RESUMEN
The plasma membrane is widely regarded as the hub of the numerous signal transduction activities. Yet, the fundamental biophysical mechanisms that spatiotemporally compartmentalize different classes of membrane proteins remain unclear. Using multimodal live-cell imaging, here we first show that several lipid-anchored membrane proteins are consistently depleted from the membrane regions where the Ras/PI3K/Akt/F-actin network is activated. The dynamic polarization of these proteins does not depend upon the F-actin-based cytoskeletal structures, recurring shuttling between membrane and cytosol, or directed vesicular trafficking. Photoconversion microscopy and single-molecule measurements demonstrate that these lipid-anchored molecules have substantially dissimilar diffusion profiles in different regions of the membrane which enable their selective segregation. When these diffusion coefficients are incorporated into an excitable network-based stochastic reaction-diffusion model, simulations reveal that the altered affinity mediated selective partitioning is sufficient to drive familiar propagating wave patterns. Furthermore, normally uniform integral and lipid-anchored membrane proteins partition successfully when membrane domain-specific peptides are optogenetically recruited to them. We propose "dynamic partitioning" as a new mechanism that can account for large-scale compartmentalization of a wide array of lipid-anchored and integral membrane proteins during various physiological processes where membrane polarizes.
Asunto(s)
Actinas , Proteínas de la Membrana , Proteínas de la Membrana/metabolismo , Actinas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Membrana Celular/metabolismo , Proteínas Ligadas a Lípidos/análisis , Proteínas Ligadas a Lípidos/metabolismo , Lípidos/análisis , Microdominios de Membrana/metabolismoRESUMEN
The lateral diffusion of transmembrane proteins on plasma membranes is a fundamental process for various cellular functions. Diffusion properties specific for individual protein species have been extensively studied, but the common features among protein species are poorly understood. Here, we systematically studied the lateral diffusion of various transmembrane proteins in the lower eukaryote Dictyostelium discoideum cells using a hidden Markov model for single-molecule trajectories obtained experimentally. As common features, all membrane proteins that had from one to ten transmembrane regions adopted three free diffusion states with similar diffusion coefficients regardless of their structural variability. All protein species reduced their mobility similarly upon the inhibition of microtubule or actin cytoskeleton dynamics, or myosin II. The relationship between protein size and the diffusion coefficient was consistent with the Saffman-Delbrück model, meaning that membrane viscosity is a major determinant of lateral diffusion, but protein size is not. These protein species-independent properties of multistate free diffusion were explained simply and quantitatively by free diffusion on the three membrane regions with different viscosities, which is in sharp contrast to the complex diffusion behavior of transmembrane proteins in higher eukaryotes.
Asunto(s)
Dictyostelium , Dictyostelium/metabolismo , Proteínas de la Membrana/metabolismo , Membrana Celular/metabolismo , Difusión , Membranas/metabolismoRESUMEN
The plasma membrane is widely regarded as the hub of the signal transduction network activities that drives numerous physiological responses, including cell polarity and migration. Yet, the symmetry breaking process in the membrane, that leads to dynamic compartmentalization of different proteins, remains poorly understood. Using multimodal live-cell imaging, here we first show that multiple endogenous and synthetic lipid-anchored proteins, despite maintaining stable tight association with the inner leaflet of the plasma membrane, were unexpectedly depleted from the membrane domains where the signaling network was spontaneously activated such as in the new protrusions as well as within the propagating ventral waves. Although their asymmetric patterns resembled those of standard peripheral "back" proteins such as PTEN, unlike the latter, these lipidated proteins did not dissociate from the membrane upon global receptor activation. Our experiments not only discounted the possibility of recurrent reversible translocation from membrane to cytosol as it occurs for weakly bound peripheral membrane proteins, but also ruled out the necessity of directed vesicular trafficking and cytoskeletal supramolecular structure-based restrictions in driving these dynamic symmetry breaking events. Selective photoconversion-based protein tracking assays suggested that these asymmetric patterns instead originate from the inherent ability of these membrane proteins to "dynamically partition" into distinct domains within the plane of the membrane. Consistently, single-molecule measurements showed that these lipid-anchored molecules have substantially dissimilar diffusion profiles in different regions of the membrane. When these profiles were incorporated into an excitable network-based stochastic reaction-diffusion model of the system, simulations revealed that our proposed "dynamic partitioning" mechanism is sufficient to give rise to familiar asymmetric propagating wave patterns. Moreover, we demonstrated that normally uniform integral and lipid-anchored membrane proteins in Dictyostelium and mammalian neutrophil cells can be induced to partition spatiotemporally to form polarized patterns, by optogenetically recruiting membrane domain-specific peptides to these proteins. Together, our results indicate "dynamic partitioning" as a new mechanism of plasma membrane organization, that can account for large-scale compartmentalization of a wide array of lipid-anchored and integral membrane proteins in different physiological processes.
