AJP - Renal Physiology, Vol 250, Issue 6 1073-F1081, Copyright © 1986 by American Physiological Society
Phorbol ester-stimulated phosphorylation of basolateral membranes from canine kidney
M. R. Hammerman, S. Rogers, J. J. Morrissey and J. R. Gavin 3rd
To determine whether protein kinase C is present in the basolateral
membrane of the renal proximal tubular cell, we performed experiments to
ascertain whether specific binding of [3H]phorbol 12,13-dibutyrate could be
demonstrated in basolateral membranes isolated from canine kidney. Specific
binding was demonstrable that was half maximal at between 10(-7) and 10(-8)
M phorbol 12,13-dibutyrate. Binding was inhibited by
12-O-tetradecanoylphorbol-13-acetate (TPA) and other tumor-promoting
phorbol esters, but not by inactive phorbol esters, including 4
alpha-phorbol. Incubation of basolateral membranes with TPA and phorbol
12,13-dibutyrate, but not with 4 alpha-phorbol, in the presence of
submicromolar concentrations of free calcium, enhanced phosphorylation of
several proteins demonstrable in autoradiograms of sodium dodecyl
sulfate-polyacrylamide gels originating from membranes subsequently exposed
to [gamma-32P]ATP for 30 s. Dephosphorylation of [32P]phosphoproteins was
observed in gels from membranes incubated with [gamma-32P]ATP over time.
TPA-stimulated phosphorylation of one protein band with Mr 135,000 was
quantitated and was found to increase as a function of [TPA]. Half-maximal
TPA-stimulated phosphorylation of this protein band occurred at slightly
less than 10(-9) M TPA. Our findings are consistent with a role for protein
kinase C-effected phosphorylation of basolateral membrane proteins in the
mediation or modulation of hormonal actions in the proximal tubular cell.
