RESUMEN
Activated Cdc42-associated kinase (ACK), a non-receptor tyrosine kinase, is an effector for the small GTPase Cdc42. ACK is emerging as an important component of the cancer landscape and thus, a promising target for the treatment of many malignancies. ACK is also being increasingly recognized as a potentially influential player in the regulation of protein homoeostasis. The delicate equilibrium between protein synthesis and protein degradation is crucial for healthy cell function and dysregulation of protein homoeostasis is a common occurrence in human disease. Here, we review the molecular mechanisms by which ACK regulates the stability of diverse cellular proteins (e.g. EGFR, p27, p53, p85 isoforms and RhoGDI-3), some of which rely on the kinase activity of ACK while others, interestingly, do not. Ultimately, further research will be required to bridge our knowledge gaps and determine if ACK regulates the stability of further cellular proteins but collectively, such mechanistic interrogation would contribute to determining whether ACK is a promising target for anti-cancer therapy. In therapeutics, proteasome inhibitors are an efficacious but problematic class of drugs. Targeting other modulators of proteostasis, like ACK, could open novel avenues for intervention.
Asunto(s)
Neoplasias , Proteínas Tirosina Quinasas , Humanos , Proteína de Unión al GTP cdc42/metabolismo , Fosforilación , Estabilidad Proteica , Proteínas Tirosina Quinasas/metabolismoRESUMEN
Activated Cdc42-associated kinase (ACK) is an oncogenic nonreceptor tyrosine kinase associated with poor prognosis in several human cancers. ACK promotes proliferation, in part by contributing to the activation of Akt, the major effector of class 1A phosphoinositide 3-kinases (PI3Ks), which transduce signals via membrane phosphoinositol lipids. We now show that ACK also interacts with other key components of class 1A PI3K signaling, the PI3K regulatory subunits. We demonstrate ACK binds to all five PI3K regulatory subunit isoforms and directly phosphorylates p85α, p85ß, p50α, and p55α on Tyr607 (or analogous residues). We found that phosphorylation of p85ß promotes cell proliferation in HEK293T cells. We demonstrate that ACK interacts with p85α exclusively in nuclear-enriched cell fractions, where p85α phosphorylated at Tyr607 (pTyr607) also resides, and identify an interaction between pTyr607 and the N-terminal SH2 domain that supports dimerization of the regulatory subunits. We infer from this that ACK targets p110-independent p85 and further postulate that these regulatory subunit dimers undertake novel nuclear functions underpinning ACK activity. We conclude that these dimers represent a previously undescribed mode of regulation for the class1A PI3K regulatory subunits and potentially reveal additional avenues for therapeutic intervention.
Asunto(s)
Fosfatidilinositol 3-Quinasas , Proteínas Tirosina Quinasas , Núcleo Celular/enzimología , Células HEK293 , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Multimerización de Proteína , Proteínas Tirosina Quinasas/metabolismo , Transducción de SeñalRESUMEN
Both RalA and RalB interact with the ubiquitous calcium sensor, calmodulin (CaM). New structural and biophysical characterisation of these interactions strongly suggests that, in the native membrane-associated state, only RalA can be extracted from the membrane by CaM and this non-canonical interaction could underpin the divergent signalling roles of these closely related GTPases. The isoform specificity for RalA exhibited by CaM is hypothesised to contribute to the disparate signalling roles of RalA and RalB in mitochondrial dynamics. This would lead to CaM shuttling RalA to the mitochondrial membrane but leaving RalB localisation unperturbed, and in doing so triggering mitochondrial fission pathways rather than mitophagy.
Asunto(s)
Calmodulina , Transducción de Señal , Calmodulina/metabolismo , GTP Fosfohidrolasas/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
RalA is a small GTPase and a member of the Ras family. This molecular switch is activated downstream of Ras and is widely implicated in tumor formation and growth. Previous work has shown that the ubiquitous Ca2+-sensor calmodulin (CaM) binds to small GTPases such as RalA and K-Ras4B, but a lack of structural information has obscured the functional consequences of these interactions. Here, we have investigated the binding of CaM to RalA and found that CaM interacts exclusively with the C terminus of RalA, which is lipidated with a prenyl group in vivo to aid membrane attachment. Biophysical and structural analyses show that the two RalA membrane-targeting motifs (the prenyl anchor and the polybasic motif) are engaged by distinct lobes of CaM and that CaM binding leads to removal of RalA from its membrane environment. The structure of this complex, along with a biophysical investigation into membrane removal, provides a framework with which to understand how CaM regulates the function of RalA and sheds light on the interaction of CaM with other small GTPases, including K-Ras4B.
