Activated [alpha].sub.2-Macroglobulin Binding to Cell Surface GRP78 Induces T-Loop Phosphorylation of Akt1 by PDK1 in Association with Raptor

PDK1 phosphorylates multiple substrates including Akt by PIP.sub.3 -dependent mechanisms. In this report we provide evidence that in prostate cancer cells stimulated with activated [alpha].sub.2 -macroglobulin ([alpha].sub.2 M*) PDK1 phosphorylates Akt in the T-loop at Thr.sup.308 by using Raptor in the mTORC1 complex as a scaffold protein. First we demonstrate that PDK1, Raptor, and mTOR co-immunoprecipitate. Silencing the expression, not only of PDK1, but also Raptor by RNAi nearly abolished Akt phosphorylation at Akt.sup.Thr308 in Raptor-immunoprecipitates of [alpha].sub.2 M*-stimulated prostate cancer cells. Immunodepleting Raptor or PDK from cell lysates of cells treated with [alpha].sub.2 M* drastically reduced Akt phosphorylation at Thr.sup.308, which was recovered by adding the supernatant of Raptor- or PDK1-depleted cell lysates, respectively. Studies of insulin binding to its receptor on prostate cancer cells yielded similar results. We thus demonstrate that phosphorylating the T-loop Akt residue Thr.sup.308 by PDK1 requires Raptor of the mTORC1 complex as a platform or scaffold protein.