# Multisite phosphorylated S6K1 directs a regulatory module determining adipocyte lipid metabolism

> **NIH NIH R01** · CLEVELAND CLINIC LERNER COM-CWRU · 2022 · $467,490

## Abstract

Project Summary/Abstract
Obesity is an epidemic-scale problem in the U.S. and worldwide with enormous health and economic costs. The
mTORC1-S6 kinase 1 (S6K1) axis drives anabolic pathways determining obesity. We recently identified
glutamyl-prolyl tRNA synthetase (EPRS) as an mTORC1-S6K1 target that contributes to mouse adiposity.
Insulin-stimulated EPRS phosphorylation at Ser999 by S6K1 in adipocytes induces its binding to fatty acid
transport protein 1 (FATP1) and translocation to the plasma membrane to increase long-chain fatty acid (LCFA)
uptake. Recent studies reveal that phosphorylation of S6K1 by cyclin-dependent kinase 5 (Cdk5) at Ser424 and
Ser429 in the S6K1 C-terminus are required for phosphorylation of EPRS, but not for canonical substrates such
as RPS6. This unexpected finding indicates that embedded in S6K1 is a target-selective phospho-code in which
combinatorial phospho-site phosphorylation determines kinase targets. To identify additional targets of multi-phosphorylated
S6K1 (termed S6K1*) but not mTORC1-activated S6K1, we transfected HEK cells with S6K1
bearing phospho-mimetic mutations at the 3 phospho-sites, or wild-type S6K1 cDNA. Three new S6K1* targets
were identified by mass spectrometry and validated in adipocytes – coenzyme A synthase (COASY), cortactin,
and lipocalin 2. Importantly, all are implicated in adipocyte lipid metabolism: P-EPRS transports FATP1 to the
plasma membrane for increased LCFA uptake; COASY catalyzes the final two steps of synthesis of coenzyme
A, required for LCFA activation; lipocalin 2 increases LCFA β-oxidation and insulin resistance; and cortactin is
required for insulin-stimulated transport of Glut4-containing vesicles to plasma membranes. We propose that
S6K1* directs an adipocyte lipid metabolon, and is a major contributor to obesity-related phenotypes driven by
the mTORC1-S6K1 axis. We will test this hypothesis by pursuit of 3 Specific Aims: In Aim 1 we determine
S6K1*/target docking domains. By mass spectrometry and site-directed mutation analysis, we will determine
specific S6K1*-directed phosphorylation sites in the targets. In Aim 2 we determine the function of
phosphorylated S6K1* targets in adipocyte lipid metabolism. We will determine the mechanism of insulin-stimulated
transport and binding of P-EPRS to the adipocyte plasma membrane; the role of phosphorylation in
COASY catalytic activity and localization; whether P-cortactin transports P-EPRS/FATP1-containing vesicles to
the plasma membrane; and extracellular secretion and intracellular localization of P-lipocalin 2, and its role in
LCFA oxidation. In Aim 3 we elucidate In vivo role of S6K1* in lipid metabolism and obesity. We will determine
the effect of diet-induced obesity on the S6K1* activation pathway and on target phosphorylation in mice.
Taking advantage of our new mouse model (generated by Crispr-Cas9 technology) bearing a Ser429-to-Ala
mutation in Rps6kb1 (mouse gene encoding S6K1) that lack S6K1* activity, while retainin...

## Key facts

- **NIH application ID:** 10349543
- **Project number:** 5R01DK123236-03
- **Recipient organization:** CLEVELAND CLINIC LERNER COM-CWRU
- **Principal Investigator:** PAUL L FOX
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $467,490
- **Award type:** 5
- **Project period:** 2020-04-09 → 2024-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10349543

## Citation

> US National Institutes of Health, RePORTER application 10349543, Multisite phosphorylated S6K1 directs a regulatory module determining adipocyte lipid metabolism (5R01DK123236-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10349543. Licensed CC0.

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