Muscarinic receptors mediate phospholipase C-dependent activation of protein kinase B via Ca2+, ErbB3, and phosphoinositide 3-kinase in 1321N1 astrocytoma cells.
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.
Tang
Xiuwen
X
Division of Cell Signalling, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland. x.tang@dundee.ac.uk
Batty
Ian H
IH
Downes
C Peter
CP
D001254:Astrocytoma / Q000201:enzymology* / Q000473:pathology; D020013:Calcium Signaling; D002217:Carbachol / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D011493:Protein Kinase C / Q000502:physiology; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D020893:Receptor, ErbB-3 / Q000502:physiology*; D011976:Receptors, Muscarinic / Q000502:physiology*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000502:physiology*
D002217:Carbachol; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D020893:Receptor, ErbB-3; D011976:Receptors, Muscarinic; D010738:Type C Phospholipases