RESUMEN
A conserved molecular machinery centered on the Cdc42 GTPase regulates cell polarity in diverse organisms. Here we review findings from budding and fission yeasts that reveal both a conserved core polarity circuit and several adaptations that each organism exploits to fulfill the needs of its lifestyle. The core circuit involves positive feedback by local activation of Cdc42 to generate a cluster of concentrated GTP-Cdc42 at the membrane. Species-specific pathways regulate the timing of polarization during the cell cycle, as well as the location and number of polarity sites.
Asunto(s)
Polaridad Celular , Saccharomyces cerevisiae/citología , Actinas/metabolismo , Ciclo Celular , Modelos BiológicosRESUMEN
Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly. This sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of the Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesions upon KANK1 activation. Other players participating in microtubule-driven, KANK-dependent focal adhesion disassembly include kinases ROCK, PAK, and FAK, as well as microtubules/focal adhesion-associated proteins kinesin-1, APC, and αTAT. Based on these data, we develop a mathematical model for a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK-dependent activation of contractility, which is consistent with experimental data.
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Adhesiones Focales , Cinesinas , Microtúbulos , Factores de Intercambio de Guanina Nucleótido Rho , Adhesiones Focales/metabolismo , Microtúbulos/metabolismo , Humanos , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Cinesinas/metabolismo , Cinesinas/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Miosina Tipo II/metabolismo , Talina/metabolismo , Talina/genética , AnimalesRESUMEN
The late stages of Golgi maturation involve a series of sequential trafficking events in which cargo-laden vesicles are produced and targeted to multiple distinct subcellular destinations. Each of these vesicle biogenesis events requires activation of an Arf GTPase by the Sec7/BIG guanine nucleotide exchange factor (GEF). Sec7 localization and activity is regulated by autoinhibition, positive feedback, and interaction with other GTPases. Although these mechanisms have been characterized biochemically, we lack a clear picture of how GEF localization and activity is modulated by these signals. Here, we report the cryogenic electron microscopy structure of full-length Sec7 in its autoinhibited form, revealing the architecture of its multiple regulatory domains. We use functional experiments to determine the basis for autoinhibition and use structural predictions to produce a model for an active conformation of the GEF that is supported empirically. This study therefore elucidates the conformational transition that Sec7 undergoes to become active on the organelle membrane surface.
Asunto(s)
GTP Fosfohidrolasas , Aparato de Golgi , Aparato de Golgi/metabolismo , Factores de Ribosilacion-ADP/metabolismoRESUMEN
The endosomal system of eukaryotic cells represents a central sorting and recycling compartment linked to metabolic signaling and the regulation of cell growth. Tightly controlled activation of Rab GTPases is required to establish the different domains of endosomes and lysosomes. In metazoans, Rab7 controls endosomal maturation, autophagy, and lysosomal function. It is activated by the guanine nucleotide exchange factor (GEF) complex Mon1-Ccz1-Bulli (MCBulli) of the tri-longin domain (TLD) family. While the Mon1 and Ccz1 subunits have been shown to constitute the active site of the complex, the role of Bulli remains elusive. We here present the cryo-electron microscopy (cryo-EM) structure of MCBulli at 3.2 Å resolution. Bulli associates as a leg-like extension at the periphery of the Mon1 and Ccz1 heterodimers, consistent with earlier reports that Bulli does not impact the activity of the complex or the interactions with recruiter and substrate GTPases. While MCBulli shows structural homology to the related ciliogenesis and planar cell polarity effector (Fuzzy-Inturned-Wdpcp) complex, the interaction of the TLD core subunits Mon1-Ccz1 and Fuzzy-Inturned with Bulli and Wdpcp, respectively, is remarkably different. The variations in the overall architecture suggest divergent functions of the Bulli and Wdpcp subunits. Based on our structural analysis, Bulli likely serves as a recruitment platform for additional regulators of endolysosomal trafficking to sites of Rab7 activation.
