Role of Magnesium in Nucleotide Exchange on the Small G Protein Rac Investigated Using Novel Fluorescent Guanine Nucleotide Analogues

Shutes, Adam; Phillips, Robert; Corrie, John E. T.; Webb, Martin R.

doi:10.1021/bi0119464

Cited by 18 publications

(21 citation statements)

References 27 publications

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“…), direct displacement of Mg 2ϩ or loss of its coordination (36) by EHT 1864, or potentially through an allosteric mechanism caused by EHT 1864 binding at another site on the small GTPase. Our preliminary observations 5 indicate that nucleotide release is more rapid when Rac is associated with the triphosphate nucleotide, suggesting the involvement of Mg 2ϩ as a key step in the release mechanism (36). Further biochemical analyses, such as the use of NMR to identify Rac1 residues whose coordinates are altered by EHT 1864 binding, crystallization of the inhibitor in complex with Rac1 for a full structural determination, and direct measurement of inhibitor affinity in the presence or absence of nucleotide or Mg 2ϩ via SPR (such as Biacore), will be required to completely elucidate this mechanism.…”

Section: Discussionmentioning

confidence: 78%

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Specificity and Mechanism of Action of EHT 1864, a Novel Small Molecule Inhibitor of Rac Family Small GTPases

Shutes

Onesto

Picard

et al. 2007

Journal of Biological Chemistry

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287

263

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There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes the uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However, to date, most efforts have focused on inhibitors that indirectly block Rho GTPase function, by targeting either enzymes involved in post-translational processing or downstream protein kinase effectors. We recently determined that the EHT 1864 small molecule can inhibit Rac function in vivo. In this study, we evaluated the biological and biochemical specificities and biochemical mechanism of action of EHT 1864. We determined that EHT 1864 specifically inhibited Rac1-dependent platelet-derived growth factor-induced lamellipodia formation. Furthermore, our biochemical analyses with recombinant Rac proteins found that EHT 1864 possesses high affinity binding to Rac1, as well as the related Rac1b, Rac2, and Rac3 isoforms, and this association promoted the loss of bound nucleotide, inhibiting both guanine nucleotide association and Tiam1 Rac guanine nucleotide exchange factor-stimulated exchange factor activity in vitro. EHT 1864 therefore places Rac in an inert and inactive state, preventing its engagement with downstream effectors. Finally, we evaluated the ability of EHT 1864 to block Rac-dependent growth transformation, and we determined that EHT 1864 potently blocked transformation caused by constitutively activated Rac1, as well as Rac-dependent transformation caused by Tiam1 or Ras. Taken together, our results suggest that EHT 1864 selectively inhibits Rac downstream signaling and transformation by a novel mechanism involving guanine nucleotide displacement.Rho family proteins (20 human members) comprise a major branch of the Ras superfamily of small GTPases (1, 2). Like Ras, Rho GTPases function as GDP/GTP-regulated binary on-off switches. In resting, quiescent cells, Rho GTPases exist predominately in their inactive, GDP-bound state. Upon growth factor stimulation, Rho-specific guanine nucleotide exchange factors (RhoGEFs) 4 are activated, and they stimulate the intrinsic guanine nucleotide exchange activity to promote formation of the active GTP-bound protein. Rho-GTP binds preferentially to downstream effector proteins. Rho-specific GTPaseactivating proteins then stimulate the intrinsic GTP hydrolysis activity of Rho GTPases, returning the protein to its inactive state and terminating effector interaction.RhoA, Rac1, and Cdc42 are the best studied and understood members of the Rho GTPase family (1, 2). Rho GTPases are regulators of a diverse variety of cellular processes that include regulation of cell proliferation, actin cytoskeleton reorganization, and gene expression (3). Like Ras and other members of the Ras superfamily (4, 5), the aberrant activity of Rho GTPases has been implicated in cancer and other human diseases (6 -10). In particular, the Rac subfamily has also been linked to c...

