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 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.
Peyrollier
K
K
School of Life Sciences and the Division of Signal Transduction Therapy, Dow Street, University of Dundee, Dundee DD1 5EH, UK.
Hajduch
E
E
Gray
A
A
Litherland
G J
GJ
Prescott
A R
AR
Leslie
N R
NR
Hundal
H S
HS
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000199:Actins / Q000378:metabolism*; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3; D006133:Growth Substances / Q000494:pharmacology*; D006728:Hormones / Q000494:pharmacology*; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D009132:Muscles / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D051059:PTEN Phosphohydrolase; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D025521:Tumor Suppressor Proteins
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D015638:Cytochalasin D; D038362:Glycogen Synthase Kinase 3; D006133:Growth Substances; D006728:Hormones; D007328:Insulin; D051059:PTEN Phosphohydrolase; D019869:Phosphatidylinositol 3-Kinases; D010716:Phosphatidylinositols; D010744:Phosphoric Monoester Hydrolases; D010982:Platelet-Derived Growth Factor; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins; D013844:Thiazoles; D053778:Thiazolidines; D025521:Tumor Suppressor Proteins; C494929:PTEN protein, human; C037068:latrunculin B