RESUMEN
Liprin-α1 is a widely expressed scaffolding protein responsible for regulating cellular processes such as focal adhesion, cell motility, and synaptic transmission. Liprin-α1 interacts with many proteins including ELKS, GIT1, liprin-ß, and LAR-family receptor tyrosine protein phosphatase. Through these protein-protein interactions, liprin-α1 assembles large higher-order molecular complexes; however, the regulation of this complex assembly/disassembly is unknown. Liquid-liquid phase separation (LLPS) is a process that concentrates proteins within cellular nano-domains to facilitate efficient spatiotemporal signaling in response to signaling cascades. While there is no report that liprin-α1 spontaneously undergoes LLPS, we found that GFP-liprin-α1 expressed in HEK293 cells occasionally forms droplet-like condensates. MS-based interactomics identified Protein Phosphatase 2A (PP2A)/B56δ (PPP2R5D) trimers as specific interaction partners of liprin-α1 through a canonical Short Linear Interaction Motif (SLiM) in its N-terminal dimerization domain. Mutation of this SLiM nearly abolished PP2A interaction, and resulted in significantly increased LLPS. GFP-liprin-α1 showed significantly increased droplet formation in HEK293 cells devoid of B56δ (PPP2R5D knockout), suggesting that PPP2R5D/PP2A holoenzyme inhibits liprin-α1 LLPS. Guided by reported liprin-α1 Ser/Thr phosphorylation sites, we found liprin-α1 phospho-mimetic mutant at serine 763 (S763E) is sufficient to drive its LLPS. Domain mapping studies of liprin-α1 indicated that the intrinsically disordered region, the N-terminal dimerization domain, and the SAM domains are all necessary for liprin-α1 LLPS. Finally, expression of p.E420K, a human PPP2R5D variant causing Houge-Janssens Syndrome type 1 (also known as Jordan's Syndrome), significantly compromised suppression of liprin-α1 LLPS. Our work identified B56δ-PP2A holoenzyme as an inhibitor of liprin-α1 LLPS via regulation at multiple phosphorylation sites.
RESUMEN
Precise regulation of protein phosphorylation is critical for many cellular processes, and dysfunction in this process has been linked to various neurological disorders and diseases. Protein phosphatase 1 (PP1) is a ubiquitously expressed serine/threonine phosphatase with three major isoforms, (α, ß, γ) and hundreds of known substrates. Previously, we reported that PP1α and PP1γ are essential for the known role of PP1 in synaptic physiology and learning/memory, while PP1ß displayed a surprising opposing function. De novo mutations in PP1ß cause neurodevelopmental disorders in humans, but the mechanisms involved are currently unknown. A Cre-Lox system was used to delete PP1ß specifically in neurons in order to study its effects on developing mice. These animals fail to survive to 3 postnatal weeks, and exhibit deficits in cortical myelination and glutamate release. There was defective compound action potential (CAP) propagation in the optic nerve of the null mice, which was traced to a deficit in the formation of nodes of Ranvier. Finally, it was found that phosphorylation of the PP1ß-specific substrate, myosin light chain 2 (MLC2), is significantly enhanced in PP1ß null optic nerves. Several novel important in vivo roles of PP1ß in neurons were discovered, and these data will aid future investigations in delineating the mechanisms by which de novo mutations in PP1ß lead to intellectual and developmental delays in patients.
RESUMEN
Protein phosphatase 1 (PP1) regulates synaptic plasticity and has been described as a molecular constraint on learning and memory. There are three neuronal isoforms, PP1α, PP1ß, and PP1γ, but little is known about their individual functions. PP1α and PP1γ are assumed to mediate the effects of PP1 on learning and memory based on their enrichment at dendritic spines and their preferential binding to neurabin and spinophilin, major PP1 synaptic scaffolding proteins. However, it was recently discovered that human de novo PP1ß mutations cause intellectual disability, suggesting an important but ill-defined role for PP1ß. In this study, we investigated the functions of each PP1 isoform in hippocampal synaptic physiology using conditional CA1-specific knockout mice. In stark contrast to classic PP1 function, we found that PP1ß promotes synaptic plasticity as well as spatial memory. These changes in synaptic plasticity and memory are accompanied by changes in GluA1 phosphorylation, GluN2A levels, and dendritic spine density and morphology, including silent synapse number. These functions of PP1ß reveal a previously unidentified signaling pathway regulating spine maturation and plasticity, broadening our understanding of the complex role of PP1 in synaptic physiology.
