Biomedical subjects

Sharon L Campbell

Publications and source records attributed to Sharon L Campbell.

At least 19 recordsLinked to original sources

PubMed2006

Redox regulation of RhoA.

We have previously shown that redox agents including superoxide anion radical and nitrogen dioxide can react with GXXXXGK(S/T)C motif-containing GTPases (i.e., Rac1, Cdc42, and RhoA) to stimulate guanine nucleotide release. We now show that the reaction of RhoA with redox agents leads to different functional consequences from that of Rac1 and Cdc42 due to the presence of an additional cysteine (GXXXCGK(S/T)C) in the RhoA redox-active motif. While reaction of redox agents with RhoA stimulates guanine nucleotide dissociation, RhoA is subsequently inactivated through formation of an intramolecular disulfide that prevents guanine nucleotide binding thereby causing RhoA inactivation. Thus, redox agents may function to downregulate RhoA activity under conditions that stimulate Rac1 and Cdc42 activity. The opposing functions of these GTPases may be due in part to their differential redox regulation. In addition, the results presented herein suggest that the platinated-chemotherapeutic agent, cisplatin, which is known for targeting nucleic acids, reacts with RhoA to produce a RhoA thiol-cisplatin-thiol adduct, leading to inactivation of RhoA. Similarly, certain arsenic complexes (i.e., arsenate and arsenic trioxide) may inactivate RhoA by bridging the cysteine residues in the GXXXCGK(S/T)C motif. Thus, in addition to redox agents, platinated-chemotherapeutic agents and arsenic complexes may modulate the activity of GTPases containing the GXXXCGK(S/T)C motif (i.e., RhoA and RhoB).

Amino Acid Sequence↗

PubMed2006

Ras regulation by reactive oxygen and nitrogen species.

Ras GTPases cycle between inactive GDP-bound and active GTP-bound states to modulate a diverse array of processes involved in cellular growth control. We have previously shown that both NO/O(2) (via nitrogen dioxide, (*)NO(2)) and superoxide radical anion (O(2)(*)(-)) promote Ras guanine nucleotide dissociation. We now show that hydrogen peroxide in the presence of transition metals (i.e., H(2)O(2)/transition metals) and peroxynitrite also trigger radical-based Ras guanine nucleotide dissociation. The primary redox-active reaction species derived from H(2)O(2)/transition metals and peroxynitrite is O(2)(*)(-) and (*)NO(2), respectively. A small fraction of hydroxyl radical (OH(*)) is also present in both. We also show that both carbonate radical (CO(3)(*)(-)) and (*)NO(2), derived from the mixture of peroxynitrite and bicarbonate, facilitate Ras guanine nucleotide dissociation. We further demonstrate that NO/O(2) and O(2)(*)(-) promote Ras GDP exchange with GTP in the presence of a radical-quenching agent, ascorbate, or NO, and generation of Ras-GTP promotes high-affinity binding of the Ras-binding domain of Raf-1, a downstream effector of Ras. S-Nitrosylated Ras (Ras-SNO) can be formed when NO serves as a radical-quenching agent, and hydroxyl radical but not (*)NO(2) or O(2)(*)(-) can further react with Ras-SNO to modulate Ras activity in vitro. However, given the lack of redox specificity associated with the high redox potential of OH(*), it is unclear whether this reaction occurs under physiological conditions.

Copper↗

PubMed2006

Topological determinants of protein domain swapping.

Protein domain swapping has been repeatedly observed in a variety of proteins and is believed to result from destabilization due to mutations or changes in environment. Based on results from our studies and others, we propose that structures of the domain-swapped proteins are mainly determined by their native topologies. We performed molecular dynamics simulations of seven different proteins, known to undergo domain swapping experimentally, under mildly denaturing conditions and found in all cases that the domain-swapped structures can be recapitulated by using protein topology in a simple protein model. Our studies further indicated that, in many cases, domain swapping occurs at positions around which the protein tends to unfold prior to complete unfolding. This, in turn, enabled prediction of protein structural elements that are responsible for domain swapping. In particular, two distinct domain-swapped dimer conformations of the focal adhesion targeting domain of focal adhesion kinase were predicted computationally and were supported experimentally by data obtained from NMR analyses.

