The identity of specific serine phosphorylation residues of insulin receptor substrate (IRS)-2 and their impact on insulin signal transduction are largely unknown. Ser 675 and Ser 907 of mouse IRS-2 are adjacent to PI3 kinase or Grb2 binding domains, respectively. Using monoclonal phospho-site 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 si-RNA mediated knockdown of ERK1/2 indicated that the insulin-induced phosphorylation of Ser 907 was ERK-dependent. Phosphorylation of Ser 907 did not influence the insulin-induced association of IRS-2 with Grb2, but phosphorylation of the adjacent Tyr 911 was proven to be crucial in HEK 293 cells expressing IRS-2 Ala mutants. The insulin-induced phosphorylation of Ser 675 was prevented by inhibition and siRNA mediated knock-down of mTOR, but not of p70 S6K1. Mutation of Ser 675 to Ala did not affect downstream insulin signaling, but increased the half life of the protein, suggesting an involvement of phospho-Ser 675 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 PI3 kinase and Grb2 binding domains but might be implicated in the ERK and mTOR-mediated negative feedback control.