Rapid inactivation and phosphorylation of pyroglutamyl peptidase II in Y-79 human retinoblastoma cells after exposure to phorbol ester

Pyroglutamyl peptidase II (EC 3.4.19.-), a membrane-bound metalloproteinase, is a highly specific TRH-degrading enzyme. Exposure of Y-79 human retinoblastoma cells to 12-0-tetradecanoyl phorbol 13-acetate (TPA) decreased the activity of this enzyme in a time- and concentration-dependent manner (IC₅₀ 5 × 10^-9 M). After 15 min of TPA treatment, only 10% of pyroglutamyl peptidase II activity remained. TPA treatment did not affect the activity of the cytosolic enzyme pyroglutamyl peptidase I (EC 3.4.19.3) or the membrane-bound enzyme dipeptidyl peptidase IV (EC 3.4.19.3). Pretreatment of the cells with the protein kinase C inhibitors H-7 or sphingosine prevented the inactivation of pyroglutamyl peptidase II by TPA. The time course of the TPA-mediated effect paralleled the time course of translocation and activation of protein kinase C in this cell line. Immunoblot analysis demonstrated that inactivation of pyroglutamyl peptidase II was not due to dissociation or internalization of this enzyme molecule. Incubation of TPA-activated Y-79 cell membranes with γ-[³²P]-ATP followed by immunoprecipitation revealed a time-dependent phosphorylation of a 48 kilodalton subunit of pyroglutamyl peptidase II. These studies indicate that the phorbol ester effect is mediated by protein kinase C, and reveal a mechanism for potentiation of the action of TRH at its target sites.