Thermodynamic and kinetic characterization of the interaction between the Ras binding domain of AF6 and members of the Ras subfamily

Cellular signaling downstream of Ras is highly diversified and may involve many different effector molecules. A potential candidate is AF6 which was o riginally identified as a fusion to ALL-1 in acute myeloid leukemia. In the present work the interaction between Ras and AF6 is characterized and comp ared with other effecters. The binding characteristics are quite similar to Raf and RalGEF, i.e. nucleotide dissociation as well as GTPase-activating protein activity are inhibited, whereas the intrinsic GTPase activity of Ra s is unperturbed by AF6 binding. Particularly, the dynamics of interaction are similar to Raf and RalGEF with a lifetime of the Ras.AF6 complex in the millisecond range. As probed by P-31 NMR spectroscopy one of two major con formational states of Ras is stabilized by the interaction with AF6, Lookin g at the affinities of AF6 to a number of Pas mutants in the effector regio n, a specificity profile emerges distinct from that of other effector molec ules. This finding may be useful in defining the biological function of AF6 by selectively switching off other pathways downstream of Ras using the ap propriate effector mutant. Notably, among the Ras-related proteins AF6 bind s most tightly to Rap1A which could imply a role of Rap1A in AF6 regulation .