RESUMEN
Reversible phosphorylation is a fundamental regulatory mechanism required for many biological processes and is coordinated by the opposing actions of protein kinases and phosphatases. Protein phosphatase 1 (PP1) is a major protein phosphatase that plays an important role in many fundamental physiological processes including synaptic transmission and memory formation. Here we investigate the regulation of PP1 by prominent signaling proteins and synaptic scaffolds including GSK3ß, inhibitor-2 (I-2), neurabin (Nrb), and actin. While GSK3ß is known to regulate PP1 via phosphorylation of the PP1-binding protein I-2, we found that GSK3ß directly regulates PP1 via inhibitory phosphorylation in neurons. Additionally, using bioluminescence resonance energy transfer (BRET), we found that GSK3ß alters PP1-I-2 interaction in living cells. The effect of GSK3ß on PP1-I-2 interaction is independent of the PP1 C-terminal tail, contrary to predictions based on previous findings from purified proteins. I-2 has been shown to form a trimeric complex with PP1 and Nrb, a major synaptic scaffold for promoting PP1 localization to the actin cytoskeleton. Utilizing BRET, we found that Nrb promotes PP1-actin interaction, however no BRET was detected between I-2 and F-actin. Finally, we found that stabilizing F-actin promotes Nrb-PP1 binding and may also lead to conformational changes between Nrb-I-2 and Nrb-F-actin complexes. Overall, our findings elaborate the dynamic regulation of PP1 complexes by GSK3ß, targeting proteins, and actin polymerization.
Asunto(s)
Citoesqueleto de Actina , Actinas , Proteína Fosfatasa 1/metabolismo , Actinas/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Citoesqueleto de Actina/metabolismo , FosforilaciónRESUMEN
Inhibitor-2 (I-2) is a prototypic inhibitor of protein phosphatase-1 (PP1), a major serine-threonine phosphatase that regulates synaptic plasticity and learning and memory. Although I-2 is a potent inhibitor of PP1 in vitro, our previous work has elucidated that, in vivo, I-2 may act as a positive regulator of PP1. Here we show that I-2 and PP1γ, but not PP1α, positively regulate synaptic transmission in hippocampal neurons. Moreover, we demonstrated that I-2 enhanced PP1γ interaction with its major synaptic scaffold, neurabin, by Förster resonance energy transfer (FRET)/Fluorescence lifetime imaging microscopy (FLIM) studies, while having a limited effect on PP1 auto-inhibitory phosphorylation. Furthermore, our study indicates that the effect of I-2 on PP1 activity in vivo is dictated by I-2 threonine-72 phosphorylation. Our work thus demonstrates a molecular mechanism by which I-2 positively regulates PP1 function in synaptic transmission.
RESUMEN
Nuclear inhibitor of protein phosphatase 1 (NIPP1) is a known regulator of gene expression and plays roles in many physiological or pathological processes such as stem cell proliferation and skin inflammation. While NIPP1 has many regulatory roles in proliferating cells, its function in the central nervous system (CNS) has not been directly investigated. In the present study, we examined NIPP1 CNS function using a conditional knockout (cKO) mouse model in which the Nipp1 gene is excised from neural precursor cells. These mice exhibited severe developmental impairments that led to premature lethality. To delineate the neurological changes occurring in these animals, we first assessed microtubule-associated protein tau, a known target of NIPP1 activity. We found that phosphorylation of tau is significantly enhanced in NIPP1 cKO mice. Consistent with this, we found altered AKT and PP1 activity in NIPP1 cKO mice, suggesting that increased tau phosphorylation likely results from a shift in kinase/phosphatase activity. Secondly, we observed tremors in the NIPP1 cKO mice which prompted us to explore the integrity of the myelin sheath, an integral structure for CNS function. We demonstrated that in NIPP1 cKO mice, there is a significant decrease in MBP protein expression in the cortex, along with deficits in both the conduction of compound action potentials (CAP) and the percentage of myelinated axons in the optic nerve. Our study suggests that NIPP1 in neural precursor cells regulates phosphorylation of tau and CNS myelination and may represent a novel therapeutic target for neurodegenerative diseases.
