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 PKCγ, Thr514-phosphorylated PKCγ (pPKCγ-Thr514), and their subcellular distribution as affected by butyrate in a set of colon cancer cell lines. Thr514-phosphorylation of de novo synthesized PKCγ is the first step in priming of the inactive PKCγ 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 pPKCγ-Thr514, and a third against pPKCγ-pan-Thr514. The antibody against an unmodified C-terminal peptide epitope did not recognize pPKCγ-Thr514, suggesting that phosphorylation at this site interferes with the binding of the antibody to the C-terminus. Marked butyrate-induced upregulation of PKCγ 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), PKCγ was insensitive to upregulation, and present exclusively as pPKCγ-Thr514. Lovo and SW480 expressed higher levels of PKCγ. In HT29 cells, butyrate-induced upregulation of the non-phosphorylated PKCγ was observed in both the membrane and the cytosolic fraction. In Caco2 cells, the Thr514-phosphorylated form was present at high levels in both fractions. The presence of unphosphorylated PKCγ in HT29 cells, and its complete absence in Caco2 cells demonstrates a cell type-dependent differential coupling of Thr514-phosphorylation with de novo synthesis of PKCγ in colon cancer cells.