RESUMEN
The Small GTPase Rac1 is critical for various fundamental cellular processes, including cognitive functions. The cyclical activation and inactivation of Rac1, mediated by Rac guanine nucleotide exchange factors (RacGEFs) and Rac GTPase-activating proteins (RacGAPs), respectively, are essential for activating intracellular signaling pathways and controlling cellular processes. We have recently shown that the Alzheimer's disease (AD) therapeutic drug donepezil activates the Rac1-PAK pathway in the nucleus accumbens (NAc) for enhanced aversive learning. Also, PAK activation itself in the NAc enhances aversive learning. As aversive learning allows short-term preliminary AD drug screening, here we tested whether sustained Rac1 activation by RacGAP inhibition can be used as an AD therapeutic strategy for improving AD-learning deficits based on aversive learning. We found that the RacGAP domain of breakpoint cluster region protein (Bcr) (Bcr-GAP) efficiently inhibited Rac1 activity in a membrane ruffling assay. We also found that, in striatal/accumbal primary neurons, Bcr knockdown by microRNA mimic-expressing adeno-associated virus (AAV-miRNA mimic) activated Rac1-PAK signaling, while Bcr-GAP-expressing AAV inactivated it. Furthermore, conditional knockdown of Bcr in the NAc of wild-type adult mice enhanced aversive learning, while Bcr-GAP expression in the NAc inhibited it. The findings indicate that Rac1 activation by RacGAP inhibition enhances aversive learning, implying the AD therapeutic potential of Rac1 signaling.
Asunto(s)
Enfermedad de Alzheimer , MicroARNs , Animales , Ratones , Enfermedad de Alzheimer/tratamiento farmacológico , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Transducción de SeñalRESUMEN
The nucleus accumbens (NAc) plays critical roles in emotional behaviors, including aversive learning. Aversive stimuli such as an electric foot shock increase acetylcholine (ACh) in the NAc, and muscarinic signaling appears to increase neuronal excitability and aversive learning. Muscarinic signaling inhibits the voltage-dependent potassium KCNQ current which regulates neuronal excitability, but the regulatory mechanism has not been fully elucidated. Phosphorylation of KCNQ2 at threonine 217 (T217) and its inhibitory effect on channel activity were predicted. However, whether and how muscarinic signaling phosphorylates KCNQ2 in vivo remains unclear. Here, we found that PKC directly phosphorylated KCNQ2 at T217 in vitro. Carbachol and a muscarinic M1 receptor (M1R) agonist facilitated KCNQ2 phosphorylation at T217 in NAc/striatum slices in a PKC-dependent manner. Systemic administration of the cholinesterase inhibitor donepezil, which is commonly used to treat dementia, and electric foot shock to mice induced the phosphorylation of KCNQ2 at T217 in the NAc, whereas phosphorylation was suppressed by an M1R antagonist. Conditional deletion of Kcnq2 in the NAc enhanced electric foot shock induced aversive learning. Our findings indicate that muscarinic signaling induces the phosphorylation of KCNQ2 at T217 via PKC activation for aversive learning.
Asunto(s)
Reacción de Prevención/fisiología , Canal de Potasio KCNQ2/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Núcleo Accumbens/metabolismo , Sistema Nervioso Parasimpático/fisiología , Proteína Quinasa C/metabolismo , Receptores Muscarínicos/fisiología , Animales , Carbacol/farmacología , Inhibidores de la Colinesterasa/farmacología , Donepezilo/farmacología , Canal de Potasio KCNQ2/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Proteínas del Tejido Nervioso/genética , Fosforilación , Receptor Muscarínico M2/efectos de los fármacosRESUMEN
The structural plasticity of dendritic spines plays a critical role in NMDA-induced long-term potentiation (LTP) in the brain. The small GTPases RhoA and Ras are considered key regulators of spine morphology and enlargement. However, the regulatory interaction between RhoA and Ras underlying NMDA-induced spine enlargement is largely unknown. In this study, we found that Rho-kinase/ROCK, an effector of RhoA, phosphorylated SynGAP1 (a synaptic Ras-GTPase activating protein) at Ser842 and increased its interaction with 14-3-3ζ, thereby activating Ras-ERK signaling in a reconstitution system in HeLa cells. We also found that the stimulation of NMDA receptor by glycine treatment for LTP induction stimulated SynGAP1 phosphorylation, Ras-ERK activation, spine enlargement and SynGAP1 delocalization from the spines in striatal neurons, and these effects were prevented by Rho-kinase inhibition. Rho-kinase-mediated phosphorylation of SynGAP1 appeared to increase its dissociation from PSD95, a postsynaptic scaffolding protein located at postsynaptic density, by forming a complex with 14-3-3ζ. These results suggest that Rho-kinase phosphorylates SynGAP1 at Ser842, thereby activating the Ras-ERK pathway for NMDA-induced morphological changes in dendritic spines.
