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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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Interaction of Gem and ROK. (A) Interactions of Gem and Rad with ROK in vitro. Lysates from Cos cells transiently transfected with myc-tagged full-length ROKα or ROKβ were incubated with purified recombinant GST–Gem (GTPγS) or –Rad (GTPγS) bound to glutathione-Sepharose beads. ROK that cosedimented with Gem or Rad was shown by Western blot analysis with anti-myc antibody. (B) Interaction of Gem and ROKβ in vivo. Cos7 or N1E-115 cells were cotransfected with ROKβ and Gem. Cell lysates were subjected to immunoprecipitation with either anti-Gem monoclonal antibody or mouse IgG as a control for nonspecificity. Coprecipitated ROKβ was revealed by Western blot using anti-myc antibody.
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fig2: Interaction of Gem and ROK. (A) Interactions of Gem and Rad with ROK in vitro. Lysates from Cos cells transiently transfected with myc-tagged full-length ROKα or ROKβ were incubated with purified recombinant GST–Gem (GTPγS) or –Rad (GTPγS) bound to glutathione-Sepharose beads. ROK that cosedimented with Gem or Rad was shown by Western blot analysis with anti-myc antibody. (B) Interaction of Gem and ROKβ in vivo. Cos7 or N1E-115 cells were cotransfected with ROKβ and Gem. Cell lysates were subjected to immunoprecipitation with either anti-Gem monoclonal antibody or mouse IgG as a control for nonspecificity. Coprecipitated ROKβ was revealed by Western blot using anti-myc antibody.

Mentions: We also investigated the physical association of Gem and Rad with ROK (Fig. 2) and determined that binding can be observed. As shown in Fig. 2 A, recombinant glutathione-S-transferase (GST)–Gem and recombinant GST–Rad bound ROKβ and to a lesser extent ROKα in whole-cell extracts derived from transfected COS7 cells. The presence of GTP-γS did not effect the binding efficiency (unpublished data). In addition, coprecipitation of ROKβ with Gem was observed from extracts of either transfected COS7 or N1E-115 cells (Fig. 2 B), consistent with the interaction seen in the yeast two-hybrid analyses.


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)

Interaction of Gem and ROK. (A) Interactions of Gem and Rad with ROK in vitro. Lysates from Cos cells transiently transfected with myc-tagged full-length ROKα or ROKβ were incubated with purified recombinant GST–Gem (GTPγS) or –Rad (GTPγS) bound to glutathione-Sepharose beads. ROK that cosedimented with Gem or Rad was shown by Western blot analysis with anti-myc antibody. (B) Interaction of Gem and ROKβ in vivo. Cos7 or N1E-115 cells were cotransfected with ROKβ and Gem. Cell lysates were subjected to immunoprecipitation with either anti-Gem monoclonal antibody or mouse IgG as a control for nonspecificity. Coprecipitated ROKβ was revealed by Western blot using anti-myc antibody.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Interaction of Gem and ROK. (A) Interactions of Gem and Rad with ROK in vitro. Lysates from Cos cells transiently transfected with myc-tagged full-length ROKα or ROKβ were incubated with purified recombinant GST–Gem (GTPγS) or –Rad (GTPγS) bound to glutathione-Sepharose beads. ROK that cosedimented with Gem or Rad was shown by Western blot analysis with anti-myc antibody. (B) Interaction of Gem and ROKβ in vivo. Cos7 or N1E-115 cells were cotransfected with ROKβ and Gem. Cell lysates were subjected to immunoprecipitation with either anti-Gem monoclonal antibody or mouse IgG as a control for nonspecificity. Coprecipitated ROKβ was revealed by Western blot using anti-myc antibody.
Mentions: We also investigated the physical association of Gem and Rad with ROK (Fig. 2) and determined that binding can be observed. As shown in Fig. 2 A, recombinant glutathione-S-transferase (GST)–Gem and recombinant GST–Rad bound ROKβ and to a lesser extent ROKα in whole-cell extracts derived from transfected COS7 cells. The presence of GTP-γS did not effect the binding efficiency (unpublished data). In addition, coprecipitation of ROKβ with Gem was observed from extracts of either transfected COS7 or N1E-115 cells (Fig. 2 B), consistent with the interaction seen in the yeast two-hybrid analyses.

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