Presence Of Raf Research Articles

Membrane Composition and Raf[CRD]-Membrane Attachment Are Driving Forces for K-Ras4B Dimer Stability.

Ras proteins are membrane-anchored GTPases that regulate key cellular signaling networks. It has been recently shown that different anionic lipid types can affect the properties of Ras in terms of dimerization/clustering on the cell membrane. To understand the effects of anionic lipids on key spatiotemporal properties of dimeric K-Ras4B, we perform all-atom molecular dynamics simulations of the dimer K-Ras4B in the presence and absence of Raf[RBD/CRD] effectors on two model anionic lipid membranes: one containing 78% mol DOPC, 20% mol DOPS, and 2% mol PIP2 and another one with enhanced concentration of anionic lipids containing 50% mol DOPC, 40% mol DOPS, and 10% mol PIP2. Analysis of our results unveils the orientational space of dimeric K-Ras4B and shows that the stability of the dimer is enhanced on the membrane containing a high concentration of anionic lipids in the absence of Raf effectors. This enhanced stability is also observed in the presence of Raf[RBD/CRD] effectors although it is not influenced by the concentration of anionic lipids in the membrane, but rather on the ability of Raf[CRD] to anchor to the membrane. We generate dominant K-Ras4B conformations by Markov state modeling and yield the population of states according to the K-Ras4B orientation on the membrane. For the membrane containing anionic lipids, we observe correlations between the diffusion of K-Ras4B and PIP2 and anchoring of anionic lipids to the Raf[CRD] domain. We conclude that the presence of effectors with the Raf[CRD] domain anchoring on the membrane as well as the membrane composition both influence the conformational stability of the K-Ras4B dimer, enabling the preservation of crucial interface interactions.

Determining conformational dynamics of the allosteric site in HRas R97G

HRas is a small GTPase that acts as a molecular switch for the Raf/MEK/ERK pathway that leads to cell proliferation, survival, and differentiation. Oncogenic mutations in HRas renders it unable to hydrolyze GTP to GDP, resulting in constitutive activation of HRas and cell proliferation. Additionally, HRas contains an allosteric site that binds Ca2+ and a negatively charged ligand, promoting helix 3 to shift towards helix 4 and the ordering of switch II. This conformation is associated with intrinsic hydrolysis of HRas in the presence of Raf. Experiments have been performed in our lab to study the effects of point mutations in the allosteric site of HRas on intrinsic hydrolysis constants. When compared to wild‐type, HRas R97G demonstrated increased intrinsic hydrolysis when alone and a significant decrease when in the presence of Raf. Molecular dynamics simulations were utilized to further gain insight on how the dynamics of the allosteric site differed in HRas R97G compared to the wild‐type. These simulations demonstrated increased flexibility in the upper region of helix 3, with greater accessibility to the catalytic conformation for GTP hydrolysis.Support or Funding InformationNSF MCB 1244203

Transition State Mimic of Intrinsic Hydrolysis in Ras GTPase

Ras GTPases are involved in signal transduction pathways that control fundamental cellular processes such as proliferation, cycling, survival, and apoptosis. Ras functions via an on/off switch mechanism, where Ras is active in downstream signaling when bound to GTP and inactive upon GTP hydrolysis to GDP. Our lab discovered an allosteric site that is proposed to interact with the membrane, where binding in this allosteric site promotes ordering of the switch II region, favoring a conformation conducive to hydrolysis of GTP to GDP. When Ca2+ and acetate bind at this site in Ras crystals there is a disorder to order transition in switch II, positioning catalytic residues in place for hydrolysis. Interestingly, mutations of R97 to bulky aliphatic residues, such as L and M, mimic this effect, increasing hydrolysis rate constants relative to wild type. We had previously shown that in the presence of AlF3 RasR97L‐GDP binds to the Ras binding domain of Raf (Raf‐RBD), mimicking the γ‐phosphate of GTP. Here we present gel filtration studies of the R97M‐GDP mutant in the presence of Raf and AlF3 showing complex formation. The fact that Raf‐RBD binds to the R97L and R97M mutants, but not to wild type Ras, suggests that in these mutants the conformation associated with hydrolysis is more populated than in the wild type. AlFx has been used extensively to visualize the transition state of the GAP‐catalyzed GTP hydrolysis on Ras and its homologues. We are currently working on obtaining crystals of the H‐RasR97M‐Raf‐AlFx complex as a transition state model to elucidate the mechanism of intrinsic hydrolysis in Ras.Support or Funding InformationNSF MCB‐1517295

