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 ?-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 retaining canonical S6K1 activity, we will test the role of S6K1* in target phosphorylation in vivo, in lipid metabolism, and in diet-induced obesity.
Obesity is a worldwide, epidemic-scale problem with enormous health and economic costs. We have discovered a new signaling pathway that directs fat accumulation in adipocytes (fat cells). We will investigate the role of this pathway in fat synthesis in adipose tissue in mice, and anticipate insights that will permit development of new therapeutic targets and new drugs against obesity in humans.