The Regulation of Cellular Localization of both Active and Inactive ERK1/2

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The Regulation of Cellular Localization of both Active and Inactive ERK1/2

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Title: The Regulation of Cellular Localization of both Active and Inactive ERK1/2
Author: Goad, Daryl Len
Abstract: 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 ?1 and ?2, 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 ?1 and ?2. 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.
URI: http://hdl.handle.net/2152.5/316
Date: 2009-01-14

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