Animals↗

PubMed2005

Mechanism of redox-mediated guanine nucleotide exchange on redox-active Rho GTPases.

Rho GTPases regulate multiple cellular processes including actin cytoskeletal rearrangements, transcriptional regulation, and oxidant production. The studies described herein demonstrate that small molecule redox agents, in addition to protein regulatory factors, can regulate the activity of redox-active Rho GTPases. A novel (GXXXXGK(S/T)C) motif, conserved in a number of Rho GTPases, appears critical for redox-mediated guanine nucleotide dissociation in vitro. A detailed molecular mechanism for redox regulation of GXXXXGK(S/T)C motif-containing Rho GTPases is proposed.

Amino Acid Motifs↗

PubMed2005

Recognition and activation of Rho GTPases by Vav1 and Vav2 guanine nucleotide exchange factors.

Vav proteins are Rho GTPase-specific guanine nucleotide exchange factors (GEFs) that are distinguished by the tandem arrangement of Dbl homology (DH), Pleckstrin homology (PH), and cysteine rich domains (CRD). Whereas the tandem DH-PH arrangement is conserved among Rho GEFs, the presence of the CRD is unique to Vav family members and is required for efficient nucleotide exchange. We provide evidence that Vav2-mediated nucleotide exchange of Rho GTPases follows the Theorell-Chance mechanism in which the Vav2.Rho GTPase complex is the major species during the exchange process and the Vav2.GDP-Mg(2+).Rho GTPase ternary complex is present only transiently. The GTPase specificity for the DH-PH-CRD Vav2 in vitro follows this order: Rac1 > Cdc42 > RhoA. Results obtained from fluorescence anisotropy and NMR chemical shift mapping experiments indicate that the isolated Vav1 CRD is capable of directly associating with Rac1, and residues K116 and S83 that are in the proximity of the P-loop and the guanine base either are part of this binding interface or undergo a conformational change in response to CRD binding. The NMR studies are supported by kinetic measurements on Rac1 mutants S83A, K116A, and K116Q and Vav2 CRD mutant K533A in that these mutants affect both the initial binding event of Vav2 with Rac1 (k(on)) and the rate-limiting dissociation of Vav2 from the Vav2.Rac1 binary complex (thereby influencing the enzyme turnover number, k(cat)). The results suggest that the CRD domain in Vav proteins plays an active role, affecting both the k(on) and the k(cat) for Vav-mediated nucleotide exchange on Rho GTPases.

Amino Acid Sequence↗

PubMed2005

Superoxide anion radical modulates the activity of Ras and Ras-related GTPases by a radical-based mechanism similar to that of nitric oxide.

Ras GTPases cycle between inactive GDP-bound and active GTP-bound states to modulate a diverse array of processes involved in cellular growth control. The activity of Ras is up-regulated by cellular agents, including both protein (guanine nucleotide exchange factors) and redox-active agents (nitric oxide (NO) and superoxide anion radical (O2*). We have recently elucidated the mechanism by which NO promotes guanine nucleotide dissociation of redox-active NKCD motif-containing Ras and Ras-related GTPases. In this study, we show that guanine nucleotide dissociation is enhanced upon exposure of the redox-active GTPases, Ras and Rap1A, to O2* and provide evidence for the efficient guanine nucleotide reassociation in the presence of the radical quenching agent ascorbate to complete guanine nucleotide exchange. In vivo, guanine nucleotide reassociation is necessary to populate Ras in its biologically active GTP-bound form after the dissociation of GDP. We further show that treatment of the redox-active GTPases with O2* releases GDP in form of an unstable the oxygenated GDP adduct, putatively assigned as 5-oxo-GDP. 5-Oxo-GDP was not produced from either the C118S or the F28L Ras variants upon the treatment of O2*, supporting the involvement of residues Cys118 and Phe28 in O2*-mediated Ras guanine nucleotide dissociation. These results indicate that the mechanism of O2*-mediated Ras guanine nucleotide dissociation is similar to that of NO/O2-mediated Ras guanine nucleotide dissociation.

