Molecular decipherment of Rho effector pathways regulating tight-junction permeability.
We reported recently that the activation of RhoA induced an increase in transepithelial electrical resistance (TER). To clarify effectors of Rho for this RhoA-induced regulation of tight-junction permeability, we introduced two effector-loop mutants of constitutively active RhoA(V14), RhoA(V14/L40) and RhoA(V14/C42), into Mardin-Darby canine kidney cells in an isopropyl beta-D-thiogalactoside-inducible expression system. RhoA(V14) and the two effector-loop mutants interacted in vitro with the Rho-binding domain of Rho-associated kinase, ROKalpha. Next we examined two parameters of Rho functions, stress-fibre formation and TER elevation, induced by RhoA(V14). Stress-fibre formation was induced by RhoA(V14/C42) but not by RhoA(V14/L40). On the other hand, TER elevation was induced by neither RhoA(V14/L40) nor RhoA(V14/C42). RhoA-associated kinase inhibitor, Y-27632, inhibited both stress-fibre formation and TER elevation induced by RhoA(V14). These results demonstrated that RhoA-induced regulation of tight-junction permeability is mediated by Rho-associated kinase and at least one other unidentified effector, the coupling to RhoA being disrupted by mutation at position 40 or 42 in the effector loop.
Fujita
H
H
Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Katoh
H
H
Hasegawa
H
H
Yasui
H
H
Aoki
J
J
Yamaguchi
Y
Y
Negishi
M
M
D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000502:physiology*; D004285:Dogs; D047908:Intracellular Signaling Peptides and Proteins; D008856:Microscopy, Fluorescence; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000502:physiology*
D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein