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

Karine Peyrollier, Eric Hajduch, Alexander Gray, Gary Litherland, Alan R. Prescott, Nick R. Leslie, Harinder S. Hundal

Research output: Contribution to journalArticlepeer-review

51 Citations (Scopus)

Abstract

We show here that cytochalasin D-induced depolymerization of actin filaments markedly reduces the stimulus-dependent activation of protein kinase B (PKB) in four different cell types (HEK-293 cells, L6 myotubes, 3T3-L1 adipocytes 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-phosphoinositides in the plasma membrane. Disassembly of the actin cytoskeleton significantly reduced the insulin-mediated translocation of both PKB-PH-GFP and GRP1-PH-GFP to the plasma membrane, consistent with diminished synthesis of 3-phosphoinositides. Actin depolymerization did not affect the hormonal activation of phosphoinositide 3-kinase (PI 3-kinase), and since cytochalasin D treatment also led to reduced platelet-derived growth factor (PDGF)-induced phosphorylation of PKB in U87MG cells, a PTEN (phosphatase and tensin homologue deleted on chromosome 10) null cell line, lipid phosphatase activity was unlikely to account for any reduction in cellular 3-phosphoinositides. Withdrawal of cytochalasin D from the extracellular medium induced actin filament repolymerization, and reinstated both the recruitment of PH-GFP fusion proteins to the plasma membrane and PKB activation in response to insulin and PDGF. Our findings indicate that an intact actin network is a crucial requirement for PI 3-kinase-mediated production of 3-phosphoinositides and, therefore, for the activation of PKB.
Original languageEnglish
Pages (from-to)617-622
Number of pages6
JournalBiochemical Journal
Volume352
Issue number3
DOIs
Publication statusPublished - 2000
Externally publishedYes

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