Phospholipase C, protein kinase C, Ca(2+)/calmodulin-dependent protein kinase II, and tyrosine phosphorylation are involved in carbachol-induced phospholipase D activation in Chinese hamster ovary cells expressing muscarinic acetylcholine receptor of Caenorhabditis elegans.
Recently, we have isolated a cDNA encoding a muscarinic acetylcholine receptor (mAChR) from Caenorhabditis elegans. To investigate the regulation of phospholipase D (PLD) signaling via a muscarinic receptor, we generated stable transfected Chinese hamster ovary (CHO) cells that overexpress the mAChR of C. elegans (CHO-GAR-3). Carbachol (CCh) induced inositol phosphate formation and a significantly higher Ca(2+) elevation and stimulated PLD activity through the mAChR; this was insensitive to pertussis toxin, but its activity was abolished by the phospholipase C (PLC) inhibitor U73122. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after CCh treatment. The CCh-induced PLD activation and tyrosine phosphorylation were significantly reduced by the protein kinase C (PKC) inhibitor calphostin C and down-regulation of PKC and the tyrosine kinase inhibitor genistein. Moreover, the Ca(2+)-calmodulin-dependent protein kinase II (CaM kinase II) inhibitor KN62, in addition to chelation of extracellular or intracellular Ca(2+) by EGTA and BAPTA/AM, abolished CCh-induced PLD activation and protein tyrosine phosphorylation. Taken together, these results suggest that the PLC/PKC-PLD pathway and the CaM kinase II/tyrosine kinase-PLD pathway are involved in the activation of PLD through mAChRs of C. elegans.
Min
D S
DS
Departments of Physiology. Pharmacology, College of Medicine, The Catholic University of Korea, Seoul, Korea. dsmin@cmc.cuk.ac.kr
Cho
N J
NJ
Yoon
S H
SH
Lee
Y H
YH
Hahn
S J
SJ
Lee
K H
KH
Kim
M S
MS
Jo
Y H
YH
D000818:Animals; D016466:CHO Cells; D017173:Caenorhabditis elegans / Q000235:genetics*; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology*; D006224:Cricetinae; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D037342:Pertussis Toxin; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000235:genetics* / Q000502:physiology; D015398:Signal Transduction; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D004791:Enzyme Inhibitors; D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; D019000:Phosphotyrosine; D002217:Carbachol; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010738:Type C Phospholipases; D010739:Phospholipase D; D002118:Calcium