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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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Effect of Gem and Rad on the actin cytoskeleton. HeLa and NIH-3T3 cells were transfected with Gem or cotransfected with pEGFP-N1 and Rad. The cells were analyzed via confocal microscopy. Gem was stained with polyclonal anti-Gem antibody and FITC-conjugated goat anti–rabbit. Focal adhesions were visualized by staining with antivinculin antibody and stress fibers were identified with rhodamine-conjugated phalloidin.
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fig5: Effect of Gem and Rad on the actin cytoskeleton. HeLa and NIH-3T3 cells were transfected with Gem or cotransfected with pEGFP-N1 and Rad. The cells were analyzed via confocal microscopy. Gem was stained with polyclonal anti-Gem antibody and FITC-conjugated goat anti–rabbit. Focal adhesions were visualized by staining with antivinculin antibody and stress fibers were identified with rhodamine-conjugated phalloidin.

Mentions: ROK has been shown to play a fundamental role in the regulation of the actinomyosin cytoskeleton, including the formation of stress fibers and focal adhesions. Therefore, we analyzed the effect of Gem and Rad expression in epithelial cells and fibroblasts on the cytoskeleton as judged by staining for F-actin and vinculin (Fig. 5). As shown for HeLa cells in Fig. 5, transient Gem or Rad overexpression inhibited the presence of focal adhesions in the main cell body while leaving peripheral focal complexes intact, in agreement with previous reports that ROK activity is required for the maintenance of central but not peripheral focal contacts (Totsukawa et al., 2000). Gem or Rad overexpression in fibroblasts often induced an unusual dendritic morphology (Fig. 5) characterized by abnormal cellular elongation or the presence of branching filopodial structures and rounding or retraction of the cell body. Gem or Rad expression was accompanied by loss of central but not peripheral focal contacts and loss of stress fibers. In addition, enhanced lamellipodia formation was evident in Gem-transfected cells (unpublished data). Interestingly, a dendritic morphology is induced in BALB/c 3T3 cells after prolonged inhibition of RhoA or ROK (Hirose et al., 1998). Low levels of Gem expression generally did not result in loss of focal adhesions or stress fibers or induction of a dendritic morphology, possibly as a result of residual ROK activity.


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)

Effect of Gem and Rad on the actin cytoskeleton. HeLa and NIH-3T3 cells were transfected with Gem or cotransfected with pEGFP-N1 and Rad. The cells were analyzed via confocal microscopy. Gem was stained with polyclonal anti-Gem antibody and FITC-conjugated goat anti–rabbit. Focal adhesions were visualized by staining with antivinculin antibody and stress fibers were identified with rhodamine-conjugated phalloidin.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Effect of Gem and Rad on the actin cytoskeleton. HeLa and NIH-3T3 cells were transfected with Gem or cotransfected with pEGFP-N1 and Rad. The cells were analyzed via confocal microscopy. Gem was stained with polyclonal anti-Gem antibody and FITC-conjugated goat anti–rabbit. Focal adhesions were visualized by staining with antivinculin antibody and stress fibers were identified with rhodamine-conjugated phalloidin.
Mentions: ROK has been shown to play a fundamental role in the regulation of the actinomyosin cytoskeleton, including the formation of stress fibers and focal adhesions. Therefore, we analyzed the effect of Gem and Rad expression in epithelial cells and fibroblasts on the cytoskeleton as judged by staining for F-actin and vinculin (Fig. 5). As shown for HeLa cells in Fig. 5, transient Gem or Rad overexpression inhibited the presence of focal adhesions in the main cell body while leaving peripheral focal complexes intact, in agreement with previous reports that ROK activity is required for the maintenance of central but not peripheral focal contacts (Totsukawa et al., 2000). Gem or Rad overexpression in fibroblasts often induced an unusual dendritic morphology (Fig. 5) characterized by abnormal cellular elongation or the presence of branching filopodial structures and rounding or retraction of the cell body. Gem or Rad expression was accompanied by loss of central but not peripheral focal contacts and loss of stress fibers. In addition, enhanced lamellipodia formation was evident in Gem-transfected cells (unpublished data). Interestingly, a dendritic morphology is induced in BALB/c 3T3 cells after prolonged inhibition of RhoA or ROK (Hirose et al., 1998). Low levels of Gem expression generally did not result in loss of focal adhesions or stress fibers or induction of a dendritic morphology, possibly as a result of residual ROK activity.

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