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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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ROK fragments used in two-hybrid assays and referred to in Table I. ROKβ: a (787–1027), b (2–421), c (422–1097), d (1098–1354), e (422–933), f (422–727). ROKα: a (807–1056), b (2–438), c (439–1126), d (1127–1388).
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fig1: ROK fragments used in two-hybrid assays and referred to in Table I. ROKβ: a (787–1027), b (2–421), c (422–1097), d (1098–1354), e (422–933), f (422–727). ROKα: a (807–1056), b (2–438), c (439–1126), d (1127–1388).

Mentions: To gain insight into its biochemical function, we used Gem as bait in a yeast two-hybrid analysis of a human B cell library. A clone encoding a fragment of ROKβ (also called ROCK I) was identified as interacting strongly with Gem and was of particular interest in light of various reports suggesting an association between RGK family members and the cytoskeleton (Dorin et al., 1995; Bilan et al., 1998; Pan et al., 2000). The ROKβ clone (amino acids [aa] 787–1027) spanned the COOH-terminal half of the coiled-coil domain and the p21 Rho binding domain (PBD) (Leung et al., 1996). The interaction of Gem with various regions of ROKβ was tested further in a two-hybrid assay (Table I and Fig. 1). Gem did not bind the PBD, PH/CRD, or NH2-terminal kinase domains (Table I) but interacted with the coiled-coil domain exclusive of the PBD (Table II). In addition, point mutations introduced at aa 1004 and 1005 in the PBD were shown to inhibit interaction with Rho, whereas Gem binding remained intact (Table II). Therefore, Gem binds to a region of ROKβ adjacent to but distinct from Rho binding, and the interaction of Gem with ROKβ does not require Rho binding.


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)

ROK fragments used in two-hybrid assays and referred to in Table I. ROKβ: a (787–1027), b (2–421), c (422–1097), d (1098–1354), e (422–933), f (422–727). ROKα: a (807–1056), b (2–438), c (439–1126), d (1127–1388).
© Copyright Policy
Related In: Results  -  Collection

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

fig1: ROK fragments used in two-hybrid assays and referred to in Table I. ROKβ: a (787–1027), b (2–421), c (422–1097), d (1098–1354), e (422–933), f (422–727). ROKα: a (807–1056), b (2–438), c (439–1126), d (1127–1388).
Mentions: To gain insight into its biochemical function, we used Gem as bait in a yeast two-hybrid analysis of a human B cell library. A clone encoding a fragment of ROKβ (also called ROCK I) was identified as interacting strongly with Gem and was of particular interest in light of various reports suggesting an association between RGK family members and the cytoskeleton (Dorin et al., 1995; Bilan et al., 1998; Pan et al., 2000). The ROKβ clone (amino acids [aa] 787–1027) spanned the COOH-terminal half of the coiled-coil domain and the p21 Rho binding domain (PBD) (Leung et al., 1996). The interaction of Gem with various regions of ROKβ was tested further in a two-hybrid assay (Table I and Fig. 1). Gem did not bind the PBD, PH/CRD, or NH2-terminal kinase domains (Table I) but interacted with the coiled-coil domain exclusive of the PBD (Table II). In addition, point mutations introduced at aa 1004 and 1005 in the PBD were shown to inhibit interaction with Rho, whereas Gem binding remained intact (Table II). Therefore, Gem binds to a region of ROKβ adjacent to but distinct from Rho binding, and the interaction of Gem with ROKβ does not require Rho binding.

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