Substrate Interaction and Sub-Cellular Localization in Map Kinase Pathways
Protein kinase cascades control responses to extracellular cues by transmitting signals throughout the cell. Prominent among multifunctional enzymes in kinase cascades are the mitogen-activated protein kinases (MAPKs). Among the various MAPKs identified, the extracellular signal-regulated kinases, ERK2 and ERK5 are two closely related enzymes that have overlapping functions in a number of cellular pathways. Sub-cellular localization and specificity towards substrates are two mechanisms of controlling the function of an enzyme in the cell. My dissertation discusses the insights we have gained into both these regulated processes through our studies on ERK2 and ERK5. Sub-cellular localization of ERK2 is a tightly regulated process. The current model for sub-cellular localization of ERK2 suggests that there is continuous nuclear-cytoplasmic shuttling of the free pool of ERK2. Anchoring of ERK2 in the different compartments of the cell plays a critical role in determining its location. Entry of inactive ERK2 into the nucleus has been reported to occur by an energy- and carrier-independent mechanism. However, export of inactive ERK2 and import of active ERK2 in intact cells seem to occur by an active process. The mechanisms governing these processes have not been investigated. We have used an in vitro permeabilized-cell reconstitution assay in HeLa and BJ fibroblast cells to explore the mechanism of GFP-ERK2 export and His6-tagged thiophosphorylated ERK2 import. Our results identify more levels of regulation within this model. The ERK5 pathway is triggered in response to various stimuli including growth factors and cellular stresses. Compared to other MAPKs, little is known about ERK5 substrate specificity. Our lab had shown previously that ERK5 is capable of stimulating nuclear factor-κΒ (NF-κΒ ). Our data suggested that this function might be attributed in part to ribosomal protein S6 kinase (known as RSK or p90RSK), which was activated by coexpression with ERK5 and a constitutively active form of its MAP2K, MEK5DD. Here we demonstrate that RSK, among the first known substrates of the ERK1/2 MAPKs, is also directly phosphorylated and activated by ERK5. We have used RSK to explore the basis of substrate recognition by ERK5.