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Péptidos y Proteínas de Señalización Intracelular , Células-Madre Neurales , Ratones , Animales , Proteína Fosfatasa 1/metabolismo , Fosforilación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células-Madre Neurales/metabolismo , Sistema Nervioso Central/metabolismo , Vaina de Mielina/metabolismoRESUMEN
Arsenic is a well-established carcinogen known to increase mortality, but its effects on the central nervous system are less well understood. Epidemiological studies suggest that early life exposure is associated with learning deficits and behavioral changes. Studies in arsenic-exposed rodents have begun to shed light on potential mechanistic underpinnings, including changes in synaptic transmission and plasticity. However, previous studies relied on extended exposure into adulthood, and little is known about the effect of arsenic exposure in early development. Here, we studied the effects of early developmental arsenic exposure in juvenile mice on synaptic transmission and plasticity in the hippocampus. C57BL/6J females were exposed to arsenic (0, 50 ppb, 36 ppm) via drinking water two weeks prior to mating, with continued exposure throughout gestation and parturition. Electrophysiological recordings were then performed on juvenile offspring prior to weaning. In this paradigm, the offspring are exposed to arsenic indirectly, via the mother. We found that high (36 ppm) and relatively low (50 ppb) arsenic exposure both decreased basal synaptic transmission. A compensatory increase in pre-synaptic vesicular release was only observed in the high-exposure group. These results suggest that indirect, ecologically relevant arsenic exposure in early development impacts hippocampal synaptic transmission and plasticity that could underlie learning deficits reported in epidemiological studies.
RESUMEN
Protein phosphatases, by counteracting protein kinases, regulate the reversible phosphorylation of many substrates involved in synaptic plasticity, a cellular model for learning and memory. A prominent phosphatase regulating synaptic plasticity and neurologic disorders is the serine/threonine protein phosphatase 1 (PP1). PP1 has three isoforms (α, ß, and γ, encoded by three different genes), which are regulated by a vast number of interacting subunits that define their enzymatic substrate specificity. In this review, we discuss evidence showing that PP1 regulates synaptic transmission and plasticity, as well as presenting novel models of PP1 regulation suggested by recent experimental evidence. We also outline the required targeting of PP1 by neurabin and spinophilin to achieve substrate specificity at the synapse to regulate AMPAR and NMDAR function. We then highlight the role of inhibitor-2 in regulating PP1 function in plasticity, including its positive regulation of PP1 function in vivo in memory formation. We also discuss the distinct function of the three PP1 isoforms in synaptic plasticity and brain function, as well as briefly discuss the role of inhibitory phosphorylation of PP1, which has received recent emphasis in the regulation of PP1 activity in neurons.
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Plasticidad Neuronal/fisiología , Proteína Fosfatasa 1/fisiología , Transmisión Sináptica/fisiología , Animales , Humanos , Proteína Fosfatasa 1/química , Estructura Terciaria de Proteína , Receptores de N-Metil-D-Aspartato/fisiología , Transducción de Señal/fisiologíaRESUMEN
Extracellular magnesium ion ([Mg2+]) is a well-known voltage-dependent blocker of NMDA receptors, which plays a critical role in the regulation of neuronal plasticity, learning, and memory. It is generally believed that NMDA receptor activation involves in Mg2+ being removed into extracellular compartment from the channel pore. On the other hand, Mg2+ is one of the most abundant intracellular cations, and involved in numerous cellular functions. However, we do not know if extracellular magnesium ions can influx into neurons to affect intracellular signaling pathways. In our current study, we found that extracellular [Mg2+] elevation enhanced CREB activation by NMDA receptor signaling in both mixed sex rat cultured neurons and brain slices. Moreover, we found that extracellular [Mg2+] led to CREB activation by NMDA application, albeit in a delayed manner, even in the absence of extracellular calcium, suggesting a potential independent role of magnesium in CREB activation. Consistent with this, we found that NMDA application leads to an NMDAR-dependent increase in intracellular-free [Mg2+] in cultured neurons in the absence of extracellular calcium. Chelating this magnesium influx or inhibiting P38 mitogen-activated protein kinase (p38 MAPK) blocked the delayed pCREB by NMDA. Finally, we found that NMDAR signaling in the absence of extracellular calcium activates p38 MAPK. Our studies thus indicate that magnesium influx, dependent on NMDA receptor opening, can transduce a signaling pathway to activate CREB in neurons.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Magnesio/metabolismo , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacos , Animales , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Masculino , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
Protein phosphatase-1 (PP1) constrains learning and memory formation in part through its effects on the induction threshold of long-term potentiation (LTP) and depression (LTD). LTD induction requires both the enzymatic activity of PP1 and its proper anchoring to synaptic spines. We have shown previously that neurabin, a major synaptic scaffolding protein, targets PP1 to synapses for LTD induction. Here, we show that PP1 bound on spinophilin, a close homolog of neurabin and another major synaptic PP1 anchoring protein, does not play a role in LTD induction, which suggests that neurabin plays a privileged role in nanodomain targeting of PP1 in LTD induction. We found that protein kinase A can significantly weaken the neurabin-PP1 interaction in neurons via phosphorylation of neurabin at serine 461, a phosphorylation site adjacent to the PP1-binding motif that is not conserved in spinophilin. Finally, we found that a neurabin mutation (S461E), which mimics phosphorylation, blocked AMPA receptor endocytosis and LTD induction. The results indicate the critical importance of nanodomain targeting of PP1 within synaptic spines and its regulation in LTD induction.
