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1.
Hum Mol Genet ; 2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-31943058

RESUMO

The link between mutations associated with intellectual disability (ID) and the mechanisms underlying cognitive dysfunctions remains largely unknown. Here, we focused on PAK3, a serine/threonine kinase whose gene mutations cause X-linked ID. We generated a new mutant mouse model bearing the missense R67C mutation of the Pak3 gene (Pak3-R67C), known to cause moderate to severe ID in humans without other clinical signs and investigated hippocampal-dependent memory and adult hippocampal neurogenesis. Adult male Pak3-R67C mice exhibited selective impairments in long-term spatial memory and pattern separation function, suggestive of altered hippocampal neurogenesis. A delayed-non-matching-to-place paradigm testing memory flexibility and proactive interference, reported here as being adult neurogenesis-dependent, revealed a hypersensitivity to high interference in Pak3-R67C mice. Analyzing adult hippocampal neurogenesis in Pak3-R67C mice reveals no alteration in the first steps of adult neurogenesis, but an accelerated death of a population of adult-born neurons during the critical period of 18-28 days after their birth. We then investigated the recruitment of hippocampal adult-born neurons after spatial memory recall. Post-recall activation of mature dentate granule cells in Pak3-R67C mice was unaffected, but a complete failure of activation of young DCX+ newborn neurons was found, suggesting they were not recruited during the memory task. Decreased expression of the KCC2b chloride co-transporter and altered dendritic development indicate that young adult-born neurons are not fully functional in Pak3-R67C mice. We suggest that these defects in the dynamics and learning-associated recruitment of newborn hippocampal neurons may contribute to the selective cognitive deficits observed in this mouse model of ID.

2.
Neurobiol Dis ; 136: 104709, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31843706

RESUMO

Corpus callosum agenesis (CCA) is a brain malformation associated with a wide clinical spectrum including intellectual disability (ID) and an etiopathological complexity. We identified a novel missense G424R mutation in the X-linked p21-activated kinase 3 (PAK3) gene in a boy presenting with severe ID, microcephaly and CCA and his fetal sibling with CCA and severe hydrocephaly. PAK3 kinase is known to control synaptic plasticity and dendritic spine dynamics but its implication is less characterized in brain ontogenesis. In order to identify developmental functions of PAK3 impacted by mutations responsible for CCA, we compared the biochemical and biological effects of three PAK3 mutations localized in the catalytic domain. These mutations include two "severe" G424R and K389N variants (responsible for severe ID and CCA) and the "mild" A365E variant (responsible for nonsyndromic mild ID). Whereas they suppressed kinase activity, only the two severe variants displayed normal protein stability. Furthermore, they increased interactions between PAK3 and the guanine exchange factor αPIX/ARHGEF6, disturbed adhesion point dynamics and cell spreading, and severely impacted cell migration. Our findings highlight new molecular defects associated with mutations responsible for severe clinical phenotypes with developmental brain defects.

3.
Neurobiol Dis ; 98: 137-148, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27940202

RESUMO

Oligodendrocyte and myelin deficits have been reported in mental/psychiatric diseases. The p21-activated kinase 3 (PAK3), a serine/threonine kinase, whose activity is stimulated by the binding of active Rac and Cdc42 GTPases is affected in these pathologies. Indeed, many mutations of Pak3 gene have been described in non-syndromic intellectual disability diseases. Pak3 is expressed mainly in the brain where its role has been investigated in neurons but not in glial cells. Here, we showed that PAK3 is highly expressed in oligodendrocyte precursors (OPCs) and its expression decreases in mature oligodendrocytes. In the developing white matter of the Pak3 knockout mice, we found defects of oligodendrocyte differentiation in the corpus callosum and to a lesser extent in the anterior commissure, which were compensated at the adult stage. In vitro experiments in OPC cultures, derived from Pak3 knockout and wild type brains, support a developmental and cell-autonomous role for PAK3 in regulating OPC differentiation into mature oligodendrocytes. Moreover, we did not detect any obvious alterations of the proliferation or migration of Pak3 null OPCs compared to wild type. Overall, our data highlight PAK3 as a new regulator of OPC differentiation.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Neurais/metabolismo , Oligodendroglia/metabolismo , Quinases Ativadas por p21/metabolismo , Animais , Comissura Anterior/citologia , Comissura Anterior/crescimento & desenvolvimento , Comissura Anterior/metabolismo , Movimento Celular/fisiologia , Células Cultivadas , Corpo Caloso/citologia , Corpo Caloso/crescimento & desenvolvimento , Corpo Caloso/metabolismo , Masculino , Camundongos Knockout , Células-Tronco Neurais/citologia , Oligodendroglia/citologia , Substância Branca/citologia , Substância Branca/crescimento & desenvolvimento , Substância Branca/metabolismo , Quinases Ativadas por p21/genética
4.
Neurotox Res ; 30(1): 76-87, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26966010

