CONSTRUCTION AND CHARACTERIZATION OF A CONDITIONALLY ACTIVE VERSION OF THE SERINE THREONINE KINASE AKT/

Akt is a serine/threonine kinase that requires a functional phosphatid ylinositol 3-kinase to be stimulated by insulin and other growth facto rs. 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 o f a mutant murine estrogen receptor that selectively binds 4-hydroxyta moxifen. The chimeric protein was expressed in NIH3T3 cells and was sh own to be stimulated by hormone treatment 17-fold after only a 20-min treatment. This hormone treatment also stimulated an approximate 3-fol d 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 co nditionally active Akt was also found to rapidly stimulate in these ce lls the phosphorylation of properties of PHAS-I, a key protein in the regulation of protein synthesis, The conditionally active Akt, when ex pressed in 3T3-L1 adipocytes, was also stimulated, although its rate a nd extent of activation was less then in the NIH3T3 cells, Its stimula tion was shown to be capable of inducing glucose uptake into adipocyte s by stimulating translocation of the insulin-responsive glucose trans porter GLUT4 to the plasma membrane.