Carbachol stimulates transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T84 cells. Implications for carbachol-stimulated chloride secretion.
We have examined the role of tyrosine phosphorylation in regulation of calcium-dependent chloride secretion across T84 colonic epithelial cells. The calcium-mediated agonist carbachol (CCh, 100 microM) stimulated a time-dependent increase in tyrosine phosphorylation of a range of proteins (with molecular masses ranging up to 180 kDa) in T84 cells. The tyrosine kinase inhibitor, genistein (5 microM), significantly potentiated chloride secretory responses to CCh, indicating a role for CCh-stimulated tyrosine phosphorylation in negative regulation of CCh-stimulated secretory responses. Further studies revealed that CCh stimulated an increase in both phosphorylation and activity of the extracellular signal-regulated kinase (ERK) isoforms of mitogen-activated protein kinase. Chloride secretory responses to CCh were also potentiated by the mitogen-activated protein kinase inhibitor, PD98059 (20 microM). Phosphorylation of ERK in response to CCh was mimicked by the protein kinase C (PKC) activator, phorbol myristate acetate (100 nM), but was not altered by the PKC inhibitor GF 109203X (1 microM). ERK phosphorylation was also induced by epidermal growth factor (EGF) (100 ng/ml). Immunoprecipitation/Western blot studies revealed that CCh stimulated tyrosine phosphorylation of the EGF receptor (EGFr) and increased co-immunoprecipitation of the adapter proteins, Shc and Grb2, with the EGFr. An inhibitor of EGFr phosphorylation, tyrphostin AG1478 (1 microM), reversed CCh-stimulated phosphorylation of both EGFr and ERK. Tyrphostin AG1478 also potentiated chloride secretory responses to CCh. We conclude that CCh activates ERK in T84 cells via a mechanism involving transactivation of the EGFr, and that this pathway constitutes an inhibitory signaling pathway by which chloride secretory responses to CCh may be negatively regulated.
Keely
S J
SJ
Department of Medicine, University of California, San Diego, School of Medicine, San Diego, California 92103, USA.
Uribe
J M
JM
Barrett
K E
KE
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D001692:Biological Transport / Q000187:drug effects; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology*; D002712:Chlorides / Q000378:metabolism*; D003106:Colon / Q000378:metabolism*; D066246:ErbB Receptors / Q000819:agonists*; D051380:GRB2 Adaptor Protein; D007413:Intestinal Mucosa / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011799:Quinazolines; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D015533:Transcriptional Activation; D020032:Tyrphostins / Q000494:pharmacology
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D002712:Chlorides; D051380:GRB2 Adaptor Protein; D011506:Proteins; D011799:Quinazolines; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D055765:Shc Signaling Adaptor Proteins; D020032:Tyrphostins; C101044:RTKI cpd; D002217:Carbachol; D066246:ErbB Receptors; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases