Christoph Block

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

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.

Quantitative structure-activity analysis correlating Ras/Raf interaction in vitro to Raf activation in vivo

Binding of Ras to c-Raf-1 is a pivotal step of many mitogenic signalling pathways. Based on the recent crystal structure of the complex of Rap1 A with the Ras-binding domain of Raf, mutations were introduced in c-Raf-1 and their effects on Ras/Raf binding affinity in vitro and Ras/Raf regulated gene expression in vivo were analysed. Our data reveal an empirical semi-logarithmic correlation between dissociation constants and Raf-induced gene activity. The functional epitope that primarily determines binding affinity consists of residues Gin 66, Lys 84 and Arg 89 in Raf. This quantitative structure-activity investigation may provide a general approach to correlate structure-guided biochemical analysis with biological function of protein–protein interactions.

Sulindac sulfide inhibits Ras signaling.

The non-steroidal anti-inflammatory drug sulindac is used in cancer prevention and therapy, but the molecular aspects of its anti-tumor effect remain unresolved. In vivo the prodrug sulindac, is converted into the metabolite sulindac sulfide. We found that sulindac sulfide strongly inhibits Ras induced malignant transformation and Ras/Raf dependent transactivation. Sulindac sulfide decreases the Ras induced activation of its main effector, the c-Raf-1 kinase. In vitro sulindac sulfide directly binds to the Ras gene product p21ras in a non-covalent manner. Moreover, we can show that sulindac sulfide inhibits the interaction of p21ras with the p21ras binding domain of the Raf protein. In addition, sulindac sulfide can impair the nucleotide exchange on p21ras by CDC25 as well as the acceleration of the p21ras GTPase reaction by p120GAP. Due to its action at the most critical site in Ras signaling we propose sulindac sulfide as a lead compound in the search for novel anti-cancer drugs which directly inhibit Ras mediated cell proliferation and malignant transformation.

Mitogenic signaling of Ras is regulated by differential interaction with Raf isozymes

In the mitogenic signaling cascade interaction of Ras with Raf represents a critical step for the regulation of cell growth and differentiation. The major effector of Ras, the serine/threonine kinase Raf exists as three isoforms with different tissue distributions. We demonstrate that transient transfection of oncogenic Ha-Ras leads to a preferential activation of endogenous c-Raf-1 in HEK 293 cells as opposed to A-Raf. In vitro binding studies using purified Ras binding domains of Raf as well as in vivo bindings tests with full length molecules reveals significantly lower binding affinities of A-Raf to Ha-Ras as compared to other Raf isoforms. The Ras-binding interface of c-Raf differs from A-Raf by a conservative Arg to Lys exchange at residue 59 or 22 respectively. Mutational analysis reveals that this residue represents a point of isozyme discrimination: c-Raf-R59K binds Ha-Ras weaker than the wildtype, likewise A-Raf-K22R increases its affinity to Ha-Ras in vivo and in vitro. Differential binding affinities are reflected in downstream signaling. Immunecomplex kinase assays reveal that Ha-Ras mediated Raf activation is decreased for c-Raf-R59K and increased for A-Raf-K22R when compared to the respective wildtype forms. Thus our observations introduce a new level of isoform discrimination in Ras/Raf signaling as a functional consequence of a conservative amino acid exchange in the Ras binding domains.

The RafC1 Cysteine-Rich Domain Contains Multiple Distinct Regulatory Epitopes Which Control Ras-Dependent Raf Activation

ABSTRACT Activation of c-Raf-1 (referred to as Raf) by Ras is a pivotal step in mitogenic signaling. Raf activation is initiated by binding of Ras to the regulatory N terminus of Raf. While Ras binding to residues 51 to 131 is well understood, the role of the RafC1 cysteine-rich domain comprising residues 139 to 184 has remained elusive. To resolve the function of the RafC1 domain, we have performed an exhaustive surface scanning mutagenesis. In our study, we defined a high-resolution map of multiple distinct functional epitopes within RafC1 that are required for both negative control of the kinase and the positive function of the protein. Activating mutations in three different epitopes enhanced Ras-dependent Raf activation, while only some of these mutations markedly increased Raf basal activity. One contiguous inhibitory epitope consisting of S177, T182, and M183 clearly contributed to Ras-Raf binding energy and represents the putative Ras binding site of the RafC1 domain. The effects of all RafC1 mutations on Ras binding and Raf activation were independent of Ras lipid modification. The inhibitory mutation L160A is localized to a position analogous to the phorbol ester binding site in the protein kinase C C1 domain, suggesting a function in cofactor binding. Complete inhibition of Ras-dependent Raf activation was achieved by combining mutations K144A and L160A, which clearly demonstrates an absolute requirement for correct RafC1 function in Ras-dependent Raf activation.

Ras-binding domains: predicting function versus folding

Ras interacts with a number of effector molecules to achieve its prolific signalling. Based on iterative sequence profile and motif searches of databases a novel family of Ras‐binding domains was recently identified (Ponting and Benjamin, Trends Biochem. Sci. 21: 422–425, 1996). Among them the rat unconventional myosin and Rho‐GTPase‐activating protein myr 5 was predicted to contain a Ras‐binding domain at its N‐terminus. Here we report that direct binding experiments between the proposed Ras‐binding domain of myr 5 and Ras failed to demonstrate any interaction. Molecular modelling suggests that this domain in myr 5 adopts a similar folding topology as the Ras‐binding domain of Raf kinase. However, unlike the Ras‐binding domain of Raf kinase, the myr 5 domain lacks the positive surface charges necessary for binding the negatively charged Ras contact site. This result exemplifies the functional diversity of similar structures and suggests that the identified Ras‐binding motif does not reliably predict Ras‐binding domains.

