Allosteric Modulation of Ras-GTP Is Linked to Signal Transduction through RAF Kinase

Abstract

Ras is a key signal transduction protein in the cell. Mutants of Gly 12 and Gln 61 impair GTPase activity and are found prominently in cancers. In wild type Ras-GTP, an allosteric switch promotes disorder to order transition in switch II, placing Gln 61 in the active site. We show that the "on" and "off" conformations of the allosteric switch can also be attained in RasG12V and RasQ61L. Although both mutants have similarly impaired active sites in the on state, RasQ61L stabilizes an anti-catalytic conformation of switch II in the off state of the allosteric switch when bound to Raf. This translates into more potent activation of the MAPK pathway involving Ras, Raf kinase, MEK, and ERK (Ras/Raf/MEK/ERK) in cells transfected with RasQ61L relative to RasG12V. This differential is not observed in the Raf-independent pathway involving Ras, phosphoinositide 3-kinase (PI3K), and Akt (Ras/PI3K/Akt). Using a combination of structural analysis, hydrolysis rates, and experiments in NIH-3T3 cells, we link the allosteric switch to the control of signaling in the Ras/Raf/MEK/ERK pathway, supporting a GTPase-activating protein-independent model for duration of the Ras-Raf complex.Ras GTPase is a central protein in signal transduction pathways in the cell that mediate the control of cell proliferation as well as many other functions (1, 2). It is isoprenylated at the C terminus through which it is anchored to the cell membrane (3). When bound to GTP, Ras propagates its signal by interacting with effector proteins such as Raf (4) and phosphoinositide 3-kinase (PI3K) (5) among others (6, 7). Once GTP is hydrolyzed to GDP on Ras, complexes with effectors are no longer favored, and signaling is turned off. The signal is thus directly connected to the level of Ras-GTP, which in turn is kept in check by the opposing actions of guanine nucleotide exchange factors that catalyze the loading of GTP (8), and of GTPase-activating proteins (GAPs) 2 that increase the intrinsically slow GTPase activity of Ras for timely depletion of Ras-GTP (9). We have recently discovered a mechanism through which the active site of Ras can be modulated by ligand binding at an allosteric site near the Ras-membrane interface, suggesting a GAP-independent mechanism through which hydrolysis rates could be increased (10). The binding of calcium and a negatively charged ligand, mimicked in our Ras-GppNHp crystal structure by an acetate molecule, promote a network of Hbonding interactions that connect the allosteric site to switch II, resulting in a disorder to order transition that positions key residues for catalysis (10). Thus, an "on" state of the allosteric switch implies increased hydrolysis rates associated with signaling being turned off. When the allosteric switch is "off," GTP hydrolysis is impaired and signaling remains on.The active site residues are situated primarily in the socalled switch I, switch II, and the phosphate-binding loop (Ploop) composed of residues 30 -40, 60 -76, and 10 -17, respectively (11). In particular, mutants of residues Gly 1...

Direct Attack on RAS: Intramolecular Communication and Mutation-Specific Effects

“…The G12D and Q61 mutants directly impair the chemistry of GTP hydrolysis by either placing a negative charge right over the nucleotide or removing an important catalytic residue. Furthermore, Q61L is also unique in that it has a strong tendency to stabilize the T state in the anticatalytic conformation when bound to RAF, abolishing even the slow hydrolysis rate observed in the uncomplexed protein (14,15,73). In the absence of binding partners, both switch I and switch II are disordered in solution (11,61), and they become stabilized by interactions with other proteins (15,30,76,77).…”

“…5A). It has been shown that both G12V and Q61L can attain the R and T states of the allosteric switch under circumstances where switch I is in state 2 (14), as it is when bound to RAF, and it is likely that other oncogenic mutants can as well.…”

“…This is a difficult problem to overcome, as the mutations themselves severely perturb the active site and impair the ability of GAPs to enhance GTP hydrolysis even upon binding RAS (50,71). If it is indeed the case that the duration of the RAS-RAF interaction is regulated by intrinsic hydrolysis activated by membrane interaction and calcium binding at the allosteric site (13,14,16,56), there an exciting window of opportunity exists for targeting signaling through RAS-RAF enhanced by some oncogenic mutants. This is of interest because the RAS-RAF-MEK-ERK pathway is a potent mitogenic pathway and an aggressive driver of several types of cancers (72).…”

“…In structures available for the G12 and Q61 mutants, those in which switch I is in state 2 and the allosteric switch is in the R state (14,34,35,67,73,74), with the exception of G12D, the bridging water molecule seen in the wild-type protein is not present and Y32 Protein residues that interact with inhibitors between switch II and helix 3 (a3) are colored blue, and those that interact on the outer side of switch II and the b-sheet are colored orange. B, binding sites for inhibitors that bind to RAS-GTP.…”

“…Switch II contains the catalytic residue Q61 and is normally highly disordered (11), resulting in a very slow hydrolysis rate in the absence of GAPs (12). We have shown an association between allosteric modulation and intrinsic hydrolysis in the presence of RAF that would be expected to take place independent of GAPs (13)(14)(15)(16). In our current model, the GTP hydrolysis rate is expected to be increased in the RAS/RAF complex by binding of Ca 2þ at a remote allosteric site, resulting in a conformational shift from a catalytically impaired T state (disordered active site) to the competent R state in which the catalytic residue Q61 is ordered in the active site (Fig.…”