Functional and biochemical analysis of ERK2 in mouse embryonic stem cells

Abstract

The ERK-MAPK pathway is a dynamic signaling module, conserved across Eukarya, and capable of processing a myriad of environmental and cellular signals. It has been implicated in controlling important cell fate decisions in many cell types and species. In mES cells, growth factor activation of the ERK-MAPK pathway is involved in the earliest stages of lineage segregation, however very little is currently known about the mechanism by which this is accomplished. Taking a loss-of-function gene targeting approach I have reexamined the relative contribution of ERK2 activity to FGF-ERK signaling. Although ERK2 depletion results in an attenuation of the combined ERK1/2 activity, this is compensated for by the hyperactivation of the remaining ERK1 isozyme. Normal ERK1/2 function can be restored to ERK2 deficient cells by transgenic expression of either ERK1 or ERK2, indicating a degree of functional redundancy between both isoforms. When subjected to the appropriate cues, lineage commitment proceeded normally in ERK2 deficient cells, however increased self-renewal was observed under standard culture conditions. Several attempts were made to further probe ERK1/2 function by siRNA depletion, and dominant negative inhibition of ERK1 in Erk2 knockout cells, however both approaches failed to provide further insight. Furthermore, taking a candidate approach, the role of Srf, a canonical target of ERK1/2 signaling, was examined. Initial experiments indicated a role for SRK in neural differentiation, however due to issues of culture adaptation and instability in several cell lines it was not possible to conclude this line of research within the time frame of this thesis. IP-MS/MS analysis identified several proteins known to interact with ERK2 and indicated an involvement in nuclear pore function through TPR as well as transcriptional and translational regulation through RSK proteins. Moreover, this study identified DUSP6 and DUSP9 as the primary induced dual specificity phosphatases that regulate ERK2 activity in mES cells. To further probe the functional significance of the ERK:p90RSK interaction I examined a mES cell line genetically depleted for PDK1, a crucial regulator of p90RSK function. This cell line exhibits no detectable p90RSK activity, however in contrast to studies in other cell lines, p90RSK activity is dispensable for mitogen-induced cFos expression in mES cells. Subsequent experiments demonstrated a requirement for PDK1 activity in either the specification or maintenance of mES cell derived neurons. Further analysis indicated that p90RSK may be involved in a negative feedback loop regulating ERK1/2 activity, and if so may represent a point whereby ERK1/2 activity can be manipulated. To examine this I determined the effect pharmacological inhibition of p90RSK has on ERK1/2 activity and self-renewal using a novel p90Rsk inhibitor, BI-D1870. Although treatment with BI-D1870 correlated with enhanced ERK1/2 phosphorylation, the offtarget effects this molecule exhibits made it impossible to draw any firm conclusions from these experiments. Overall this study has demonstrated a degree of redundancy between ERK1/2 isozymes in mES cells. It has highlighted the complex nature of ERK1/2 regulation as well as the robustness of this pathway to perturbations in ERK dose. Furthermore, it has underscored some of the common pitfalls encountered when studying differentiation phenotypes in mES cells. Although this study failed to highlight anything more than a coincidental relationship between ERK1/2 activity and self-renewal capacity of mES cells, it has helped to highlight some important behavioral characteristics of the FGF-MAPK pathway in mES cells and provide a platform for further study.