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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 RGK family members and ROK on N1E-115 mouse neuroblastoma phenotype. N1E-115 mouse neuroblastoma cells were cotransfected with pEGFP-N1 and the DNA indicated. The total amount of DNA was normalized with vector DNA. Data presented in the bar graphs with standard deviations are the average of at least four independent experiments. (A) Representative round (ROKβ-transfected), flattened/short neurite (Gem-transfected), and long neurite (Gem-transfected) morphologies in N1E-115 cells. (B) Gem reverses ROKβ-, whereas Rad reverses ROKα-dependent neurite retraction. Rem1 and Rem2 are not involved in neurite induction. (C) Gem expression opposes neurite retraction stimulated by constitutively active RhoA(63L) but has little effect on cell flattening caused by the dominant-negative RhoA(19N). Dominant-negative Rac(17N) prevents Gem from stimulating cell flattening and neurite extension.
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fig3: Effect of RGK family members and ROK on N1E-115 mouse neuroblastoma phenotype. N1E-115 mouse neuroblastoma cells were cotransfected with pEGFP-N1 and the DNA indicated. The total amount of DNA was normalized with vector DNA. Data presented in the bar graphs with standard deviations are the average of at least four independent experiments. (A) Representative round (ROKβ-transfected), flattened/short neurite (Gem-transfected), and long neurite (Gem-transfected) morphologies in N1E-115 cells. (B) Gem reverses ROKβ-, whereas Rad reverses ROKα-dependent neurite retraction. Rem1 and Rem2 are not involved in neurite induction. (C) Gem expression opposes neurite retraction stimulated by constitutively active RhoA(63L) but has little effect on cell flattening caused by the dominant-negative RhoA(19N). Dominant-negative Rac(17N) prevents Gem from stimulating cell flattening and neurite extension.

Mentions: The functional effect observed after transient transfection of Gem or Rad suggested an inhibition of ROK activity (Fig. 3, A and B). That is, Gem or Rad individually stimulated flattening and neurite extension of N1E-115 cells, a phenotype that is observed after transfection of dominant-negative ROK or with the ROK inhibitor Y-27632 (see Fig. 8, A and B) (Hirose et al., 1998). Correlative with ROK binding in the yeast two-hybrid assay, Gem or Rad displayed functional activity but Rem1 and Rem2 did not. In addition, an interference assay was used to investigate the specificity of Gem and Rad for the ROK isoforms. Transfection of either ROKα or ROKβ into N1E-115 cells caused enhanced cell rounding (Fig. 3, A and B). Previous investigations have shown that transfected ROK is active in the absence of Rho binding (Leung et al., 1996). Cotransfection of Gem opposed the effects of ROKβ but not ROKα. Surprisingly, cotransfection of Rad fully reversed the activity of ROKα and only weakly effected ROKβ. Therefore, in N1E-115 cells, Rad appears to have functional specificity for full-length ROKα, as compared with ROKβ. Western blots were used to verify equivalent expression levels for transfected Gem, ROKβ, and ROKα in the various experimental conditions depicted in Fig. 3 (unpublished data). The Gem(S89N) mutant stimulated cell flattening and neurite extension, consistent with its ability to bind ROKβ. Additionally, Gem and Rad expression in N1E-115 inhibited lysphosphatidic acid–induced cell rounding (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)

Effect of RGK family members and ROK on N1E-115 mouse neuroblastoma phenotype. N1E-115 mouse neuroblastoma cells were cotransfected with pEGFP-N1 and the DNA indicated. The total amount of DNA was normalized with vector DNA. Data presented in the bar graphs with standard deviations are the average of at least four independent experiments. (A) Representative round (ROKβ-transfected), flattened/short neurite (Gem-transfected), and long neurite (Gem-transfected) morphologies in N1E-115 cells. (B) Gem reverses ROKβ-, whereas Rad reverses ROKα-dependent neurite retraction. Rem1 and Rem2 are not involved in neurite induction. (C) Gem expression opposes neurite retraction stimulated by constitutively active RhoA(63L) but has little effect on cell flattening caused by the dominant-negative RhoA(19N). Dominant-negative Rac(17N) prevents Gem from stimulating cell flattening and neurite extension.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Effect of RGK family members and ROK on N1E-115 mouse neuroblastoma phenotype. N1E-115 mouse neuroblastoma cells were cotransfected with pEGFP-N1 and the DNA indicated. The total amount of DNA was normalized with vector DNA. Data presented in the bar graphs with standard deviations are the average of at least four independent experiments. (A) Representative round (ROKβ-transfected), flattened/short neurite (Gem-transfected), and long neurite (Gem-transfected) morphologies in N1E-115 cells. (B) Gem reverses ROKβ-, whereas Rad reverses ROKα-dependent neurite retraction. Rem1 and Rem2 are not involved in neurite induction. (C) Gem expression opposes neurite retraction stimulated by constitutively active RhoA(63L) but has little effect on cell flattening caused by the dominant-negative RhoA(19N). Dominant-negative Rac(17N) prevents Gem from stimulating cell flattening and neurite extension.
Mentions: The functional effect observed after transient transfection of Gem or Rad suggested an inhibition of ROK activity (Fig. 3, A and B). That is, Gem or Rad individually stimulated flattening and neurite extension of N1E-115 cells, a phenotype that is observed after transfection of dominant-negative ROK or with the ROK inhibitor Y-27632 (see Fig. 8, A and B) (Hirose et al., 1998). Correlative with ROK binding in the yeast two-hybrid assay, Gem or Rad displayed functional activity but Rem1 and Rem2 did not. In addition, an interference assay was used to investigate the specificity of Gem and Rad for the ROK isoforms. Transfection of either ROKα or ROKβ into N1E-115 cells caused enhanced cell rounding (Fig. 3, A and B). Previous investigations have shown that transfected ROK is active in the absence of Rho binding (Leung et al., 1996). Cotransfection of Gem opposed the effects of ROKβ but not ROKα. Surprisingly, cotransfection of Rad fully reversed the activity of ROKα and only weakly effected ROKβ. Therefore, in N1E-115 cells, Rad appears to have functional specificity for full-length ROKα, as compared with ROKβ. Western blots were used to verify equivalent expression levels for transfected Gem, ROKβ, and ROKα in the various experimental conditions depicted in Fig. 3 (unpublished data). The Gem(S89N) mutant stimulated cell flattening and neurite extension, consistent with its ability to bind ROKβ. Additionally, Gem and Rad expression in N1E-115 inhibited lysphosphatidic acid–induced cell rounding (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