RESUMO

In humans, Down syndrome (DS) is caused by the presence of an extra copy of autosome 21. The most striking finding in DS patients is intellectual disability and the onset of Alzheimer's disease (AD)-like neuropathology in adulthood. Gene overdose is most likely to underlie both developmental impairments, as well as altered neuronal function in DS. Lately, the disruption of cellular signaling and regulatory pathways has been implicated in DS pathophysiology, and many of such pathways may represent common targets for diverse DS-related genes, which could in turn represent attractive therapeutical targets. In this regard, one DS-related gene Down Syndrome Cell Adhesion Molecule (DSCAM), has important functions in neuronal proliferation, maturation, and synaptogenesis. p21-associated kinases (PAKs) appear as a most interesting possibility for study, as DSCAM is known to regulate the PAKs pathway. Hence, in DS, overexpressed DSCAM could deregulate PAKs activity and affect signaling pathways that regulate synaptic plasticity such as dendritic spine dynamics and axon guidance and growth. In the present work, we used an immortalized cell line derived from the cerebral cortex of an animal model of DS such as the trisomy 16 (Ts16) fetal mouse (named CTb), and a similar cell line established from a normal littermate (named CNh), to study the effect of DSCAM in the PAKs pathway. The present study shows that DSCAM is overexpressed in CTb cells by approximately twofold, compared to CNh cells. Congruently, PAK1, as well as its downstream effectors LIMK and cofilin, stay phosphorylated for longer periods after DSCAM activation in the CTb cells, leading to an altered actin dynamics, expressed as an increased basal F/G ratio and reduced neurite growth, in the trisomic condition. The present work presents the correlation between DSCAM gene overexpression and a dysregulation of the PAK pathway, resulting in altered morphological parameters of neuronal plasticity in the trisomic cell line, namely decreased number and length of processes.


Assuntos
Moléculas de Adesão Celular/metabolismo , Síndrome de Down/metabolismo , Neurônios/citologia , Quinases Ativadas por p21/metabolismo , Actinas/metabolismo , Animais , Moléculas de Adesão Celular/genética , Células Cultivadas , Cofilina 1/metabolismo , Modelos Animais de Doenças , Síndrome de Down/genética , Quinases Lim/metabolismo , Camundongos , Fosforilação
5.
Front Cell Neurosci ; 10: 289, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28066185

RESUMO

Interneurons are essential modulators of brain activity and their abnormal maturation may lead to neural and intellectual disabilities. Here we show that cultures derived from murine medial ganglionic eminences (MGEs) produce virtually pure, polarized γ-aminobutyric acid (GABA)-ergic interneurons that can form morphologically identifiable inhibitory synapses. We show that Rac GTPases and a protein complex including the GIT family scaffold proteins are expressed during maturation in vitro, and are required for the normal development of neurites. GIT1 promotes neurite extension in a conformation-dependent manner, while affecting its interaction with specific partners reduces neurite branching. Proteins of the GIT network are concentrated at growth cones, and interaction mutants may affect growth cone behavior. Our findings identify the PIX/GIT1/liprin-α1/ERC1 network as critical for the regulation of interneuron neurite differentiation in vitro, and show that these cultures represent a valuable system to identify the molecular mechanisms driving the maturation of cortical/hippocampal interneurons.

6.
Diabetes ; 63(1): 203-15, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24163148

RESUMO

The transcription factor neurogenin3 (Ngn3) triggers islet cell differentiation in the developing pancreas. However, little is known about the molecular mechanisms coupling cell cycle exit and differentiation in Ngn3(+) islet progenitors. We identified a novel effector of Ngn3 endocrinogenic function, the p21 protein-activated kinase Pak3, known to control neuronal differentiation and implicated in X-linked intellectual disability in humans. We show that Pak3 expression is initiated in Ngn3(+) endocrine progenitor cells and next maintained in maturing hormone-expressing cells during pancreas development as well as in adult islet cells. In Pak3-deficient embryos, the proliferation of Ngn3(+) progenitors and ß-cells is transiently increased concomitantly with an upregulation of Ccnd1. ß-Cell differentiation is impaired at E15.5 but resumes at later stages. Pak3-deficient mice do not develop overt diabetes but are glucose intolerant under high-fat diet (HFD). In the intestine, Pak3 is expressed in enteroendocrine cells but is not necessary for their differentiation. Our results indicate that Pak3 is a novel regulator of ß-cell differentiation and function. Pak3 acts downstream of Ngn3 to promote cell cycle exit and differentiation in the embryo by a mechanism that might involve repression of Ccnd1. In the adult, Pak3 is required for the proper control of glucose homeostasis under challenging HFD.


Assuntos
Glicemia/metabolismo , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Células Secretoras de Insulina/citologia , Pâncreas/citologia , Quinases Ativadas por p21/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Células Enteroendócrinas/citologia , Células Enteroendócrinas/metabolismo , Homeostase/fisiologia , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Pâncreas/embriologia , Pâncreas/metabolismo , Quinases Ativadas por p21/genética
7.
J Biol Chem ; 287(36): 30084-96, 2012 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-22815483

RESUMO

p21-activated kinase 1 (PAK1) and PAK3 belong to group I of the PAK family and control cell movement and division. They also regulate dendritic spine formation and maturation in the brain, and play a role in synaptic transmission and synaptic plasticity. PAK3, in particular, is known for its implication in X-linked intellectual disability. The pak3 gene is expressed in neurons as a GTPase-regulated PAK3a protein and also as three splice variants which display constitutive kinase activity. PAK1 regulation is based on its homodimerization, forming an inactive complex. Here, we analyze the PAK3 capacity to dimerize and show that although PAK3a is able to homodimerize, it is more likely to form heterodimeric complexes with PAK1. We further show that two intellectual disability mutations impair dimerization with PAK1. The b and c inserts present in the regulatory domain of PAK3 splice variants decrease the dimerization but retain the capacity to form heterodimers with PAK1. PAK1 and PAK3 are co-expressed in neurons, are colocalized within dendritic spines, co-purify with post-synaptic densities, and co-immunoprecipitate in brain lysates. Using kinase assays, we demonstrate that PAK1 inhibits the activity of PAK3a but not of the splice variant PAK3b in a trans-regulatory manner. Altogether, these results show that PAK3 and PAK1 signaling may be coordinated by heterodimerization.


Assuntos
Espinhas Dendríticas/enzimologia , Proteínas do Tecido Nervoso/metabolismo , Densidade Pós-Sináptica/enzimologia , Multimerização Proteica , Quinases Ativadas por p21/metabolismo , Processamento Alternativo/genética , Animais , Ativação Enzimática/genética , Regulação Enzimológica da Expressão Gênica/genética , Doenças Genéticas Ligadas ao Cromossomo X/enzimologia , Doenças Genéticas Ligadas ao Cromossomo X/genética , Células HeLa , Humanos , Deficiência Intelectual/enzimologia , Deficiência Intelectual/genética , Camundongos , Mutação , Proteínas do Tecido Nervoso/genética , Estrutura Terciária de Proteína , Transdução de Sinais/genética , Quinases Ativadas por p21/genética
8.
J Neurosci ; 32(2): 519-27, 2012 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-22238087

RESUMO

Several gene mutations linked to intellectual disability in humans code for synaptic molecules implicated in small GTPase signaling. This is the case of the Rac/Cdc42 effector p21-activated kinase 3 (PAK3). The mechanisms responsible for the intellectual defects and the consequences of the mutation on the development and wiring of brain networks remain unknown. Here we show that expression of PAK3 mutants, suppression of PAK3, or inhibition of PAK3 function in rat hippocampal slice cultures interfere with activity-mediated spine dynamics. Inhibition of PAK3 resulted in two main alterations: (1) an increased growth of new, unstable spines, occurring in clusters, and mediated by activity; and (2) an impairment of plasticity-mediated spine stabilization interfering with the formation of persistent spines. Additionally, we find that PAK3 is specifically recruited by activity from dendrites into spines, providing a new mechanism through which PAK3 could participate in the control of both spine stabilization and local spine growth. Together, these data identify a novel function of PAK3 in regulating activity-mediated rearrangement of synaptic connectivity associated with learning and suggest that defects in spine formation and refinement during development could account for intellectual disability.


