Regulation of extracellular-signal regulated kinase and c-Jun N-terminal kinase by G-protein-linked muscarinic acetylcholine receptors.
Extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs, or stress-activated protein kinases) are activated by diverse extracellular signals and mediate a variety of cellular responses, including mitogenesis, differentiation, hypertrophy, inflammatory reactions and apoptosis. We have examined the involvement of Ca2+ and protein kinase C (PKC) in ERK and JNK activation by the human G-protein-coupled m2 and m3 muscarinic acetylcholine receptors (mAChR) expressed in Chinese hamster ovary (CHO) cells. We show that the Ca2+-mobilizing m3 AChR is efficiently coupled to JNK and ERK activation, whereas the m2 AChR activates ERK but not JNK. Activation of JNK in CHO-m3 cells by the agonist methacholine (MCh) was delayed in onset and more sustained relative to that of ERK in either CHO-m2 or CHO-m3 cells. The EC50 values for MCh-induced ERK activation in both cell types were essentially identical and similar to that for JNK activation in CHO-m3 cells, suggesting little amplification of the response. Agonist-stimulated Ins(1,4,5)P3 accumulation in CHO-m3 cells was insensitive to pertussis toxin (PTX), consistent with a Gq/phosphoinositide-specific phospholipase C-beta mediated pathway, whereas a significant component of ERK and JNK activation in CHO-m3 cells was PTX-sensitive, indicating Gi/o involvement. Using manipulations that prevent receptor-mediated extracellular Ca2+ influx and intracellular Ca2+-store release, we also show that ERK activation by m2 and m3 receptors is Ca2+-independent. In contrast, a significant component (>50%) of JNK activation mediated by the m3 AChR was dependent on Ca2+, mainly derived from extracellular influx. PKC inhibition and down-regulation studies suggested that JNK activation was negatively regulated by PKC. Conversely, ERK activation by both m2 and m3 AChRs required PKC, suggesting a novel mechanism for PKC activation by PTX-sensitive m2 AChRs. In summary, mAChRs activate JNK and ERK via divergent mechanisms involving either Ca2+ or PKC respectively.
Department of Cell Physiology and Pharmacology, University of Leicester School of Medicine, P.O. Box 138, Medical Sciences Building, University Road, Leicester LE1 9HN, UK.
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Wylie
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Challiss
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D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D006224:Cricetinae; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D037342:Pertussis Toxin; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000081082:Phosphoinositide-3 Kinase Inhibitors; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D019204:GTP-Binding Proteins; D002118:Calcium