Phorbol ester-stimulated phosphorylation of basolateral membranes from canine kidney.
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.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D004285:Dogs; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology / Q000378:metabolism*; D008566:Membranes / Q000201:enzymology / Q000378:metabolism; D008871:Microvilli / Q000201:enzymology; D009994:Osmolar Concentration; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000378:metabolism / Q000494:pharmacology*; D010743:Phospholipids / Q000378:metabolism; D010766:Phosphorylation; D013268:Stimulation, Chemical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014316:Tritium; D000520:alpha-Glucosidases / Q000378:metabolism
D000596:Amino Acids; D010703:Phorbol Esters; D010743:Phospholipids; D014316:Tritium; D015240:Phorbol 12,13-Dibutyrate; D000255:Adenosine Triphosphate; D000520:alpha-Glucosidases; D013755:Tetradecanoylphorbol Acetate