Insulin-induced serine phosphorylation of IRS-2 via ERK1/2 and mTOR: studies on the function of Ser675 and Ser907

L Fritsche, SS Neukamm, R Lehmann… - American Journal …, 2011 - journals.physiology.org
L Fritsche, SS Neukamm, R Lehmann, E Kremmer, AM Hennige, A Hunder-Gugel, M Schenk…
American Journal of Physiology-Endocrinology and Metabolism, 2011journals.physiology.org
The identity of specific serine phosphorylation residues of insulin receptor substrate (IRS)-2
and their impact on insulin signal transduction are largely unknown. Ser675 and Ser907 of
mouse IRS-2 are adjacent to PI 3-kinase or Grb2 binding domains, respectively. Using
monoclonal phosphosite-specific antibodies, we demonstrated the phosphorylation of both
serines after stimulation of Fao hepatoma cells with insulin, anisomycin, or phorbol esters.
Phosphorylation of both sites was a late and prolonged event during insulin treatment and …
The identity of specific serine phosphorylation residues of insulin receptor substrate (IRS)-2 and their impact on insulin signal transduction are largely unknown. Ser675 and Ser907 of mouse IRS-2 are adjacent to PI 3-kinase or Grb2 binding domains, respectively. Using monoclonal phosphosite-specific antibodies, we demonstrated the phosphorylation of both serines after stimulation of Fao hepatoma cells with insulin, anisomycin, or phorbol esters. Phosphorylation of both sites was a late and prolonged event during insulin treatment and was also detected in liver tissue of insulin-treated as well as refed mice. Inhibition and siRNA-mediated knockdown of ERK1/2 indicated that the insulin-induced phosphorylation of Ser907 was ERK dependent. Phosphorylation of Ser907 did not prevent the insulin-induced association of IRS-2 with Grb2, but phosphorylation of the adjacent Tyr911 was proved to be crucial in HEK 293 cells expressing IRS-2 Ala mutants. The insulin-induced phosphorylation of Ser675 was prevented by inhibition and siRNA-mediated knockdown of mTOR but not of p70S6K1. Mutation of Ser675 to Ala did not affect downstream insulin signaling but increased the half-life of the protein, suggesting an involvement of phospho-Ser675 in an accelerated degradation of IRS-2. Moreover, the insulin-induced degradation of IRS-2 was blocked by inhibition of mTOR. We conclude that the two novel insulin-dependent serine phosphorylation sites of IRS-2 were not involved in the regulation of the adjacent PI 3-kinase and Grb2 binding domains but might be implicated in the ERK- and mTOR-mediated negative feedback control.
American Physiological Society