AJP - Cell Physiology, Vol 261, Issue 3 C543-C549, Copyright © 1991 by American Physiological Society
Carbachol-stimulated phosphorylation of a 170-kDa endogenous protein in avian salt gland cells
J. Torchia, Y. Qu, J. Francis, D. J. Pon and A. K. Sen
Department of Pharmacology, University of Toronto, Ontario, Canada.
The effect of cholinergic stimulation of cellular protein phosphorylation
was studied using an intact cell preparation isolated from the avian salt
gland. Isolated cells were allowed to incorporate 32Pi into the cellular
ATP pool and then challenged with compounds known to induce ion secretion
in this tissue. Addition of carbachol resulted in a time- and
concentration-dependent (EC50 = 500 nM) increase in 32Pi content of a
170-kDa protein (pp170). The stimulated phosphorylation could be blocked by
the inclusion of atropine (100 microM). Subcellular fractionation studies
localized pp170 to the plasma membrane fraction of the tissue. The integral
nature of this protein was demonstrated by detergent-solubilization
experiments with Triton X-100. The possibility that carbachol stimulates
phosphorylation of pp170 via activation of protein kinase C (PKC) was
investigated. Incubating salt gland cells with 4 beta-phorbol 12-myristate
13-acetate (PMA; 1 microM) or carbachol (100 microM) resulted in a
translocation of soluble PKC from the cytosol to a plasma membrane
fraction. Addition of either PMA (1 microM) or ionomycin (1 microM) alone
did not enhance phosphorylation of pp170. A 4.5-fold increase in the
phosphorylation state of pp170 was only observed when PMA and ionomycin
were added concurrently. Preincubation of salt gland cells with PKC
inhibitors H-7 (50 microM) or staurosporine (10 microM) inhibited the
carbachol-stimulated phosphorylation of pp170. These findings suggest that
carbachol mediates its secretomimetic effects via activation of PKC and
that pp170 may represent a novel integral membrane PKC substrate protein.
