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Published 15 April 2002. doi:10.1083/jcb.200111026
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© The Rockefeller University Press, 0021-9525/2002/4/291 $5.00
The Journal of Cell Biology, Volume 157, Number 2, April 15, 2002 291-302

Article

The GTP binding proteins Gem and Rad are negative regulators of the Rho–Rho kinase pathway



Yvona Ward1, Seow-Fong Yap1, V. Ravichandran1, Fumio Matsumura2, Masaaki Ito3, Beth Spinelli1 and Kathleen Kelly1

1 Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892
2 Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08855
3 First Department of Internal Medicine, Mie University School of Medicine, Mie 514-8507 Japan

Address correspondence to Kathleen Kelly, Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, Building 10, Room 3B43, Bethesda, MD 20892. Tel.: (301) 435-4651. Fax: (301) 435-4655. E-mail: kkelly{at}helix.nih.gov

The cytoskeletal changes that alter cellular morphogenesis and motility depend upon a complex interplay among molecules that regulate actin, myosin, and other cytoskeletal components. The Rho family of GTP binding proteins are important upstream mediators of cytoskeletal organization. Gem and Rad are members of another family of small GTP binding proteins (the Rad, Gem, and Kir family) for which biochemical functions have been mostly unknown. Here we show that Gem and Rad interface with the Rho pathway through association with the Rho effectors, Rho kinase (ROK) {alpha} and ß. Gem binds ROKß independently of RhoA in the ROKß coiled-coil region adjacent to the Rho binding domain. Expression of Gem inhibited ROKß-mediated phosphorylation of myosin light chain and myosin phosphatase, but not LIM kinase, suggesting that Gem acts by modifying the substrate specificity of ROKß. Gem or Rad expression led to cell flattening and neurite extension in N1E-115 neuroblastoma cells. In interference assays, Gem opposed ROKß- and Rad opposed ROK{alpha}-mediated cell rounding and neurite retraction. Gem did not oppose cell rounding initiated by ROKß containing a deletion of the Gem binding region, demonstrating that Gem binding to ROKß is required for the effects observed. In epithelial or fibroblastic cells, Gem or Rad expression resulted in stress fiber and focal adhesion disassembly. In addition, Gem reverted the anchorage-independent growth and invasiveness of Dbl-transformed fibroblasts. These results identify physiological roles for Gem and Rad in cytoskeletal regulation mediated by ROK.

Key Words: Gem; Rad; Rho kinase; myosin light chain; neuroblastoma


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