Construction and characterization of a conditionally active version of the serine/threonine kinase Akt

Akt is a serine/threonine kinase that requires a functional phosphatidylinositol 3-kinase to be stimulated by insulin and other growth factors. When directed to membranes by the addition of a src myristoylation sequence, Akt becomes constitutively active. In the present study, a conditionally active version of Akt was constructed by fusing the Akt containing the myristoylation sequence to the hormone binding domain of a mutant murine estrogen receptor that selectively binds 4-hydroxytamoxifen. The chimeric protein was expressed in NIH3T3 cells and was shown to be stimulated by hormone treatment 17-fold after only a 20-min treatment. This hormone treatment also stimulated an approximate 3-fold increase in the phosphorylation of the chimeric protein and a shift in its migration on SDS gels. Activation of this conditionally active Akt resulted in the rapid stimulation of the 70-kDa S6 kinase, This conditionally active Akt was also found to rapidly stimulate in these cells the phosphorylation of properties of PHAS-I, a key protein in the regulation of protein synthesis, The conditionally active Akt, when expressed in 3T3-L1 adipocytes, was also stimulated, although its rate and extent of activation was less then in the NIH3T3 cells, Its stimulation was shown to be capable of inducing glucose uptake into adipocytes by stimulating translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane.