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 types
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Research Support, Non-U.S. Gov't
MeSH terms
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3-Phosphoinositide-Dependent Protein Kinases
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Actins / metabolism*
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Adipocytes / cytology
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Adipocytes / drug effects
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Adipocytes / enzymology
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Adipocytes / metabolism
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Animals
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Bridged Bicyclo Compounds, Heterocyclic / pharmacology
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Calcium-Calmodulin-Dependent Protein Kinases / metabolism
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Cell Line
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Cytochalasin D / pharmacology
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Cytoskeleton / drug effects*
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Cytoskeleton / metabolism
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Enzyme Activation / drug effects
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Glycogen Synthase Kinase 3
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Growth Substances / pharmacology*
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Hormones / pharmacology*
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Humans
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Insulin / pharmacology
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Mice
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Muscles / cytology
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Muscles / drug effects
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Muscles / enzymology
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Muscles / metabolism
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PTEN Phosphohydrolase
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Phosphatidylinositol 3-Kinases / metabolism
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Phosphatidylinositols / metabolism
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Phosphoric Monoester Hydrolases / physiology
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Phosphorylation / drug effects
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Platelet-Derived Growth Factor / pharmacology
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Protein Serine-Threonine Kinases / metabolism
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Protein Transport / drug effects
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Proto-Oncogene Proteins / metabolism*
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Proto-Oncogene Proteins c-akt
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Recombinant Fusion Proteins / metabolism
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Thiazoles / pharmacology
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Thiazolidines
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Tumor Suppressor Proteins*
Substances
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Actins
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Bridged Bicyclo Compounds, Heterocyclic
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Growth Substances
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Hormones
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Insulin
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Phosphatidylinositols
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Platelet-Derived Growth Factor
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Proto-Oncogene Proteins
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Recombinant Fusion Proteins
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Thiazoles
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Thiazolidines
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Tumor Suppressor Proteins
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Cytochalasin D
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3-Phosphoinositide-Dependent Protein Kinases
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-akt
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Calcium-Calmodulin-Dependent Protein Kinases
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Glycogen Synthase Kinase 3
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Phosphoric Monoester Hydrolases
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PTEN Phosphohydrolase
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PTEN protein, human
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latrunculin B