IκB Kinase γ/Nuclear Factor-κB-Essential Modulator (IKKγ/NEMO) Facilitates RhoA GTPase Activation, which, in Turn, Activates Rho-associated Kinase ( ROCK) to Phosphorylate IKKβ in Response to Transforming Growth Factor (TGF)-β1

Background: TGF-1 activates RhoA and nuclear factor-B (NF-B), but the activation mechanism was not clearly elucidated. Results: IKK disrupts RhoA-Rho guanine nucleotide dissociation inhibitor (RhoGDI) complex, facilitating GTP binding to RhoA, resulting in IKK phosphorylation by ROCK. Conclusion: IKK facilitates RhoA activation, which in turn activates NF-B. Significance: We found the new mechanism of IKK to activate RhoA and NF-B by TGF-1. Transforming growth factor (TGF)-1 plays several roles in a variety of cellular functions. TGF-1 transmits its signal through Smad transcription factor-dependent and -independent pathways. It was reported that TGF-1 activates NF-B and RhoA, and RhoA activates NF-B in several kinds of cells in a Smad-independent pathway. However, the activation molecular mechanism of NF-B by RhoA upon TGF-1 has not been clearly elucidated. We observed that RhoA-GTP level was increased by TGF-1 in RAW264.7 cells. RhoA-GDP and RhoGDI were bound to N- and C-terminal domains of IKK, respectively. Purified IKK facilitated GTP binding to RhoA complexed with RhoGDI. Furthermore, Dbs, a guanine nucletotide exchange factor of RhoA much more enhanced GTP binding to RhoA complexed with RhoGDI in the presence of IKK. Indeed, si-IKK abolished RhoA activation in response to TGF-1 in cells. However, TGF-1 stimulated the release of RhoA-GTP from IKK and Rho-associated kinase (ROCK), an active RhoA effector protein, directly phosphorylated IKK in vitro, whereas TGF-1-activated kinase 1 activated RhoA upon TGF-1 stimulation. Taken together, our data indicate that IKK facilitates RhoA activation via a guanine nucletotide exchange factor, which in turn activates ROCK to phosphorylate IKK, leading to NF-B activation that induced the chemokine expression and cell migration upon TGF-1.