This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Background: This project is designed to investigate signaling from the chemokine, CX3CL1, through ROCK, to its effects on cytoskeletal elements in the context of leukocyte recruitment across the endothelium. Many extracellular stimuli use the RhoA/ROCK signaling module to phosphorylate a subset of a large number of potential substrates, and lead to selective reorganization of cytoskeletal elements, to elicit the appropriate biological response. Two potential mechanisms for determining the specific ROCK signaling events is by the formation of multi-protein complexes, and restricting signaling to particular subcellular locations.Objective: The first objective is to clearly identify mechanisms for specifying ROCK signaling, such as the composition of multi-protein complexes containing ROCK. Previously, we used a proteomics based screen to identify 38 potential binding partners. This year, we have followed up on these initial results to verify and characterize these interactions to be able to build more specific hypotheses for the regulation of ROCK activity.Results: Careful analysis of the proteomics results showed that many of the potential ROCK binding partners are unexpectedly associated with membrane dynamics. We have followed this up in two ways. First, we have been able to confirm with co-immunoprecipitation experiments that ROCK interacts with Dynamin, an important protein for regulating membrane trafficking, and a novel interaction for ROCK. Second, we have performed subcellular fractionation studies that show ROCK resides in multiple membrane compartment, and identified endosomes as a novel location for ROCK. Finally, we have initiated studies to understand what sequences within ROCK regulate its localization. We found that the C-terminal PH domain contains information to target proteins to internal membrane compartments. This is different from what was predicted in the literature, but consistent with both our proteomics and subcellular fractionation data.Discussion: Our proteomics studies were designed to identify components of multi-protein complexes containing ROCK, as a mechanism for determining signaling specificity. Interestingly, we found membrane regulatory proteins, such as dynamin, in complex with ROCK. This led us to follow the hypothesis that ROCK subcellular localization is an important mechanism for determining context specific signaling. Thus, while we began with the idea that there were two potential mechanisms for regulating ROCK signaling the formation of mulit-protein complexes and subcellular localization, our data now indicate that the binding partners are likely important for regulating its subcellular localization, combining the two regulatory mechanisms into one.
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