Serine 302 Phosphorylation of Mouse Insulin Receptor Substrate 1 (Irs1) Is Dispensable For Normal Insulin Signaling and Feedback Regulation by Hepatic S6 Kinase

Kyle D, etc
JBC, 2016

Constitutive activation of the mammalian target of rapamycin complex 1 and S6 kinase (mTORC1¿S6K) attenuates insulin-stimulated Akt activity in certain tumors, in part through 'feedback' phosphorylation of upstream insulin receptor substrate IRS1. However, the significance of this mechanism for regulating insulin sensitivity in normal tissue remains unclear. We investigated the function of S302 in mouse Irs1---the major site of its phosphorylation by S6K in vitro---through genetic knock-in of a serine-to-alanine mutation (A302). Although insulin rapidly stimulated feedback phosphorylation of S302 in mouse liver and muscle, homozygous A302 mice (A/A) and their knock-in controls (S/S) exhibited similar glucose homeostasis and muscle insulin signaling. Further, both A302 and control primary hepatocytes from which Irs2 was deleted showed marked inhibition of insulin-stimulated Irs1 tyrosine phosphorylation and PI 3-kinase (PI3K) binding after emetine treatment to raise intracellular amino acids and activate mTORC1¿S6K signaling. To specifically activate mTORC1 in mouse tissue, we deleted hepatic Tsc1 using Cre adenovirus. Though it moderately decreased Irs1·PI3K association and Akt phosphorylation in liver, Tsc1 deletion failed to cause glucose intolerance or promote hyperinsulinemia in mixed-background A/A or S/S mice. Moreover, Tsc1 deletion failed to stimulate phospho-S302 or other putative S6K sites within Irs1, whereas ribosomal S6 protein was constitutively phosphorylated. Following acute Tsc1 deletion from hepatocytes, Akt phosphorylation---though not Irs1·PI3K association---was rapidly restored by treatment with mTORC1 inhibitor rapamycin. Thus, within the hepatic compartment, mTORC1¿S6K signaling regulates Akt largely through IRS-independent means, with little effect upon physiologic insulin sensitivity.

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doi: 10.1074/jbc.M116.71491
Boston Children's Hospital,