Amino Acid Motifs↗

PubMed2005

Novel C-Raf phosphorylation sites: serine 296 and 301 participate in Raf regulation.

The C-Raf kinase is regulated by numerous phosphorylation steps. To quantify the most prominent phosphorylation sites of C-Raf, we performed mass spectrometry analysis of wild-type C-Raf and the constitutively active C-Raf mutant C-Raf-Y340D/Y341D. We confirmed phosphorylation of most of the sites reported in the literature with the exception that we did not detect phosphorylation of threonine 268/269 (autophosphorylation sites) and threonine 491/serine 494 (kinase activation loop). Importantly, we detected novel phosphorylation sites at the positions of serine 296 and 301. The degree of phosphorylation in these positions depends on the level of activation of C-Raf. Furthermore, we show here, using point mutant forms of C-Raf kinases with serine to alanine and serine to aspartic acid substitution, that serines 296 and 301 contribute to negative regulation of C-Raf.

Amino Acid Sequence↗

PubMed2005

Mechanism of free radical nitric oxide-mediated Ras guanine nucleotide dissociation.

Ras proteins cycle between GDP-bound and GTP-bound states to modulate a diverse array of cellular growth processes. In this study, we have elucidated a mechanism by which nitric oxide, in the presence of oxygen (NO/O2), regulates Ras activity. We show that treatment of Ras with NO/O2 causes conversion of Ras-bound GDP into a free 463.3 Da nucleotide-nitration product. Mass and UV/visible spectroscopic analyses suggest that this nitration product is 5-guanidino-4-nitroimidazole diphosphate (NIm-DP), a degradation product of 5-nitro-GDP. These results indicate that NO/O2 mediates Ras guanine nucleotide exchange (GNE) by conversion of Ras-bound GDP into an unstable 5-nitro-GDP. 5-Nitro-GDP can be produced by radical-based reaction of the GDP guanine base with nitrogen dioxide (*NO2). We also provide evidence that the Ras Phe28 side-chain plays a key role in the formation of a NO/O2-induced Ras 5-nitro-GDP product. We previously proposed a mechanism of NO/O2-mediated Ras GNE, in which *NO2, formed by the reaction of NO with O2, generates a Ras Cys118 thiyl radical (Ras-S118) intermediate. In the present study, we provide evidence for a radical-based mechanism of NO/O2-mediated Ras GNE. According to this mechanism, reaction of NO with O2 produces *NO2. *NO2 then reacts with Ras to produce Ras-S118, which withdraws an electron from the Ras-bound guanine nucleotide base to produce a guanine nucleotide diphosphate cation radical (G(+)-DP) via the Phe28 side-chain. G(+)-DP is subsequently converted to a neutral radical, and can react with another *NO2 to produce 5-nitro-GDP. This radical-based reaction process disrupts key binding interactions between Ras and the guanine base, resulting in release of GDP from Ras and its conversion to free 5-nitro-GDP. This mechanism is likely to be common to other NKCD motif-containing Ras superfamily GTPases, as NO/O2 also facilitates GNE on the redox-active Rap1A and Rab3A GTPases.

Binding Sites↗

PubMed2005

Recognition and processing of cisplatin- and oxaliplatin-DNA adducts.