Allosteric Control of Conformational States in Ras Regulate the Intrinsic Hydrolysis Reaction in the Complex with Raf

Ras GTPases are important signaling proteins that act as a hub in many intracellular pathways, including cell proliferation and survival. Ras signaling is dependent on its architecture, which consists of a catalytic N-terminal G-domain, and a C-terminal post-translationally modified hyper-variable region (HVR). Ras acts as a molecular switch by cycling between an active GTP bound state, and an inactive GDP-bound state. In addition to the regulation of Ras by GAPs and GEFs, intrinsic hydrolysis appears to have a role in regulating the timing of the Raf-Ras complex through an allosteric switch mechanism that helps transition Ras from a disordered T-state, to a catalytically competent R-state. Ordering of switch II (R state) in the presence of Raf, is the result of ligand binding to R97 in the allosteric site. Using X-ray crystallography, size-exclusion and single-turnover hydrolysis experiments, we have explored the structural consequences of R97 mutation, and find that while they all preferentially stabilize the R- or T-state to some degree the R97L and R97G mutants show significant differences in the presence of Raf-RBD. R97L stabilizes the R state sufficiently to capture the transition state mimic AlFx in the presence of Raf-RBD in solution, while wild-type Ras under the same conditions does not bind AlFx. R97G on the other hand, appears to selectively stabilize the T-state when bound to Raf, as shown by a decrease in hydrolysis in the presence of Raf-RBD. Our results provide the first direct evidence that the allosteric site is associated with regulation of intrinsic hydrolysis. Our work is supported by the National Science Foundation (NSF MCB-1244203).

Allosteric Effects of the Oncogenic RasQ61L Mutant on Raf-RBD

The Ras/Raf/MEK/ERK signal transduction pathway is a major regulator of cell proliferation activated by Ras-guanosine triphosphate (GTP). The oncogenic mutant RasQ61L is not able to hydrolyze GTP in the presence of Raf and thus is a constitutive activator of this mitogenic pathway. The Ras/Raf interaction is essential for the activation of the Raf kinase domain through a currently unknown mechanism. We present the crystal structures of the Ras-GppNHp/Raf-RBD and RasQ61L-GppNHp/Raf-RBD complexes, which, in combination with MD simulations, reveal differences in allosteric interactions leading from the Ras/Raf interface to the Ras calcium-binding site and to the remote Raf-RBD loop L4. In the presence of Raf, the RasQ61L mutant has a rigid switch II relative to the wild-type and increased flexibility at the interface with switch I, which propagates across Raf-RBD. We show that in addition to local perturbations on Ras, RasQ61L has substantial long-range effects on the Ras allosteric lobe and on Raf-RBD.

Temperature dependence of the rigid amorphous fraction in poly(ethylene terephthalate)

The analysis of the cp melting curve of poly(ethylene terephthalate) (PET) after non-isothermal crystallization, according to a three-phase enthalpy-based procedure, has allowed to monitor the correct crystalline and rigid amorphous fractions evolution during fusion at low heating rate and to identify the temperature of complete mobilization of the rigid amorphous fraction, located in the proximity of 215°C. This limit temperature for the disappearance of the rigid amorphous fraction in PET corresponds to the transition between the triple and the double melting behaviour displayed by PET after isothermal crystallization. The multiple melting behaviour of PET is usually attributed to the fusion of original crystals recrystallized during the heating at conventional scanning rate, and to the presence of dual lamellar stacks. In the present study, PET was isothermally crystallized at various temperatures (Tc) ranging from 185°C to 225°C, and the double and triple melting profile that is observed at conventional DSC heating rates, after isothermal crystallization at Tc higher and lower than 215°C respectively, has been put in relation with the absence and the presence of RAF around the original crystal lamellae. All the experimental evidences here reported indicate that the critical temperature for the formation and the disappearance of the rigid amorphous fraction in the PET sample analyzed in the present study at conventional DSC scanning rates, is around 215°C.

