This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Activation of Epidermal Growth Factor (EGF) Receptor triggers a network of signal transduction events including activating the MAPK cascade along with accelerating endocytosis of EGF-receptor complexes. MEK and ERK proteins are localized on endosomes, however the precise mechanism and the role of endosomal targeting are yet to be understood. In addition, members of the Ras-RAF-MEK-ERK cascade are reported to be organized into signal-transducing modules regulated by scaffold proteins. The suggested role of scaffold proteins is to enhance the specificity and the selectivity of interactions by bringing kinase-substrate partners into these modules. We seek to elucidate endocytosis involvement in regulating of the MAPK signaling pathway by the scaffold protein Shoc2. Shoc2 is a positive regulator of ERK1/2 activation and signaling. We demonstrated that Shoc2 relocates to a subset of endosomes upon EGFR activation. This protein is unique in its ability to control MAPK activation that is also regulated by endocytic trafficking. However, the mechanism is poorly understood. Essentially, our goal will be to define the functional importance of endosomal localization of Shoc2 for MAPK activity by utilizing various biochemical assays and advanced methods of fluorescence microscopy. We will analyze the mechanism of the spatial regulation of Shoc2 and reconstitute Shoc2 in cancer cells to study the contribution of Shoc2 to MAPK activation in tumor cells. A number of reagents and newly developed methodologies will allow us to pursue this goal.
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