Abstract
In 1321N1 astrocytoma cells, heterotrimeric G-protein-coupled receptors that activate phosphoinositide-specific phospholipase Cbeta (PLCbeta) isoforms via G(q), induced a prolonged activation of protein kinase B (PKB) after a short delay. For example, the effect of carbachol acting on M3 muscarinic receptors is blocked by wortmannin, suggesting it is mediated via a phosphoinositide 3-kinase (PI 3-kinase). In support of this, carbachol increased PI 3-kinase activity in PI 3-kinase (p85) immunoprecipitates. The pathway linking PLC-coupled receptors to PI 3-kinase was deduced to involve phosphoinositide hydrolysis and Ca2+-dependent ErbB3 transactivation but not protein kinase C on the basis of the following evidence: (i) inhibition of carbachol stimulated PLC by pretreatment with the phorbol ester phorbol 12-myristate 13-acetate concomitantly reduced PKB activity, whereas stimulation of other PLC-coupled receptors also activated PKB; (ii) Ca2+ ionophores and thapsigargin stimulated PKB activity in a wortmannin-sensitive manner, whereas bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid blocked carbachol-stimulated PKB activity; (iii) phorbol 12-myristate 13-acetate alone did not activate PKB, whereas a protein kinase C inhibitor did not prevent the activation of PKB by carbachol; and (iv) carbachol stimulated ErbB3-tyrosine phosphorylation and association with p85, and both these and PKB activity were blocked by tyrphostin AG1478, an epidermal growth factor receptor-tyrosine kinase inhibitor. These experiments define a novel pathway linking G(q)-coupled G-protein-coupled receptors to the activation of PI 3-kinase and PKB.
Highlights
Interest in protein kinase B (PKB) stems from the discovery that this kinase is rapidly activated in response to a wide variety of receptortyrosine kinases and that such activation is prevented by strategies that inhibit or prevent the activation of phosphoinositide 3-kinases (PI 3-kinases; Refs. 2, 3, 5, 6)
Muscarinic Receptor Stimulation Activates PKB—In previous work using 1321N1 cells to study cross talk between PI 3-kinase and phospholipase C (PLC)-dependent signaling pathways, we showed that insulin-stimulated production of PtdIns [3,4,5]P3 was inhibited by agonists acting on PLC-coupled receptors
It was unexpected that PLC-coupled receptors would prove capable of stimulating PKB activity themselves
Summary
Materials—1321N1 cells were obtained from the European Tissue Culture Collection, and materials for cell culture were from Invitrogen or Sigma. Antibodies to the following were obtained from the sources indicated: phosphotyrosine, Upstate Biotechnology; insulin receptor substrate-1, Upstate Biotechnology or Santa Cruz Biotechnology; PKB isoforms (␣, , and ␥) and phospho-Ser473 PKB, Medical Research Council Protein Phosphorylation Unit, University of Dundee; and phospho-Thr308 PKB antibodies, Cell Signaling. Cell debris was collected, transferred onto ice, and subsequently centrifuged for 5 min at 20,000 ϫ g and 4 °C to remove insoluble material. Immunoprecipitates were collected by brief centrifugation and washed and assayed for PI 3-kinase activity as described previously [25, 26]. PKB Assay—The PKB isoforms (␣, , and ␥) were immunoprecipitated from cell lysates (ϳ300 g protein) using the isoform-specific antibodies, and the immunoprecipitates were assayed as described previously [2, 27], except that assays were continued for 45 min at 30 °C. SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting— SDS-polyacrylamide gel electrophoresis and Western blotting were performed as described previously [26]
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