Construction and Characterization of a Conditionally Active Version of the Serine/Threonine Kinase Akt

• Published in: The Journal of biological chemistry Vol. 273; no. 19; pp. 11937 - 11943
• Main Authors: Kohn, A D, Barthel, A, Kovacina, K S, Boge, A, Wallach, B, Summers, S A, Birnbaum, M J, Scott, P H, Lawrence, Jr, J C, Roth, R A
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 08-05-1998
• Subjects: 3T3 Cells; Adaptor Proteins, Signal Transducing; Adipocytes - metabolism; Animals; Carrier Proteins; Cell Cycle Proteins; Enzyme Activation; Eukaryotic Initiation Factor-4E; Eukaryotic Initiation Factors; Glucose - metabolism; Glucose Transporter Type 4; Mice; Monosaccharide Transport Proteins - metabolism; Muscle Proteins; Myristates - metabolism; Peptide Initiation Factors - metabolism; Phosphoproteins - metabolism; Protein-Serine-Threonine Kinases; Proto-Oncogene Proteins - chemistry; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases - metabolism; Structure-Activity Relationship
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.19.11937

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.