Asunto(s)
Calmodulina/metabolismo , Membrana Dobles de Lípidos/metabolismo , Proteínas de Unión al GTP ral/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Calmodulina/química , Membrana Celular/metabolismo , Humanos , Membrana Dobles de Lípidos/química , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Unión Proteica , Prenilación de Proteína , Serina/metabolismo , Proteínas de Unión al GTP ral/químicaRESUMEN
Cdc42 is a member of the Rho family of small GTPases and a master regulator of the actin cytoskeleton, controlling cell motility, polarity and cell cycle progression. This small G protein and its regulators have been the subject of many years of fruitful investigation and the advent of functional genomics and proteomics has opened up new avenues of exploration including how it functions at specific locations in the cell. This has coincided with the introduction of new structural techniques with the ability to study small GTPases in the context of the membrane. The role of Cdc42 in cancer is well established but the molecular details of its action are still being uncovered. Here we review alterations found to Cdc42 itself and to key components of the signal transduction pathways it controls in cancer. Given the challenges encountered with targeting small G proteins directly therapeutically, it is arguably the regulators of Cdc42 and the effector signalling pathways downstream of the small G protein which will be the most tractable targets for therapeutic intervention. These will require interrogation in order to fully understand the global signalling contribution of Cdc42, unlock the potential for mapping new signalling axes and ultimately produce inhibitors of Cdc42 driven signalling.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Mutación , Neoplasias/genética , Transducción de Señal/genética , Proteína de Unión al GTP cdc42/genética , Citoesqueleto de Actina/metabolismo , Animales , Humanos , Microdominios de Membrana/metabolismo , Microtúbulos/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Unión Proteica , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Proteína de Unión al GTP cdc42/química , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
RLIP76/RalBP1 is an ATP-dependent transporter of glutathione conjugates, which is overexpressed in various human cancers, but its diverse functions in normal cells, which include endocytosis, stress response and mitochondrial dynamics, are still not fully understood. The protein can be divided into three distinct regions, each with its own structural properties. At the centre of the protein are two well-defined domains, a GTPase activating protein domain targeting Rho family small G proteins and a small coiled-coil that binds to the Ras family small GTPases RalA and RalB. In engaging with Rho and Ral proteins, RLIP76 bridges these two distinct G protein families. The N-terminal region is predicted to be disordered and is rich in basic amino acids, which may mediate membrane association, consistent with its role in transport. RLIP76 is an ATP-dependent transporter with ATP-binding sites within the N-terminus and the Ral binding domain. Furthermore, RLIP76 is subject to extensive phosphorylation, particularly in the N-terminal region. In contrast, the C-terminal region is thought to form an extensive coiled-coil that could mediate dimerization. Here, we review the structural features of RLIP76, including experimental data and computational predictions, and discuss the implications of its various post-translational modifications.