Study of the effect of Ca(OAc)2 concentration on the state of the Allosteric Switch in Ras GTPase

Ras GTPase cycles between GDP inactive and GTP active forms and is of great importance in signal transduction that regulates cell proliferation, differentiation and survival. Although signaling is largely attenuated by the presence of GTPase activating proteins (GAPs), our group has recently discovered and allosteric mechanism that may promote hydrolysis of GTP in the presence of Raf and possibly other tight binding effectors, demonstrating subtle levels of regulation of the GTP‐bound protein. The allosteric switch is ON, with complete ordering of the active site, in crystals soaked in 100mM Ca(OAc)2, which binds to an allosteric site remote from the catalytic center. In the presence of CaCl2 the allosteric switch is OFF. Ca(OAc)2 binding is mediated by arginine 97 (R97) through salt bridge interactions with acetate. Here we show that R97A, which cannot bind acetate, is in the OFF state in the presence of 245 mM Ca(OAc)2 but switches to the ON state at 365 mM Ca(OAc)2. The R97F mutant is observed in the OFF state in the presence of 200mM Ca(OAc)2. It is clear that the bulk solvent composition has an important effect in the state of the allosteric switch in R97A. The soaking of crystals in 365 mM Ca(OAc)2 will test whether this is also the case for R97F and other allosteric site mutants.

Chapter Three - The Allosteric Switch and Conformational States in Ras GTPase Affected by Small Molecules

Ras is a hub protein in signal transduction pathways leading to the control of cell proliferation, migration, and survival and a major target for drug discovery due to the presence of its mutants in about 20% of human cancers. Yet, the discovery of small molecules that can directly interfere with its function has been elusive in spite of intense efforts. This is most likely due to its highly flexible nature and the lack of a well-ordered active site. This chapter contains a discussion of our current understanding of conformational states in Ras-GTP, with focus on a recently discovered allosteric switch mechanism that may promote intrinsic hydrolysis of GTP in the presence of Raf. We discuss the manner in which small molecules are known to affect the equilibrium of states in Ras-GTP and suggest novel strategies to go forward in the search for inhibitors of this master signaling protein.

Mechanism of intrinsic hydrolysis in Ras and the effect of Raf on oncogenic Ras mutants

The Ras GTPase and its effector Raf are key mediators of the Raf/MEK/ERK signal transduction pathway, which plays a central role in cell growth and malignant transformation. While it is well accepted that signaling through the Ras/Raf interaction is critical in the development of many cancers, most studies to date have focused on the properties of the individual components of this complex. Thus, the biochemical properties of Ras and its oncogenic mutants have been well characterized in vitro in the absence of Raf and numerous structures of wild type and oncogenic Ras mutants have been used to study the possible mechanisms through which Ras becomes defective in its ability to hydrolyze GTP. We show that in the context of the Ras/Raf‐RBD‐CRD complex the highly oncogenic mutant RasQ61L is unable to hydrolyze GTP and that the high transformation efficiency in RasQ61L is correlated with an anti‐catalytic conformation of the active site in this and other highly transforming mutants. We propose a new mechanism through which intrinsic hydrolysis occurs in Ras and explain the structural features that render the RasQ61L mutant a severely impaired enzyme in the presence of Raf. We also present crystal structures supporting a role for Gln61 in our proposed mechanism and show how the G12V mutant disrupts this structure while in the non‐transforming G12P mutant it is well tolerated.

Transformation Efficiency of RasQ61 Mutants Linked to Structural Features of the Switch Regions in the Presence of Raf

Transformation efficiencies of Ras mutants at residue 61 range over three orders of magnitude, but the in vitro GTPase activity decreases 10-fold for all mutants. We show that Raf impairs the GTPase activity of RasQ61L, suggesting that the Ras/Raf complex differentially modulates transformation. Our crystal structures show that, in transforming mutants, switch II takes part in a network of hydrophobic interactions burying the nucleotide and precatalytic water molecule. Our results suggest that Y32 and a water molecule bridging it to the gamma-phosphate in the wild-type structure play a role in GTP hydrolysis in lieu of the Arg finger in the absence of GAP. The bridging water molecule is absent in the transforming mutants, contributing to the burying of the nucleotide. We propose a mechanism for intrinsic hydrolysis in Raf-bound Ras and elucidate structural features in the Q61 mutants that correlate with their potency to transform cells.

Germline mutations of MEK in cardio-facio-cutaneous syndrome are sensitive to MEK and RAF inhibition: implications for therapeutic options

Cardio-facio-cutaneous (CFC) syndrome is a sporadic developmental disorder characterized by distinctive craniofacial features, heart defects, mental retardation and ectodermal abnormalities. We recently reported missense germline mutations in the genes MEK1 and MEK2 in patients with CFC. These mutations, including F53S and Y130C MEK1, and F57C MEK2, are the first naturally occurring mutations to be identified in these genes. This study reports data concerning the biochemical functions of the novel mutants, as well as the roles of these MEK genes in the MAPK signaling cascade. Our CFC MEK variants cannot induce ERK unless they are phosphorylated by RAF at two key serine residues in the regulatory loop. When we replaced the serine residues with alanines, ERK phosphorylation was significantly reduced in the presence of RAF. We did find that F57C MEK2 activation was less dependent on RAF signaling than the other mutants. This difference results in F57C MEK2 being resistant to the selective RAF inhibitor SB-590885. All three mutants are sensitive to the MEK inhibitor U0126. The majority of CFC cases result from mutations in B-RAF. A recent report indicates the possibility that cancer cells with activated B-RAF have enhanced, selective sensitivity to MEK inhibitors. Thus, regardless of mutations identified in an individual with CFC, MEK inhibition is a potential therapeutic approach for this population.