Assuntos
Deficiência Intelectual/metabolismo , Rede Nervosa/metabolismo , Transmissão Sináptica/genética , Quinases Ativadas por p21/genética , Animais , Células HeLa , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/fisiopatologia , Aprendizagem/fisiologia , Camundongos , Rede Nervosa/anormalidades , Rede Nervosa/fisiopatologia , Técnicas de Cultura de Órgãos , Ratos , Quinases Ativadas por p21/deficiência
9.
J Biol Chem ; 286(46): 40044-59, 2011 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-21949127

RESUMO

Mutations in the p21-activated kinase 3 gene (pak3) are responsible for nonsyndromic forms of mental retardation. Expression of mutated PAK3 proteins in hippocampal neurons induces abnormal dendritic spine morphology and long term potentiation anomalies, whereas pak3 gene invalidation leads to cognitive impairments. How PAK3 regulates synaptic plasticity is still largely unknown. To better understand how PAK3 affects neuronal synaptic plasticity, we focused on its interaction with the Nck adaptors that play a crucial role in PAK signaling. We report here that PAK3 interacts preferentially with Nck2/Grb4 in brain extracts and in transfected cells. This interaction is independent of PAK3 kinase activity. Selective uncoupling of the Nck2 interactions in acute cortical slices using an interfering peptide leads to a rapid increase in evoked transmission to pyramidal neurons. The P12A mutation in the PAK3 protein strongly decreases the interaction with Nck2 but only slightly with Nck1. In transfected hippocampal cultures, expression of the P12A-mutated protein has no effect on spine morphogenesis or synaptic density. The PAK3-P12A mutant does not affect synaptic transmission, whereas the expression of the wild-type PAK3 protein decreases the amplitude of spontaneous miniature excitatory currents. Altogether, these data show that PAK3 down-regulates synaptic transmission through its interaction with Nck2.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Oncogênicas/metabolismo , Transmissão Sináptica/fisiologia , Quinases Ativadas por p21/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Mutação de Sentido Incorreto , Proteínas Oncogênicas/genética , Quinases Ativadas por p21/genética
10.
Cell Signal ; 21(3): 384-93, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19036346

RESUMO

Group I p21-activated kinases are a family of key effectors of Rac1 and Cdc42 and they regulate many aspects of cellular function, such as cytoskeleton dynamics, cell movement and cell migration, cell proliferation and differentiation, and gene expression. The three genes PAK1/2/3 are expressed in brain and recent evidence indicates their crucial roles in neuronal cell fate, in axonal guidance and neuronal polarisation, and in neuronal migration. Moreover they are implicated in neurodegenerative diseases and play an important role in synaptic plasticity, with PAK3 being specifically involved in mental retardation. The main goal of this review is to describe the molecular mechanisms that govern the different functions of group I PAK in neuronal signalling and to discuss the specific functions of each isoform.


Assuntos
Encéfalo/enzimologia , Neurogênese/fisiologia , Neurônios/enzimologia , Quinases Ativadas por p21/metabolismo , Animais , Encéfalo/citologia , Encéfalo/embriologia , Sobrevivência Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Transtornos Neurocognitivos/enzimologia , Transtornos Neurocognitivos/genética , Transtornos Neurocognitivos/fisiopatologia , Doenças Neurodegenerativas/enzimologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/fisiopatologia , Plasticidade Neuronal/fisiologia , Quinases Ativadas por p21/genética
11.
J Neurochem ; 106(3): 1184-97, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18507705

