Kinetic analysis of the interaction of actin-depolymerizing factor (ADF)/cofilin with G- and F-actins - Comparison of plant and human ADFs and effect of phosphorylation

The thermodynamics and kinetics of actin interaction with Arabidopsis thaliana actin-depolymerizing factor (ADF)(1), human ADF, and S6D mutant ADF(1) protein mimicking phosphorylated (inactive) ADF are examined comparatively. ADFs interact with ADP.G-actin in rapid equilibrium (k(+) = 155 mu M-1.s(-l) and k(-) = 16 s(-1) at 4 degrees C under physiological ionic conditions). The kinetics of interaction of plant and human ADFs with F-actin are slower and exhibit kinetic cooperativity, consistent with a scheme in which the initial binding of ADF to two adjacent subunits of the filament nucleates a structural change that propagates along the filament, allowing faster binding of ADF in a "zipper" mode. ADF binds in a non-cooperative faster process to gelsolin-capped filaments or to subtilisin-cleaved F-actin, which are structurally different from standard filaments (Orlova, A, Prochniewicz, E,, and Egelman, E, H. (1995) J. Mel. Biol. 245, 598-607), In contrast, the binding of phalloidin to F-actin cooperatively inhibits its interaction with ADF. The ADF-facilitated nucleation of ADP actin self-assembly indicates that ADF stabilizes lateral interactions in the filament, Plant and human ADFs cause only partial depolymerization of F-actin at pH 8, consistent with identical functions in enhancing F-actin dynamics, Phosphorylation does not affect ADF activity per se, but decreases its affinity for actin by 20-fold.