RESUMEN
Genome-wide screening is powerful method used to identify genes and pathways associated with a phenotype of interest. The simple eukaryote Dictyostelium discoideum has a unique life cycle and is often used as a crucial research model for a wide range of biological processes and rare metabolites. To address the inadequacies of conventional genetic screening approaches, we developed a highly efficient CRISPR/Cas9-based genome-wide screening system for Dictyostelium. A genome-wide library of 27,405 gRNAs and a kinase library of 4,582 gRNAs were compiled and mutant pools were generated. The resulting mutants were screened for defects in cell growth and more than 10 candidate genes were identified. Six of these were validated and five recreated mutants presented with growth abnormalities. Finally, the genes implicated in developmental defects were screened to identify the unknown genes associated with a phenotype of interest. These findings demonstrate the potential of the CRISPR/Cas9 system as an efficient genome-wide screening method.
Asunto(s)
Dictyostelium , Sistemas CRISPR-Cas/genética , Dictyostelium/genética , Biblioteca de Genes , Genoma , ARN Guía de Kinetoplastida/genéticaRESUMEN
The development of new techniques to create gene knockouts and knock-ins is essential for successful investigation of gene functions and elucidation of the causes of diseases and their associated fundamental cellular processes. In the biomedical model organism Dictyostelium discoideum, the methodology for gene targeting with homologous recombination to generate mutants is well-established. Recently, we have applied CRISPR/Cas9-mediated approaches in Dictyostelium, allowing the rapid generation of mutants by transiently expressing sgRNA and Cas9 using an all-in-one vector. CRISPR/Cas9 techniques not only provide an alternative to homologous recombination-based gene knockouts but also enable the creation of mutants that were technically unfeasible previously. Herein, we provide a detailed protocol for the CRISPR/Cas9-based method in Dictyostelium. We also describe new tools, including double knockouts using a single CRISPR vector, drug-inducible knockouts, and gene knockdown using CRISPR interference (CRISPRi). We demonstrate the use of these tools for some candidate genes. Our data indicate that more suitable mutants can be rapidly generated using CRISPR/Cas9-based techniques to study gene function in Dictyostelium.
RESUMEN
Chemotaxis describes directional motility along ambient chemical gradients and has important roles in human physiology and pathology. Typical chemotactic cells, such as neutrophils and Dictyostelium cells, can detect spatial differences in chemical gradients over a background concentration of a 105 scale. Studies of Dictyostelium cells have elucidated the molecular mechanisms of gradient sensing involving G protein coupled receptor (GPCR) signaling. GPCR transduces spatial information through its cognate heterotrimeric G protein as a guanine nucleotide change factor (GEF). More recently, studies have revealed unconventional regulation of heterotrimeric G protein in the gradient sensing. In this review, we explain how multiple mechanisms of GPCR signaling ensure the broad range sensing of chemical gradients in Dictyostelium cells as a model for eukaryotic chemotaxis.
RESUMEN
GPCR signaling is the most prevailing molecular mechanism for detecting ambient signals in eukaryotes. Chemotactic cells use GPCR signaling to process chemical cues for directional migration over a broad concentration range and with high sensitivity. Dictyostelium discoideum is a classical model, in which the molecular mechanism underlying eukaryotic chemotaxis has been well studied. Here, we describe protocols to evaluate the spatiotemporal chemotactic responses of Dictyostelium discoideum by different microscopic observations combined with biochemical assays. First, two different chemotaxis assays are presented to measure the dynamic concentration ranges for different cell strains or chemotactic parameters. Next, live-cell imaging and biochemical assays are provided to detect the activities of GPCR and its partner heterotrimeric G proteins upon chemoattractant stimulation. Finally, a method for detecting how a cell deciphers chemical gradients is described.
