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J Biol Chem, Vol. 273, Issue 42, 27111-27117, October 16, 1998
From the Department of Medicine, University of California, San
Diego, School of Medicine, San Diego, California 92103
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 µM)
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 µM), 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 µM). 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 µM). 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 µM), 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.
Carbachol Stimulates Transactivation of Epidermal Growth Factor
Receptor and Mitogen-activated Protein Kinase in T84
Cells
IMPLICATIONS FOR CARBACHOL-STIMULATED CHLORIDE SECRETION
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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