Global Phosphoproteomic
Analysis of Insulin/Akt/mTORC1/S6K
Signaling in Rat Hepatocytes
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Abstract
Insulin resistance is a hallmark
of type 2 diabetes. Although multiple
genetic and physiological factors interact to cause insulin resistance,
deregulated signaling by phosphorylation is a common underlying mechanism.
In particular, the specific phosphorylation-dependent regulatory mechanisms
and signaling outputs of insulin are poorly understood in hepatocytes,
which represents one of the most important insulin-responsive cell
types. Using primary rat hepatocytes as a model system, we performed
reductive dimethylation (ReDi)-based quantitative mass spectrometric
analysis and characterized the phosphoproteome that is regulated by
insulin as well as its key downstream kinases including Akt, mTORC1,
and S6K. We identified a total of 12 294 unique, confidently
localized phosphorylation sites and 3805 phosphorylated proteins in
this single cell type. Detailed bioinformatic analysis on each individual
data set identified both known and previously unrecognized targets
of this key insulin downstream effector pathway. Furthermore, integrated
analysis of the hepatic Akt/mTORC1/S6K signaling axis allowed the
delineation of the substrate specificity of several close-related
kinases within the insulin signaling pathway. We expect that the data
sets will serve as an invaluable resource, providing the foundation
for future hypothesis-driven research that helps delineate the molecular
mechanisms that underlie the pathogenesis of type 2 diabetes and related
metabolic syndrome