Asunto(s)
Espinas Dendríticas , Potenciación a Largo Plazo , Proteínas Activadoras de ras GTPasa , Proteínas 14-3-3/metabolismo , Animales , Espinas Dendríticas/metabolismo , Células HeLa , Hipocampo/metabolismo , Humanos , Potenciación a Largo Plazo/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , N-Metilaspartato/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Proteína de Unión al GTP rhoARESUMEN
The N-methyl-D-aspartate receptor (NMDAR)-mediated structural plasticity of dendritic spines plays an important role in synaptic transmission in the brain during learning and memory formation. The Rho family of small GTPase RhoA and its downstream effector Rho-kinase/ROCK are considered as one of the major regulators of synaptic plasticity and dendritic spine formation, including long-term potentiation (LTP). However, the mechanism by which Rho-kinase regulates synaptic plasticity is not yet fully understood. Here, we found that Rho-kinase directly phosphorylated discs large MAGUK scaffold protein 2 (DLG2/PSD-93), a major postsynaptic scaffold protein that connects postsynaptic proteins with NMDARs; an ionotropic glutamate receptor, which plays a critical role in synaptic plasticity. Stimulation of striatal slices with an NMDAR agonist induced Rho-kinase-mediated phosphorylation of PSD-93 at Thr612. We also identified PSD-93-interacting proteins, including DLG4 (PSD-95), NMDARs, synaptic Ras GTPase-activating protein 1 (SynGAP1), ADAM metallopeptidase domain 22 (ADAM22), and leucine-rich glioma-inactivated 1 (LGI1), by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among them, Rho-kinase increased the binding of PSD-93 to PSD-95 and NMDARs. Furthermore, we found that chemical-LTP induced by glycine, which activates NMDARs, increased PSD-93 phosphorylation at Thr612, spine size, and PSD-93 colocalization with PSD-95, while these events were blocked by pretreatment with a Rho-kinase inhibitor. These results indicate that Rho-kinase phosphorylates PSD-93 downstream of NMDARs, and suggest that Rho-kinase mediated phosphorylation of PSD-93 increases the association with PSD-95 and NMDARs to regulate structural synaptic plasticity.
Asunto(s)
Receptores de N-Metil-D-Aspartato , Quinasas Asociadas a rho , Quinasas Asociadas a rho/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Cromatografía Liquida , Espectrometría de Masas en Tándem , Plasticidad Neuronal/fisiología , Transmisión Sináptica/fisiología , Homólogo 4 de la Proteína Discs Large/metabolismo , Sinapsis/metabolismo , Hipocampo/metabolismoRESUMEN
In Drosophila, dopamine signaling to the mushroom body intrinsic neurons, Kenyon cells (KCs), is critical to stabilize olfactory memory. Little is known about the downstream intracellular molecular signaling underlying memory stabilization. Here we address this question in the context of sugar-rewarded olfactory long-term memory (LTM). We show that associative training increases the phosphorylation of MAPK in KCs, via Dop1R2 signaling. Consistently, the attenuation of Dop1R2, Raf, or MAPK expression in KCs selectively impairs LTM, but not short-term memory. Moreover, we show that the LTM deficit caused by the knockdown of Dop1R2 can be rescued by expressing active Raf in KCs. Thus, the Dop1R2/Raf/MAPK pathway is a pivotal downstream effector of dopamine signaling for stabilizing appetitive olfactory memory.SIGNIFICANCE STATEMENT Dopaminergic input to the Kenyon cells (KCs) is pivotal to stabilize memory in Drosophila This process is mediated by dopamine receptors like Dop1R2. Nevertheless, little is known for its underlying molecular mechanism. Here we show that the Raf/MAPK pathway is specifically engaged in appetitive long-term memory in KCs. With combined biochemical and behavioral experiments, we reveal that activation of the Raf/MAPK pathway is regulated through Dop1R2, shedding light on how dopamine modulates intracellular signaling for memory stabilization.