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Proteínas de Transporte Vesicular , Proteínas de Unión al GTP rab , Animales , Proteínas de Transporte Vesicular/metabolismo , Microscopía por Crioelectrón , Transporte de Proteínas , Proteínas de Unión al GTP rab/metabolismo , Endosomas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismoRESUMEN
Accurate sensing and responding to physical microenvironment are crucial for cell function and survival, but the underlying molecular mechanisms remain elusive. Pollen tube (PT) provides a perfect single-cell model for studying mechanobiology since it's naturally subjected to complex mechanical instructions from the pistil during invasive growth. Recent reports have revealed discrepant PT behaviors between in vivo and flat, two-dimensional in vitro cultures. Here, we established the Stigma-style-transmitting tract (TT) Physical microenvironment Assay (SPA) to recapitulate pressure changes in the pistil. This biomimetic assay has enabled us to swiftly identify highly redundant genes, GEF8/9/11/12/13, as new regulators for maintaining PTs integrity during style-to-TT emergence. In contrast to normal growth on solid medium, SPA successfully phenocopied gef8/9/11/12/13 PT in vivo growth-arrest deficiency. Our results suggest the existence of distinct signaling pathways regulating in vivo and in vitro PT integrity maintenance, underscoring the necessity of faithfully mimicking the physical microenvironment for studying plant cell biology.
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Tubo Polínico , Polen , Tubo Polínico/metabolismo , Polen/metabolismo , Flores/genética , Polinización , FenotipoRESUMEN
Normal receptor tyrosine kinases (RTKs) need to reach the plasma membrane (PM) for ligand-induced activation, whereas its cancer-causing mutants can be activated before reaching the PM in organelles, such as the Golgi/trans-Golgi network (TGN). Inhibitors of protein export from the endoplasmic reticulum (ER), such as brefeldin A (BFA) and 2-methylcoprophilinamide (M-COPA), can suppress the activation of mutant RTKs in cancer cells, suggesting that RTK mutants cannot initiate signaling in the ER. BFA and M-COPA block the function of ADP-ribosylation factors (ARFs) that play a crucial role in ER-Golgi protein trafficking. However, among ARF family proteins, the specific ARFs inhibited by BFA or M-COPA, that is, the ARFs involved in RTKs transport from the ER, remain unclear. In this study, we showed that M-COPA blocked the export of not only KIT but also PDGFRA/EGFR/MET RTKs from the ER. ER-retained RTKs could not fully transduce anti-apoptotic signals, thereby leading to cancer cell apoptosis. Moreover, a single knockdown of ARF1, ARF3, ARF4, ARF5, or ARF6 could not block ER export of RTKs, indicating that BFA/M-COPA treatment cannot be mimicked by the knockdown of only one ARF member. Interestingly, simultaneous transfection of ARF1, ARF4, and ARF5 siRNAs mirrored the effect of BFA/M-COPA treatment. Consistent with these results, in vitro pulldown assays showed that BFA/M-COPA blocked the function of ARF1, ARF4, and ARF5. Taken together, these results suggest that BFA/M-COPA targets at least ARF1, ARF4, and ARF5; in other words, RTKs require the simultaneous activation of ARF1, ARF4, and ARF5 for their ER export.
