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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 on ROK activity. (A) Gem inhibits ROKβ- but not ROKα-dependent phosphorylation of MLC. Cos7 cells were transfected with flag-tagged MLC and myc-tagged ROKα, ROKβ, or the constitutively active kinase domain of ROKβ. The ability of Gem to inhibit MLC phosphorylation was demonstrated by cotransfection with full-length wild-type Gem. Western blots generated with antibodies specific for MLC phosphorylated on serine 19, total transfected MLC detected with antiflag antibodies, or ROK detected with anti-myc antibodies are shown. (B) Gem-mediated inhibition of MLC phosphatase phosphorylation by ROKβ was demonstrated by transfection of the MBS into Cos7 cells together with ROKβ and/or Gem. Western blots generated with antibodies specific for MBS phosphorylated on threonine 695 as well as total MBS and ROK are shown. (C) Gem has no effect on ROKβ-dependent LIMK activation. HA-tagged LIMK was cotransfected into Cos7 cells with ROKβ and/or Gem and then immunoprecipitated using anti-HA antibody. An in vitro kinase assay was performed using cofilin and [γ32P]ATP as substrates for the phosphoryl transfer. Phosphorylated cofilin was revealed by autoradiography and ROK protein levels were assayed by Western blots.
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fig7: Effect of Gem on ROK activity. (A) Gem inhibits ROKβ- but not ROKα-dependent phosphorylation of MLC. Cos7 cells were transfected with flag-tagged MLC and myc-tagged ROKα, ROKβ, or the constitutively active kinase domain of ROKβ. The ability of Gem to inhibit MLC phosphorylation was demonstrated by cotransfection with full-length wild-type Gem. Western blots generated with antibodies specific for MLC phosphorylated on serine 19, total transfected MLC detected with antiflag antibodies, or ROK detected with anti-myc antibodies are shown. (B) Gem-mediated inhibition of MLC phosphatase phosphorylation by ROKβ was demonstrated by transfection of the MBS into Cos7 cells together with ROKβ and/or Gem. Western blots generated with antibodies specific for MBS phosphorylated on threonine 695 as well as total MBS and ROK are shown. (C) Gem has no effect on ROKβ-dependent LIMK activation. HA-tagged LIMK was cotransfected into Cos7 cells with ROKβ and/or Gem and then immunoprecipitated using anti-HA antibody. An in vitro kinase assay was performed using cofilin and [γ32P]ATP as substrates for the phosphoryl transfer. Phosphorylated cofilin was revealed by autoradiography and ROK protein levels were assayed by Western blots.

Mentions: An important question is the mechanism of action whereby Gem functionally opposes ROKβ. We considered the possibilities that Gem (a) inhibits ROK kinase activity or (b) redirects ROK localization and/or substrate specificity. We have obtained no evidence suggesting a direct effect of Gem upon the kinase activity of ROKβ. For example, no change in the level of in vitro kinase activity was observed in immunoprecipitated ROKβ relative to the presence or absence of coexpressed Gem (unpublished data). Therefore, in order to test the second possibility, the effect of Gem expression upon the in vivo activity of ROKβ was investigated in COS cells for the substrates MLC, MBS, and LIMK. ROK-dependent phosphorylation of MLC and MBS was assayed with phosphospecific antibodies. LIMK phosphorylation was measured indirectly by an immune complex kinase activity assay using cofilin as the substrate. As shown in Fig. 7 A, ROKβ stimulated increased phosphorylation of MLC, which was reversed in the presence of coexpressed Gem. ROKα-mediated phosphorylation was unaffected by Gem as was phosphorylation mediated by the kinase domain of ROKβ in the absence of the Gem binding region. Similarly, ROKβ-dependent phosphorylation of MBS was inhibited by Gem coexpression (Fig. 7 B). By contrast, as shown in Fig. 7 C, immunoprecipitated LIMK demonstrated a ROK-dependent increase in cofilin-directed kinase activity, which was essentially unaffected by coexpressed Gem. Therefore, Gem had a selective effect on ROK-mediated phosphorylation, inhibiting MLC and myosin phosphatase phosphorylation, consistent with the opposition by Gem of ROK-activated actinomyosin contractility. These data suggest that Gem most likely differentially modifies the access of ROK to its substrates.