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Section: Discussionmentioning

confidence: 78%

“…Rac proteins were prepared similarly as described elsewhere (30,35,36). Briefly, BL21(DE3) cells expressing Rac1, Rac1b, Rac2, and Cdc42, respectively, were incubated at 37°C, before induction with 1 mM isopropyl 1-thio-␤-D-galactopyranoside for 4 h at 37°C (Rac3 required overnight induction at room temperature).…”

Section: Methodsmentioning

confidence: 99%

Specificity and Mechanism of Action of EHT 1864, a Novel Small Molecule Inhibitor of Rac Family Small GTPases

Shutes

Onesto

Picard

et al. 2007

Journal of Biological Chemistry

Self Cite

287

263

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“…In ATP-bound kinases, the DFG aspartate interacts with a catalytically essential magnesium ion (Mg 2ϩ ) 2.3 Å away, which in turn coordinates the ␤ and ␥ phosphates of ATP (5). Encouraged by observations on small G proteins (45,46) showing that Mg 2ϩ binding is weakened by Ϸ3 orders of magnitude after transfer of the ␥ phosphate, we speculate that in kinases, this Mg 2ϩ may likewise be released after phosphate transfer, thus reducing the positive charge in the immediate vicinity of the DFG aspartate. If this is the case, we predict that this change in the electrostatic environment of the DFG aspartate would lead to its protonation-either from the solvent or through proton transfer from the catalytic base Asp-363-causing the motif to flip to a DFG-out conformation and thus facilitating ADP release.…”

Section: Conservation Of the Dfg Motif And Its Role In Kinase Catalysismentioning

confidence: 99%

A conserved protonation-dependent switch controls drug binding in the Abl kinase

Shan

Seeliger

Eastwood

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

239

314

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In many protein kinases, a characteristic conformational change (the ''DFG flip'') connects catalytically active and inactive conformations. Many kinase inhibitors-including the cancer drug imatinib-selectively target a specific DFG conformation, but the function and mechanism of the flip remain unclear. Using long molecular dynamics simulations of the Abl kinase, we visualized the DFG flip in atomiclevel detail and formulated an energetic model predicting that protonation of the DFG aspartate controls the flip. Consistent with our model's predictions, we demonstrated experimentally that the kinetics of imatinib binding to Abl kinase have a pH dependence that disappears when the DFG aspartate is mutated. Our model suggests a possible explanation for the high degree of conservation of the DFG motif: that the flip, modulated by electrostatic changes inherent to the catalytic cycle, allows the kinase to access flexible conformations facilitating nucleotide binding and release.conformational change ͉ DFG motif ͉ imatinib ͉ molecular dynamics simulation ͉ pH dependence

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“…Furthermore, this model would suggest that the transition from "GTP-like" to "GDP-like" structure of Ras is associated with cleavage, resulting in weakened NF1 binding. Other evidence suggests that the conformation change may occur associated with P i release (45). So although the dissociation of NF1 before P i cannot be excluded, it seems less likely than the reverse order.…”

Section: Rate Constants For a Minimal Reaction Schemementioning

confidence: 99%

The Mechanism of Ras GTPase Activation by Neurofibromin

Phillips¹,

Hunter²,

Eccleston³

et al. 2003

Biochemistry

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Individual rate constants have been determined for each step of the Ras.GTP hydrolysis mechanism, activated by neurofibromin. Fluorescence intensity and anisotropy stopped-flow measurements used the fluorescent GTP analogue, mantGTP (2'(3')-O-(N-methylanthraniloyl)GTP), to determine rate constants for binding and release of neurofibromin. Quenched flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor, MDCC-PBP was used to measure phosphate release kinetics. Phosphate-water oxygen exchange, using (18)O-substituted GTP and inorganic phosphate (P(i)), was used to determine the extent of reversal of the hydrolysis step and of P(i) binding. The data show that neurofibromin and P(i) dissociate from the NF1.Ras.GDP.P(i) complex with identical kinetics, which are 3-fold slower than the preceding cleavage step. A model is presented in which the P(i) release is associated with the change of Ras from "GTP" to "GDP" conformation. In this model, the conformation change on P(i) release causes the large change in affinity of neurofibromin, which then dissociates rapidly.

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Role of Magnesium in Nucleotide Exchange on the Small G Protein Rac Investigated Using Novel Fluorescent Guanine Nucleotide Analogues

Cited by 18 publications

References 27 publications

Specificity and Mechanism of Action of EHT 1864, a Novel Small Molecule Inhibitor of Rac Family Small GTPases

Specificity and Mechanism of Action of EHT 1864, a Novel Small Molecule Inhibitor of Rac Family Small GTPases

A conserved protonation-dependent switch controls drug binding in the Abl kinase

The Mechanism of Ras GTPase Activation by Neurofibromin

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