Asunto(s)
Conducta Apetitiva/fisiología , Proteínas de Drosophila/metabolismo , Memoria a Largo Plazo/fisiología , Neuronas/metabolismo , Receptores de Dopamina D1/metabolismo , Transducción de Señal/fisiología , Animales , Drosophila , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Masculino , Cuerpos Pedunculados/fisiología , Quinasas raf/metabolismoRESUMEN
Dopamine type 1 receptor (D1R) signaling activates protein kinase A (PKA), which then activates mitogen-activated protein kinase (MAPK) through Rap1, in striatal medium spiny neurons (MSNs). MAPK plays a pivotal role in reward-related behavior through the activation of certain transcription factors. How D1R signaling regulates behavior through transcription factors remains largely unknown. CREB-binding protein (CBP) promotes transcription through hundreds of different transcription factors and is also important for reward-related behavior. To identify transcription factors regulated by dopamine signaling in MSNs, we performed a phosphoproteomic analysis using affinity beads coated with CBP. We obtained approximately 40 novel candidate proteins in the striatum of the C57BL/6 mouse brain after cocaine administration. Among them, the megakaryoblastic leukemia-2 (MKL2) protein, a transcriptional coactivator of serum response factor (SRF), was our focus. We found that the interaction between CBP and MKL2 was increased by cocaine administration. Additionally, MKL2, CBP and SRF formed a ternary complex in vivo. The C-terminal domain of MKL2 interacted with CBP-KIX and was phosphorylated by MAPK in COS7 cells. The activation of PKA-MAPK signaling induced the nuclear localization of MKL2 and increased SRF-dependent transcriptional activity in neurons. These results demonstrate that dopamine signaling regulates the interaction of MKL2 with CBP in a phosphorylation-dependent manner and thereby controls SRF-dependent gene expression. Cover Image for this issue: https://doi.org/10.1111/jnc.15067.
Asunto(s)
Cuerpo Estriado/metabolismo , Espacio Intracelular/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor de Respuesta Sérica/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional/fisiología , Animales , Células COS , Chlorocebus aethiops , Cocaína/farmacología , Cuerpo Estriado/química , Cuerpo Estriado/efectos de los fármacos , Inhibidores de Captación de Dopamina/farmacología , Femenino , Células HEK293 , Humanos , Espacio Intracelular/química , Espacio Intracelular/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Proteínas Quinasas Activadas por Mitógenos/análisis , Técnicas de Cultivo de Órganos , Embarazo , Factor de Respuesta Sérica/análisis , Factores de Transcripción/análisis , Activación Transcripcional/efectos de los fármacos , XenopusRESUMEN
BACKGROUND: Heart failure is a complex syndrome that results from structural or functional impairment of ventricular filling or blood ejection. Protein phosphorylation is a major and essential intracellular mechanism that mediates various cellular processes in cardiomyocytes in response to extracellular and intracellular signals. The RHOA-associated protein kinase (ROCK/Rho-kinase), an effector regulated by the small GTPase RHOA, causes pathological phosphorylation of proteins, resulting in cardiovascular diseases. RHOA also activates protein kinase N (PKN); however, the role of PKN in cardiovascular diseases remains unclear. METHODS: To explore the role of PKNs in heart failure, we generated tamoxifen-inducible, cardiomyocyte-specific PKN1- and PKN2-knockout mice by intercrossing the αMHC-CreERT2 line with Pkn1flox/flox and Pkn2flox/flox mice and applied a mouse model of transverse aortic constriction- and angiotensin II-induced heart failure. To identify a novel substrate of PKNs, we incubated GST-tagged myocardin-related transcription factor A (MRTFA) with recombinant GST-PKN-catalytic domain or GST-ROCK-catalytic domain in the presence of radiolabeled ATP and detected radioactive GST-MRTFA as phosphorylated MRTFA. RESULTS: We demonstrated that RHOA activates 2 members of the PKN family of proteins, PKN1 and PKN2, in cardiomyocytes of mice with cardiac dysfunction. Cardiomyocyte-specific deletion of the genes encoding Pkn1 and Pkn2 (cmc-PKN1/2 DKO) did not affect basal heart function but protected mice from pressure overload- and angiotensin II-induced cardiac dysfunction. Furthermore, we identified MRTFA as a novel substrate of PKN1 and PKN2 and found that MRTFA phosphorylation by PKN was considerably more effective than that by ROCK in vitro. We confirmed that endogenous MRTFA phosphorylation in the heart was induced by pressure overload- and angiotensin II-induced cardiac dysfunction in wild-type mice, whereas cmc-PKN1/2 DKO mice suppressed transverse aortic constriction- and angiotensin II-induced phosphorylation of MRTFA. Although RHOA-mediated actin polymerization accelerated MRTFA-induced gene transcription, PKN1 and PKN2 inhibited the interaction of MRTFA with globular actin by phosphorylating MRTFA, causing increased serum response factor-mediated expression of cardiac hypertrophy- and fibrosis-associated genes. CONCLUSIONS: Our results indicate that PKN1 and PKN2 activation causes cardiac dysfunction and is involved in the transition to heart failure, thus providing unique targets for therapeutic intervention for heart failure.
