FIG. 7.

Integrin recycling and cell spreading are suppressed by active GSK-3β. (A and B) NIH 3T3 fibroblasts were transfected with human αvβ3 (hαvβ3) in combination with wt-PKB, PKB-AAA, Mu6pro, Mu6pro-akt1/2, GSK-3β-F216, or GSK-3β-A9. Serum-starved cells were surface labeled with 0.2 mg of N-hydroxysuccinimide-S-S-biotin/ml for 30 min at 4°C, and internalization was allowed to proceed for 15 min at 22°C (A) or for 30 min at 37°C (B). Recycling was determined as for Fig. 3; recycling conditions were 10 min at 37°C in the absence (open bars; control) and presence (solid bars) of PDGF-BB (10 ng/ml) (A) or for 30 min at 37°C in the absence of PDGF (B). One hundred nanomolar wortmannin (WMN), 20 mM LiCl, 10 μM SB216763, and 30 μM SB415286 were included during the recycling period where indicated. Values are means ± standard errors of the means (SEM; n > 10). CON, control. (C and D) NIH 3T3 fibroblasts were transfected with wt-PKB, PKB-AAA, Mu6pro, Mu6pro-akt1/2, GSK-3β-F216, or GSK-3β-A9 in combination with a β-galactosidase transfection marker, and cell spreading on vitronectin was determined as for Fig. 6. Cells were spread in the presence of 100 nM wortmannin, 20 mM LiCl, and 30 μM SB415286 as indicated. Values are means ± SEM (n > 80 cells).