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Factor 1 de Ribosilacion-ADP , Factores de Ribosilacion-ADP , Brefeldino A , Retículo Endoplásmico , Transporte de Proteínas , Humanos , Factores de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/genética , Retículo Endoplásmico/metabolismo , Factor 1 de Ribosilacion-ADP/metabolismo , Factor 1 de Ribosilacion-ADP/genética , Brefeldino A/farmacología , Transporte de Proteínas/efectos de los fármacos , Receptores ErbB/metabolismo , Receptores ErbB/genética , Células HeLaRESUMEN
Cell polarity oscillations in Myxococcus xanthus motility are driven by a prokaryotic small Ras-like GTPase, mutual gliding protein A (MglA), which switches from one cell pole to the other in response to extracellular signals. MglA dynamics is regulated by MglB, which functions both as a GTPase activating protein (GAP) and a guanine nucleotide exchange factor (GEF) for MglA. With an aim to dissect the asymmetric role of the two MglB protomers in the dual GAP and GEF activities, we generated a functional MglAB complex by coexpressing MglB with a linked construct of MglA and MglB. This strategy enabled us to generate mutations of individual MglB protomers (MglB1 or MglB2 linked to MglA) and delineate their role in GEF and GAP activities. We establish that the C-terminal helix of MglB1, but not MglB2, stimulates nucleotide exchange through a site away from the nucleotide-binding pocket, confirming an allosteric mechanism. Interaction between the N-terminal ß-strand of MglB1 and ß0 of MglA is essential for the optimal GEF activity of MglB. Specific residues of MglB2, which interact with Switch-I of MglA, partially contribute to its GAP activity. Thus, the role of the MglB2 protomer in the GAP activity of MglB is limited to restricting the conformation of MglA active site loops. The direct demonstration of the allosteric mechanism of GEF action provides us new insights into the regulation of small Ras-like GTPases, a feature potentially present in many uncharacterized GEFs.
Asunto(s)
Proteínas Bacterianas , Proteínas Activadoras de GTPasa , Myxococcus xanthus , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Activación Enzimática , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Myxococcus xanthus/metabolismo , Myxococcus xanthus/genética , Myxococcus xanthus/enzimología , Multimerización de Proteína , Modelos Moleculares , Estructura Cuaternaria de ProteínaRESUMEN
Highly homologous members of the Gαi family, Gαi1-3, have distinct tissue distributions and physiological functions, yet their biochemical and functional properties are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gαi1 effector that is poorly activated by Gαi2. In a proteomic proximity labeling screen we observed a strong preference for Gαi1 relative to Gαi2 with respect to engagement of a broad range of potential targets. We investigated the mechanistic basis for this selectivity using PRG as a representative target. Substitution of either the helical domain (HD) from Gαi1 into Gαi2 or substitution of a single amino acid, A230 in Gαi2 with the corresponding D in Gαi1, largely rescues PRG activation and interactions with other potential Gαi targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, separation at the HD-Ras-like domain (RLD) interface is more pronounced in Gαi2 than Gαi1. Mutation of A230 to D in Gαi2 stabilizes HD-RLD interactions via ionic interactions with R145 in the HD which in turn modify the conformation of Switch III. These data support a model where D229 in Gαi1 interacts with R144 and stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gαi2 is unable to stabilize this network leading to an overall lower efficacy with respect to target interactions. This study reveals distinct mechanistic properties that could underly differential biological and physiological consequences of activation of Gαi1 or Gαi2 by G protein-coupled receptors.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Transducción de Señal , Humanos , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Simulación de Dinámica Molecular , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidad alfa de la Proteína de Unión al GTP Gi2/química , Células HEK293 , Dominios Proteicos , Estabilidad Proteica , Unión ProteicaRESUMEN
