Phospholipase C, protein kinase C, Ca2+/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
Ds. Min et al., Phospholipase C, protein kinase C, Ca2+/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, J NEUROCHEM, 75(1), 2000, pp. 274-281
Recently, we have isolated a cDNA encoding a muscarinic acetylcholine recep
tor (mAChR) from Caenorhabditis elegans. To investigate the regulation of p
hospholipase D (PLD) signaling via a muscarinic receptor, we generated stab
le transfected Chinese hamster ovary (CHO) cells that overexpress the mAChR
of C. elegans (CHO-GAR-3). Carbachol (CCh) induced inositol phosphate form
ation and a significantly higher Ca2+ elevation and stimulated PLD activity
through the mAChR; this was insensitive to pertussis toxin, but its activi
ty was abolished by the phospholipase C (PLC) inhibitor U73122. Western blo
t analysis revealed several apparent tyrosine-phosphorylated protein bands
after CCh treatment. The CCh-induced PLD activation and tyrosine phosphoryl
ation were significantly reduced by the protein kinase C (PKC) inhibitor ca
lphostin C and down-regulation of PKC and the tyrosine kinase inhibitor gen
istein. Moreover, the Ca2+/calmodulin-dependent protein kinase If (CaM kina
se II) inhibitor KN62, in addition to chelation of extracellular or intrace
llular Ca2+ by EGTA and BAPTA/AM, abolished CCh-induced PLD activation and
protein tyrosine phosphorylation. Taken together, these results suggest tha
t the PLC/PKC-PLD pathway and the CaM kinase Il/tyrosine kinase-PLD pathway
are involved in the activation of PLD through mAChRs of C. elegans.