Asunto(s)
Factores Quimiotácticos/farmacología , Quimiotaxis/efectos de los fármacos , Dictyostelium/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , AMP Cíclico/metabolismo , Dictyostelium/efectos de los fármacos , Inmunoprecipitación , Transducción de Señal , Análisis Espacio-TemporalRESUMEN
Collective cell migration is a key process during the development of multicellular organisms, in which the migrations of individual cells are coordinated through chemical guidance and physical contact between cells. Talin has been implicated in mechanical linkage between actin-based motile machinery and adhesion molecules, but how talin contributes to collective cell migration is unclear. Here we show that talin B is involved in chemical coordination between cells for collective cell migration at the multicellular mound stage in the development of Dictyostelium discoideum. From early aggregation to the mound formation, talB-null cells exhibited collective migration normally with cAMP relay. Subsequently, talB-null cells showed developmental arrest at the mound stage, and at the same time, they had impaired collective migration and cAMP relay, while wild-type cells exhibited rotational cell migration continuously in concert with cAMP relay during the mound stage. Genetic suppression of PI3K activity partially restored talB-null phenotypes in collective cell migration and cAMP relay. Overall, our observations suggest that talin B regulates chemical coordination via PI3K-mediated signaling in a stage-specific manner for the multicellular development of Dictyostelium cells.
Asunto(s)
Movimiento Celular , Dictyostelium/citología , Fosfatidilinositol 3-Quinasas/metabolismo , Talina/fisiología , Agregación Celular , AMP Cíclico/metabolismo , Dictyostelium/metabolismo , Proteínas ProtozoariasRESUMEN
mTORC2 plays critical roles in metabolism, cell survival and actin cytoskeletal dynamics through the phosphorylation of AKT. Despite its importance to biology and medicine, it is unclear how mTORC2-mediated AKT phosphorylation is controlled. Here, we identify an unforeseen principle by which a GDP-bound form of the conserved small G protein Rho GTPase directly activates mTORC2 in AKT phosphorylation in social amoebae (Dictyostelium discoideum) cells. Using biochemical reconstitution with purified proteins, we demonstrate that Rho-GDP promotes AKT phosphorylation by assembling a supercomplex with Ras-GTP and mTORC2. This supercomplex formation is controlled by the chemoattractant-induced phosphorylation of Rho-GDP at S192 by GSK-3. Furthermore, Rho-GDP rescues defects in both mTORC2-mediated AKT phosphorylation and directed cell migration in Rho-null cells in a manner dependent on phosphorylation of S192. Thus, in contrast to the prevailing view that the GDP-bound forms of G proteins are inactive, our study reveals that mTORC2-AKT signalling is activated by Rho-GDP.
Asunto(s)
Movimiento Celular/fisiología , Dimerización , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Citoesqueleto/metabolismo , Proteínas de Unión al GTP/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Guanosina Difosfato/metabolismo , Humanos , Fosforilación/fisiologíaRESUMEN
The wide range sensing of extracellular signals is a common feature of various sensory cells. Eukaryotic chemotactic cells driven by GPCRs and their cognate G proteins are one example. This system endows the cells directional motility towards their destination over long distances. There are several mechanisms to achieve the long dynamic range, including negative regulation of the receptors upon ligand interaction and spatial regulation of G proteins, as we found recently. However, these mechanisms are insufficient to explain the 105-fold range of chemotaxis seen in Dictyostelium. Here, we reveal that the receptor-mediated activation, recruitment, and capturing of G proteins mediate chemotactic signaling at the lower, middle and higher concentration ranges, respectively. These multiple mechanisms of G protein dynamics can successfully cover distinct ranges of ligand concentrations, resulting in seamless and broad chemotaxis. Furthermore, single-molecule imaging analysis showed that the activated Gα subunit forms an unconventional complex with the agonist-bound receptor. This complex formation of GPCR-Gα increased the membrane-binding time of individual Gα molecules and therefore resulted in the local accumulation of Gα. Our findings provide an additional chemotactic dynamic range mechanism in which multiple G protein dynamics positively contribute to the production of gradient information.