Heterotrimeric G proteins (Gαßγ) are molecular switches that relay signals from 7-transmembrane receptors located at the cell surface to the cytoplasm. The function of these receptors is so intimately linked to heterotrimeric G proteins that they are named G protein-coupled receptors (GPCRs), showcasing the interdependent nature of this archetypical receptor-transducer axis of transmembrane signaling in eukaryotes. It is generally assumed that activation of heterotrimeric G protein signaling occurs exclusively by the action of GPCRs, but this idea has been challenged by the discovery of alternative mechanisms by which G proteins can propagate signals in the cell. This review will focus on a general principle of G protein signaling that operates without the direct involvement of GPCRs. The mechanism of G protein signaling reviewed here is mediated by a class of G protein regulators defined by containing an evolutionarily conserved sequence named the Gα-binding-and-activating (GBA) motif. Using the best characterized proteins with a GBA motif as examples, Gα-interacting vesicle-associated protein (GIV)/Girdin and dishevelled-associating protein with a high frequency of leucine residues (DAPLE), this review will cover (i) the mechanisms by which extracellular cues not relayed by GPCRs promote the coupling of GBA motif-containing regulators with G proteins, (ii) the structural and molecular basis for how GBA motifs interact with Gα subunits to facilitate signaling, (iii) the relevance of this mechanism in different cellular and pathological processes, including cancer and birth defects, and (iv) strategies to manipulate GBA-G protein coupling for experimental therapeutics purposes, including the development of rationally engineered proteins and chemical probes.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas , Receptores Acoplados a Proteínas G , Secuencias de Aminoácidos , Membrana Celular/metabolismo , Proteínas de Unión al GTP Heterotriméricas/química , Proteínas de Unión al GTP Heterotriméricas/genética , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Humanos , Animales , Ingeniería de ProteínasRESUMEN
The dedicator of cytokinesis (DOCK)/engulfment and cell motility (ELMO) complex serves as a guanine nucleotide exchange factor (GEF) for the GTPase Rac. RhoG, another GTPase, activates the ELMO-DOCK-Rac pathway during engulfment and migration. Recent cryo-EM structures of the DOCK2/ELMO1 and DOCK2/ELMO1/Rac1 complexes have identified closed and open conformations that are key to understanding the autoinhibition mechanism. Nevertheless, the structural details of RhoG-mediated activation of the DOCK/ELMO complex remain elusive. Herein, we present cryo-EM structures of DOCK5/ELMO1 alone and in complex with RhoG and Rac1. The DOCK5/ELMO1 structure exhibits a closed conformation similar to that of DOCK2/ELMO1, suggesting a shared regulatory mechanism of the autoinhibitory state across DOCK-A/B subfamilies (DOCK1-5). Conversely, the RhoG/DOCK5/ELMO1/Rac1 complex adopts an open conformation that differs from that of the DOCK2/ELMO1/Rac1 complex, with RhoG binding to both ELMO1 and DOCK5. The alignment of the DOCK5 phosphatidylinositol (3,4,5)-trisphosphate binding site with the RhoG C-terminal lipidation site suggests simultaneous binding of RhoG and DOCK5/ELMO1 to the plasma membrane. Structural comparison of the apo and RhoG-bound states revealed that RhoG facilitates a closed-to-open state conformational change of DOCK5/ELMO1. Biochemical and surface plasmon resonance (SPR) assays confirm that RhoG enhances the Rac GEF activity of DOCK5/ELMO1 and increases its binding affinity for Rac1. Further analysis of structural variability underscored the conformational flexibility of the DOCK5/ELMO1/Rac1 complex core, potentially facilitating the proximity of the DOCK5 GEF domain to the plasma membrane. These findings elucidate the structural mechanism underlying the RhoG-induced allosteric activation and membrane binding of the DOCK/ELMO complex.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Factores de Intercambio de Guanina Nucleótido , Proteína de Unión al GTP rac1 , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Unión Proteica , Conformación Proteica , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/química , Proteínas de Unión al GTP rho/metabolismo , Proteínas de Unión al GTP rho/químicaRESUMEN
The small GTPase Rab22A is an important regulator of the formation of tubular endosomes, which are one of the types of recycling endosome compartments of the clathrin-independent endocytosis pathway. In order to regulate tubular endosome formation, Rab22A must be activated by a specific guanine-nucleotide-exchange factor (GEF); however, all of the GEFs that have been reported to exhibit Rab22A-GEF activity in vitro also activate Rab5A, an essential regulator of the clathrin-mediated endocytosis pathway, and no Rab22A-specific GEF has ever been identified. Here, we identified Vps9d1, a previously uncharacterized vacuolar protein sorting 9 (VPS9) domain-containing protein, as a novel Rab22A-GEF. The formation of tubular endosome structures was found to be severely impaired in Vps9d1-depleted HeLa cells, but Rab5A localization was unaffected. Expression of a constitutively active Rab22A mutant in Vps9d1-depleted HeLa cells restored tubular endosomes, but expression of a GEF-activity-deficient Vps9d1 mutant did not. Moreover, Vps9d1 depletion altered the distribution of clathrin-independent endocytosed cargos and impaired their recycling. Our findings indicate that Vps9d1 promotes tubular endosome formation by specifically activating Rab22A.