Asunto(s)
Actinas/metabolismo , Insuficiencia Cardíaca/enzimología , Miocitos Cardíacos/enzimología , Proteína Quinasa C/metabolismo , Transactivadores/metabolismo , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos Cardíacos/patología , Fosforilación , Unión Proteica , Proteína Quinasa C/deficiencia , Proteína Quinasa C/genética , Transducción de Señal , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Genetically-encoded biosensors based on Förster/fluorescence resonance energy transfer (FRET) are versatile tools for studying the spatio-temporal regulation of signaling molecules within not only the cells but also tissues. Perhaps the hardest task in the development of a FRET biosensor for protein kinases is to identify the kinase-specific substrate peptide to be used in the FRET biosensor. To solve this problem, we took advantage of kinase-interacting substrate screening (KISS) technology, which deduces a consensus substrate sequence for the protein kinase of interest. Here, we show that a consensus substrate sequence for ROCK identified by KISS yielded a FRET biosensor for ROCK, named Eevee-ROCK, with high sensitivity and specificity. By treating HeLa cells with inhibitors or siRNAs against ROCK, we show that a substantial part of the basal FRET signal of Eevee-ROCK was derived from the activities of ROCK1 and ROCK2. Eevee-ROCK readily detected ROCK activation by epidermal growth factor, lysophosphatidic acid, and serum. When cells stably-expressing Eevee-ROCK were time-lapse imaged for three days, ROCK activity was found to increase after the completion of cytokinesis, concomitant with the spreading of cells. Eevee-ROCK also revealed a gradual increase in ROCK activity during apoptosis. Thus, Eevee-ROCK, which was developed from a substrate sequence predicted by the KISS technology, will pave the way to a better understanding of the function of ROCK in a physiological context.
Asunto(s)
Técnicas Biosensibles , Quinasas Asociadas a rho/metabolismo , Secuencia de Aminoácidos , Western Blotting , Transferencia Resonante de Energía de Fluorescencia , Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Microscopía Fluorescente , Fosforilación , Plásmidos/genética , Plásmidos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Especificidad por Sustrato , Imagen de Lapso de Tiempo , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genéticaRESUMEN
Protein phosphorylation plays an important role in the physiological regulation of cardiac function. Myocardial contraction and pathogenesis of cardiac diseases have been reported to be associated with adaptive or maladaptive protein phosphorylation; however, phosphorylation signaling in the heart is not fully elucidated. We recently developed a novel kinase-interacting substrate screening (KISS) method for exhaustive screening of protein kinase substrates, using mass spectrometry and affinity chromatography. First, we examined protein phosphorylation by extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), which has been relatively well studied in cardiomyocytes. The KISS method showed that ERK and PKA mediated the phosphorylation of known cardiac-substrates of each kinase such as Rps6ka1 and cTnI, respectively. Using this method, we found about 330 proteins as Rho-kinase-mediated substrates, whose substrate in cardiomyocytes is unknown. Among them, CARP/Ankrd1, a muscle ankyrin repeat protein, was confirmed as a novel Rho-kinase-mediated substrate. We also found that non-phosphorylatable form of CARP repressed cardiac hypertrophy-related gene Myosin light chain-2v (MLC-2v) promoter activity, and decreased cell size of heart derived H9c2 myoblasts more efficiently than wild type-CARP. Thus, focused proteomics enable us to reveal a novel signaling pathway in the heart.
