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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

View larger version (8K): [in a new window]   Fig. 1. Cell-surface expression of HA-GLUT4-Akt2 fusion constructs in rat adipose cells. Isolated cells were transfected with HA-GLUT4, HA-GLUT4-Akt2-wt, and HA-GLUT4-Akt2-K179A (KD), and cultured for 20 hours. After harvesting the cells, cell-surface levels of the constructs were determined in the transfected cells in the basal (white) and insulin-stimulated states (67 nM, black) using an HA antibody binding assay and the cell-surface-associated radioactivity was expressed relative to the basal HA-GLUT4 value in each experiment (1668, 1558, 2311 cpm). Relative cell-surface levels were then normalized to the total expression level of each construct determined by immunoblotting as described in Materials and Methods. Results are the means ± s.e.m. of the means obtained from at least duplicate determinations in each of 3-5 independent experiments.

View larger version (58K): [in a new window]   Fig. 2. Phosphorylation of HA-GLUT4-Akt2 fusion proteins at Ser474 and Thr309 in rat adipose cells. Isolated cells were transfected with HA-GLUT4-Akt2-wt (WT) and HA-GLUT4-Akt2-K179A (KD), and cultured for 20 hours. After harvesting the cells, total cell membrane proteins were prepared from the transfected cells in the basal and insulin-stimulated states (67 nM) by differential centrifugation. 40 µg of protein were subjected to immunoblotting with anti-Akt-phospho-Ser473 or -phospho-Thr308 antibody. These data are representative of 3 independent experiments.

View larger version (34K): [in a new window]   Fig. 3. Effects of dominant negative dynamin on cell-surface expression (A) and Ser474 phosphorylation (B) of HA-GLUT4-Akt2-KD in rat adipose cells. Isolated cells were transfected with HA-GLUT4-Akt2-KD alone or co-transfected with HA-GLUT4-Akt2-KD and dominant negative dynamin, and cultured for 20 hours. After harvesting the cells, cell-surface levels of HA-GLUT4-Akt2-KD (A), and Ser474 phosphorylation of Akt2 in membrane fractions (B) were detected in the transfected cells in the basal (white) and insulin-stimulated states (67 nM, black) using an anti-HA antibody binding assay and immunoblotting, respectively. Results in A are the means ± s.e.m. of the means obtained from at least duplicate determinations in 3 independent experiments; results in B are representative of 2 independent experiments.

View larger version (33K): [in a new window]   Fig. 4. Cell-surface expression (A) and Ser474-phosphorylation (B) of HA-GLUT4-Akt2-S474A and -T309A constructs in rat adipose cells. Isolated cells were transfected with HA-GLUT4, HA-GLUT4-Akt2-wt, HA-GLUT4-Akt2-S474A, and HA-GLUT4-Akt2-T309A, and cultured for 20 hours. After harvesting the cells, cell-surface levels of the constructs were determined in the transfected cells in the basal (white) and insulin-stimulated states (67 nM, black) using an HA antibody binding assay and the cell-surface-associated radioactivity was expressed relative to the basal HA-GLUT4 value in each experiment (1668, 1558, 2311 cpm). Relative cell-surface levels were then normalized to the total expression level of each construct determined by immunoblotting as described in Materials and Methods. Results are the means ± s.e.m. of the means obtained from at least duplicate determinations in each of 3 independent experiments (A). Results in B are immunoblotting of total membrane proteins prepared from the transfected cells with anti-Akt-phospho-Ser473 antibody. These data are representative of 3 independent experiments.

View larger version (9K): [in a new window]   Fig. 5. Effects of HA-GLUT4-Akt2 fusion proteins on cell-surface expression of myc-GLUT4 in rat adipose cells. Isolated cells were co-transfected with myc-GLUT4 and either nothing, HA-GLUT4-Akt2-wt, HA-GLUT4-Akt2-S474A, or HA-GLUT4-Akt2-KD, and cultured for 20 h. After harvesting the cells, cell-surface levels of myc-GLUT4 were determined in the transfected cells in the basal (white) and insulin-stimulated states (67 nM, black) using a myc antibody binding assay, and all of the values were expressed relative to the mean basal myc-GLUT4 value in each experiment as described in Materials and Methods. Results are the means ± s.e.m. of the means obtained from at least duplicate determinations in each of 3-5 independent experiments.

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