Asunto(s)
Endosomas , Proteínas de Unión al GTP rab , Humanos , Células HeLa , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Endosomas/metabolismo , Endocitosis/fisiología , Transporte de Proteínas/fisiología , Clatrina/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismoRESUMEN
Fibronectin (FN)-binding integrins control a variety of cellular responses through Rho GTPases. The FN-binding integrins, αvß3 and α5ß1, are known to induce different effects on cell morphology and motility. Here, we report that FN-bound αvß3 integrin, but not FN-bound α5ß1 integrin, triggers the dissociation of the RhoA GEF Lfc (also known as GEF-H1 and ARHGEF2 in humans) from microtubules (MTs), leading to the activation of RhoA, formation of stress fibres and maturation of focal adhesions (FAs). Conversely, loss of Lfc expression decreases RhoA activity, stress fibre formation and FA size, suggesting that Lfc is the major GEF downstream of FN-bound αvß3 that controls RhoA activity. Mechanistically, FN-engaged αvß3 integrin activates a kinase cascade involving MARK2 and MARK3, which in turn leads to phosphorylation of several phospho-sites on Lfc. In particular, S151 was identified as the main site involved in the regulation of Lfc localization and activity. Our findings indicate that activation of Lfc and RhoA is orchestrated in FN-adherent cells in an integrin-specific manner.
Asunto(s)
Integrina alfa5beta1 , Integrina alfaV , Humanos , Integrina alfaV/metabolismo , Integrina alfa5beta1/metabolismo , Integrinas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Fosforilación , Proteína de Unión al GTP rhoA/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismoRESUMEN
Within a cell, vesicles play a crucial role in the transport of membrane material and proteins to a given target membrane, and thus regulate a variety of cellular functions. Vesicular transport occurs by means of, among others, endocytosis, where cargoes are taken up by the cell and are processed further upon vesicular trafficking, i.e. transported back to the plasma membrane via recycling endosomes or the degraded by fusion of the vesicles with lysosomes. During evolution, a variety of vesicles with individual functions arose, with some of them building up highly specialised subcellular compartments. In this study, we have analysed the biosynthesis of a new vesicular compartment present in the valve cells of Drosophila melanogaster. We show that the compartment is formed by invaginations of the plasma membrane and grows via re-routing of the recycling endosomal pathway. This is achieved by inactivation of other membrane-consuming pathways and a plasma membrane-like molecular signature of the compartment in these highly specialised heart cells.
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Drosophila melanogaster , Endosomas , Animales , Membrana Celular/metabolismo , Drosophila melanogaster/metabolismo , Endocitosis , Endosomas/metabolismo , Válvulas Cardíacas/metabolismo , Transporte de Proteínas , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Activation of the GTPase Rab7/Ypt7 by its cognate guanine nucleotide exchange factor (GEF) Mon1-Ccz1 marks organelles such as endosomes and autophagosomes for fusion with lysosomes/vacuoles and degradation of their content. Here, we present a high-resolution cryogenic electron microscopy structure of the Mon1-Ccz1 complex that reveals its architecture in atomic detail. Mon1 and Ccz1 are arranged side by side in a pseudo-twofold symmetrical heterodimer. The three Longin domains of each Mon1 and Ccz1 are triangularly arranged, providing a strong scaffold for the catalytic center of the GEF. At the opposite side of the Ypt7-binding site, a positively charged and relatively flat patch stretches the Longin domains 2/3 of Mon1 and functions as a phosphatidylinositol phosphate-binding site, explaining how the GEF is targeted to membranes. Our work provides molecular insight into the mechanisms of endosomal Rab activation and serves as a blueprint for understanding the function of members of the Tri Longin domain Rab-GEF family.
