The long term goal of this proposal is to understand molecular mechanisms that regulate microfilament reorganization during mitosis, as well as during oncogenic transformation of cultured cells. We will focus our studies on the biological significance of the mitosis-specific phosphorylation of caldesmon. We have demonstrated, for the first time, that nonmuscle caldesmon, a protein of relative molecular mass Mr = 83,000 which binds to actin and calmodulin, is disassociated from microfilaments during mitosis, apparently as a consequence of phosphorylation. We believe that this process may contribute to the changes of shape and structure seen in cells undergoing mitosis because caldesmon inhibits actomyosin ATPase, and because caldesmon, together with tropomyosin, regulates the actin severing and capping activities of gelsolin. We will biochemically characterize how the mitosis-specific phosphorylation of caldesmon changes microfilament assembly and actomyosin interactions in vitro. In vivo functions of mitosis-specific phosphorylation of caldesmon will be explored through microinjection of phosphorylated caldesmon, antibodies that block phosphorylation or dephosphorylation, and mutant caldesmons lacking the phosphorylation sites, into cultured cells. We will observe whether the microinjection will perturb the assembly of microfilaments during mitosis and other cell cycle stages. We will examine changes in the phosphorylation state of caldesmon during cell cycle progression in detail to see whether the phosphorylation of caldesmon correlates with changes in microfilament assembly. Because transformed cells show morphological alterations similar to those found in mitotic cells, we will examine, using ts Rat-I cells, whether caldesmon is phosphorylated during cell transformation. Finally, because we have found that cdc2 kinase (mitosis promotion factor) is one of two kinase activities that phosphorylate caldesmon during mitosis, we will characterize how cdc2 kinase and the other kinase activity phosphorylate caldesmon. We will also search for other microfilament-associated proteins that are phosphorylated by these kinases during mitosis. Because cell cycle control is intimately related to cell transformation, the proposed studies will help us understand not only how cells divide, but also why cancer cells lose control of cell division.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37CA042742-08
Application #
3482526
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1985-11-01
Project End
1995-12-31
Budget Start
1992-01-01
Budget End
1992-12-31
Support Year
8
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Rutgers University
Department
Type
Schools of Arts and Sciences
DUNS #
038633251
City
New Brunswick
State
NJ
Country
United States
Zip Code
08901
Matsumura, Fumio; Yamakita, Yoshihiko; Yamashiro, Shigeko (2011) Myosin phosphatase-targeting subunit 1 controls chromatid segregation. J Biol Chem 286:10825-33
Yamakita, Yoshihiko; Matsumura, Fumio; Yamashiro, Shigeko (2009) Fascin1 is dispensable for mouse development but is favorable for neonatal survival. Cell Motil Cytoskeleton 66:524-34
Yamashiro, Shigeko; Yamakita, Yoshihiko; Totsukawa, Go et al. (2008) Myosin phosphatase-targeting subunit 1 regulates mitosis by antagonizing polo-like kinase 1. Dev Cell 14:787-97
Takiguchi, Kingo; Matsumura, Fumio (2005) Role of the basic C-terminal half of caldesmon in its regulation of F-actin: comparison between caldesmon and calponin. J Biochem 138:805-13
Totsukawa, Go; Wu, Yue; Sasaki, Yasuharu et al. (2004) Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts. J Cell Biol 164:427-39
Yamashiro, Shigeko; Totsukawa, Go; Yamakita, Yoshihiko et al. (2003) Citron kinase, a Rho-dependent kinase, induces di-phosphorylation of regulatory light chain of myosin II. Mol Biol Cell 14:1745-56
Ward, Yvona; Yap, Seow-Fong; Ravichandran, V et al. (2002) The GTP binding proteins Gem and Rad are negative regulators of the Rho-Rho kinase pathway. J Cell Biol 157:291-302
Yamashiro, S; Chern, H; Yamakita, Y et al. (2001) Mutant Caldesmon lacking cdc2 phosphorylation sites delays M-phase entry and inhibits cytokinesis. Mol Biol Cell 12:239-50
Cohan, C S; Welnhofer, E A; Zhao, L et al. (2001) Role of the actin bundling protein fascin in growth cone morphogenesis: localization in filopodia and lamellipodia. Cell Motil Cytoskeleton 48:109-20
Totsukawa, G; Yamakita, Y; Yamashiro, S et al. (2000) Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts. J Cell Biol 150:797-806

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