A role for the actin cytoskeleton in the hormonal and growth-factor-mediated activation of protein kinase B

We show here that cytochalasin D-induced depolymerization of actin filament s markedly reduces the stimulus-dependent activation of protein kinase B (P KB) in four different cell types (HEK-293 cells, L6 myotubes, 3T3-L1 adipoc ytes and U87MG cells). HEK-293 cells expressing the pleckstrin homology (PH ) domains of PKB and general receptor for phosphoinositides-1 (GRP1) fused to green fluorescent protein (GFP) were used to monitor production of 3-pho sphoinositides in the plasma membrane. Disassembly of the actin cytoskeleto n significantly reduced the insulin-mediated translocation of both PKB-PH-G FP and GRP1-PH-GFP to the plasma membrane, consistent with diminished synth esis of 3-phosphoinositides. Actin de polymerization did not affect the hor monal activation of phosphoinositide 3-kinase (PI 3-kinase), and since cyto chalasin D treatment also led to reduced platelet-derived growth factor (PD GF)-induced phosphorylation of PKB in U87MG cells, a PTEN (phosphatase and tensin homologue deleted on chromosome 10) null cell line, lipid phosphatas e activity was unlikely to account for any reduction in cellular 3-phosphoi nositides. Withdrawal of cytochalasin D from the extracellular medium induc ed actin filament repolymerization, and reinstated both the recruitment of PH-GFP fusion proteins to the plasma membrane and PKB activation in respons e to insulin and PDGF. Our findings indicate that an intact actin network i s a crucial requirement for PI 3-kinase-mediated production of 3-phosphoino sitides and, therefore, for the activation of PKB.