RESUMEN
Cdc42 is a member of the Rho family of small GTPases and a key regulator of the actin cytoskeleton, controlling cell motility, polarity and cell cycle progression. It signals downstream of the master regulator Ras and is essential for cell transformation by this potent oncogene. Overexpression of Cdc42 is observed in several cancers, where it is linked to poor prognosis. As a regulator of both cell architecture and motility, deregulation of Cdc42 is also linked to tumour metastasis. Like Ras, Cdc42 and other components of the signalling pathways it controls represent important potential targets for cancer therapeutics. In this review, we consider the progress that has been made targeting Cdc42, its regulators and effectors, including new modalities and new approaches to inhibition. Strategies under consideration include inhibition of lipid modification, modulation of Cdc42-GEF, Cdc42-GDI and Cdc42-effector interactions, and direct inhibition of downstream effectors.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Neoplasias/metabolismo , Transducción de Señal/fisiología , Proteína de Unión al GTP cdc42/metabolismo , Aminoquinolinas/uso terapéutico , Animales , Benzamidas/uso terapéutico , Benzazepinas/uso terapéutico , Humanos , Terapia Molecular Dirigida/métodos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Oximas/uso terapéutico , Unión Proteica/efectos de los fármacos , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sulfonamidas/uso terapéutico , Tiourea/análogos & derivados , Tiourea/uso terapéutico , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Proteína de Unión al GTP cdc42/genéticaRESUMEN
There are three RhoGDIs in mammalian cells, which were initially defined as negative regulators of Rho family small GTPases. However, it is now accepted that RhoGDIs not only maintain small GTPases in their inactive GDP-bound form but also act as chaperones for small GTPases, targeting them to specific intracellular membranes and protecting them from degradation. Studies to date with RhoGDIs have usually focused on the interactions between the "typical" or "classical" small GTPases, such as the Rho, Rac, and Cdc42 subfamily members, and either the widely expressed RhoGDI-1 or the hematopoietic-specific RhoGDI-2. Less is known about the third member of the family, RhoGDI-3 and its interacting partners. RhoGDI-3 has a unique N-terminal extension and is found to localize in both the cytoplasm and the Golgi. RhoGDI-3 has been shown to target RhoB and RhoG to endomembranes. In order to facilitate a more thorough understanding of RhoGDI function, we undertook a systematic study to determine all possible Rho family small GTPases that interact with the RhoGDIs. RhoGDI-1 and RhoGDI-2 were found to have relatively restricted activity, mainly binding members of the Rho and Rac subfamilies. RhoGDI-3 displayed wider specificity, interacting with the members of Rho, Rac, and Cdc42 subfamilies but also forming complexes with "atypical" small Rho GTPases such as Wrch2/RhoV, Rnd2, Miro2, and RhoH. Levels of RhoA, RhoB, RhoC, Rac1, RhoH, and Wrch2/RhoV bound to GTP were found to decrease following coexpression with RhoGDI-3, confirming its role as a negative regulator of these small Rho GTPases.
Asunto(s)
Inhibidor alfa de Disociación del Nucleótido Guanina rho/metabolismo , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , Inhibidor gamma de Disociación del Nucleótido Guanina rho/metabolismo , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Proteínas de Unión al GTP/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/química , Células HEK293 , Humanos , Proteínas de Unión al GTP Monoméricas/metabolismo , Unión Proteica , Proteínas de Unión al GTP rho/química , Inhibidor alfa de Disociación del Nucleótido Guanina rho/fisiología , Inhibidor beta de Disociación del Nucleótido Guanina rho/fisiología , Inhibidor gamma de Disociación del Nucleótido Guanina rho/fisiología , Inhibidores de la Disociación del Nucleótido Guanina rho-Específico/metabolismo , Inhibidores de la Disociación del Nucleótido Guanina rho-Específico/fisiologíaRESUMEN
The protein kinase C-related kinase (PRK) family of serine/threonine kinases, PRK1, PRK2 and PRK3, are effectors for the Rho family small G proteins. An array of studies have linked these kinases to multiple signalling pathways and physiological roles, but while PRK1 is relatively well-characterized, the entire PRK family remains understudied. Here, we provide a holistic overview of the structure and function of PRKs and describe the molecular events that govern activation and autoregulation of catalytic activity, including phosphorylation, protein interactions and lipid binding. We begin with a structural description of the regulatory and catalytic domains, which facilitates the understanding of their regulation in molecular detail. We then examine their diverse physiological roles in cytoskeletal reorganization, cell adhesion, chromatin remodelling, androgen receptor signalling, cell cycle regulation, the immune response, glucose metabolism and development, highlighting isoform redundancy but also isoform specificity. Finally, we consider the involvement of PRKs in pathologies, including cancer, heart disease and bacterial infections. The abundance of PRK-driven pathologies suggests that these enzymes will be good therapeutic targets and we briefly report some of the progress to date.