Asunto(s)
Miocardio/enzimología , Proteómica , Transducción de Señal , Quinasas Asociadas a rho/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Animales , Encéfalo/metabolismo , Células Cultivadas , Cromatografía de Afinidad , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Inmunohistoquímica , Espectrometría de Masas , Microscopía Fluorescente , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosforilación , Unión Proteica , Ratas , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Especificidad por Sustrato , Quinasas Asociadas a rho/químicaRESUMEN
Protein phosphorylation is a major and essential post-translational modification in eukaryotic cells that plays a critical role in various cellular processes. Recent progresses in mass spectrometry techniques have enabled the effective identification and analysis of protein phosphorylation. Mass spectrometry-based approaches in investigating protein phosphorylation are very powerful and informative and can further improve our understanding of protein phosphorylation as a whole, but they cannot determine the upstream kinases involved. We introduce several studies that attempted to uncover the relationships between various kinases of interest and substrates, including two methods we developed: an in vitro approach termed the kinase-interacting substrate screening (KISS) method and an in vivo approach termed the phosphatase inhibitor and kinase inhibitor substrate screening (PIKISS) method. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.
Asunto(s)
Inhibidores de Proteínas Quinasas/química , Proteómica , Quinasas Asociadas a rho/metabolismo , Secuencia de Aminoácidos , Espectrometría de Masas , Fosforilación/efectos de los fármacos , Especificidad por Sustrato , Quinasas Asociadas a rho/químicaRESUMEN
Protein phosphorylation plays a key role in regulating nearly all intracellular biological events. However, poorly developed phospho-specific antibodies and low phosphoprotein abundance make it difficult to study phosphoproteins. Cellular protein phosphorylation data have been obtained using phosphoproteomic approaches, but the detection of low-abundance or fast-cycling phosphorylation sites remains a challenge. Enrichment of phosphoproteins together with phosphopeptides may greatly enhance the spectrum of low-abundance but biologically important phosphoproteins. Previously, we used 14-3-3ζ to selectively enrich for HeLa cell lysate phosphoproteins. However, because 14-3-3 does not isolate phosphoproteins lacking the 14-3-3-binding motif, we looked for other domains that could complementarily enrich for phosphoproteins. We here assessed and characterized the phosphoprotein binding domains Pin1-WW, CHEK2-FHA, and DLG1-GK. Using a strategy based on affinity chromatography, phosphoproteins were collected from the lysates of HeLa cells treated with phosphatase inhibitor or cAMP activator. We identified different subsets of phosphoproteins associated with WW or FHA after calyculin A, okadaic acid, or forskolin treatment. Our Kinase-Oriented Substrate Screening (KiOSS) method, which used phosphoprotein-binding domains, showed that WW and FHA are applicable and useful for the identification of novel phospho-substrates for kinases and can therefore be used as biological filters for comprehensive phosphoproteome analysis.
Asunto(s)
Proteínas 14-3-3/química , Cromatografía de Afinidad/métodos , Fosfoproteínas/aislamiento & purificación , Proteómica/métodos , Secuencia de Aminoácidos , Extractos Celulares/química , Colforsina/farmacología , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células HeLa , Humanos , Toxinas Marinas , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Ácido Ocadaico/farmacología , Oxazoles/farmacología , Fosforilación/efectos de los fármacos , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
Protein kinase A (PKA) is a serine/threonine kinase whose activity depends on the levels of cyclic AMP (cAMP). PKA plays essential roles in numerous cell types such as myocytes and neurons. Numerous substrate screens have been attempted to clarify the entire scope of the PKA signaling cascade, but it is still underway. Here, we performed a comprehensive screen that consisted of immunoprecipitation and mass spectrometry, with a focus on the identification of PKA substrates. The lysate of HeLa cells treated with Forskolin (FSK)/3-isobutyl methyl xanthine (IBMX) and/or H-89 was subjected to immunoprecipitation using anti-phospho-PKA substrate antibody. The identity of the phosophoproteins and phosphorylation sites in the precipitants was determined using liquid chromatography tandem mass spectrometry (LC/MS/MS). We obtained 112 proteins as candidate substrates and 65 candidate sites overall. Among the candidate substrates, Rho-kinase/ROCK2 was confirmed to be a novel substrate of PKA both in vitro and in vivo. In addition to Rho-kinase, we found more than a hundred of novel candidate substrates of PKA using this screen, and these discoveries provide us with new insights into PKA signaling.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteómica , 1-Metil-3-Isobutilxantina/farmacología , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Colforsina/farmacología , Células HeLa , Humanos , Inmunoprecipitación , Isoquinolinas/farmacología , Espectrometría de Masas , Fosforilación/efectos de los fármacos , Unión Proteica , Sulfonamidas/farmacología , Quinasas Asociadas a rho/química , Quinasas Asociadas a rho/metabolismoRESUMEN
