Protein kinase Cgamma in colon cancer cells: expression, Thr514 phosphorylation and sensitivity to butyrate-mediated upregulation as related to the degree of differentiation.

Protein kinase C (PKC) isoenzymes are expressed and activated in a cell type-specific manner, and play an essential role in tissue-specific signal transduction. The presence of butyrate at millimolar concentrations in the colon raises the question of whether it affects the expression of PKC isoenzymes in the different cell types of the colonic epithelium. We investigated the protein expression levels of PKCgamma, Thr(514)-phosphorylated PKCgamma (pPKCgamma-Thr(514)), and their subcellular distribution as affected by butyrate in a set of colon cancer cell lines. Thr(514)-phosphorylation of de novo synthesized PKCgamma is the first step in priming of the inactive PKCgamma before its release into the cytoplasm. For immunoblot analysis, we employed three antibodies, one against an unmodified sequence, mapping within 50 amino acids at its C-terminus, a second against pPKCgamma-Thr(514), and a third against pPKCgamma-pan-Thr(514). The antibody against an unmodified C-terminal peptide epitope did not recognize pPKCgamma-Thr(514), suggesting that phosphorylation at this site interferes with the binding of the antibody to the C-terminus. Marked butyrate-induced upregulation of PKCgamma occurred in HT29 cells (model for colonocyte stem cells) and HT29-derived cell lines. However, in Caco2 and IEC-18 cells (models for differentiated intestinal epithelial cells), PKCgamma was insensitive to upregulation, and present exclusively as pPKCgamma-Thr(514). Lovo and SW480 expressed higher levels of PKCgamma. In HT29 cells, butyrate-induced upregulation of the non-phosphorylated PKCgamma was observed in both the membrane and the cytosolic fraction. In Caco2 cells, the Thr(514)-phosphorylated form was present at high levels in both fractions. The presence of unphosphorylated PKCgamma in HT29 cells, and its complete absence in Caco2 cells demonstrates a cell type-dependent differential coupling of Thr(514)-phosphorylation with de novo synthesis of PKCgamma in colon cancer cells.