Asunto(s)
Proteína Quinasa C/química , Proteínas Quinasas/química , Proteínas Quinasas/fisiología , Animales , Catálisis , Humanos , Conformación Proteica , Transducción de Señal/fisiología , Relación Estructura-ActividadRESUMEN
Ral GTPases have been implicated as critical drivers of cell growth and metastasis in numerous Ras-driven cancers. We have previously reported stapled peptides, based on the Ral effector RLIP76, that can disrupt Ral signaling. Stapled peptides are short peptides that are locked into their bioactive form using a synthetic brace. Here, using an affinity maturation of the RLIP76 Ral-binding domain, we identified several sequence substitutions that together improve binding to Ral proteins by more than 20-fold. Hits from the selection were rigorously analyzed to determine the contributions of individual residues and two 1.5 Å cocrystal structures of the tightest-binding mutants in complex with RalB revealed key interactions. Insights gained from this maturation were used to design second-generation stapled peptides based on RLIP76 that exhibited vastly improved selectivity for Ral GTPases when compared with the first-generation lead peptide. The binding of second-generation peptides to Ral proteins was quantified and the binding site of the lead peptide on RalB was determined by NMR. Stapled peptides successfully competed with multiple Ral-effector interactions in cellular lysates. Our findings demonstrate how manipulation of a native binding partner can assist in the rational design of stapled peptide inhibitors targeting a protein-protein interaction.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas de Unión al GTP ral/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Calorimetría , Dicroismo Circular , Fluorescencia , Proteínas Activadoras de GTPasa/química , Humanos , Espectroscopía de Resonancia Magnética , Unión Proteica , Transducción de Señal , Proteínas de Unión al GTP ral/químicaRESUMEN
The structure-function paradigm has guided investigations into the molecules involved in cellular signalling for decades. The peripheries of this paradigm, however, start to unravel when considering the co-operation between proteins and the membrane in signalling processes. Intrinsically disordered regions hold distinct advantages over folded domains in terms of their binding promiscuity, sensitivity to their particular environment and their ease of modulation through post-translational modifications. Low sequence complexity and bias towards charged residues are also favourable for the multivalent electrostatic interactions that occur at the surfaces of lipid bilayers. This review looks at the principles behind the successful marriage between protein disorder and membranes in addition to the role of this partnership in modifying and regulating signalling in cellular processes. The HVR (hypervariable region) of small GTPases is highlighted as a well-studied example of the nuanced role a short intrinsically disordered region can play in the fine-tuning of signalling pathways.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Membrana Dobles de Lípidos/química , Proteínas/química , Electricidad Estática , Animales , Sitios de Unión , Fenómenos Bioquímicos , Biofisica/métodos , Cardiolipinas/química , Membrana Celular/metabolismo , Dimerización , GTP Fosfohidrolasas/química , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lípidos/química , Mitocondrias/metabolismo , Conformación Molecular , Unión Proteica , Conformación Proteica , Dominios Proteicos , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Transducción de SeñalRESUMEN
The Rho-family of small GTPases are biological molecular switches that are best known for their regulation of the actin cytoskeleton. Through their activation and stimulation of downstream effectors, the Rho-family control pathways involved in cellular morphology, which are commonly activated in cancer cell invasion and metastasis. While this makes them excellent potential therapeutic targets, a deeper understanding of the downstream signalling pathways they influence will be required for successful drug targeting. Signal transducers and activators of transcription (STATs) are a family of transcription factors that are hyper-activated in most cancer types and while STATs are widely understood to be activated by the JAK family of kinases, many additional activators have been discovered. A growing number of examples of Rho-family driven STAT activation, largely of the oncogenic family members, STAT3 and STAT5, are being identified. Cdc42, Rac1, RhoA, RhoC and RhoH have all been implicated in STAT activation, contributing to Rho GTPase-driven changes in cellular morphology that lead to cell proliferation, invasion and metastasis. This highlights the importance and therapeutic potential of the Rho-family as regulators of non-canonical activation of STAT signalling.