Cdk5 is a member of the cyclin-dependent kinase (Cdk) family that plays a role in various neuronal activities including brain development, synaptic regulation, and neurodegeneration. Cdk5 requires the neuronal specific activators, p35 and p39 for subcellular compartmentalization. However, it is not known how active Cdk5 is recruited to F-actin cytoskeleton, which is a Cdk5 target. Here we found p35 and p39 localized to F-actin rich regions of the plasma membrane and investigated the underlying targeting mechanism in vitro by expressing them with Rho family GTPases in Neuro2A cells. Both p35 and p39 accumulated at the cell peripheral lamellipodia and perinuclear regions, where active Rac1 is localized. Interestingly, p35 and p39 displayed different localization patterns as p35 was found more at the perinuclear region and p39 was found more in peripheral lamellipodia. We then confirmed this distinct localization in primary hippocampal neurons. We also determined that the localization of p39 to lamellipodia requires myristoylation and Lys clusters within the N-terminal p10 region. Additionally, we found that p39-Cdk5, but not p35-Cdk5 suppressed lamellipodia formation by reducing Rac1 activity. These results suggest that p39-Cdk5 has a dominant role in Rac1-dependent lamellipodial activity.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Seudópodos/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Actinas/metabolismo , Animales , Células Cultivadas , Quinasa 5 Dependiente de la Ciclina/metabolismo , Embrión de Mamíferos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Conos de Crecimiento/metabolismo , Hipocampo/citología , Inmunoprecipitación , Ratones , Mutación/genética , Proteínas del Tejido Nervioso/genética , Neuroblastoma/patología , Neuronas/citología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ratas , TransfecciónRESUMEN
Protein phosphorylation, a key regulator of cellular processes, plays a central role in brain function and is implicated in neurological disorders. Information on protein phosphorylation is expected to be a clue for understanding various neuropsychiatric disorders and developing therapeutic strategies. Nonetheless, existing databases lack a specific focus on phosphorylation events in the brain, which are crucial for investigating the downstream pathway regulated by neurotransmitters. To overcome the gap, we have developed a web-based database named "Kinase-Associated Neural PHOspho-Signaling (KANPHOS)." This paper presents the design concept, detailed features, and a series of improvements for KANPHOS. KANPHOS is designed to support data-driven research by fulfilling three key objectives: (1) enabling the search for protein kinases and their substrates related to extracellular signals or diseases; (2) facilitating a consolidated search for information encompassing phosphorylated substrate genes, proteins, mutant mice, diseases, and more; and (3) offering integrated functionalities to support pathway and network analysis. KANPHOS is also equipped with API functionality to interact with external databases and analysis tools, enhancing its utility in data-driven investigations. Those key features represent a critical step toward unraveling the complex landscape of protein phosphorylation in the brain, with implications for elucidating the molecular mechanisms underlying neurological disorders. KANPHOS is freely accessible to all researchers at https://kanphos.jp.
RESUMEN
A polarity complex of PAR-3, PAR-6, and atypical protein kinase C (aPKC) functions in various cell polarization events. PAR-3 directly interacts with Tiam1/Taim2 (STEF), Rac1-specific guanine nucleotide exchange factors, and forms a complex with aPKC-PAR-6-Cdc42*GTP, leading to Rac1 activation. RhoA antagonizes Rac1 in certain types of cells. However, the relationship between RhoA and the PAR complex remains elusive. We found here that Rho-kinase/ROCK/ROK, the effector of RhoA, phosphorylated PAR-3 at Thr833 and thereby disrupted its interaction with aPKC and PAR-6, but not with Tiam2. Phosphorylated PAR-3 was observed in the leading edge, and in central and rear portions of migrating cells having front-rear polarity. Knockdown of PAR-3 by small interfering RNA (siRNA) impaired cell migration, front-rear polarization, and PAR-3-mediated Rac1 activation, which were recovered with siRNA-resistant PAR-3, but not with the phospho-mimic PAR-3 mutant. We propose that RhoA/Rho-kinase inhibits PAR complex formation through PAR-3 phosphorylation, resulting in Rac1 inactivation.