The cytotoxicity of platinum compounds is thought to be determined primarily by their DNA adducts. Cisplatin and oxaliplatin are structurally distinct, but form the same types of adducts at the same sites on DNA. However, the DNA adducts are differentially recognized by a number of cellular proteins. For example, mismatch repair proteins and some damage-recognition proteins bind to cisplatin-GG adducts with higher affinity than to oxaliplatin-GG adducts, and this differential recognition of cisplatin- and oxaliplatin-GG adducts is thought to contribute to the differences in cytotoxicity and tumor range of cisplatin and oxaliplatin. A detailed kinetic analysis of the insertion and extension steps of dNTP incorporation in the vicinity of the adduct shows that both DNA polymerase beta (pol beta) and DNA polymerase eta (pol eta) catalyze translesion synthesis past oxaliplatin-GG adducts with greater efficiency than past cisplatin-GG adducts. In the case of pol eta, the efficiency and fidelity of translesion synthesis in vitro is very similar to that previously observed with cyclobutane TT dimers, suggesting that pol eta is likely to be involved in error-free bypass of Pt adducts in vivo. This has been confirmed for cisplatin by comparing the cisplatin-induced mutation frequency in human fibroblast cell lines with and without pol eta. Thus, the greater efficiency of bypass of oxaliplatin-GG adducts by pol eta may explain the lower mutagenicity of oxaliplatin compared to cisplatin. The ability of these cellular proteins to discriminate between cisplatin and oxaliplatin adducts suggest that there exist significant conformational differences between the adducts, yet the crystal structures of the cisplatin- and oxaliplatin-GG adducts were very similar. We have recently solved the solution structure of the oxaliplatin-GG adduct and have shown that it is significantly different from the previously published solution structures of the cisplatin-GG adducts. Furthermore, the observed differences in conformation provide a logical explanation for the differential recognition of cisplatin and oxaliplatin adducts by mismatch repair and damage-recognition proteins.

Base Pair Mismatch↗

PubMed2004

NMR characterization of full-length farnesylated and non-farnesylated H-Ras and its implications for Raf activation.

The C terminus, also known as the hypervariable region (residues 166-189), of H-, N-, and K-Ras proteins has sequence determinants necessary for full activation of downstream effectors such as Raf kinase and PI-3 kinase as well as for the correct targeting of Ras proteins to lipid rafts and non-raft membranes. There is considerable interest in understanding how residues in the extreme C terminus of the different Ras proteins and farnesylation of the CaaX box cysteine affect Ras membrane localization and allosteric activation of Raf kinase. To provide insights into the structural and dynamic changes that occur in Ras upon farnesylation, we have used NMR spectroscopy to compare the properties of truncated H-Ras (1-166), to non-processed full-length H-Ras (residues 1-185) and full-length (1-189) farnesylated H-Ras. We report that the C-terminal helix alpha-5 extends to residue N172, and the remaining 17 amino acid residues in the C terminus are conformationally averaged in solution. Removal of either 23 or 18 amino acid residues from the C terminus of full length H-Ras generates truncated H-Ras (1-166) and H-Ras (1-171) proteins, respectively, that have been structurally characterized and are biochemical active. Here we report that C-terminal truncation of H-Ras results in minor structural and dynamic perturbations that are propagated throughout the H-Ras protein including increased flexibility of the central beta-sheet and the C-terminal helix alpha-5. Ordering of residues in loop-2, which is involved in Raf CRD binding is also observed. Farnesylation of full-length H-Ras at C186 does not result in detectable conformational changes in H-Ras. Chemical shift mapping studies of farnesylated and non-farnesylated forms of H-Ras with the Raf-CRD show that the farnesyl moiety, the extreme H-Ras C terminus and residues 23-30, contribute to H-Ras:Raf-CRD interactions, thereby increasing the affinity of H-Ras for the Raf-CRD.

Cell Membrane↗

PubMed2004

NMR solution structure of an oxaliplatin 1,2-d(GG) intrastrand cross-link in a DNA dodecamer duplex.

