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

Ward Y, Yap SF, Ravichandran V, Matsumura F, Ito M, Spinelli B, Kelly K - J. Cell Biol. (2002)

Bottom Line: Here we show that Gem and Rad interface with the Rho pathway through association with the Rho effectors, Rho kinase (ROK) alpha and beta.Gem did not oppose cell rounding initiated by ROKbeta containing a deletion of the Gem binding region, demonstrating that Gem binding to ROKbeta is required for the effects observed.These results identify physiological roles for Gem and Rad in cytoskeletal regulation mediated by ROK.

View Article: PubMed Central - PubMed

Affiliation: Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.

ABSTRACT
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 beta. Gem binds ROKbeta independently of RhoA in the ROKbeta coiled-coil region adjacent to the Rho binding domain. Expression of Gem inhibited ROKbeta-mediated phosphorylation of myosin light chain and myosin phosphatase, but not LIM kinase, suggesting that Gem acts by modifying the substrate specificity of ROKbeta. Gem or Rad expression led to cell flattening and neurite extension in N1E-115 neuroblastoma cells. In interference assays, Gem opposed ROKbeta- and Rad opposed ROKalpha-mediated cell rounding and neurite retraction. Gem did not oppose cell rounding initiated by ROKbeta containing a deletion of the Gem binding region, demonstrating that Gem binding to ROKbeta 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.

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Gem inhibits Rho-dependent transformation and invasion. NIH-3T3 cells were permanently transfected with Dbl and further cotransfected with Gem or Gem(S89N). (A) The ability of the cells to form colonies in soft agar (average of three experiments shown) and (B) the invasiveness of the cells through matrigel (results are the average of five microscopic fields with background subtracted) were determined.
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fig6: Gem inhibits Rho-dependent transformation and invasion. NIH-3T3 cells were permanently transfected with Dbl and further cotransfected with Gem or Gem(S89N). (A) The ability of the cells to form colonies in soft agar (average of three experiments shown) and (B) the invasiveness of the cells through matrigel (results are the average of five microscopic fields with background subtracted) were determined.

Mentions: Rho-dependent transformation has been shown to require ROK signaling for its establishment and maintenance (Sahai et al., 1998). As one example, 3T3 cells transformed by Dbl (a Rho guanine nucleotide exchange factor) are inhibited in anchorage-independent growth by the ROK inhibitor Y-27632 (Sahai et al., 1999). Therefore, we investigated the effect of constitutively expressed wild-type and mutant (S89N) Gem on the ability of Dbl-transformed 3T3 cells to grow in soft agar. Polyclonal expression of Gem reduced the ability of Dbl-transformed cells to produce colonies in soft agar by ∼60% (Fig. 6 A), consistent with the inhibition of ROK activity by Gem. Inhibition was observed for both wild-type and mutant (S89N) Gem, in accordance with their similar functional activities in neuroblastoma cells. Gem expression did not affect the growth rate of the Dbl-transformed cells (unpublished data).


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

Ward Y, Yap SF, Ravichandran V, Matsumura F, Ito M, Spinelli B, Kelly K - J. Cell Biol. (2002)

Gem inhibits Rho-dependent transformation and invasion. NIH-3T3 cells were permanently transfected with Dbl and further cotransfected with Gem or Gem(S89N). (A) The ability of the cells to form colonies in soft agar (average of three experiments shown) and (B) the invasiveness of the cells through matrigel (results are the average of five microscopic fields with background subtracted) were determined.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199248&req=5

fig6: Gem inhibits Rho-dependent transformation and invasion. NIH-3T3 cells were permanently transfected with Dbl and further cotransfected with Gem or Gem(S89N). (A) The ability of the cells to form colonies in soft agar (average of three experiments shown) and (B) the invasiveness of the cells through matrigel (results are the average of five microscopic fields with background subtracted) were determined.
Mentions: Rho-dependent transformation has been shown to require ROK signaling for its establishment and maintenance (Sahai et al., 1998). As one example, 3T3 cells transformed by Dbl (a Rho guanine nucleotide exchange factor) are inhibited in anchorage-independent growth by the ROK inhibitor Y-27632 (Sahai et al., 1999). Therefore, we investigated the effect of constitutively expressed wild-type and mutant (S89N) Gem on the ability of Dbl-transformed 3T3 cells to grow in soft agar. Polyclonal expression of Gem reduced the ability of Dbl-transformed cells to produce colonies in soft agar by ∼60% (Fig. 6 A), consistent with the inhibition of ROK activity by Gem. Inhibition was observed for both wild-type and mutant (S89N) Gem, in accordance with their similar functional activities in neuroblastoma cells. Gem expression did not affect the growth rate of the Dbl-transformed cells (unpublished data).

Bottom Line: Here we show that Gem and Rad interface with the Rho pathway through association with the Rho effectors, Rho kinase (ROK) alpha and beta.Gem did not oppose cell rounding initiated by ROKbeta containing a deletion of the Gem binding region, demonstrating that Gem binding to ROKbeta is required for the effects observed.These results identify physiological roles for Gem and Rad in cytoskeletal regulation mediated by ROK.

View Article: PubMed Central - PubMed

Affiliation: Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.

ABSTRACT
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 beta. Gem binds ROKbeta independently of RhoA in the ROKbeta coiled-coil region adjacent to the Rho binding domain. Expression of Gem inhibited ROKbeta-mediated phosphorylation of myosin light chain and myosin phosphatase, but not LIM kinase, suggesting that Gem acts by modifying the substrate specificity of ROKbeta. Gem or Rad expression led to cell flattening and neurite extension in N1E-115 neuroblastoma cells. In interference assays, Gem opposed ROKbeta- and Rad opposed ROKalpha-mediated cell rounding and neurite retraction. Gem did not oppose cell rounding initiated by ROKbeta containing a deletion of the Gem binding region, demonstrating that Gem binding to ROKbeta 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.

Show MeSH
Related in: MedlinePlus