Asunto(s)
Proteínas Portadoras/metabolismo , Complejos Multiproteicos/metabolismo , Quinasas Asociadas a rho/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Proteínas Portadoras/química , Movimiento Celular , Polaridad Celular , Chlorocebus aethiops , Activación Enzimática , Células HeLa , Humanos , Datos de Secuencia Molecular , Fosforilación , Fosfotreonina/metabolismo , Seudópodos/metabolismo , Ratas , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Dopamine regulates psychomotor function by D1 receptor/PKA-dependent phosphorylation of DARPP-32. DARPP-32, phosphorylated at Thr34 by PKA, inhibits protein phosphatase 1 (PP1), and amplifies the phosphorylation of other PKA/PP1 substrates following D1 receptor activation. In addition to the D1 receptor/PKA/DARPP-32 signaling pathway, D1 receptor stimulation is known to activate Rap1/ERK signaling. Rap1 activation is mediated through the phosphorylation of Rasgrp2 (guanine nucleotide exchange factor; activation) and Rap1gap (GTPase-activating protein; inhibition) by PKA. In this study, we investigated the role of PP1 inhibition by phospho-Thr34 DARPP-32 in the D1 receptor-induced phosphorylation of Rasgrp2 and Rap1gap at PKA sites. The analyses in striatal and NAc slices from wild-type and DARPP-32 knockout mice revealed that the phosphorylation of Rasgrp2 at Ser116/Ser117 and Ser586, but not of Rasgrp2 at Ser554 or Rap1gap at Ser441 or Ser499 induced by a D1 receptor agonist, is under the control of the DARPP-32/PP1. The results were supported by pharmacological analyses using a selective PP1 inhibitor, tautomycetin. In addition, analyses using a PP1 and PP2A inhibitor, okadaic acid, revealed that all sites of Rasgrp2 and Rap1gap were regulated by PP2A. Thus, the interactive machinery of DARPP-32/PP1 may contribute to efficient D1 receptor signaling via Rasgrp2/Rap1 in the striatum.
Asunto(s)
Cuerpo Estriado , Neostriado , Animales , Ratones , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 1/farmacología , Cuerpo Estriado/metabolismo , Neostriado/metabolismo , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Transducción de Señal , Fosforilación , Receptores de Dopamina D1/metabolismoRESUMEN
The small GTPase RhoA is a molecular switch in various extracellular signals. Rho-kinase/ROCK/ROK, a major effector of RhoA, regulates diverse cellular functions by phosphorylating cytoskeletal proteins, endocytic proteins, and polarity proteins. More than twenty Rho-kinase substrates have been reported, but the known substrates do not fully explain the Rho-kinase functions. Herein, we describe the comprehensive screening for Rho-kinase substrates by treating HeLa cells with Rho-kinase and phosphatase inhibitors. The cell lysates containing the phosphorylated substrates were then subjected to affinity chromatography using beads coated with 14-3-3 protein, which interacts with proteins containing phosphorylated serine or threonine residues, to enrich the phosphorylated proteins. The identities of the molecules and phosphorylation sites were determined by liquid chromatography tandem mass spectrometry (LC/MS/MS) after tryptic digestion and phosphopeptide enrichment. The phosphorylated proteins whose phosphopeptide ion peaks were suppressed by treatment with the Rho-kinase inhibitor were regarded as candidate substrates. We identified 121 proteins as candidate substrates. We also identified phosphorylation sites in Partitioning defective 3 homolog (Par-3) at Ser143 and Ser144. We found that Rho-kinase phosphorylated Par-3 at Ser144 both in vitro and in vivo. The method used in this study would be applicable and useful to identify novel substrates of other kinases.
