The GTP binding proteins Gem and Rad are negative regulators of the Rho-Rho kinase pathway.
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.
Ward
Yvona
Y
Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.
Yap
Seow-Fong
SF
Ravichandran
V
V
Matsumura
Fumio
F
Ito
Masaaki
M
Spinelli
Beth
B
Kelly
Kathleen
K
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002471:Cell Transformation, Neoplastic; D003599:Cytoskeleton / Q000378:metabolism; D025461:Feedback, Physiological; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism; D009361:Neoplasm Invasiveness; D016501:Neurites / Q000201:enzymology / Q000378:metabolism; D009447:Neuroblastoma / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D018631:ras Proteins / Q000378:metabolism*; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000199:Actins; D017874:Immediate-Early Proteins; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; C084355:RRAD protein, human; C490030:Rrad protein, mouse; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; C490245:GEM protein, human; C088119:Gem protein, mouse; D020559:Monomeric GTP-Binding Proteins; D018631:ras Proteins; D020742:rhoA GTP-Binding Protein