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Potenciación a Largo Plazo , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteína Fosfatasa 1/metabolismo , Receptores AMPA/metabolismo , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Endocitosis , Activación Enzimática , Células HEK293 , Humanos , Proteínas Mutantes/metabolismo , Neuronas/metabolismo , Fosforilación , Fosfoserina/metabolismo , RatasRESUMEN
Reversible phosphorylation, a fundamental regulatory mechanism required for many biological processes including memory formation, is coordinated by the opposing actions of protein kinases and phosphatases. Type I protein phosphatase (PP1), in particular, has been shown to constrain learning and memory formation. However, how PP1 might be regulated in memory is still not clear. Our previous work has elucidated that PP1 inhibitor-2 (I-2) is an endogenous regulator of PP1 in hippocampal and cortical neurons (Hou et al., 2013). Contrary to expectation, our studies of contextual fear conditioning and novel object recognition in I-2 heterozygous mice suggest that I-2 is a memory suppressor. In addition, lentiviral knock-down of I-2 in the rat dorsal hippocampus facilitated memory for tasks dependent on the hippocampus. Our data indicate that I-2 suppresses memory formation, probably via negatively regulating the phosphorylation of cAMP/calcium response element-binding protein (CREB) at serine 133 and CREB-mediated gene expression in dorsal hippocampus. Surprisingly, the data from both biochemical and behavioral studies suggest that I-2, despite its assumed action as a PP1 inhibitor, is a positive regulator of PP1 function in memory formation. SIGNIFICANCE STATEMENT: We found that inhibitor-2 acts as a memory suppressor through its positive functional influence on type I protein phosphatase (PP1), likely resulting in negative regulation of cAMP/calcium response element-binding protein (CREB) and CREB-activated gene expression. Our studies thus provide an interesting example of a molecule with an in vivo function that is opposite to its in vitro function. PP1 plays critical roles in many essential physiological functions such as cell mitosis and glucose metabolism in addition to its known role in memory formation. PP1 pharmacological inhibitors would thus not be able to serve as good therapeutic reagents because of its many targets. However, identification of PP1 inhibitor-2 as a critical contributor to suppression of memory formation by PP1 may provide a novel therapeutic target for memory-related diseases.
