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 x 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 gamma-[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 of potentiation of the action of TRH at its target sites.
Suen
C S
CS
Department of Pharmacology, Mount Sinai School of Medicine, City University of New York, New York 10029.
Wilk
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S
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D002460:Cell Line; D004837:Epinephrine / Q000494:pharmacology; D005134:Eye Neoplasms; D006801:Humans; D007106:Immune Sera; D015151:Immunoblotting; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011751:Pyroglutamyl-Peptidase I / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D012175:Retinoblastoma; D013110:Sphingosine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D007106:Immune Sera; D007546:Isoquinolines; D010879:Piperazines; D015240:Phorbol 12,13-Dibutyrate; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D011751:Pyroglutamyl-Peptidase I; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D004837:Epinephrine