Asunto(s)
Proteínas 14-3-3/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteína de Unión al GTP rhoA/metabolismo , Proteínas 14-3-3/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Cromatografía de Afinidad , Regulación de la Expresión Génica , Células HeLa , Humanos , Proteínas de la Membrana/genética , Ratones , Microesferas , Fosfoproteínas/genética , Fosforilación , Plásmidos , Proteómica , Ratas , Transducción de Señal , Transfección , Proteína de Unión al GTP rhoA/genéticaRESUMEN
The small GTP-binding protein Rho plays a crucial role in a wide variety of cellular functions through various effector proteins. Rho-kinase is a key effector protein of Rho, which is composed of two isoforms, ROCK1 and ROCK2. To clarify the site of action of ROCK1 and ROCK2, we performed immunofluorescence and immunoelectron microscopic analyses using isoform-specific antibodies in mouse tissues. In the large and small intestines, ROCK1 immunoreactivity was predominantly identified in epithelial cells, and ROCK2 immunoreactivity was negligible. In these epithelial cells, ROCK1 immunoreactivity was distributed on plasma membranes, while ROCK1 immunogold signals were localized at cell-cell contacts and cell adhesion sites, especially at the adherens junctions at the ultrastructural level. In the bladder epithelium, however, ROCK1 and ROCK2 signals were identified at intermediate filaments, and ROCK2 signals were also observed in nuclei. In the three types of muscular cells-smooth, cardiac, and skeletal muscle cells-ROCK1 and ROCK2 also showed differential distribution. ROCK1 signals were localized at actin filaments, plasma membranes, and vesicles near plasma membranes in smooth muscle cells; at the lysosomes in skeletal muscle cells; and were undetectable in cardiac muscle cells. ROCK2 signals were localized at actin filaments and centrosomes in smooth muscle cells, at intercalated discs in cardiac muscle cells, and at Z-discs and sarcoplasmic reticulum in skeletal muscle cells. In the brain, ROCK1 immunoreactivity was distributed in glia, whereas ROCK2 immunoreactivity was observed in neurons. These results indicate that the two isoforms of Rho-kinase distribute differentially to accomplish their specific functions.
Asunto(s)
Encéfalo/enzimología , Epitelio/enzimología , Músculos/enzimología , Quinasas Asociadas a rho/metabolismo , Animales , Encéfalo/citología , Células HeLa , Humanos , Espacio Intracelular/enzimología , Ratones , Ratones Endogámicos C57BL , Músculos/citología , Especificidad de Órganos , Transporte de ProteínasRESUMEN
Protein kinases exert physiological functions through phosphorylating their specific substrates; however, the mode of kinase-substrate recognition is not fully understood. Rho-kinase is a Ser/Thr protein kinase that regulates cytoskeletal reorganization through phosphorylating myosin light chain (MLC) and the myosin phosphatase targeting subunit 1 (MYPT1) of MLC phosphatase (MLCP) and is involved in various diseases, due to its aberrant cellular contraction, morphology, and movement. Despite the importance of the prediction and identification of substrates and phosphorylation sites, understanding of the precise regularity in phosphorylation preference of Rho-kinase remains far from satisfactory. Here we analyzed the Rho-kinase-MYPT1 interaction, to understand the mode of Rho-kinase substrate recognition and found that the three short regions of MYPT1 close to phosphorylation sites (referred to as docking motifs (DMs); DM1 (DLQEAEKTIGRS), DM2 (KSQPKSIRERRRPR), and DM3 (RKARSRQAR)) are important for interactions with Rho-kinase. The phosphorylation levels of MYPT1 without DMs were reduced, and the effects were limited to the neighboring phosphorylation sites. We further demonstrated that the combination of pseudosubstrate (PS) and DM of MYPT1 (PS1 + DM3 and PS2 + DM2) serves as a potent inhibitor of Rho-kinase. The present information will be useful in identifying new substrates and developing selective Rho-kinase inhibitors.
Asunto(s)
Cadenas Ligeras de Miosina , Quinasas Asociadas a rho , Cadenas Ligeras de Miosina/metabolismo , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Fosforilación , Quinasas Asociadas a rho/metabolismoRESUMEN
Dysfunctional dopamine signaling is implicated in various neuropsychological disorders. Previously, we reported that dopamine increases D1 receptor (D1R)-expressing medium spiny neuron (MSN) excitability and firing rates in the nucleus accumbens (NAc) via the PKA/Rap1/ERK pathway to promote reward behavior. Here, the results show that the D1R agonist, SKF81297, inhibits KCNQ-mediated currents and increases D1R-MSN firing rates in murine NAc slices, which is abolished by ERK inhibition. In vitro ERK phosphorylates KCNQ2 at Ser414 and Ser476; in vivo, KCNQ2 is phosphorylated downstream of dopamine signaling in NAc slices. Conditional deletion of Kcnq2 in D1R-MSNs reduces the inhibitory effect of SKF81297 on KCNQ channel activity, while enhancing neuronal excitability and cocaine-induced reward behavior. These effects are restored by wild-type, but not phospho-deficient KCNQ2. Hence, D1R-ERK signaling controls MSN excitability via KCNQ2 phosphorylation to regulate reward behavior, making KCNQ2 a potential therapeutical target for psychiatric diseases with a dysfunctional reward circuit.