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Memoria/fisiología , Proteína Fosfatasa 1/antagonistas & inhibidores , Proteína Fosfatasa 1/fisiología , Proteínas/fisiología , Animales , Células Cultivadas , Femenino , Hipocampo/fisiología , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , RatasRESUMEN
NMDA receptor signaling plays a complex role in CREB activation and CREB-mediated gene transcription, depending on the subcellular location of NMDA receptors, as well as how strongly they are activated. However, it is not known whether Rac1, the prototype of Rac GTPase, plays a role in neuronal CREB activation induced by NMDA receptor signaling. Here, we report that NSC23766, a widely used specific Rac1 inhibitor, inhibits basal CREB phosphorylation at S133 (pCREB) and antagonizes changes in pCREB levels induced by NMDA bath application in rat cortical neurons. Unexpectedly, we found that NSC23766 affects the levels of neuronal pCREB in a Rac1-independent manner. Instead, our results indicate that NSC23766 can directly regulate NMDA receptors as indicated by their strong effects on both exogenous and synaptically evoked NMDA receptor-mediated currents in mouse and rat neurons, respectively. Our findings strongly suggest that Rac1 does not affect pCREB signaling in cortical neurons and reveal that NSC23766 could be a novel NMDA receptor antagonist.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Sistemas de Liberación de Medicamentos/métodos , Receptores de N-Metil-D-Aspartato/fisiología , Transducción de Señal/fisiología , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Aminoquinolinas/farmacología , Animales , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Femenino , Masculino , Técnicas de Cultivo de Órganos , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The functional stability of neurons in the face of large variations in both activity and efficacy of synaptic connections suggests that neurons possess intrinsic negative feedback mechanisms to balance and tune excitability. While NMDA receptors have been established to play an important role in glutamate receptor-dependent plasticity through protein dephosphorylation, the effects of synaptic activation on intrinsic excitability are less well characterized. We show that increases in synaptic activity result in dephosphorylation of the potassium channel subunit Kv2.1. This dephosphorylation is induced through NMDA receptors and is executed through protein phosphatase-1 (PP1), an enzyme previously established to play a key role in regulating ligand gated ion channels in synaptic plasticity. Dephosphorylation of Kv2.1 by PP1 in response to synaptic activity results in substantial shifts in the inactivation curve of IK, resulting in a reduction in intrinsic excitability, facilitating negative feedback to neuronal excitability.
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Neuronas/metabolismo , Proteína Fosfatasa 1/metabolismo , Canales de Potasio Shab/metabolismo , Sinapsis/fisiología , Animales , Retroalimentación Fisiológica , Fosforilación , Cultivo Primario de Células , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Homeostatic synaptic downscaling is a negative feedback response to chronic elevated network activity to reduce the firing rate of neurons. This form of synaptic plasticity decreases the strength of individual synapses to the same proportion, or in a multiplicative manner. Because of this, synaptic downscaling has been hypothesized to counter the potential run-away excitation due to Hebbian type of long term potentiation (LTP), while preserving relative synaptic weight encoded in individual synapses and thus memory information. In this article, we will review the current knowledge on the signaling and molecular mechanisms of synaptic downscaling. Specifically, we focus on three general areas. First the functional roles of several immediate early genes such as Plk2, Homer1a, Arc and Narp are discussed. Secondly, we examine the current knowledge on the regulation of synaptic protein levels by ubiquitination and transcriptional repression in synaptic downscaling. Thirdly, we review the dynamics of signaling molecules such as kinases and phosphatases critical for synaptic downscaling, and their regulation of synaptic scaffolding proteins. Finally we briefly discuss the heterogeneity of homeostatic synaptic downscaling mechanisms. This article is part of the Special Issue entitled 'Homeostatic Synaptic Plasticity'.
Asunto(s)
Plasticidad Neuronal , Sinapsis/metabolismo , Animales , Genes Inmediatos-Precoces/fisiología , Homeostasis , Humanos , UbiquitinaciónRESUMEN
Protein phosphorylation plays a critical role in neuronal transcription, translation, cell viability, and synaptic plasticity. In neurons, phospho-enzymes and specific substrates directly link glutamate release and post-synaptic depolarization to these cellular functions; however, many of these enzymes and their protein substrates remain uncharacterized or unidentified. In this article, we identify a novel, synaptically driven neuronal phosphoproteome characterized by a specific motif of serine/threonine-glutamine ([S/T]-Q, abbreviated as SQ). These SQ-containing substrates are predominantly localized to dendrites, synapses, the soma; and activation of this SQ phosphoproteome by bicuculline application is induced via calcium influx through L-type calcium channels. On the other hand, acute application of NMDA can inactivate this SQ phosphoproteome. We demonstrate that the SQ motif kinase Ataxia-telangiectasia mutated can also localize to dendrites and dendritic spines, in addition to other subcellular compartments, and is activated by bicuculline application. Pharmacology studies indicate that Ataxia-telangiectasia mutated and its sister kinase ataxia telangiectasia mutated and Rad3-related up-regulate these neuronal SQ substrates. Phosphoproteomics identified over 150 SQ-containing substrates whose phosphorylation is bidirectionally regulated by synaptic activity.
