Adducin is a membrane skeletal protein that binds to actin filaments (F-actin) and thereby promotes the association of spectrin with F-actin to form a spectrin-actin meshwork beneath plasma membranes such as ruffling membranes. Rho-associated kinase (Rho- kinase), which is activated by the small guanosine triphosphatase Rho, phosphorylates α-adducin and thereby enhances the F-actin–binding activity of α-adducin in vitro. Here we identified the sites of phosphorylation of α-adducin by Rho-kinase as Thr445 and Thr480. We prepared antibody that specifically recognized α-adducin phosphorylated at Thr445, and found by use of this antibody that Rho-kinase phosphorylated α-adducin at Thr445 in COS7 cells in a Rho-dependent manner. Phosphorylated α-adducin accumulated in the membrane ruffling area of Madin-Darby canine kidney (MDCK) epithelial cells and the leading edge of scattering cells during the action of tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF). The microinjection of Botulinum C3 ADP-ribosyl-transferase, dominant negative Rho-kinase, or α-adducinT445A,T480A (substitution of Thr445 and Thr480 by Ala) inhibited the TPA-induced membrane ruffling in MDCK cells and wound-induced migra- tion in NRK49F cells. α-AdducinT445D,T480D (substi- tution of Thr445 and Thr480 by Asp), but not α-adducinT445A,T480A, counteracted the inhibitory effect of the dominant negative Rho-kinase on the TPA-induced membrane ruffling in MDCK cells. Taken together, these results indicate that Rho-kinase phosphorylates α-adducin downstream of Rho in vivo, and that the phosphorylation of adducin by Rho-kinase plays a crucial role in the regulation of membrane ruffling and cell motility.

Initiation of nasopharyngeal-associated lymphoid tissue (NALT) development is independent of the programmed cytokine cascade necessary for the formation of Peyer's patches (PP) and peripheral lymph nodes (PLN), a cytokine cascade which consists of IL-7R, LTalpha1beta2/LTbetaR, and NIK. However, the subsequent organization of NALT seems to be controlled by these cytokine signaling cascades since the maturation of NALT structure is generally incomplete in those cytokine cascade-deficient mice. NALT as well as PP and PLN are completely absent in Id2(-/-) mice. NALT organogenesis is initiated following the adoptive transfer of CD3(-)CD4(+)CD45(+) cells into Id2(-/-) mice, constituting direct evidence that CD3(-)CD4(+)CD45(+) inducer cells can provide an IL-7R-, LTalpha1beta2/LTbetaR-, and NIK-independent tissue organogenesis pathway for secondary lymphoid tissue development.