Functional Interactions of Raf and MEK with Jun-N-Terminal Kinase (JNK) Result in a Positive Feedback Loop on the Oncogenic Ras Signaling Pathway

In previous studies we have found that oncogenic (Val 12)-ras-p21 induces Xenopus laevis oocyte maturation that is selectively blocked by two ras-p21 peptides, 35-47, also called PNC-7, that blocks its interaction with raf, and 96-110, also called PNC-2, that blocks its interaction with jun-N-terminal kinase (JNK). Each peptide blocks activation of both JNK and MAP kinase (MAPK or ERK) suggesting interaction between the raf-MEK-ERK and JNK-jun pathways. We further found that dominant negative raf blocks JNK induction of oocyte maturation, again suggesting cross-talk between pathways. In this study, we have undertaken to determine where these points of cross-talk occur. First, we have immunoprecipitated injected Val 12-Ha-ras-p21 from oocytes and found that a complex forms between ras-p21 raf, MEK, MAPK, and JNK. Co-injection of either peptide, but not a control peptide, causes diminished binding of ras-p21, raf, and JNK. Thus, one site of interaction is cooperative binding of Val 12-ras-p21 to raf and JNK. Second, we have injected JNK, c-raf, and MEK into oocytes alone and in the presence of raf and MEK inhibitors and found that JNK activation is independent of the raf-MEK-MAPK pathway but that activated JNK activates raf, allowing for activation of ERK. Furthermore, we have found that constitutively activated MEK activates JNK. We have corroborated these findings in studies with isolated protein components from a human astrocyte (U-251) cell line; that is, JNK phosphorylates raf but not the reverse; MEK phosphorylates JNK but not the reverse. We further have found that JNK does not phosphorylate MAPK and that MAPK does not phosphorylate JNK. The stress-inducing agent, anisomycin, causes activation of JNK, raf, MEK, and ERK in this cell line; activation of JNK is not inhibitable by the MEK inhibitor, U0126, while activation of raf, MEK, and ERK are blocked by this agent. These results suggest that activated JNK can, in turn, activate not only jun but also raf that, in turn, activates MEK that can then cross-activate JNK in a positive feedback loop.

The insert region of RhoA is essential for Rho kinase activation and cellular transformation.

RhoA is involved in multiple cellular processes, including cytoskeletal organization, gene expression, and transformation. These processes are mediated by a variety of downstream effector proteins. However, which effectors are involved in cellular transformation and how these proteins are activated following interaction with Rho remains to be established. A unique feature that distinguishes the Rho family from other Ras-related GTPases is the insert region, which may confer Rho-specific signaling events. Here we report that deletion of the insert region does not result in impaired effector binding. Instead, this insert deletion mutant (RhoDeltaRas, in which the insert helix has been replaced with loop 8 of Ras) acted in a dominant inhibitory fashion to block RhoA-induced transformation. Since RhoDeltaRas failed to promote stress fiber formation, we examined the ability of this mutant to bind to and subsequently activate Rho kinase. Surprisingly, RhoDeltaRas-GTP coprecipitated with Rho kinase but failed to activate it in vivo. These data suggested that the insert domain is not required for Rho kinase binding but plays a role in its activation. The constitutively active catalytic domain of Rho kinase did not promote focus formation alone or in the presence of Raf(340D) but cooperated with RhoDeltaRas to induce cellular transformation. This suggests that Rho kinase needs to cooperate with additional Rho effectors to promote transformation. Further, the Rho kinase catalytic domain reversed the inhibitory effect of RhoDeltaRas on Rho-induced transformation, suggesting that one of the downstream targets of Rho-induced transformation abrogated by RhoDeltaRas is indeed Rho kinase. In conclusion, we have demonstrated that the insert region of RhoA is required for Rho kinase activation but not for binding and that this kinase activity is required to induce morphologic transformation of NIH 3T3 cells.