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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 appears to inhibit ROKβ through a direct interaction. N1E-115 mouse neuroblastoma cells were cotransfected with pEGFP-N1 and the DNA indicated above. The total amount of DNA was normalized with vector DNA. Data presented with standard deviations are the average of at least three independent experiments. (A) ROK isoforms missing the Gem/Rad binding domains are not inhibited by coexpression of Gem or Rad. (B) Gem- and Rad-induced cell flattening is inhibited by the ROKα (aa 807–1006) and ROKβ (aa 787–976) coiled-coil (CC) isolated domains.
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fig4: Gem appears to inhibit ROKβ through a direct interaction. N1E-115 mouse neuroblastoma cells were cotransfected with pEGFP-N1 and the DNA indicated above. The total amount of DNA was normalized with vector DNA. Data presented with standard deviations are the average of at least three independent experiments. (A) ROK isoforms missing the Gem/Rad binding domains are not inhibited by coexpression of Gem or Rad. (B) Gem- and Rad-induced cell flattening is inhibited by the ROKα (aa 807–1006) and ROKβ (aa 787–976) coiled-coil (CC) isolated domains.

Mentions: An important question is whether the inhibitory effects of Gem and Rad on ROK-mediated functions require the interaction of Gem/Rad with ROK, or, alternatively, are an indirect effect. To address this question, we have assayed the ability of Gem or Rad to interfere with cell rounding initiated by ROK mutants missing Gem/Rad binding domains (Fig. 4 A). Constitutively active ROKβ or ROKα truncated shortly after the kinase domain robustly stimulated rounding of N1E-115 cells that was unaffected by Gem or Rad expression (Fig. 4 A), suggesting that Gem/Rad binding to ROK is required for inhibition. An additional ROKβ mutant, ROKβ(Δ787–906) was constructed by deleting the Gem binding domain but leaving other regulatory domains (including the Rho binding domain) intact. This mutant form of ROKβ was stably expressed in COS and N1E-115 cells (unpublished data). As shown in Fig. 4 A, ROKβ(Δ787−906) was effective at mediating neurite retraction that could not be reversed by Gem expression, further supporting the conclusion that Gem and ROKβ interact directly.


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 appears to inhibit ROKβ through a direct interaction. N1E-115 mouse neuroblastoma cells were cotransfected with pEGFP-N1 and the DNA indicated above. The total amount of DNA was normalized with vector DNA. Data presented with standard deviations are the average of at least three independent experiments. (A) ROK isoforms missing the Gem/Rad binding domains are not inhibited by coexpression of Gem or Rad. (B) Gem- and Rad-induced cell flattening is inhibited by the ROKα (aa 807–1006) and ROKβ (aa 787–976) coiled-coil (CC) isolated domains.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Gem appears to inhibit ROKβ through a direct interaction. N1E-115 mouse neuroblastoma cells were cotransfected with pEGFP-N1 and the DNA indicated above. The total amount of DNA was normalized with vector DNA. Data presented with standard deviations are the average of at least three independent experiments. (A) ROK isoforms missing the Gem/Rad binding domains are not inhibited by coexpression of Gem or Rad. (B) Gem- and Rad-induced cell flattening is inhibited by the ROKα (aa 807–1006) and ROKβ (aa 787–976) coiled-coil (CC) isolated domains.
Mentions: An important question is whether the inhibitory effects of Gem and Rad on ROK-mediated functions require the interaction of Gem/Rad with ROK, or, alternatively, are an indirect effect. To address this question, we have assayed the ability of Gem or Rad to interfere with cell rounding initiated by ROK mutants missing Gem/Rad binding domains (Fig. 4 A). Constitutively active ROKβ or ROKα truncated shortly after the kinase domain robustly stimulated rounding of N1E-115 cells that was unaffected by Gem or Rad expression (Fig. 4 A), suggesting that Gem/Rad binding to ROK is required for inhibition. An additional ROKβ mutant, ROKβ(Δ787–906) was constructed by deleting the Gem binding domain but leaving other regulatory domains (including the Rho binding domain) intact. This mutant form of ROKβ was stably expressed in COS and N1E-115 cells (unpublished data). As shown in Fig. 4 A, ROKβ(Δ787−906) was effective at mediating neurite retraction that could not be reversed by Gem expression, further supporting the conclusion that Gem and ROKβ interact directly.

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