title: Activity, phosphorylation state and subcellular distribution of GLUT4-targeted Akt2 in rat adipose cells.
abstract: In this study, fusion of the kinase domain of Akt2 to the cytosolic C terminus of exofacially-HA-tagged GLUT4 is used to investigate the activity, phosphorylation state and subcellular localization of Akt2 specifically targeted to the GLUT4-trafficking pathway in rat adipose cells. Fusion of wild-type (wt) Akt2, but not a kinase-dead (KD) mutant results in constitutive targeting of the HA-GLUT4 fusion protein to the cell surface to a level similar to that of HA-GLUT4 itself in the insulin-stimulated state. Insulin does not further enhance the cell-surface level of HA-GLUT4-Akt2-wt, but does stimulate the translocation of HA-GLUT4-Akt2-KD. Cell-surface HA-GLUT4-Akt2-wt is found to be phosphorylated on Ser474 in both the absence and presence of insulin, and mutation of Ser474 to Ala reduces the increased basal cell-surface localization of the fusion protein. While Ser474 phosphorylation of HA-GLUT4-Akt2-KD is detected only in the insulin-stimulated state, trapping this fusion protein on the cell surface by coexpression of a dominant negative mutant dynamin does not induce Ser474 phosphorylation. Phosphorylation on Thr309 is not detectable in either HA-GLUT4-Akt2-wt or HA-GLUT4-Akt2-KD, in either the basal or insulin-stimulated state, and mutation of Thr309 to Ala does not influence the insulin-independent increases in cell-surface localization and Ser474 phosphorylation. Expression of HA-GLUT4-Akt2-wt stimulates the translocation of cotransfected myc-GLUT4 to a level similar to that in the insulin-stimulated state; this increase is moderately reduced by mutation of Ser474 to Ala and absent with the kinase-dead mutant. These results demonstrate that targeting Akt2 to the GLUT4-trafficking pathway induces Akt2 activation and GLUT4 translocation. Ser474 phosphorylation is an autocatalytic reaction requiring an active kinase, and kinase activity is associated with a plasma membrane localization. Fusion of Akt2 to the C terminus of GLUT4 appears to substitute for Thr309 phosphorylation in activating the autocatalytic process.
authors.affiliation: EDMNS/DB, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 8 Center Dr MSC 0842, Bethesda, MD 20892-0842, USA.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Chen
authors.forename: Hadi
authors.initials: H
authors.lastname: Al-Hasani
authors.forename: Torbjorn
authors.initials: T
authors.lastname: Olausson
authors.forename: Ann-Marie
authors.initials: AM
authors.lastname: Wenthzel
authors.forename: Ulf
authors.initials: U
authors.lastname: Smith
authors.forename: Samuel W
authors.initials: SW
authors.lastname: Cushman
mesh_terms: D017667:Adipocytes / Q000378:metabolism*; D000273:Adipose Tissue / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D034281:Dynamins / Q000378:metabolism*; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000378:metabolism; D008297:Male; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions
chemical_list: D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C495694:Slc2a4 protein, rat; C494923:Akt2 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D034281:Dynamins