Asunto(s)
Quimiotaxis , Dictyostelium/citología , Dictyostelium/metabolismo , Proteínas de Unión al GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , AMP Cíclico/metabolismo , Espacio Intracelular/metabolismo , Proteínas Protozoarias/metabolismo , Transducción de SeñalRESUMEN
In eukaryotic chemotaxis, parallel signaling pathways regulate the spatiotemporal pseudopod dynamics at the leading edge of a motile cell through the characteristic dynamics of an excitable system; however, differences in the excitability and the physiological roles of individual pathways remain to be elucidated. Here, we found that two different pathways, mediated by soluble guanylyl cyclase (sGC) and phosphoinositide 3-kinase (PI3K), caused similar all-or-none responses for sGC localization and phosphatidylinositol 3,4,5-trisphosphate production but with different refractory periods, by undertaking simultaneous observations of the excitable properties of the two pathways in Dictyostelium cells. Owing to the shorter refractory period, sGC signaling responded more frequently to chemoattractants, leading to pseudopod formation with higher frequency. sGC excitability was regulated negatively by its product cGMP and by cGMP-binding protein C (GbpC) through the suppression of F-actin polymerization, providing the underlying delayed negative-feedback mechanism for the cyclical pseudopod formation. These results suggest that parallel pathways respond to environmental cues on different timescales in order to mediate chemotactic motility in a manner based on their intrinsic excitability.
Asunto(s)
Eucariontes/metabolismo , Animales , Quimiotaxis/fisiología , Transducción de SeñalRESUMEN
G protein interacting protein 1 (Gip1) binds and sequesters heterotrimeric G proteins in the cytosolic pool, thus regulating G protein-coupled receptor (GPCR) signalling for eukaryotic chemotaxis. Here, we report the underlying structural basis of Gip1 function. The crystal structure reveals that the region of Gip1 that binds to the G protein has a cylinder-like fold with a central hydrophobic cavity composed of six α-helices. Mutagenesis and biochemical analyses indicate that the hydrophobic cavity and the hydrogen bond network at the entrance of the cavity are essential for complex formation with the geranylgeranyl modification on the Gγ subunit. Mutations of the cavity impair G protein sequestration and translocation to the membrane from the cytosol upon receptor stimulation, leading to defects in chemotaxis at higher chemoattractant concentrations. These results demonstrate that the Gip1-dependent regulation of G protein shuttling ensures wide-range gradient sensing in eukaryotic chemotaxis.
Asunto(s)
Quimiotaxis/fisiología , Citosol/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP Heterotriméricas/química , Receptores Acoplados a Proteínas G/química , Factores Quimiotácticos/química , Cristalografía por Rayos X , Dictyostelium , Eucariontes , Proteínas de Unión al GTP/genética , Enlace de Hidrógeno , Modelos Moleculares , Mutagénesis , Mutación , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína Fosfatasa 1 , Proteínas de Saccharomyces cerevisiae , Transducción de SeñalRESUMEN
Chemotactic eukaryote cells can sense chemical gradients over a wide range of concentrations via heterotrimeric G-protein signaling; however, the underlying wide-range sensing mechanisms are only partially understood. Here we report that a novel regulator of G proteins, G protein-interacting protein 1 (Gip1), is essential for extending the chemotactic range ofDictyosteliumcells. Genetic disruption of Gip1 caused severe defects in gradient sensing and directed cell migration at high but not low concentrations of chemoattractant. Also, Gip1 was found to bind and sequester G proteins in cytosolic pools. Receptor activation induced G-protein translocation to the plasma membrane from the cytosol in a Gip1-dependent manner, causing a biased redistribution of G protein on the membrane along a chemoattractant gradient. These findings suggest that Gip1 regulates G-protein shuttling between the cytosol and the membrane to ensure the availability and biased redistribution of G protein on the membrane for receptor-mediated chemotactic signaling. This mechanism offers an explanation for the wide-range sensing seen in eukaryotic chemotaxis.
Asunto(s)
Membrana Celular/metabolismo , Quimiotaxis/fisiología , Dictyostelium/metabolismo , Reguladores de Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Transducción de Señal/fisiología , Membrana Celular/genética , Dictyostelium/genética , Reguladores de Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP Heterotriméricas/genéticaRESUMEN
Directional cell migration in an electric field, a phenomenon called galvanotaxis or electrotaxis, occurs in many types of cells, and may play an important role in wound healing and development. Small extracellular electric fields can guide the migration of amoeboid cells, and we established a large-scale screening approach to search for mutants with electrotaxis phenotypes from a collection of 563 Dictyostelium discoideum strains with morphological defects. We identified 28 strains that were defective in electrotaxis and 10 strains with a slightly higher directional response. Using plasmid rescue followed by gene disruption, we identified some of the mutated genes, including some previously implicated in chemotaxis. Among these, we studied PiaA, which encodes a critical component of TORC2, a kinase protein complex that transduces changes in motility by activating the kinase PKB (also known as Akt). Furthermore, we found that electrotaxis was decreased in mutants lacking gefA, rasC, rip3, lst8, or pkbR1, genes that encode other components of the TORC2-PKB pathway. Thus, we have developed a high-throughput screening technique that will be a useful tool to elucidate the molecular mechanisms of electrotaxis.
