Myosin II plays fundamental roles in cytokinesis, cell migration, and cell shape changes during development. In all these settings, it is well established that dynamic localized assembly of myosin into the cytoskeleton is critical for its cellular contractile roles. Despite the importance of spatially and temporally regulated assembly, the signaling mechanisms that control localized assembly and disassembly of myosin II are not understood in any system. We are using the simple amoeba Dictyostelium discoideum as a model system for identifying signaling pathways that regulate myosin assembly. This simple amoeba displays forms of cellular motility, chemotaxis, and second messenger signaling similar to those displayed by motile mammalian cells such as neutrophils or macrophages. Myosin II assembly in this system is regulated by phosphorylation/dephosphorylation of a set of mapped threonine residues that lie near the tip of the myosin tail. We have previously identified a set of novel myosin heavy chain kinases (MHCKs) in this system, that participate in the in vivo control of myosin assembly via phosphorylation of the mapped target sites in the myosin tail. These enzymes are now recognized as the prototype for a highly novel family of protein kinases present in Dictyostelium and throughout the animal kingdom known as """"""""Alpha kinases"""""""". Recently, 1 mammalian alpha kinase (TRPM7) has been implicated in apoptotic pathway activation in response to oxidative stress, but roles of other mammalian alpha kinases are largely unknown. Our molecular and genomic approaches in Dictyostelium have revealed a total of 6 alpha kinases in this simple model organism. At least 2 of the Dictyostelium alpha kinases appear NOT to be MHC kinases, but likely serve other roles in the cell. We propose a series of complementary approaches to gain insights into the roles of all of these enzymes. In this proposal we will: (1) use biochemistry/cellular analysis to elucidate the role of autophosphorylation in MHCK activation, (2) perform studies to test the cellular role of an MHC phosphatase that we identified biochemically in earlier work, (3) perform further cellular/biochemical analysis of VwkA and AK1, 2 remaining Dictyostelium alpha kinases for which cellular roles are not well understood, and (4) perform genetic screens to identify new genes involved in myosin II-related cellular functions in settings ranging from cell division to osmotic protective responses. Our studies will provide an important foundation of direct relevance for understanding myosin II regulation in mammalian systems. The studies we propose addressing the full complement of Dictyostelium alpha kinases will also provide a powerful platform for comparisons and insights into possible roles of alpha kinases in mammalian systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM050009-18S1
Application #
7931578
Study Section
Special Emphasis Panel (ZRG1-CB-H (02))
Program Officer
Gindhart, Joseph G
Project Start
2009-09-30
Project End
2010-08-31
Budget Start
2009-09-30
Budget End
2010-08-31
Support Year
18
Fiscal Year
2009
Total Cost
$84,074
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
135781701
City
Cleveland
State
OH
Country
United States
Zip Code
44195
Rai, Vandana; Thomas, Dustin G; Beach, Jordan R et al. (2017) Myosin IIA Heavy Chain Phosphorylation Mediates Adhesion Maturation and Protrusion in Three Dimensions. J Biol Chem 292:3099-3111
Thomas, Dustin; Thiagarajan, Praveena S; Rai, Vandana et al. (2016) Increased cancer stem cell invasion is mediated by myosin IIB and nuclear translocation. Oncotarget 7:47586-47592
Gupta, Sounak; Hau, Andrew M; Al-Ahmadie, Hikmat A et al. (2016) Transforming Growth Factor-? Is an Upstream Regulator of Mammalian Target of Rapamycin Complex 2-Dependent Bladder Cancer Cell Migration and Invasion. Am J Pathol 186:1351-60
Southern, Brian D; Grove, Lisa M; Rahaman, Shaik O et al. (2016) Matrix-driven Myosin II Mediates the Pro-fibrotic Fibroblast Phenotype. J Biol Chem 291:6083-95
Pasapera, Ana M; Plotnikov, Sergey V; Fischer, Robert S et al. (2015) Rac1-dependent phosphorylation and focal adhesion recruitment of myosin IIA regulates migration and mechanosensing. Curr Biol 25:175-186
Thomas, Dustin G; Yenepalli, Aishwarya; Denais, Celine Marie et al. (2015) Non-muscle myosin IIB is critical for nuclear translocation during 3D invasion. J Cell Biol 210:583-94
Thiagarajan, Praveena S; Hitomi, Masahiro; Hale, James S et al. (2015) Development of a Fluorescent Reporter System to Delineate Cancer Stem Cells in Triple-Negative Breast Cancer. Stem Cells 33:2114-2125
Chandrasekharan, Unni M; Dechert, Lisa; Davidson, Uchechukwu I et al. (2013) Release of nonmuscle myosin II from the cytosolic domain of tumor necrosis factor receptor 2 is required for target gene expression. Sci Signal 6:ra60
Gupta, Sounak; Hau, Andrew M; Beach, Jordan R et al. (2013) Mammalian target of rapamycin complex 2 (mTORC2) is a critical determinant of bladder cancer invasion. PLoS One 8:e81081
Crish, James; Conti, Mary Anne; Sakai, Takao et al. (2013) Keratin 5-Cre-driven excision of nonmuscle myosin IIA in early embryo trophectoderm leads to placenta defects and embryonic lethality. Dev Biol 382:136-48

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