# Role of Tuberin S1365 Phosphorylation in mTORC1 Regulation

> **NIH NIH F31** · JOHNS HOPKINS UNIVERSITY · 2020 · $43,622

## Abstract

Project Summary
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.

## Key facts

- **NIH application ID:** 9960578
- **Project number:** 5F31HL143905-03
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Brittany Dunkerly-Eyring
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $43,622
- **Award type:** 5
- **Project period:** 2018-07-23 → 2021-06-25

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9960578

## Citation

> US National Institutes of Health, RePORTER application 9960578, Role of Tuberin S1365 Phosphorylation in mTORC1 Regulation (5F31HL143905-03). Retrieved via AI Analytics 2026-06-01 from https://api.ai-analytics.org/grant/nih/9960578. Licensed CC0.

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