Asunto(s)
Membrana Celular/metabolismo , Chaetomium/metabolismo , Proteínas Fúngicas/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Unión a GTP rab7/metabolismo , Membrana Celular/genética , Chaetomium/genética , Proteínas Fúngicas/genética , Complejos Multiproteicos/genética , Proteínas de Unión a GTP rab7/genéticaRESUMEN
High-resolution structural studies on G-protein-coupled receptors (GPCRs) have flourished recently, providing long-sought insights into the dynamic process of guanine nucleotide-binding protein (G-protein) activation. In parallel, analogous studies are starting to shed light on how the same G-proteins are activated by non-GPCR proteins. Can we learn about common themes and variations in G-protein activation from them?
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/química , Humanos , Conformación Proteica , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
ARL13B is a small GTPase enriched in cilia. Deletion of Arl13b in mouse kidney results in renal cysts and an associated absence of primary cilia. Similarly, ablation of cilia leads to kidney cysts. To investigate whether ARL13B functions from within cilia to direct kidney development, we examined kidneys of mice expressing an engineered cilia-excluded ARL13B variant, ARL13BV358A. These mice retained renal cilia and developed cystic kidneys. Because ARL13B functions as a guanine nucleotide exchange factor (GEF) for ARL3, we examined kidneys of mice expressing an ARL13B variant that lacks ARL3 GEF activity, ARL13BR79Q. We found normal kidney development with no evidence of cysts in these mice. Taken together, our results show that ARL13B functions within cilia to inhibit renal cystogenesis during mouse development, and that this function does not depend on its role as a GEF for ARL3.
Asunto(s)
Enfermedades Renales Quísticas , Riñón , Animales , Ratones , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Cilios/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Riñón/metabolismo , Enfermedades Renales Quísticas/genéticaRESUMEN
TRIO encodes a cytoskeletal regulatory protein with three catalytic domains-two guanine exchange factor (GEF) domains, GEF1 and GEF2, and a kinase domain-as well as several accessory domains that have not been extensively studied. Function-damaging variants in the TRIO gene are known to be enriched in individuals with neurodevelopmental disorders (NDDs). Disease variants in the GEF1 domain or the nine adjacent spectrin repeats (SRs) are enriched in NDDs, suggesting that dysregulated GEF1 activity is linked to these disorders. We provide evidence here that the Trio SRs interact intramolecularly with the GEF1 domain to inhibit its enzymatic activity. We demonstrate that SRs 6-9 decrease GEF1 catalytic activity both in vitro and in cells and show that NDD-associated variants in the SR8 and GEF1 domains relieve this autoinhibitory constraint. Our results from chemical cross-linking and bio-layer interferometry indicate that the SRs primarily contact the pleckstrin homology region of the GEF1 domain, reducing GEF1 binding to the small GTPase Rac1. Together, our findings reveal a key regulatory mechanism that is commonly disrupted in multiple NDDs and may offer a new target for therapeutic intervention for TRIO-associated NDDs.