Asunto(s)
Factor de Transcripción STAT3/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular , Progresión de la Enfermedad , Proteínas de Unión al GTP/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias/metabolismo , Dominios Proteicos , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The phosphatidylinositol 3-kinase (PI3K) pathway is a critical regulator of many cellular processes including cell survival, growth, proliferation and motility. Not surprisingly therefore, the PI3K pathway is one of the most frequently mutated pathways in human cancers. In addition to their canonical role as part of the PI3K holoenzyme, the class IA PI3K regulatory subunits undertake critical functions independent of PI3K. The PI3K regulatory subunits exist in excess over the p110 catalytic subunits and therefore free in the cell. p110-independent p85 is unstable and exists in a monomer-dimer equilibrium. Two conformations of dimeric p85 have been reported that are mediated by N-terminal and C-terminal protein domain interactions, respectively. The role of p110-independent p85 is under investigation and it has been found to perform critical adaptor functions, sequestering or influencing compartmentalisation of key signalling proteins. Free p85 has roles in glucose homeostasis, cellular stress pathways, receptor trafficking and cell migration. As a regulator of fundamental pathways, the amount of p110-independent p85 in the cell is critical. Factors that influence the monomer-dimer equilibrium of p110-independent p85 offer additional control over this system, disruption to which likely results in disease. Here we review the current knowledge of the structure and functions of p110-independent class IA PI3K regulatory subunits.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Movimiento Celular , Dimerización , Humanos , Insulina/metabolismo , Mutación , Neoplasias/enzimología , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/genética , Conformación Proteica , Transporte de Proteínas , Transducción de SeñalRESUMEN
PRK1 is a member of the protein kinase C-related kinase (PRK) family of serine/threonine kinases and a downstream effector of Rho GTPases. PRK1 has three N-terminal Homology Region 1 (HR1) domains (HR1a, HR1b and HR1c), which form antiparallel coiled coils that interact with Rho family GTPases. PRK1 also has a C2-like domain that targets it to the plasma membrane and a kinase domain, which is a member of the protein kinase C superfamily. PRK1 is involved in cytoskeletal regulation, cell adhesion, cell cycle progression and the immune response, and is implicated in cancer. There is currently no structural information for the HR1c domain. The 1H, 15N and 13C NMR backbone and sidechain resonance assignment of the HR1c domain presented here forms the basis for this domain's structural characterisation. This work will also enable studies of interactions between the three HR1 domains in an effort to obtain structural insight into the regulation of PRK1 activity.
Asunto(s)
Espectroscopía de Resonancia Magnética con Carbono-13 , Proteína Quinasa C/química , Espectroscopía de Protones por Resonancia Magnética , Secuencia de Aminoácidos , Humanos , Isótopos de Nitrógeno , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
The Ral proteins (RalA and RalB) are small G proteins of the Ras family that have been implicated in exocytosis, endocytosis, transcriptional regulation and mitochondrial fission, as well as having a role in tumourigenesis. RalA and RalB are activated downstream of the master regulator, Ras, which causes the nucleotide exchange of GDP for GTP. Here we report the 1H, 15 N and 13C resonance assignments of RalA in its active form bound to the GTP analogue GMPPNP. We also report the backbone assignments of RalA in its inactive, GDP-bound form. The assignments give insight into the switch regions, which change conformation upon nucleotide exchange. These switch regions are invisible in the spectra of the active, GMPPNP bound form but the residues proximal to the switches can be monitored. RalA is also an important drug target due to its over activation in some cancers and these assignments will be extremely useful for NMR-based screening approaches.
Asunto(s)
Proteínas de Unión al GTP Monoméricas/química , Resonancia Magnética Nuclear Biomolecular , Proteínas de Unión al GTP ral/química , Guanosina Difosfato/química , Conformación ProteicaRESUMEN
Aberrant Ras signaling drives 30% of cancers, and inhibition of the Rho family small GTPase signaling has been shown to combat Ras-driven cancers. Here, we present the discovery of a 16-mer cyclic peptide that binds to Cdc42 with nanomolar affinity. Affinity maturation of this sequence has produced a panel of derived candidates with increased affinity and modulated specificity for other closely-related small GTPases. The structure of the tightest binding peptide was solved by NMR, and its binding site on Cdc42 was determined. Addition of a cell-penetrating sequence allowed the peptides to access the cell interior and engage with their target(s), modulating signaling pathways. In Ras-driven cancer cell models, the peptides have an inhibitory effect on proliferation and show suppression of both invasion and motility. As such, they represent promising candidates for Rho-family small GTPase inhibitors and therapeutics targeting Ras-driven cancers. Our data add to the growing literature demonstrating that peptides are establishing their place in the biologics arm of drug discovery.