We have determined, at high resolution, the NMR solution structure of an oxaliplatin-GG DNA dodecamer in the AGGC sequence context by 2D NMR studies. Homonuclear assignment strategies resulted in unambiguous assignment of 203 out of 249 protons, which corresponds to assignment of approximately 81% of the protons. Assignments of H5' and H5" protons were tentative due to resonance overlap. The structure of the oxaliplatin duplex was calculated using the program CNS with a simulated annealing protocol. A total of 510 experimental restraints were employed in the structure calculation. Of 20 calculated structures, the 15 with the lowest energy were accepted as a family. The RMSD of the 15 lowest energy structures was 0.68 A, indicating good structural convergence. The theoretical NOESY spectrum obtained by back-calculation from the final average structure showed excellent agreement with the experimental data, indicating that the final structure was in good agreement with the experimental NMR data. Significant conformational differences were observed between the oxaliplatin-GG 12-mer DNA we studied and all previous solution structures of cisplatin-GG DNA duplexes. For example, the oxaliplatin-GG adduct shows much less distortion at the AG base-pair step than the cisplatin-GG adducts. In addition, the oxaliplatin-GG structure also has a narrow minor groove and an overall axis bend of about 31 degrees, both of which are very different from the recent NMR structures for the cisplatin-GG adducts. These structural differences may explain some of the biological differences between oxaliplatin- and cisplatin-GG adducts.

Antineoplastic Agents↗

PubMed2004

pH-dependent perturbation of Ras-guanine nucleotide interactions and Ras guanine nucleotide exchange.

p21Ras (Ras) proteins cycle between active GTP-bound and inactive GDP-bound states to mediate signal transduction pathways that promote cell growth, differentiation, and apoptosis. To better understand how cellular regulatory factors, such as guanine nucleotide exchange factors (GEFs) and nitric oxide (NO), modulate Ras-guanine nucleotide binding interactions, we have conducted NMR and kinetic studies to investigate the pH dependence of Ras-GDP interactions and Ras-guanine nucleotide exchange (GNE). pH-sensitive amide protons were identified and found to be associated with residues in the switch I (Phe28-Asp30) and switch II (Asp57 and Thr58) regions of Ras. Furthermore, most of the residues that interact with Mg2+ exhibit pH-sensitive amide proton chemical shifts which appear to be coupled to pH-dependent Ras Mg2+ binding and guanine nucleotide binding affinity. These results suggest that perturbation of Mg2+ interactions within the Ras-guanine nucleotide complex is critical for pH-dependent dissociation of guanine nucleotide ligands from Ras. Notably, these same regions undergo conformational changes upon association with the Ras GEF, SOS. In addition, although we have recently shown that addition of NO to Ras in the presence of oxygen produces a Ras thiyl radical intermediate that promotes Ras GNE, we have also postulated that another byproduct of this reaction, a H+, may contribute to NO-mediated GNE. However, the results presented herein suggest that the H+ byproduct of the reaction is unlikely to be involved in the NO-mediated Ras GNE.

Amino Acid Sequence↗

PubMed2004

Requirement for C-terminal sequences in regulation of Ect2 guanine nucleotide exchange specificity and transformation.

Ect2 was identified originally as a transforming protein and a member of the Dbl family of Rho guanine nucleotide exchange factors (GEFs). Like all Dbl family proteins, Ect2 contains a tandem Dbl homology (DH) and pleckstrin homology (PH) domain structure. Previous studies demonstrated that N-terminal deletion of sequences upstream of the DH domain created a constitutively activated, transforming variant of Ect2 (designated DeltaN-Ect2 DH/PH/C), indicating that the N terminus served as a negative regulator of DH domain function in vivo. The role of sequences C-terminal to the DH domain has not been established. Therefore, we assessed the consequences of mutation of C-terminal sequences on Ect2-transforming activity. Surprisingly, in contrast to observations with other Dbl family proteins, we found that mutation of the invariant tryptophan residue in the PH domain did not impair DeltaN-Ect2 DH/PH/C transforming activity. Furthermore, although the sequences C-terminal to the PH domain lack any known functional domains or motifs, deletion of these sequences (DeltaN-Ect2 DH/PH) resulted in a dramatic reduction in transforming activity. Whereas DeltaN-Ect2 caused formation of lamellipodia, DeltaN-Ect2 DH/PH enhanced actin stress fiber formation, suggesting that C-terminal sequences influenced Ect2 Rho GTPase specificity. Consistent with this possibility, we determined that DeltaN-Ect2 DH/PH activated RhoA, but not Rac1 or Cdc42, whereas DeltaN-Ect2 DH/PH/C activated all three Rho GTPases in vivo. Taken together, these observations suggest that regions of Ect2 C-terminal to the DH domain alter the profile of Rho GTPases activated in vivo and consequently may contribute to the enhanced transforming activity of DeltaN-Ect2 DH/PH/C.

