PKG), which suppressed hormonal growth factor (PE or ET1)-stimulated mTORC1 activity to attenuate pathological cardiomyocyte hypertrophy. This was recapitulated during growth factor stimulation with expression of a phospho-null (S1365A) or a phospho-mimetic (S1365E) TSC2 that exacerbated or blunted mTORC1 activation, respectively. The nature of TSC2 pS1365 as a potential metabolic sensor is unknown and will provide mechanistic insight into the TSC2 kinase input hierarchy that regulates the homeostatic function of mTORC1. Objective: To determine how pS1365 affects the ability of TSC2 to integrate metabolic dependent signals to regulate mTORC1. Methods/Results: TSC2 KO MEFs were infected with TSC2 WT or S1365A adenovirus, and then stimulated with ET1 (hormonal stress that also activates mTORC1), and 2-DG (AMPK stimulation). Both groups responded with similar decreases in mTORC1 activation regardless of pS1365. Phosphorylation of the AMPK site on TSC2 (S1387) was increased in all groups despite the presence of a phospho-null S1365. Both MEFs and neonatal rat cardiomyocytes (NRCMs) infected with TSC2 WT, S1365A, or S1365E adenovirus similarly increased mTORC1 activation with insulin (PI3K-Akt-TSC2 pathway) treatment. Serum withdrawal from NRCMs reduced mTORC1 activation in all groups regardless of whether a WT, S1365A, or S1365E TSC2 was expressed. In NRCMs subject to hypoxia (a combination of Erk, Akt, AMPK signaling), there was a similar observation with only nominal changes between WT, S1365A, and S1365E TSC2. Conclusions: The energy and nutrient sensing role of the TSC2-mTORC1 pathway remains intact regardless of the phospho-status of TSC2 S1365. These findings provide important mechanistic insight into the function of TSC2 pS1365 as a potent suppressor of pathological mTORC1 activation while not affecting the ability of TSC2 to respond to the metabolic dependent signals – AMPK (energy), PI3K-Akt (insulin), and serum starvation.