Asunto(s)
Descubrimiento de Drogas , Péptidos Cíclicos/farmacología , Transducción de Señal/efectos de los fármacos , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Proteínas ras/metabolismo , Sitios de Unión , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Péptidos de Penetración Celular , GTP Fosfohidrolasas/antagonistas & inhibidores , Humanos , Estructura Molecular , Invasividad Neoplásica/prevención & control , Neoplasias/tratamiento farmacológico , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Inhibition of Ras signalling has been a goal almost since its central role in cell signalling and its deregulation in disease were discovered. Early attempts at inhibiting its post-translational modification using peptidomimetics were successful in cell culture but failed spectacularly in clinical trials, making industry wary of targeting this critical oncoprotein. Small molecule inhibition of the protein-protein interactions involving Ras has also been difficult due to the nature of the interaction interface. Recent improvements in design, synthesis and selection of stabilised peptides, peptidomimetics and macrocycles have suggested that these biologics may represent a new hope in Ras inhibition. Here we review the various ways in which Ras has been targeted with these molecules. We also describe work on related small G proteins of the Ras superfamily, since many of the principles may be applicable to Ras, and these also provide inhibition of pathways downstream of Ras.
Asunto(s)
Descubrimiento de Drogas , Proteínas de Unión al GTP Monoméricas/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Humanos , Proteínas de Unión al GTP Monoméricas/química , Familia de Multigenes , Péptidos/química , Péptidos/metabolismo , Unión Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteínas Son Of Sevenless/química , Proteínas Son Of Sevenless/metabolismo , Relación Estructura-Actividad , Proteínas ras/química , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
The Ras family of small guanine nucleotide-binding proteins behave as molecular switches: they are switched off and inactive when bound to GDP but can be activated by GTP binding in response to signal transduction pathways. Early structural analysis showed that two regions of the protein, which change conformation depending on the nucleotide present, mediate this switch. A large number of X-ray, NMR and simulation studies have shown that this is an over-simplification. The switch regions themselves are highly dynamic and can exist in distinct sub-states in the GTP-bound form that have different affinities for other proteins. Furthermore, regions outside the switches have been found to be sensitive to the nucleotide state of the protein, indicating that allosteric change is more widespread than previously thought. Taken together, the accrued knowledge about small G protein structures, allostery and dynamics will be essential for the design and testing of the next generation of inhibitors, both orthosteric and allosteric, as well as for understanding their mode of action.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Transducción de Señal , Sitio Alostérico , Mutación , Unión Proteica , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
Wiskott-Aldrich syndrome protein (WASP) activates the actin-related protein 2/3 homolog (Arp2/3) complex and regulates actin polymerization in a physiological setting. Cell division cycle 42 (Cdc42) is a key activator of WASP, which binds Cdc42 through a Cdc42/Rac-interactive binding (CRIB)-containing region that defines a subset of Cdc42 effectors. Here, using site-directed mutagenesis and binding affinity determination and kinetic assays, we report the results of an investigation into the energetic contributions of individual WASP residues to both the Cdc42-WASP binding interface and the kinetics of complex formation. Our results support the previously proposed dock-and-coalesce binding mechanism, initiated by electrostatic steering driven by WASP's basic region and followed by a coalescence phase likely driven by the conserved CRIB motif. The WASP basic region, however, appears also to play a role in the final complex, as its mutation affected both on- and off-rates, suggesting a more comprehensive physiological role for this region centered on the C-terminal triad of positive residues. These results highlight the expanding roles of the basic region in WASP and other CRIB-containing effector proteins in regulating complex cellular processes and coordinating multiple input signals. The data presented improve our understanding of the Cdc42-WASP interface and also add to the body of information available for Cdc42-effector complex formation, therapeutic targeting of which has promise for Ras-driven cancers. Our findings suggest that combining high-affinity peptide-binding sequences with short electrostatic steering sequences could increase the efficacy of peptidomimetic candidates designed to interfere with Cdc42 signaling in cancer.