Asunto(s)
Neuronas/fisiología , Fosfoproteínas/fisiología , Proteómica , Sinapsis/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Calcio/fisiología , Canales de Calcio Tipo L/fisiología , Corteza Cerebral/citología , Inhibidores Enzimáticos/farmacología , Femenino , Masculino , Neuronas/citología , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Fosforilación/fisiología , Embarazo , Cultivo Primario de Células , Proteoma/fisiología , Ratas , Bloqueadores de los Canales de Sodio/farmacología , Sinapsis/efectos de los fármacos , Tetrodotoxina/farmacologíaRESUMEN
The serine/threonine protein phosphatase protein phosphatase 1 (PP1) is known to play an important role in learning and memory by mediating local and downstream aspects of synaptic signaling, but how PP1 activity is controlled in different forms of synaptic plasticity remains unknown. We find that synaptic N-methyl-D-aspartate (NMDA) receptor stimulation in neurons leads to activation of PP1 through a mechanism involving inhibitory phosphorylation at Thr320 by Cdk5. Synaptic stimulation led to proteasome-dependent degradation of the Cdk5 regulator p35, inactivation of Cdk5, and increased auto-dephosphorylation of Thr320 of PP1. We also found that neither inhibitor-1 nor calcineurin were involved in the control of PP1 activity in response to synaptic NMDA receptor stimulation. Rather, the PP1 regulatory protein, inhibitor-2, formed a complex with PP1 that was controlled by synaptic stimulation. Finally, we found that inhibitor-2 was critical for the induction of long-term depression in primary neurons. Our work fills a major gap regarding the regulation of PP1 in synaptic plasticity.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteínas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Encéfalo/metabolismo , Calcineurina/metabolismo , Calcio , Células Cultivadas , Depresión Sináptica a Largo Plazo/fisiología , Plasticidad Neuronal , Neuronas/metabolismo , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño , Ratas , Transducción de Señal , Transmisión Sináptica/fisiologíaRESUMEN
Protein phosphatase-1 (PP1) activity is important for many calcium-dependent neuronal functions including Hebbian synaptic plasticity and learning and memory. PP1 activity is necessary for the induction of long-term depression, whereas downregulation of PP1 activity is required for the normal induction of long-term potentiation. However, how PP1 is activated is not clear. Moreover, it is not known whether PP1 plays a role in homeostatic synaptic scaling, another form of synaptic plasticity which functions to reset the neuronal firing rate in response to chronic neuronal activity perturbations. In this study, we found that PP1 inhibitor-2 (I-2) is phosphorylated at serine 43 (S43) in rat and mouse cortical neurons in response to bicuculine application. Expression of I-2 phosphorylation-blocking mutant I-2 (S43A) blocked the dephosphorylation of GluA2 at serine 880, AMPA receptor trafficking, and synaptic downscaling induced by bicuculline application. Our data suggest that the phosphorylation of I-2 at S43 appears to be mediated by L-type calcium channels and calcium/calmodulin-dependent myosin light-chain kinase. Our work thus reveals a novel calcium-induced PP1 activation pathway critical for homeostatic synaptic plasticity.
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Proteína Fosfatasa 1/metabolismo , Proteínas/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Animales , Animales Recién Nacidos , Canales de Calcio Tipo L/fisiología , Células Cultivadas , Homeostasis/fisiología , Ratones , Ratones Noqueados , Proteína Fosfatasa 1/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Sinapsis/enzimologíaRESUMEN
The molecular mechanism underlying the post-Golgi transport of G protein-coupled receptors (GPCRs) remains poorly understood. Here we determine the role of Rab8 GTPase, which modulates vesicular protein transport between the trans-Golgi network (TGN) and the plasma membrane, in the cell surface targeting of alpha(2B)- and beta(2)-adrenergic receptors (AR). Transient expression of GDP- and GTP-bound Rab8 mutants and short hairpin RNA-mediated knockdown of Rab8 more potently inhibited the cell surface expression of alpha(2B)-AR than beta(2)-AR. The GDP-bound Rab8(T22N) mutant attenuated ERK1/2 activation by alpha(2B)-AR, but not beta(2)-AR, and arrested alpha(2B)-AR in the TGN compartment. Co-immunoprecipitation revealed that both alpha(2B)-AR and beta(2)-AR physically interacted with Rab8 and glutathione S-transferase fusion protein pulldown assays demonstrated that Rab8 interacted with the C termini of both receptors. Interestingly, mutation of the highly conserved membrane-proximal C terminus dileucine motif selectively blocked beta(2)-AR interaction with Rab8, whereas mutation of residues Val(431)-Phe(432)-Asn(433)-Gln(434), Pro(447)-Trp(448), Gln(450)-Thr(451), and Trp(453) in the C terminus impaired alpha(2B)-AR interaction with Rab8. Furthermore, transport inhibition by Rab8(T22N) of a chimeric beta(2)-AR carrying the alpha(2B)-AR C terminus was similar to alpha(2B)-AR. These data provide strong evidence indicating that Rab8 GTPase interacts with distinct motifs in the C termini of alpha(2B)-AR and beta(2)-AR and differentially modulates their traffic from the TGN to the cell surface.
