Identifying targets and characterizing the role of aPKCs in murine embryonic stem cells

Abstract

The protein kinase C (PKC) family of isoenzymes plays a pivotal role in maintaining undifferentiated embryonic stem cells (ESC) and in differentiation to specific cell types. We saw that aPKCs are highly expressed in undifferentiated ESC, to elucidate their role in these cells we aimed at identifying direct and indirect aPKC substrates. Using 2-D phosphoproteomics and a {zeta}/{lambda} pseudo-substrate inhibitor peptide we were able to identify 68 proteins whose phosphorylation decreased in the presence of the inhibitor peptide. 34% of the identified proteins were proteins involved in protein folding, stability and synthesis, 27% in metabolism and 13% cytoskeletal elements and associated proteins involved in cell/cell adhesion processes, and cell polarization such as RhoGDI. Most of the identified proteins involved in metabolism were members of the glycolytic pathway. In addition, glucose consumption was reduced in cells treated with the pseudo-substrate peptide. Metabolomic analysis combined with our proteomics results will help elucidate metabolic processes in which aPKC is involved in undifferentiated ESCs. Together our study indicates that aPKC is involved in cell/cell adhesion and glucose metabolism in undifferentiated ESCs, processes which are known to contribute to ESC self-renewal.