Amino Acid Sequence↗

PubMed2004

Mechanism of p21Ras S-nitrosylation and kinetics of nitric oxide-mediated guanine nucleotide exchange.

Nitric oxide (NO), a highly reactive redox molecule, can react with protein thiols and protein metal centers to regulate a multitude of physiological processes. NO has been shown to promote guanine nucleotide exchange on the critical cellular signaling protein p21Ras (Ras) by S-nitrosylation of a redox-active thiol group (Cys(118)). This increases cellular Ras-GTP levels in vivo, leading to activation of downstream signaling pathways. Yet the process by which this occurs is not clear. Although several feasible mechanisms for protein S-nitrosylation with NO and NO donating have been proposed, results obtained from our studies suggest that Ras can be S-nitrosylated by direct reaction of Cys(118) with nitrogen dioxide (*NO(2)), a reaction product of NO with O(2), via a Ras thiyl-radical intermediate (Ras-S*). Results from our studies also indicate that Ras Cys(118) can be S-nitrosylated by direct reaction of Cys(118) with a glutathionyl radical (GS*), a reaction product derived from homolytic cleavage of S-nitrosoglutathione (GSNO). Moreover, we present evidence that reaction of GS* with Ras generates a Ras-S* intermediate during GSNO-mediated Ras S-nitrosylation. The Ras-S(*) radical intermediate formed from reaction of the Ras thiol with either *NO(2) or GS*, in turn, reacts with NO to complete Ras S-nitrosylation. NO and GSNO modulate Ras activity by promoting guanine nucleotide dissociation from Ras. Our results suggest that formation of the Ras radical intermediate, Ras-S*, may perturb interactions between Ras and its guanine nucleotide substrate, resulting in enhancement of guanine nucleotide dissociation from Ras.

Guanine Nucleotides↗

PubMed2004

Protein interactions with platinum-DNA adducts: from structure to function.

Because of the efficacy of cisplatin and carboplatin in a wide variety of chemotherapeutic regimens, hundreds of platinum(II) and platinum(IV) complexes have been synthesized and evaluated as anticancer agents over the past 30 years. Of the many third generation platinum compounds evaluated to date, only oxaliplatin has been approved for clinical usage in the United States. Thus, it is important to understand the mechanistic basis for the differences in efficacy, mutagenicity and tumor range between cisplatin and oxaliplatin. Cisplatin and oxaliplain form the same types of adducts at the same sites on DNA. The most abundant adduct for both compounds is the Pt-GG intrastrand diadduct. Cisplatin-GG adducts are preferentially recognized by mismatch repair proteins and some damage-recognition proteins, and this differential recognition of cisplatin- and oxaliplatin-GG adducts is thought to contribute to the differences in cytotoxicity and tumor range of cisplatin and oxaliplatin. A detailed kinetic analysis of the insertion and extension steps of dNTP incorporation in the vicinity of the adduct shows that both pol beta and pol eta catalyze translesion synthesis past oxaliplatin-GG adducts with greater efficiency than past cisplatin-GG adducts. In the case of pol eta, the efficiency and fidelity of translesion synthesis in vitro is very similar to that previously observed with cyclobutane TT dimers, suggesting that pol eta is likely to be involved in error-free bypass of Pt adducts in vivo. This has been confirmed for cisplatin by comparing the cisplatin-induced mutation frequency in human fibroblast cell lines with and without pol eta. Thus, the greater efficiency of bypass of oxaliplatin-GG adducts by pol eta is likely to explain the lower mutagenicity of oxaliplatin compared to cisplatin. The ability of these cellular proteins to discriminate between cisplatin and oxaliplatin adducts suggest that there exist significant conformational differences between the adducts, yet the crystal structures of the cisplatin- and oxaliplatin-GG adducts were very similar. We have recently solved the solution structure of the oxaliplatin-GG adduct and have shown that it is significantly different from the previously published solution structures of the cisplatin-GG adducts. Furthermore, the observed differences in conformation provide a logical explanation for the differential recognition of cisplatin and oxaliplatin adducts by mismatch repair and damage-recognition proteins. Molecular modeling studies are currently underway to analyze the mechanistic basis for the differential bypass of cisplatin and oxaliplatin adducts by DNA polymerases.

