Effect of phorbol 12, 13-dibutyrate on ligand binding, enzyme activity and translocation of protein kinase C isoforms in the alpha T3-1 gonadotrope-derived cell line.

Abstract

The effect of incubating alpha T3-1 cells with phorbol 12,13-dibutyrate (PDBu) on the protein kinase C (PKC) isoform content (predominantly alpha, epsilon and zeta isoforms) was assessed by immunoblotting, enzyme activity assay and [3H]PDBu binding. After exposure to PDBu for 17 h the immunoreactivity detected for both PKC alpha and PKC epsilon had disappeared from cytosol and had increased slightly in membranes. Immunoreactivity for PKC zeta was present as two bands in cytosol; after PDBu treatment both bands decreased in intensity, the higher molecular weight band more than the lower. The lower molecular weight band corresponded with a component of constitutive PKC activity eluting from DEAE cellulose that was defined by inhibition of basal activity with GF 109203X or H7. Investigation of very short treatment times with PDBu using binding, immunoblot and activity measurements (in the presence/absence of Ca2+) indicated that translocation of PKC alpha and epsilon was very rapid-detectable by 10 sec, maximal within minutes. Reduction of these isoforms in membranes took much longer, and was not apparent up to 150 min. The immunoblot data for PKC zeta in cytosol showed no detectable effect of PDBu treatment on the low molecular weight band up to 150 min although it was reduced at 17 h. Translocation of the upper band was detectable at 10 sec but this band may have resulted from cross-reaction with other PKC isoforms. The constitutive activity and low molecular weight ("authentic') PKC zeta immunoreactivity were partially affected after long exposure only, suggesting an action of PDBu on PKC zeta secondary to activation of the other PKC isoforms. An endogenous receptor agonist, luteinising hormone-releasing hormone (LHRH), was also used to assess by immunoblotting, translocation of the PKC isoforms. Although all the isoforms did translocate from cytosol to membrane fractions, they did so with distinctly different time courses: PKC epsilon moved more rapidly than PKC zeta which appeared to translocate more quickly than PKC alpha. After downregulation of the responsive PKC isoforms with PDBu, the remaining PKC zeta was not translocated by LHRH.