Asunto(s)
Dictyostelium , Complejos Multiproteicos , Proteínas Proto-Oncogénicas c-akt , Proteínas Protozoarias , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR , Dictyostelium/genética , Dictyostelium/metabolismo , Técnicas de Silenciamiento del Gen , Diana Mecanicista del Complejo 2 de la Rapamicina , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Many types of cells migrate directionally in direct current (DC) electric fields (EFs), a phenomenon termed galvanotaxis or electrotaxis. The directional sensing mechanisms responsible for this response to EFs, however, remain unknown. Exposing cells to an EF causes changes in plasma membrane potentials (V(m)). Exploiting the ability of Dictyostelium cells to tolerate drastic V(m) changes, we investigated the role of V(m) in electrotaxis and, in parallel, in chemotaxis. We used three independent factors to control V(m): extracellular pH, extracellular [K(+)], and electroporation. Changes in V(m) were monitored with microelectrode recording techniques. Depolarized V(m) was observed under acidic (pH 5.0) and alkaline (pH 9.0) conditions as well as under higher extracellular [K(+)] conditions. Electroporation permeabilized the cell membrane and significantly reduced the V(m), which gradually recovered over 40 min. We then recorded the electrotactic behaviors of Dictyostelium cells with a defined V(m) using these three techniques. The directionality (directedness of electrotaxis) was quantified and compared to that of chemotaxis (chemotactic index). We found that a reduced V(m) significantly impaired electrotaxis without significantly affecting random motility or chemotaxis. We conclude that extracellular pH, [K(+)], and electroporation all significantly affected electrotaxis, which appeared to be mediated by the changes in V(m). The initial directional sensing mechanisms for electrotaxis therefore differ from those of chemotaxis and may be mediated by changes in resting V(m).
Asunto(s)
Quimiotaxis/fisiología , Dictyostelium/metabolismo , Potenciales de la Membrana/fisiología , Membrana Celular/metabolismo , Movimiento Celular , Electricidad , Electroporación , Concentración de Iones de Hidrógeno , Potasio/metabolismoRESUMEN
Genetic screening of yeast for sld (synthetic lethality with dpb11) mutations has identified replication proteins, including Sld2, -3, and -5, and clarified the molecular mechanisms underlying eukaryotic chromosomal DNA replication. Here, we report a new replication protein, Sld7, identified by rescreening of sld mutations. Throughout the cell cycle, Sld7 forms a complex with Sld3, which associates with replication origins in a complex with Cdc45, binds to Dpb11 when phosphorylated by cyclin-dependent kinase, and dissociates from origins once DNA replication starts. However, Sld7 does not move with the replication fork. Sld7 binds to the nonessential N-terminal portion of Sld3 and reduces its affinity for Cdc45, a component of the replication fork. Although Sld7 is not essential for cell growth, its absence reduces the level of cellular Sld3, delays the dissociation from origins of GINS, a component of the replication fork, and slows S-phase progression. These results suggest that Sld7 is required for the proper function of Sld3 at the initiation of DNA replication.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromosomas Fúngicos/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Proteínas Portadoras/genética , Análisis Mutacional de ADN , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
By limiting phosphotidylinositol 3,4,5-triphosphate (PIP(3)) levels, tumor suppressor PTEN not only controls cell growth but also maintains cell polarity required for cytokinesis and chemotaxis. To identify the critical targets of PIP(3) that link it to the cytoskeleton, we deleted secondary genes to reverse the deficiencies of pten- cells in Dictyostelium. The polarity defects in pten- cells correlate with elevated phosphorylations of PKB substrates. Deletion of AKT orthologue, PkbA, or a subunit of its activator TORC2, reduced the phosphorylations and suppressed the cytokinesis and chemotaxis defects in pten- cells. In these double mutants, the excessive PIP(3) levels and, presumably, activation of other PIP(3)-binding proteins had little or no effect on the cytoskeleton. In bands with increased phosphorylation in pten- cells, we found PKB substrates, PI5K, GefS, GacG, and PakA. Disruption of PakA in pten- cells restored a large fraction of the cells to normal behavior. Consistently, expression of phosphomimetic PakA in pten- cells exacerbated the defects but nonphosphorylatable PakA had no effect. Thus, among many putative PTEN- and PIP(3)-dependent events, phosphorylation of PKB substrates is the key downstream regulator of cell polarity.
