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First published online 22 July 2003
doi: 10.1242/jcs.00675

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Activity, phosphorylation state and subcellular distribution of GLUT4-targeted Akt2 in rat adipose cells

¹ EDMNS/DB, NIDDK, NIH, 8 Center Dr MSC 0842, Bethesda, MD 20892-0842, USA
² German Institute for Human Nutrition, Arthur-Scheuner-Allee 114-116, 14558 Bergholz-Rehbrucke, Germany
³ Lundberg Laboratory for Diabetes Research, Department of Internal Medicine, The Sahlgrenska Academy at Gothenburg University, SE-413 45 Gothenburg, Sweden

^* Author for correspondence (e-mail: sam_cushman{at}nih.gov)

Accepted 19 May 2003

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.

Key words: PKB/Akt, GLUT4, Translocation, Phosphorylation

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