Thermodynamic and Kinetic Characterization of the Interaction between the Ras Binding Domain of AF6 and Members of the Ras Subfamily

doi:10.1074/jbc.274.19.13556

Thermodynamic and Kinetic Characterization of the Interaction between the Ras Binding Domain of AF6 and Members of the Ras Subfamily*

From the Abteilung Strukturelle Biologie, Max-Planck-Institut für Molekulare Physiologie, Postfach 102664, 44026 Dortmund and the ^‡Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Postfach, 93040 Regensburg, Germany

Abstract

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.

Footnotes

↵* This work was supported by Deutsche Forschungsgemeinschaft Grant He 2679/1.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ To whom correspondence should be addressed. Tel.: 49 231 1206 351; Fax: 49 231 1206 230; E-mail:christian.herrmann{at}mpi-dortmund.mpg.de.
↵2 T. Linnemann and C. Herrmann, unpublished observations.
Abbreviations:

PI(3)kinase

phosphatidylinositol 3-kinase

GDI

guanine nucleotide dissociation inhibition

GppNHp

guanyl-5′-yl imidodiphosphate

mant

2′,3′-(N-methylanthraniloyl), a fluorophor attached at 2′- or 3′-position to the nucleotide

HPLC

high pressure liquid chromatography

RalGEF

Ral guanine nucleotide exchange factor

RBD

Ras binding domain

X-Gal

5-bromo-4-chloro-3-indolyl-β-galactopyranoside

aa

amino acids

GAP

GTPase-activating protein
- Received October 27, 1998.
- Revision received January 13, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.