Activation of protein kinase C delta by ψδRACK peptide promotes embryonic stem cell proliferation through ERK 1/2

The protein kinase C (PKC) family of serine/threonine kinases participate in embryonic stem cell (ESC) proliferation/self-renewal. A few stimuli that induce ESC proliferation activate several PKC isoenzymes including δPKC, however, the role of this isoenzyme under basal conditions that maintain undifferentiated ESCs remains to be determined. Herewith, we aimed to characterize signaling events that occur in undifferentiated ESCs upon δPKC activation. Using phosphoproteomics and a δPKC specific activator peptide, ψδRACK, it was seen that the majority of proteins whose phosphorylation increased upon δPKC activation participate in cell proliferation. Network analysis of these proteins directly connected δPKC to Raf1 and 14-3-3. Experimental validation studies showed that activation of δPKC increased its binding to 14-3-3, transiently activated ERK1/2 and increased ESC proliferation. Independently inhibiting MEK or PI3 kinase both led to a decrease in proliferation of approximately 50%, but δPKC activation only recovered the effect of PI3 kinase inhibition suggesting that ERK1/2 activation via δPKC is probably a parallel pathway to PI3 kinase and that both pathways are necessary for undifferentiated ESC proliferation. Biological significance: The use of embryonic stem cells and induced pluripotent stem cells for regenerative therapies is still a challenge. Understanding the underlying mechanisms that keep these cells proliferating with the ability to differentiate in more than 200 cell types (self-renewal) will aid in the future use of these cells therapeutically. Using a targeted phosphoproteomics study, insights into signaling pathways involved in ESC proliferation can be obtained. Modulating these pathways will aid the obtention of a larger number of self-renewing stem cells and induced pluripotent stem cells that can be used therapeutically.