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Receptores Adrenérgicos alfa 2/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Línea Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Células Cultivadas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Immunoblotting , Microscopía Fluorescente , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mutación , Unión Proteica , Transporte de Proteínas , Interferencia de ARN , Ratas , Receptores Adrenérgicos alfa 2/genética , Receptores Adrenérgicos beta 2/genética , Transfección , Proteínas de Unión al GTP rab/genética , Red trans-Golgi/metabolismoRESUMEN
CREB activation via phosphorylation at serine 133 and resulting CREB mediated gene expression is a critical event which can have a significant effect on many cellular processes, including cell survival and plasticity. CREB can be activated by many kinases, for example, it can be phosphorylated by PKA, MAPK, and CaMKIV. The various signaling pathways leading to CREB activation have been extensively studied. On the other hand, CREB is inactivated by PP1 through dephosphorylation at S133 and not much attention has been paid to this aspect of the signaling pathway. It was shown recently that PP1 can be targeted to CREB, for efficient dephosphorylation, through PP1 binding protein HDAC1. In this study, we found that another class-I HDAC family protein, HDAC8, localized in the nucleus of HEK293 cells and also bound to both CREB and PP1. Expression of recombinant HDAC8 results in decreased CREB activation and CREB mediated gene transcription in response to forskolin application. Our study thus elucidated that more than one class-I HDAC family members can regulate the duration of CREB mediated gene transcription.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Histona Desacetilasas/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Línea Celular , Núcleo Celular/enzimología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Genes Reporteros , Histona Desacetilasas/genética , Humanos , Luciferasas/genética , Fosforilación , Proteínas Represoras/genéticaRESUMEN
Filamentous actin binding protein neurabin I (NrbI) targets protein phosphatase-1 (PP1) to specific postsynaptic microdomains, exerting critical control over AMPA receptor (AMPAR)-mediated synaptic transmission. NrbI-targeted synaptic PP1, which promotes synaptic depression upon long-term depression (LTD) stimuli, serves to prevent synaptic depression under basal conditions. The present studies investigate this opposite regulation of AMPAR trafficking during basal synaptic transmission and LTD by expressing NrbI or NrbI mutant, which is defective in PP1 binding, in hippocampal slice or neuron cultures. We find that expression of the NrbI mutant to interfere with PP1 targeting dramatically reduces basal synaptic transmission, which is correlated with the reduction in surface expression of AMPA subtype glutamate receptor (GluR) 1 and GluR2 subunits. Biochemical analysis demonstrates that the NrbI mutant selectively increases the phosphorylation of GluR2 at C-terminal consensus PKC site, serine 880, which is known to favor GluR2 interaction with PDZ (postsynaptic density 95/Discs large/zona occludens 1) protein PICK1 (protein interacting with C kinase-1). Inhibition of PKC activity or GluR2-PICK1 interaction completely reverses the synaptic depression in neurons expressing the NrbI mutant, suggesting that NrbI-targeted synaptic PP1 stabilizes the basal transmission by negatively controlling PKC phosphorylation of GluR2 and the subsequent PICK1-mediated decrease in GluR2-containing AMPAR surface expression. Distinct from basal transmission, blocking GluR2-PICK1 interaction or PKC activity produces minimal effects on LTD in NrbI-expressing neurons. Instead, NrbI-targeted PP1 facilitates LTD by dephosphorylating GluR1 at both serine 845 and serine 831, with GluR2 serine 880 phosphorylation unaltered. Our studies thus elucidate that NrbI-targeted PP1, in response to distinct synaptic activities, regulates the synaptic trafficking of specific AMPAR subunits.