Regulation of protein kinase C-δ and -ε isoforms by phorbol ester treatment of LLC-PK1 renal epithelia

Abstract

Background. LLC-PK1 renal epithelia are a widely used model for proximal tubular physiology and differentiation. Protein kinase C (PKC) has been observed to play a role in both processes. This study examines the subcellular distribution and down-regulation of PKC-δ and PKC-ε isoforms in phorbol ester-treated LLC-PK1 epithelia. Methods. Cells were treated with 10-7 mol/L 12-O-tetradecanoyl phorbol 13-acetate (TPA) for up to seven days and were extracted as total cell lysates as well as cytosolic, membrane-associated (Triton-X soluble) and a third (Triton-X insoluble) fraction. The expression and cellular localization of PKC-δ and PKC-ε isoforms were then detected using Western immunoblot and immunofluorescence. Results. Based on the use of an anti-PKC-δ monoclonal antibody, TPA was observed to cause a rapid decrease in total PKC-δ content, which then returned to near control levels by seven days of treatment. Immunofluorescence indicated that PKC-δ had a cytoskeletal localization within the cells, and a subtle cytoskeletal rearrangement occurred upon exposure to TPA. Western immunoblots showed that PKC-δ did not undergo the expected membrane translocation upon activation by TPA, but simply disappeared immediately from the cytosolic compartment. Conventional cell fractionation procedures such as homogenization and Triton extraction prior to Western immunoblot will, however, fail to evaluate completely PKC-δ in LLC-PK1 epithelia because of the highly stringent measures necessary to extract PKC-δ from the cytoskeletal compartment of these cells. Furthermore, we observed that a second (polyclonal) PKC-δ antibody may recognize phosphorylated forms of PKC-δ, which went unrecognized by the other antibody. PKC-ε was present in the cytosol, membrane, and Triton-X-insoluble fractions of the cells. TPA treatment resulted in a partial translocation of PKC-ε to both the membrane and Triton-X-insoluble fractions of the cell, but total PKC-ε remained essentially unchanged. Conclusions. The present data indicate that the localization of PKC-δ and subsequent redistribution within the LLC-PK1 cells in response to TPA treatment is highly unique and distinct from that of PKC-ε and PKC-α. An important methodological finding is that one given antibody may not recognize all phosphoproteins of a given PKC isoform.