Cardiovascular disease (CVD) is a leading cause of death worldwide, with approximately 17.7 million people dying from CVD in 2015. Patients with abnormal left ventricular hypertrophy are at higher risk for CVD. This type of muscle growth is stimulated by multiple factors, but one with a particularly central role is the protein cluster - mechanistic target of rapamycin complex 1 (mTORC1). Tuberin (TSC2), a GTPase-activating protein (GAP), is an intrinsic negative regulator of mTORC1. TSC2 is phosphorylated by many kinases, including Akt, p90RSK, AMP activated kinase (AMPK), and extracellular signaling related kinase (ERK1/2). These transduce metabolic and growth signaling to impact mTORC1 activation in one or the other direction. We recently found that cGMP-activated protein kinase G (PKG) also suppresses mTORC1 activation, and identified S1365 on TSC2 as the critical site modified for this regulation. New data with gain and loss of function S1365 phospho- mutations in vitro and in vivo support this signaling. However, many questions remain. It is unknown if PKG directly phosphorylates TSC2 and/or if other kinases are involved. While preliminary data shows S1365 modification is a potent modifier (in either direction) with growth hormone and hemodynamic stress, whether this indeed serves as a central command switch over all mTORC1 input signaling, and/or if it alters TSC2 translocation to or from the lysosome, a putative key mTORC1 control mechanism, are both unknown. Lastly, S1365 is near multiple phosphorylation sites on TSC2 targeted by AMPK, that also stimulate its GAP activity. This raises potential crosstalk between a metabolic control input to mTORC1 and that via S1365 targeting. In this project, I will address each of these questions.
In Aim 1, I use two assays developed by Kevan Shokat that detect if a selective kinase modifies TSC2 directly, or if other kinases are involved. These use a mutated kinase that can accept a bulky ATP, or an ATP-acrylate crosslinker that binds a mutated TSC2 substrate (with serine-cysteine substitution at S1365). Mutated TSC2 S1365A or S1365E KI mice or cultured cells are used to test its impact over alternative kinase inputs into TSC2 control of mTORC1.
In Aim 2, I determine if these mutations impact TSC2 translocation to the lysosome upon activation, a key element of its control over mTORC1. Studies use immunofluorescence colocalization with the various TSC2 mutations and mTORC1 stimuli.
Aim 3 is translational, and uses an in vivo model to test if a global knock-in S1365A and S1365E mouse has altered responsiveness to AMPK-related modulation of mTOR. This is performed with short term (12 or 24 hrs) food withdrawal or ischemia-reperfusion in the heart. Together, these studies will greatly advance our discovery of a novel tool to modulate mTORC1 signaling and its potential to treat cardiac disease.
Recent studies in our laboratory uncovered that serine 1365 on tuberin (TSC2) is phosphorylated following activation of protein kinase G in isolated myocytes and the intact heart. This potently alters the activation or suppression of mechanistic target of rapamycin (mTOR) and its downstream growth/autophagy signaling. In this project, I will elucidate fundamental mechanisms by which this occurs, determining if PKG is a direct modifier of TSC2, if it controls multiple signaling inputs, and in particular the role played on mTOR energy control.
