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

Abstract

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.

Fig. 1

Effect of butyrate on PKC isoenzyme protein expression levels in HT29 parental and HT29-derived cell lines. Cells were cultured under standard conditions as described in Materials and methods, and treated with 10 mM butyrate for 24 h. Lysates were subjected to Western blot analysis for the indicated PKC isoforms. Tubulin was probed as loading control. Representative blots are shown.

Fig. 2

Impact of butyrate on the level of pPKCγ-Thr⁵¹⁴ and phosphorylated forms of other PKCs in parental HT29 and HT29-derived cell lines. Cells were treated with 10 mM butyrate for 24 h, and lysates were subjected to Western blot analysis with phospho-specific antibodies against the PKC isoforms. GAPDH was probed as a loading control. Representative blots are shown.

Fig. 3

Effect of butyrate on PKC expression levels in Caco2, IEC-18, HT29R and Lovo cells. (A) The expression levels of PKCγ, pPKCγ-Thr⁵¹⁴ and pPKC(pan)(γThr⁵¹⁴) (the pan antibody detects phosphorylated sites in the activation domain of all conventional PKCs) in Caco2, IEC-18, HT29R, Lovo cells, and the impact of butyrate. Comparison with authentic PKCγ from rat brain. Cells were treated with 10 mM butyrate for 24 h. (B) The impact of butyrate on the non-modified and phospho-modified forms (pPKCs) of PKCα, PKCδ, PKCε, PKCζ. Representative blots are shown.

Fig. 4

The expression levels of PKCγ, pPKCγ-Thr⁵¹⁴ and pPKC(pan)(γThr⁵¹⁴) in Caco2, SW480, DLD-1, HCT116 and Lovo cells. Comparison with authentic PKCγ from rat brain. Cells were treated with 10 mM butyrate for 24 h. Lysates were subjected to Western blot analysis with antibodies as described in Materials and methods. GAPDH was probed as a loading control. Representative blots are shown.

Fig. 5

Kinetics of butyrate induced PKCγ expression levels. Cells were treated with 10 mM butyrate for 12, 24 and 48 h, and lysates were subjected to Western blot analysis with the antibody against non-Thr⁵¹⁴-phosphorylated PKCγ. GAPDH was used as a loading control. Representative blots are shown.

Fig. 6

Impact of butyrate on the subcellular distribution of PKCγ, pPKC-Thr⁵¹⁴ and PKCα in HT29 parental and HT29-derived cell lines. After treatment of cells with 10 mM butyrate for 24 h, and fractionation, Western blot analysis was performed as described in Materials and methods. GAPDH and IGF1-R were used as a subcellular localization-specific marker proteins for the cytosolic and plasma membrane fractions, respectively. Representative blots are shown.

Fig. 7

Impact of butyrate on the subcellular distribution of PKCγ, pPKC-Thr⁵¹⁴ and PKCα in Caco2, IEC-18, HT29R and Lovo cells. After treatment of cells with 10 mM butyrate for 24 h, and fractionation, Western blot analysis was performed as described in Materials and methods. GAPDH and IGF1-R were used as subcellular localization-specific marker proteins for the cytosolic and plasma membrane fractions, respectively. Representative blots are shown.