Asunto(s)
Polaridad Celular/genética , Fosfohidrolasa PTEN/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quimiotaxis/genética , AMP Cíclico/química , Citoesqueleto/genética , Citoesqueleto/metabolismo , Dictyostelium/citología , Dictyostelium/genética , Dictyostelium/fisiología , Eliminación de Gen , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/genéticaRESUMEN
In chemotactic cells, G protein-coupled receptors activate Ras proteins, but it is unclear how Ras-associated pathways link extracellular signaling to cell migration. We show that, in Dictyostelium discoideum, activated forms of RasC prolong the time course of TORC2 (target of rapamycin [Tor] complex 2)-mediated activation of a myristoylated protein kinase B (PKB; PKBR1) and the phosphorylation of PKB substrates, independently of phosphatidylinositol-(3,4,5)-trisphosphate. Paralleling these changes, the kinetics of chemoattractant-induced adenylyl cyclase activation and actin polymerization are extended, pseudopodial activity is increased and mislocalized, and chemotaxis is impaired. The effects of activated RasC are suppressed by deletion of the TORC2 subunit PiaA. In vitro RasC(Q62L)-dependent PKB phosphorylation can be rapidly initiated by the addition of a PiaA-associated immunocomplex to membranes of TORC2-deficient cells and blocked by TOR-specific inhibitor PP242. Furthermore, TORC2 binds specifically to the activated form of RasC. These results demonstrate that RasC is an upstream regulator of TORC2 and that the TORC2-PKB signaling mediates effects of activated Ras proteins on the cytoskeleton and cell migration.
Asunto(s)
Quimiotaxis/fisiología , Dictyostelium/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción/metabolismo , Proteínas ras/metabolismo , Animales , Dictyostelium/citología , Activación Enzimática , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/genética , Proteínas ras/genéticaRESUMEN
Eukaryotic chromosomal DNA replication requires cyclin-dependent kinase (CDK) activity. CDK phosphorylates two yeast replication proteins, Sld3 and Sld2, both of which bind to Dpb11 when phosphorylated. These phosphorylation-dependent interactions are essential and are the minimal requirements for CDK-dependent activation of DNA replication. However, how these interactions activate DNA replication has not been elucidated. Here, we show that CDK promotes the formation of a newly identified fragile complex, the preloading complex (pre-LC) containing DNA polymerase epsilon (Pol epsilon), GINS, Sld2, and Dpb11. Formation of the pre-LC requires phosphorylation of Sld2 by CDK, but is independent of DNA replication, protein association with replication origins, and Dbf4-dependent Cdc7 kinase, which is also essential for the activation of DNA replication. We also demonstrate that Pol epsilon, GINS, Dpb11, and CDK-phosphorylated Sld2 form a complex in vitro. The genetic interactions between Pol epsilon, GINS, Sld2, and Dpb11 suggest further that they form an essential complex in cells. We propose that CDK regulates the initiation of DNA replication in budding yeast through formation of the pre-LC.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , ADN Polimerasa II/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ciclo Celular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Chemotactic cells must sense shallow extracellular gradients and produce localized intracellular responses. We previously showed that the temporal and spatial activation of two protein kinase B (PKB) homologues, PkbA and PkbR1, in Dictyostelium discoideum by phosphorylation of activation loops (ALs) and hydrophobic motifs had important roles in chemotaxis. We found that hydrophobic motif phosphorylation depended on regulation of TorC2 (target of rapamycin complex 2); however, the regulation of AL phosphorylation remains to be determined at a molecular level. Here, we show that two PDK (phosphoinositide-dependent protein kinase) homologues, PdkA and PdkB, function as the key AL kinases. Cells lacking both PdkA and PdkB are defective in PKB activation, chemotaxis, and fruiting body formation upon nutrient deprivation. The pleckstrin homology domain of PdkA is sufficient to localize it to the membrane, but transient activation of PdkA is independent of PIP(3) as well as TorC2 and dispensable for full function. These results confirm the importance of the TorC2-PDK-PKB pathway in chemotaxis and point to a novel mechanism of regulation of PDKs by chemoattractant.