Cellular Regulation of Dictyostelium Myosin Heavy Chain Kinases

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DESCRIPTION (provided by applicant): The overarching goal of the studies proposed in this AREA renewal is to gain insight into the complex, and still poorly understood, processes controlling localized myosin II filament assembly and contraction in a nonmuscle cell context. The assembly of myosin II bipolar filaments results from interactions between the heavy chain "tails" of myosin II monomers. These filaments, unlike monomers, can contract actin filaments in a manner leading to localized changes in cell shape. We propose to examine further how myosin II-dependent cellular activities (specifically, cytokinesis and cellular migration) can be regulated to newly indentified candidate myosin II heavy chain kinases (MHCKs) in Dictyostelium, and by extension, how these processes can go awry in cancer cells exhibiting uncontrolled cell division and metastasis. The studies proposed here also have the potential to impact our understanding of the underlying processes driving cellular migration in other contexts such as wound healing, chemotaxis, and metazoan development. We propose to examine the cellular and enzymatic characteristics of MHCK-D and AK1, with the goal of providing not only a basis for comparison with the other Dictyostelium a- kinases, but also a framework for similar studies in higher eukaryotic systems. These studies will be driven by the broad hypothesis that since all of the Dictyostelium a-kinases studied thus far play some role in regulating myosin II, and MHCK-D and AK1 are a-kinases, then MHCK-D and AK1 should also regulate myosin II function. Thus, with our studies, we will address the following questions: 1) Does MHCK-D function in myosin II filament turnover in the cell? 2) What are the localization properties of MHCK-D? 3) What are the enzymatic and regulatory properties of MHCK-D? and 4) Does alpha kinase- 1 (AK1) play a role in regulating myosin II function? Collectively, the proposed studies are of significance since they have the potential to contribute to our understanding of how defects in human myosin II bipolar filament turnover can lead to abnormal platelet formation, glomerulonephritis, among other pathologies associated with MYH9-related disorders. In a broader context, our studies of MHCK-D and AK1 may shed light on the mechanisms for substrate targeting and subcellular localization for the mammalian a-kinase family members, transient receptor potential melastatins 6 and 7, both of which have been implicated in the regulation of myosin II bipolar filament turnover via MHC phosphorylation in mammalian systems. PUBLIC HEALTH RELEVANCE: Normal cell function relies on the ability of the cell to change its shape in highly specific and regulated ways. We are using a model organism, Dictyostelium discoideum, to study the regulation of myosin II, a protein that facilitates cell shape change by driving contraction of the cell. The overarching goal of our studies is understand how defects in myosin II regulation can compromise cellular function and perhaps contribute to the development of disease states such as cancer, or lead to defects in wound healing, cellular immune responses, and the development of atherosclerosis.