Browsing by Subject "Mitogen-Activated Protein Kinase"
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Item The Regulation of Cellular Localization of Both Active and Inactive ERK1/2(2009-01-14) Goad, Daryl Len; Cobb, Melanie H.A key question concerning the regulation and activity attributed to the extracellular signal regulated kinase1/2 (ERK1/2) cascade is how the cellular response to different ligands, cellular environment, and other cellular signals is generated. Part of the answer to the question is the ligand and context-specific spatial control of ERK1/2 in the cell. By confining activity of ERK1/2 to localized regions, cells modulate signaling output. To gain insights into the spatial control of ERK1/2 I examined nuclear import of ERK2 mutants, the interactions of ERK2 with karyopherins β1 and β2, and explored the roles of cytoskeletal elements, motor proteins and scaffolding complexes in the localization of ERK1/2. Mutation or dysregulation of the ERK1/2 signaling cascade has been identified in a host of diseases from cancer to type II diabetes [1]. The ERK1/2 proteins have well defined regions involved in protein-protein interactions. We mutated ERK2 in these regions and tested the nuclear import of the mutants. The rationale for this series of experiments was to interfere with known docking motifs on ERK2 to determine residues essential for nuclear translocation. By varying the import conditions it was possible to identify ERK2 residues that impacted nuclear import in different contexts, in particular upon activation by phosphorylation and in the presence of energy. Mutation of certain residues only affected import of phosphorylated ERK2 in the presence of energy. From these data we hypothesize there are at least two mechanisms for nuclear entry of phosphorylated ERK1/2. Since our mutational analysis of ERK2 demonstrated an energy-dependent means of ERK2 nuclear import, we examined the potential roles of karyopherins in this process. Solution binding assays showed binding of both active and inactive ERK2 to the karyopherins β1 and β2. Both forms of ERK2 were released from the karyopherins in the presence of RanGTP. Based on these and other studies, we suggest that multiple karyopherins are involved in the energy-dependent transport of active ERK1/2. In addition to their interactions with upstream activators and downstream ligands, ERK1/2 interact with scaffolds and other regulatory proteins, such as kinase suppressor of Ras 1 (KSR1) and phosphoprotein enriched in astrocytes of 15 kDa (PEA15), a protein overexpressed in type II diabetes. In this study I mutated two serine residues on PEA15. Phosphorylation of these residues is known to affect ERK1/2 binding and interaction. Knock down of PEA15 by RNAi (RNA interference) caused an increase in cellular motility. Using a variety of cell types and immunofluorescence microscopy I was able to show the heterogeneity of the endogenous total ERK1/2 pools and the active ERK1/2 pools within the cell. Additionally, I showed distinct control of nuclear localization of endogenous ERK1/2 following treatment of cells with microtubule and actin filament destabilizing drugs.Item Substrate Interaction and Sub-Cellular Localization in Map Kinase Pathways(2005-08-11) Ranganathan, Aarati; Cobb, Melanie H.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.