Abstract 4423: Methylation and acetylation are novel post-translational modifications identified for the pre-apoptotic protein kinase C (PKC) delta
Authors: Noemi Kedei, Elliott L. Paine, Lisa M. Miller Jenkins, Peter M. Blumberg
Journal: Cancer Research (2016)

Abstract Members of the PKC family of diacylglycerol (DAG) receptors play a central role in cellular signaling, affecting cell proliferation, differentiation, apoptosis, etc. Their expression and/or function are often altered in cancer. Posttranslational modifications play an important role in the regulation and intracellular localization of PKCs. For example, phosphorylation of the hydrophobic and turn motifs are required for the maturation, stability and proper folding of most isoforms, and phosphorylation of S299, Y311, and Y334 in the hinge region are well characterized agonist (phorbol ester or H2O2) induced modifications of PKC delta. We have previously reported that PMA and bryostatin 1, two PKC activators having very different biology in multiple cell lines, induced a relatively complex and different pattern of PKC delta modification in LNCaP cells as detected by charge-based separation. Moreover, bryostatin 1 failed to induce Y311 phosphorylation whereas it induced S299 and T507 phosphorylation similarly to PMA. In this study, we explored by mass spectrometry the posttranslational modifications of PKC delta induced by the two drugs. GFP-tagged PKCdelta was overexpressed in LNCaP and HEK cells, and PKC delta was immunoprecipitated from treated cells (in total lysates or in cytoplasmic and nuclear fractions) using anti-GFP antibody. Using this approach, we were able to detect the previously described phosphorylations of S302/S304, S506, S645, S664 in all samples and the agonist-induced phosphorylation of S299, while detection of phosphorylated tyrosine residues was less efficient. Novel findings include detection of agonist-induced phosphorylation of S130, S359, and S647, and acetylation or methylation of multiple K or N sites, respectively. Acetylation of K275 in the C1b domain, of K319 in the hinge region, of K475 in the catalytic domain and of K649 at the C terminus was detected in almost all samples while methylation of N278, N283 and N292 in the C1b domain was detected in control LNCaP cells only. Agonist treatment induced acetylation and methylation at additional sites in the hinge region and in the C terminus of the protein. The modifications were not identical between the cytoplasmic and nuclear PKC delta after PMA treatment: phosphorylation of T50, methylation of N292 and acetylation of K382 was detected only in the cytoplasmic fraction while phosphorylation of S359 was present in the nuclear fraction only. The newly identified acetylation and methylation add to the complexity of posttranslational modifications of PKC delta that might have regulatory roles in the localization and/or activity of the protein. Citation Format: Noemi Kedei, Elliott L. Paine, Lisa M. Miller Jenkins, Peter M. Blumberg. Methylation and acetylation are novel post-translational modifications identified for the pre-apoptotic protein kinase C (PKC) delta. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4423.