GTPase activity of Di-Ras proteins is stimulated by Rap1GAP proteins

Abstract

The Ras family is the largest and most diverse sub-group of Ras-like G proteins. This complexity is further increased by the high number of regulatory Guanine nucleotide Exchange Factors (GEFs) and GTPase activating proteins (GAPs) that target specific members of this subfamily. Di-Ras1 and Di-Ras2 are little characterized members of the Raslike sub-group with still unidentified regulatory and effector proteins. Here we determined the nucleotide binding properties of Di-Ras1/Di-Ras2. The above nanomolar affinity and the inability to react with members of the Cdc25 RasGEF family might suggest that activation does not require a GEF. We identified Rap1GAP1 and Rap1GAP2 as specific GTPase activating proteins of the Di-Ras family. Dual-specificity GAPs of the GAP1m family could not activate Di-Ras proteins, despite the presence of the required catalytic residue. Although DiRas proteins share GAPs with Rap G proteins, no common effectors could be identified in vitro.

Acknowledgements

We thank Dorothee Vogt, Patricia Stege and Carolin Koerner for help in cloning, protein purification and general lab assistance. We thank Andrea Scrima for Rheb and Tuberin constructs and Shehab Ismail for fruitful discussions. R.G. was supported by the Fonds der Chemischen Industrie and B.S. by an Alexander v. Humboldt fellowship.

Figures and Tables

Figure 1 Intrinsic nucleotide exchange and determination of nucleotide affinity. (A) Nucleotide exchange reaction of 1 µM Di-Ras proteins, prebound to mGDP or mGppNHp. The protein-bound nucleotide is reacted with excess unlabelled nucleotide. (B) Plot of pseudo-first-order rate constants for the association of 100 nM mGDP with Guanosin/GMP-bound Di-Ras2. (C) Pseudo-first-order rate constants for the association of 100 nM mGppNHp with Guanosin/GMP-bound Di-Ras2.

Figure 2 Ras- and Rap-GEFs are inactive on Di-Ras proteins. Nucleotide exchange activity of Di-Ras1 (intrinsic: red, GEF-catalysed: green), Di-Ras2 (intrinsic: magenta, GEF-catalysed: blue) and control G proteins (intrinsic: gray, GEF-catalysed: black). 1 µM of mGDP preloaded G protein were incubated with 2 µM of Sos (A), C3G (B), Epac1 (C) or Epac2 (D) and the reaction started by adding excess unlabelled nucleotide at time zero. Epac1 and Epac2 reactions were measured in the presence of 1 mM cAMP. The amount of mGDP loaded protein was set to 1.

Figure 3 Nucleotide hydrolysis of Di-Ras proteins is stimulated by Rap1GAPs. Different concentrations of GAPs were tested for activity on GTP-bound Di-Ras1 (left, red) and Di-Ras2 (right, blue). Only Rap1GAP1 and Rap1GAP2 stimulated GTPase activity of Di-Ras proteins. Control reactions of GAPs with their cognate G proteins are shown in gray. 50 µM G protein were incubated with varying GAP concentrations and the decrease in relative GTP concentration was followed by HPLC. Concentration of G protein•GTP was set to 1 at time zero for all experiments.

Figure 4 Low affinity interaction between Di-Ras1/Di-Ras2 and Ras-effectors. 1 µM mGppNHp-bound Di-Ras1 and Di-Ras2 were titrated with GST-Raf(RBD) (A) or GST-RalGDS(RA) (B). Complex formation was followed by determining fluorescence polarisation. GST-Raf(RBD) and GST-RalGDS(RA) showed very low affinity binding to Di-Ras1 or Di-Ras2. Only an approximate K_D of 120 µM could be calculated for the Raf+Di-Ras1 interaction. The K_Ds for (RalGDS+Di-Ras1), (Raf+Di-Ras2) and (RalGDS+Di-Ras2) are above 200 µM. mGppNHp-bound H-Ras and mGppNHp-bound Rap1 were used as positive controls for GST-Raf(RBD) and GST-RalGDS(RA), respectively.

Figure 5 Sequence conservation of Ras-family G proteins. (A) Sequence alignment of selected Ras-family G proteins showing 100% conservation (red), 80% conservation (orange) and 60% conservation (yellow). The non-conserved catalytic residue (Rap: T61) is shown in green, the dual-specificity GAP catalytic residue (Rap: Q63) is highlighted in blue. The catalytic residue for GEF reactions (Rap: E62) is marked in purple. Numbers are according to Di-Ras1 sequence. Residues that are labelled in (B) are further indicated with cyan (Rap(s)-Raf), green (Rap1-Rap1GAP) and magenta (Rap1-Epac) lines. (B) Surface representations of Rap1 in complex with regulatory proteins. Between Rap1 and Di-Ras1/2 conserved residues that interact with the binding partners are shown in red. Not-conserved, but interacting residues are depicted in dark gray. Conserved, not-interacting residues are shown in lightorange. Left (pdb: 3BRW): Rap1 in complex with Rap1GAP1 (not shown). Middle (pdb: 3CF6): Rap1 in complex with Epac2 (not shown). Right (pdb: 1GUA): Rap(s) (Rap E30D, K31E) in complex with Raf(RBD) (not shown).

Table 1 Nucleotide on- and off-rates

Table 2 Observed rate constants for nucleotide hydrolysis of Di-Ras proteins