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

Lookup NU author(s): Alastair Gray, Dr Gary Litherland

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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.


Publication metadata

Author(s): Litherland GJ; Gray A; Peyrollier K; Hajduch E; Prescott AR; Leslie NR; Hundal HS

Publication type: Article

Publication status: Published

Journal: Biochemical Journal

Year: 2000

Volume: 352

Issue: Pt 3

Pages: 617-622

ISSN (print): 0264-6021

Publisher: Portland Press

URL: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1221496/pdf/11104665.pdf

PubMed id: 11104665

Notes: 0264-6021 (Print) Journal Article Research Support, Non-U.S. Gov't


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