Insulin-like growth factor (IGF) binding protein-3 potentiation of IGF action is mediated through the phosphatidylinositol-3-kinase pathway and is associated with alteration in protein kinase B/AKT sensitivity

Cell-association and processing of insulin-litre growth factor binding prot ein-3 (IGFBP-3) by cultured bovine fibroblasts results in markedly enhanced type I IGF receptor signaling at a step distal to Ligand binding. The purp ose of the present study was to determine the intracellular mediators of IG FBP-3's potentiating effect. Preincubation of cultured bovine fibroblasts w ith 50 nM IGFBP-3 had no effect alone, but enhanced by 3- to 4-fold IGF-I-s timulated H-3-aminoisobutryric acid (AIB) uptake. IGFBP-3-induced potentiat ion was specifically prevented if an inhibitor of phosphatidylinositol 3 (P I3)-kinase activation (LY294002), but not an inhibitor of mitogen-activated protein kinase activation (PD98059), was present during the preincubation period. IGFBP-3 did not directly activate the downstream effector of PI3-ki nase, protein kinase B (PKB)/Akt. However, the sensitivity of PKB/Akt to ac tivation by IGF-I was increased by 2- to 4-fold with IGFBP-3 pretreatment. This increased sensitivity was accompanied by altered mobility of PKB/Akt o n SDS-polyacrylamide gels, suggestive of a diminished phosphorylation state . Consistent with this, okadaic acid, a potent serine/threonine phosphatase inhibitor, was able to block the potentiation effect of IGFBP-3 and preven t the altered mobility of the PKB/Akt molecule in response to IGFBP-3 treat ment. PKB/Akt immunoprecipitated from IGFBP-3-pretreated cells was no longe r recognized by an antibody specific for phosphorylated threonine followed by proline. These data indicate that IGFBP-3 modulates type I IGF receptor signaling through an effect on PI-3-kinase pathway substrates and suggest a novel mechanism of dephosphorylation whereby PKB/Akt is transformed into a more sensitive substrate of type I IGF receptor signaling.