Base Sequence↗

PubMed2004

The focal adhesion targeting domain of focal adhesion kinase contains a hinge region that modulates tyrosine 926 phosphorylation.

The focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK) is critical for recruitment of FAK to focal adhesions and contains tyrosine 926, which, when phosphorylated, binds the SH2 domain of Grb2. Structural studies have shown that the FAT domain is a four-helix bundle that exists as a monomer and a dimer due to domain swapping of helix 1. Here, we report the NMR solution structure of the avian FAT domain, which is similar in overall structure to the X-ray crystal structures of monomeric forms of the FAT domain, except that loop 1 is longer and less structured in solution. Residues in this region undergo temperature-dependent exchange broadening and sample aberrant phi and psi angles, which suggests that this region samples multiple conformations. We have also identified a mutant that dimerizes approximately 8 fold more than WT FAT domain and exhibits increased phosphorylation of tyrosine 926 both in vitro and in vivo.

Dimerization↗

PubMed2004

New insights into FAK signaling and localization based on detection of a FAT domain folding intermediate.

Mounting evidence suggests that the focal adhesion targeting (FAT) domain, an antiparallel four-helix bundle, exists in alternative conformations that may modulate phosphorylation, ligand binding, and the subcellular localization of focal adhesion kinase (FAK). In order to characterize the conformational dynamics of the FAT domain, we have developed a novel method for reconstructing the folding pathway of the FAT domain by using discrete molecular dynamics (DMD) simulations, with free energy constraints derived from NMR hydrogen exchange data. The DMD simulations detect a folding intermediate, in which a cooperative unfolding event causes helix 1 to lose helical character while separating from the helix bundle. The conformational dynamic features of helix 1 in the intermediate state of the FAT domain are likely to facilitate Y926 phosphorylation, yet interfere with paxillin binding. The presence of this intermediate state in vivo may promote FAK signaling via the ERK/MAPK pathway and by release of FAK from focal adhesions.

Focal Adhesion Protein-Tyrosine Kinases↗

PubMed2004

Molecular basis for Rho GTPase signaling specificity.

There is now considerable evidence for the involvement of aberrant Rho GTPase activation in breast cancer development. Like Ras, Rho GTPases function as signaling nodes regulated by diverse extracellular stimuli. Rho GTPase activation is facilitated by multiple regulatory proteins, in particular guanine nucleotide exchange factors (GEFs) such as Dbl family proteins. Activated Rho GTPases in turn interact with and regulate a spectrum of functionally diverse downstream effectors, initiating a network of cytoplasmic and nuclear signaling cascades. Thus, Rho GTPases represent points of signaling convergence as well as relay switches that disseminate signaling divergence. In this review, we highlight issues relating to the structural basis by which Dbl family GEFs facilitate signaling convergence and Rho GTPase activation, and how Rho GTPases promote signal dissemination through downstream effectors.

Breast Neoplasms↗