Inhibition of phosphatidylinositol-3-kinase enhances insulin stimulation of insulin receptor substrate 1 tyrosine phosphorylation and extracellular signal-regulated kinases in mouse R- fibroblasts

Insulin stimulates phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinases (ERK) in various mammalian cells. To study the role of PI3K in insulin stimulation of ERK, we employed PI3K inhibitor LY294002 and mouse embryonic R- fibroblasts lacking IGF-1 receptors. In these R- cells, PI3K inhibition by LY294002 enhanced insulin stimulation of ERK phosphorylation whereas LY294002 inhibited insulin stimulation of Akt phosphorylation. The enhanced insulin stimulation of ERK phosphorylation was accompanied by increased IRS-1 tyrosine phosphorylation. Insulin stimulation of insulin receptor tyrosine phosphorylation was not altered. PI3K inhibition increased IRS-1-Grb2 complex formation and ras activity following insulin treatment of cells. Increased insulin stimulation of ERK by PI3K inhibition was mediated by the MEK/ERK pathway, but did not involve inhibitory Ser(259) phosphorylation of raf that was reported to be mediated by Akt. In summary, PI3K inhibition in R- cells enhanced insulin stimulation of ERK phosphorylation by mechanisms involving enhancement of IRS-1 tyrosine phosphorylation, IRS-1-Grb2 complex formation and the ras/MEK/ERK pathway.