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 originally identified as a fusion to ALL-1 in acute myeloid leukemia. In the present work the interaction between Ras and AF6 is characterized and compared with other effectors. 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 Ras 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 31P NMR spectroscopy one of two major conformational states of Ras is stabilized by the interaction with AF6. Looking at the affinities of AF6 to a number of Ras mutants in the effector region, a specificity profile emerges distinct from that of other effector molecules. This finding may be useful in defining the biological function of AF6 by selectively switching off other pathways downstream of Ras using the appropriate effector mutant. Notably, among the Ras-related proteins AF6 binds most tightly to Rap1A which could imply a role of Rap1A in AF6 regulation.
Linnemann
T
T
Abteilung Strukturelle Biologie, Max-Planck-Institut für Molekulare Physiologie, Postfach 102664, 44026 Dortmund, Germany.
Geyer
M
M
Jaitner
B K
BK
Block
C
C
Kalbitzer
H R
HR
Wittinghofer
A
A
Herrmann
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D016547:Kinesins / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009218:Myosins / Q000378:metabolism*; D010759:Phosphorus Isotopes; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D018631:ras Proteins / Q000737:chemistry / Q000378:metabolism*
C095516:AFDN protein, human; D010759:Phosphorus Isotopes; D006160:Guanosine Triphosphate; D009218:Myosins; D016547:Kinesins; D018631:ras Proteins