The broad aim of the studies described in this AREA renewal proposal is to gain insight into the complex signaling events leading to myosin II filament assembly and localized contraction in a nonmuscle cell context. Critical cellular events, such as cell division and cell locomotion, rely on the correct localization and assembly of myosin II bipolar filaments within the cell; however, the signaling pathways regulating myosin II assembly are poorly understood. Studies in the social amoeba Dictyostelium have established that myosin II bipolar filament disassembly is driven by the phosphorylation of the myosin II heavy chain (MHC) """"""""tail"""""""" region by three structurally-related MHC kinases - MHCK A, B, and C. The functional consequence of the filament to monomer transition is the inactivation of myosin ll-mediated contraction. Despite the redundancy in their catalytic activities, recent studies indicate that the MHCK A, B, and C enzymes play different roles in regulating myosin II disassembly in different cellular contexts. MHCK A is the most extensively-studied of the MHC kinases. Recent studies, conducted by undergraduate and Master's students in my lab, have revealed that F-actin is a potent activator of MHCK A catalytic activity. These studies also led to the novel discovery that the coiled-coil domain of MHCK A bundles actin filaments. In contrast to MHCK A, there is essentially no information about the structure-function relationships that define the activities of the MHCK B and C enzymes. Thus, the following specific questions will be addressed: 1) What are the actin-binding properties of the full-length MHCK A protein? 2) Do the subdomains of MHCK B and MHCK C play a role in targeting myosin II disassembly in the cell? 3) How do the MHCK B and MHCK C subdomains contribute to the kinase activities of these enzymes? The studies described here are necessary for identifying which properties of the MHCKs might be targeted by signals that lead to very specific changes in Dictyostelium cell shape. In a broader context, our studies provide an opportunity to identify how myosin ll-mediated cellular activities (i.e. cytokinesis and cellular migration) can be regulated in a nonmuscle cell context, and by extension, how these processes can go awry in cancer cells exhibiting uncontrolled cell division and metastasis. Moreover, the studies proposed here will be important for understanding the basic mechanisms by which cellular migration is achieved in other contexts such as wound healing, chemotaxis, and metazoan development. It is particularly noteworthy that the proposed studies are likely to contribute more directly to the knowledge of how defects in myosin II bipolar filament assembly can lead to the development of pathologies such as platelet malformation and kidney function defects, associated with a set of human genetic diseases, collectively called MYH9-related disorders. ? ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Academic Research Enhancement Awards (AREA) (R15)
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Cell Structure and Function (CSF)
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Rodewald, Richard D
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University of North Carolina Greensboro
Schools of Arts and Sciences
United States
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Wessels, Deborah; Lusche, Daniel F; Steimle, Paul A et al. (2012) Myosin heavy chain kinases play essential roles in Ca2+, but not cAMP, chemotaxis and the natural aggregation of Dictyostelium discoideum. J Cell Sci 125:4934-44
Wang, Yu; Steimle, Paul A; Ren, Yixin et al. (2011) Dictyostelium huntingtin controls chemotaxis and cytokinesis through the regulation of myosin II phosphorylation. Mol Biol Cell 22:2270-81
Underwood, Julie; Greene, Jonathan; Steimle, Paul A (2010) Identification of a new mechanism for targeting myosin II heavy chain phosphorylation by Dictyostelium myosin heavy chain kinase B. BMC Res Notes 3:56
Franklin, Atiya; Hyatt, Linzi; Chowdhury, Alyssa et al. (2010) WD repeat domain of Dictyostelium myosin heavy chain kinase C functions in both substrate targeting and cellular localization. Eukaryot Cell 9:344-9
Mondal, Subhanjan; Bakthavatsalam, Deenadayalan; Steimle, Paul et al. (2008) Linking Ras to myosin function: RasGEF Q, a Dictyostelium exchange factor for RasB, affects myosin II functions. J Cell Biol 181:747-60
Russ, Misty; Croft, Daniel; Ali, Omar et al. (2006) Myosin heavy-chain kinase A from Dictyostelium possesses a novel actin-binding domain that cross-links actin filaments. Biochem J 395:373-83
Egelhoff, Thomas T; Croft, Daniel; Steimle, Paul A (2005) Actin activation of myosin heavy chain kinase A in Dictyostelium: a biochemical mechanism for the spatial regulation of myosin II filament disassembly. J Biol Chem 280:2879-87
Steimle, Paul A; Fulcher, F Kent; Patel, Yashomati M (2005) A novel role for myosin II in insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Biochem Biophys Res Commun 331:1560-5