Activation of calcium-dependent kinases and epidermal growth factor receptor regulate muscarinic acetylcholine receptor-mediated MAPK/ERK activation in thyroid epithelial cells.
We previously showed that stimulation of muscarinic acetylcholine receptors (mAChR) by carbachol (Cch) caused a time- and dose-dependent increase of mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERK) phosphorylation in thyroid epithelial cells. In this study, we demonstrated that mAChR stimulation also induced a time-dependent increase in the tyrosine phosphorylation of proline-rich tyrosine kinase 2 (Pyk2), which was prevented by pretreatment of thyroid epithelial cells with the specific Src-family tyrosine kinase inhibitor PP2. Besides, phosphorylation of Pyk2 was attenuated by chelation of extracellular Ca(2+) or inhibition of phospholipase C (PLC), and was evoked by thapsigargin, a specific microsomal Ca(2+)-ATPase inhibitor. Incorporation of Pyk2 antisense oligonucleotides in thyroid epithelial cells to down-regulated Pyk2 expression or pretreatment of cells with the Ca(2+)/calmodulin protein kinase II (CaM kinase II) inhibitor KN-62 significantly reduced Cch-induced MAPK/ERK phosphorylation. In addition, Cch-induced MAPK/ERK phosphorylation was partially inhibited by LY294002 and wortmannin, two selective inhibitors of phosphatidylinositol 3-kinase (PI3K), tyrphostin AG1478, a specific inhibitor of epidermal growth factor receptor (EGFR) kinase, and (-)-perillic acid, a post-translational inhibitor of small G-proteins isoprenylation. Taken together, our data suggest that Pyk2, CaM kinase II and Src-family tyrosine kinases are key molecules for the activation of MAPK/ERK cascade through the EGFR/Ras/Raf pathway in thyroid epithelial cells in response to mAChR stimulation.
Montiel
Mercedes
M
Department of Biochemistry and Molecular Biology, University of Malaga, Boulevard Louis Pasteur 32, 29071 Malaga, Spain.
Quesada
Juan
J
Jiménez
Eugenio
E
D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology*; D066246:ErbB Receptors / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism; D020935:MAP Kinase Signaling System; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism*; D013961:Thyroid Gland / Q000166:cytology / Q000201:enzymology*; D019061:src-Family Kinases / Q000378:metabolism
D011976:Receptors, Muscarinic; D002217:Carbachol; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D051418:Focal Adhesion Kinase 2; D019061:src-Family Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D048049:Extracellular Signal-Regulated MAP Kinases; D020559:Monomeric GTP-Binding Proteins