Discrimination of amino acids mediating Ras binding from noninteracting residues affecting Raf activation by double mutant analysis

The contribution of residues outside the Ras binding domain of Raf (RafRBD) to Ras-Raf interaction and Ras-dependent Raf activation has remained unresolved. Here, we utilize a double mutant approach to identify complementary interacting amino acids that are involved in Ras-Raf interaction and activation. Biochemical analysis demonstrates that Raf-Arg59 and Raf-Arg67 from RafRBD are interacting residues complementary to Ras-Glu37 located in the Ras effector region. Raf-Arg59 and Raf-Arg67 also mediate interaction with Ras-Glu37 in Ras-dependent Raf activation. The characteristics observed here can be used as criteria for a role of residues from other regions of Raf in Ras-Raf interaction and activation. We developed a quantitative two-hybrid system as a tool to investigate the effect of point mutations on protein-protein interactions that elude biochemical analysis of bacterially expressed proteins. This assay shows that Raf-Ser257 in the RafCR2 domain does not contribute to Ras-Raf interaction and that the Raf-S257L mutation does not restore Raf binding to Ras-E37G. Yet, Raf-S257L displays high constitutive kinase activity and further activation by Ras-G12V/E37G is still impaired as compared with activation by Ras-G12V. This strongly suggests that the RafCR2 domain is an independent domain involved in the control of Raf activity and a common mechanism for constitutively activating mutants may be the interference with the inactive ground state of the kinase.

Oncogenic insertional mutations in the P-loop of Ras are overactive in MAP kinase signaling

Mutations of Ras with three extra amino acids inserted into the phosphate-binding (P) loop have been investigated both in vitro and in vivo. Such mutants have originally been detected as oncogenes both in the ras and the TC21 genes. Biochemical experiments reveal the molecular basis of their oncogenic potential: the mutants show a strongly attenuated binding affinity for nucleotides, most notably for GDP, leading to a preference for GTP binding. Furthermore, both the intrinsic as well as the GAP-stimulated GTP hydrolysis are drastically diminished. The binding interaction with GAP is reduced, whereas binding to the Ras-binding domain of the downstream effector c-Raf1 is not altered appreciably. Microinjection into PC12 cells shows the mutants to be as potent to induce neurite outgrowth as conventional oncogenic Ras mutants. Unexpectedly, their ability to stimulate the MAP kinase pathway as measured by a reporter gene assay in RK13 cells is much higher than that of the normal oncogenic mutant G12V. This characteristic was attributed to an increased stimulation of c-Raf1 kinase activity by the insertional Ras mutants.

Quantification of the Raf-C1 interaction with solid-supported bilayers.

By use of the quartz crystal microbalance technique, the interaction of the Raf–Ras binding domain (RafRBD) and the cysteine‐rich domain Raf‐C1 with lipids was quantified by using solid‐supported bilayers immobilized on gold electrodes deposited on 5 MHz quartz plates. Solid‐supported lipid bilayers were composed of an initial octanethiol monolayer chemisorbed on gold and a physisorbed phospholipid monolayer varying in its lipid composition as the outermost layer. The integrity of bilayer preparation was monitored by impedance spectroscopy. For binding experiments, a protein construct comprising the RafRBD and Raf‐C1 linked to the maltose binding protein and a His tag, termed MBP‐Raf‐C1, was used. Dissociation constants and rate constants of the association and dissociation were obtained for various 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC)/1,2‐dimyristoyl‐sn‐glycero‐3‐phosphoserine (DMPS) lipid mixtures. Independently of the phosphatidylserine (PS) content, the dissociation constants were in the order of 5×10−7 M, while the on‐rate constants were in the range of 2×103 (M s)−1 and the off‐rate constants in the range of 1×10−3 s−1. The maximum frequency shift increased significantly with increasing amounts of DMPS; this indicates that this negatively charged lipid is the primary binding site for MBP‐Raf‐C1. Exchange of DMPS for 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphoglycerol (DMPG) did not alter the thermodynamics and kinetics of protein binding, which implies that the protein interaction is mainly electrostatically driven. Scanning force microscopy (SFM) was employed to render protein adsorption visible and to confirm the assumption of a protein monolayer on the lipid layer. SFM images clearly revealed that the protein binds preferentially, but not solely, to negatively charged phosphatidylserine headgroups. We hypothesize that PS‐enriched domains are initial binding sites with high affinity for Raf‐C1, but that lateral interactions may account for protein domain growth.

In vivo quantitative assessment of Ras/Raf interaction using the two-hybrid system

GTP-binding proteins of the Ras subfamily act as molecular switches which regulate cellular proliferation and differentiation. They are switched “ON” by binding of GTP and “OFF” by the hydrolysis of GTP to GDP. Ras acts as an oncogene when mutated to a constitutively activated form which reflects its crucial role in the control of cell proliferation and differentiation. The interaction between the GTPase switch Ras and the downstream effector protein kinase c-Raf-1 represents a paradigm for the control of mitogenic signalling. Ras, when activated, binds to and activates its downstream effector, Raf, which in turn translates the GTP-dependent “ON” signal into protein phosphorylation. This signal is propagated within a setup of kinases (MEK, ERK) which is commonly referred to as the “MAP-kinase module”, finally leading to the induction of gene expression (reviewed in Block & Wittinghofer, 1995).