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 on ROK activity. (A) Gem inhibits ROKβ- but not ROKα-dependent phosphorylation of MLC. Cos7 cells were transfected with flag-tagged MLC and myc-tagged ROKα, ROKβ, or the constitutively active kinase domain of ROKβ. The ability of Gem to inhibit MLC phosphorylation was demonstrated by cotransfection with full-length wild-type Gem. Western blots generated with antibodies specific for MLC phosphorylated on serine 19, total transfected MLC detected with antiflag antibodies, or ROK detected with anti-myc antibodies are shown. (B) Gem-mediated inhibition of MLC phosphatase phosphorylation by ROKβ was demonstrated by transfection of the MBS into Cos7 cells together with ROKβ and/or Gem. Western blots generated with antibodies specific for MBS phosphorylated on threonine 695 as well as total MBS and ROK are shown. (C) Gem has no effect on ROKβ-dependent LIMK activation. HA-tagged LIMK was cotransfected into Cos7 cells with ROKβ and/or Gem and then immunoprecipitated using anti-HA antibody. An in vitro kinase assay was performed using cofilin and [γ32P]ATP as substrates for the phosphoryl transfer. Phosphorylated cofilin was revealed by autoradiography and ROK protein levels were assayed by Western blots.
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Related In: Results  -  Collection

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fig7: Effect of Gem on ROK activity. (A) Gem inhibits ROKβ- but not ROKα-dependent phosphorylation of MLC. Cos7 cells were transfected with flag-tagged MLC and myc-tagged ROKα, ROKβ, or the constitutively active kinase domain of ROKβ. The ability of Gem to inhibit MLC phosphorylation was demonstrated by cotransfection with full-length wild-type Gem. Western blots generated with antibodies specific for MLC phosphorylated on serine 19, total transfected MLC detected with antiflag antibodies, or ROK detected with anti-myc antibodies are shown. (B) Gem-mediated inhibition of MLC phosphatase phosphorylation by ROKβ was demonstrated by transfection of the MBS into Cos7 cells together with ROKβ and/or Gem. Western blots generated with antibodies specific for MBS phosphorylated on threonine 695 as well as total MBS and ROK are shown. (C) Gem has no effect on ROKβ-dependent LIMK activation. HA-tagged LIMK was cotransfected into Cos7 cells with ROKβ and/or Gem and then immunoprecipitated using anti-HA antibody. An in vitro kinase assay was performed using cofilin and [γ32P]ATP as substrates for the phosphoryl transfer. Phosphorylated cofilin was revealed by autoradiography and ROK protein levels were assayed by Western blots.
Mentions: An important question is the mechanism of action whereby Gem functionally opposes ROKβ. We considered the possibilities that Gem (a) inhibits ROK kinase activity or (b) redirects ROK localization and/or substrate specificity. We have obtained no evidence suggesting a direct effect of Gem upon the kinase activity of ROKβ. For example, no change in the level of in vitro kinase activity was observed in immunoprecipitated ROKβ relative to the presence or absence of coexpressed Gem (unpublished data). Therefore, in order to test the second possibility, the effect of Gem expression upon the in vivo activity of ROKβ was investigated in COS cells for the substrates MLC, MBS, and LIMK. ROK-dependent phosphorylation of MLC and MBS was assayed with phosphospecific antibodies. LIMK phosphorylation was measured indirectly by an immune complex kinase activity assay using cofilin as the substrate. As shown in Fig. 7 A, ROKβ stimulated increased phosphorylation of MLC, which was reversed in the presence of coexpressed Gem. ROKα-mediated phosphorylation was unaffected by Gem as was phosphorylation mediated by the kinase domain of ROKβ in the absence of the Gem binding region. Similarly, ROKβ-dependent phosphorylation of MBS was inhibited by Gem coexpression (Fig. 7 B). By contrast, as shown in Fig. 7 C, immunoprecipitated LIMK demonstrated a ROK-dependent increase in cofilin-directed kinase activity, which was essentially unaffected by coexpressed Gem. Therefore, Gem had a selective effect on ROK-mediated phosphorylation, inhibiting MLC and myosin phosphatase phosphorylation, consistent with the opposition by Gem of ROK-activated actinomyosin contractility. These data suggest that Gem most likely differentially modifies the access of ROK to its substrates.

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