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Proteínas de Unión al GTP Monoméricas , Trastornos del Neurodesarrollo , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Guanina/metabolismo , Humanos , Proteínas de Unión al GTP Monoméricas/metabolismo , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Espectrina/metabolismoRESUMEN
Trio is a large and highly conserved metazoan signaling scaffold that contains two Dbl family guanine nucleotide exchange factor (GEF) modules, TrioN and TrioC, selective for Rac and RhoA GTPases, respectively. The GEF activities of TrioN and TrioC are implicated in several cancers, especially uveal melanoma. However, little is known about how these modules operate in the context of larger fragments of Trio. Here we show via negative stain electron microscopy that the N-terminal region of Trio is extended and could thus serve as a rigid spacer between the N-terminal putative lipid-binding domain and TrioN, whereas the C-terminal half of Trio seems globular. We found that regions C-terminal to TrioN enhance its Rac1 GEF activity and thus could play a regulatory role. We went on to characterize a minimal, well-behaved Trio fragment with enhanced activity, Trio1284-1959, in complex with Rac1 using cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry and found that the region conferring enhanced activity is disordered. Deletion of two different strongly conserved motifs in this region eliminated this enhancement, suggesting that they form transient intramolecular interactions that promote GEF activity. Because Dbl family RhoGEF modules have been challenging to directly target with small molecules, characterization of accessory Trio domains such as these may provide alternate routes for the development of therapeutics that inhibit Trio activity in human cancer.
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Factores de Intercambio de Guanina Nucleótido/química , Proteínas Serina-Treonina Quinasas/química , Factores de Intercambio de Guanina Nucleótido Rho/química , Animales , Microscopía por Crioelectrón , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal , Neoplasias de la Úvea , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Rho family small GTPases (Rho) regulate various cell motility processes by spatiotemporally controlling the actin cytoskeleton. Some Rho-specific guanine nucleotide exchange factors (RhoGEFs) are regulated via tyrosine phosphorylation by Src family tyrosine kinase (SFK). We also previously reported that PLEKHG2, a RhoGEF for the GTPases Rac1 and Cdc42, is tyrosine-phosphorylated by SRC. However, the details of the mechanisms by which SFK regulates RhoGEFs are not well understood. In this study, we found for the first time that PLEKHG1, which has very high homology to the Dbl and pleckstrin homology domains of PLEKHG2, activates Cdc42 following activation by FYN, a member of the SFK family. We also show that this activation of PLEKHG1 by FYN requires interaction between these two proteins and FYN-induced tyrosine phosphorylation of PLEKHG1. We also found that the region containing the Src homology 3 and Src homology 2 domains of FYN is required for this interaction. Finally, we demonstrated that tyrosine phosphorylation of Tyr-720 and Tyr-801 in PLEKHG1 is important for the activation of PLEKHG1. These results suggest that FYN is a regulator of PLEKHG1 and may regulate cell morphology through Rho signaling via the interaction with and tyrosine phosphorylation of PLEKHG1.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido Rho , Proteínas de Unión al GTP rho , Familia-src Quinasas , Fosforilación , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Tirosina/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Evolutionary change following gene duplication can lead to functionally divergent paralogous proteins. If comprising identical subunits their random assortment would also form potentially detrimental heteromeric proteins. In Arabidopsis, the ARF GTPase guanine-nucleotide exchange factor GNOM is essential for polar recycling of auxin-efflux transporter PIN1 from endosomes to the basal plasma membrane whereas its paralog GNL1 mediates retrograde Golgi-endoplasmic reticulum traffic. Here we show that both GNOM and GNL1 form homodimers but no heterodimers. To assess the biological significance of this, we generated transgenic plants expressing engineered heterodimer-compatible GNOM variants. Those plants showed developmental defects such as the failure to produce lateral roots. To identify mechanisms underlying heterodimer prevention, we analyzed interactions of the N-terminal dimerization and cyclophilin-binding (DCB) domain. Each DCB domain interacted with the complementary fragment (ΔDCB) both of their own and of the paralogous protein. However, only DCBGNOM interacted with itself whereas DCBGNL1 failed to interact with itself and with DCBGNOM . GNOM variants in which the DCB domain was removed or replaced by DCBGNL1 revealed a role for DCB-DCB interaction in the prevention of GNOM-GNL1 heterodimers whereas DCB-ΔDCB interaction was essential for dimer formation and GNOM function. Our data suggest a model of early DCB-DCB interaction that facilitates GNOM homodimer formation, indirectly precluding formation of detrimental heterodimers.