PROTEIN-KINASE-C AND INSULIN-RECEPTOR BETA-SUBUNIT SERINE PHOSPHORYLATION IN CULTURED FETAL-RAT HEPATOCYTES

In digitonin-permeabilized cultured foetal hepatocytes, insulin recept or beta-subunit was highly phosphorylated on serine residues in the pr esence of [gamma-P-32]ATP and Ca2+, a process enhanced after short exp osure to insulin with no detectable insulin receptor autophosphorylati on. By contrast with this situation, experiments performed with isolat ed foetal insulin receptors revealed an insulin stimulation of both se rine phosphorylation and tyrosine autophosphorylation. In permeabilize d cells, insulin receptor beta-subunit phosphorylation was increased a fter a 2-min exposure to phorbol 12-myristate 13-acetate (PMA) prior t o applying the permeabilization/phosphorylation step, while it was inh ibited by chronic treatment with PMA leading to protein kinase C (PKC) down modulation. The PKC specific inhibitor, GF109203X, strikingly re duced basal and insulin-enhanced phosphorylation of insulin receptor b eta-subunit in permeabilized cells, but failed to exert any effect wit h isolated receptors. Labelling of glycogen from [U-C-14]glucose deter mined 1 h after a 10-min transitory exposure to insulin and/or modulat ors of PKC activity showed that PMA prevented insulin glycogenic respo nse, whereas GF109203X was ineffective. Thus, although not directly re sponsible for insulin receptor serine phosphorylation in cultured foet al hepatocytes, PKC physiologically regulates this process which may i nhibit insulin receptor tyrosine kinase activity, This regulation is i ndependent of the antagonistic effect of PMA-activated PKC on insulin glycogenic response.