RESUMO

The p21-activated kinases (PAK1), PAK2, and PAK3 are members of the PAK group I and share high sequence identity and common biochemical properties. PAK3 is specifically implicated in neuronal plasticity and also regulates cell cycle progression, neuronal migration, and apoptosis. Loss of function of PAK3 is responsible for X-linked non-syndromic mental retardation whereas gain of PAK3 function is associated with cancer. To understand the functional specificities of PAK3, we analyzed the structure of PAK3 gene products. We report here the characterization of a new alternatively spliced exon called c located upstream of the previously identified exon b. Exon b is detected in all tetrapods and not in fish, exon c is only present in mammals. Mammalian PAK3 genes encode four splice variants and the corresponding proteins were detected with specific antibodies in brain extracts. All PAK3 transcripts are specifically expressed in brain and in particular in neurons. The presence of the exons b and c renders the kinase constitutively active and decreases interaction with GTPases. The expression of the new splice variants in COS7 cells alters cell morphology and modifies the structure of focal adhesions. We propose that the appearance of new alternatively spliced exons during evolution and the resulting increase of complexity of PAK3 gene products may confer new functions to this kinase and contribute to its specific roles in neuronal signaling.


Assuntos
Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Quinases Ativadas por p21/química , Quinases Ativadas por p21/genética , Processamento Alternativo/genética , Substituição de Aminoácidos/genética , Animais , Células COS , Galinhas , Chlorocebus aethiops , Humanos , Camundongos , Oryzias , Isoformas de Proteínas/biossíntese , Ratos , Especificidade da Espécie , Tetraodontiformes , Xenopus laevis , Quinases Ativadas por p21/biossíntese
12.
J Biol Chem ; 282(29): 21497-506, 2007 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-17537723

RESUMO

The p21-activated kinase 3 (PAK3) is one of the recently identified genes for which mutations lead to nonsyndromic mental retardation. PAK3 is implicated in dendritic spine morphogenesis and is a key regulator of synaptic functions. However, the underlying roles of PAK3 in these processes remain poorly understood. We report here that the three mutations R419X, A365E, and R67C, responsible for mental retardation have different effects on the biological functions of PAK3. The R419X and A365E mutations completely abrogate the kinase activity. The R67C mutation drastically decreases the binding of PAK3 to the small GTPase Cdc42 and impairs its subsequent activation by this GTPase. We also report that PAK3 binds significantly more Cdc42 than Rac1 and is selectively activated by endogenous Cdc42, suggesting that PAK3 is a specific effector of Cdc42. Interestingly, the expression of the three mutated proteins in hippocampal neurons affects spinogenesis differentially. Both kinase-dead mutants slightly decrease the number of spines but profoundly alter spine morphology, whereas expression of the R67C mutant drastically decreases spine density. These results demonstrate that the Cdc42/PAK3 is a key module in dendritic spine formation and synaptic plasticity.


Assuntos
Deficiência Intelectual/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Células COS , Chlorocebus aethiops , Hipocampo/metabolismo , Deficiência Intelectual/genética , Modelos Biológicos , Mutação , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Sinapses/metabolismo , Transmissão Sináptica , Quinases Ativadas por p21 , Proteínas rac1 de Ligação ao GTP/metabolismo
13.
Endocrinology ; 147(12): 6036-45, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16935846

RESUMO

In pituitary cells, prolactin (PRL) synthesis and release are controlled by multiple transduction pathways. In the GH4C1 somatolactotroph cell line, we previously reported that MAPK ERK-1/2 are a point of convergence between the pathways involved in the PRL gene regulation. In the present study, we focused on the involvement of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the MAPK ERK-1/2 regulation and PRL secretion in pituitary cells. Either specific pharmacological PI3K and Akt inhibitors (LY294002, Akt I, and phosphoinositide analog-6) or Akt dominant-negative mutant (K179M) enhanced ERK-1/2 phosphorylation in unstimulated GH4C1 cells. Under the same conditions, PI3K and Akt inhibition also both increased Raf-1 kinase activity and the levels of GTP-bound (active form) monomeric G protein Rap1, which suggests that a down-regulation of the ERK-1/2 cascade is induced by the PI3K/Akt signaling pathway in unstimulated cells. On the contrary, ERK-1/2 phosphorylation, Raf-1 activity, and Rap1 activation were almost completely blocked in IGF-I-stimulated cells previously subjected to PI3K or Akt inhibition. Although the PRL promoter was not affected by either PI3K/Akt inhibition or activation, PRL release increased in response to the pharmacological PI3K/Akt inhibitors in unstimulated GH4C1 and rat pituitary primary cells. The IGF-I-stimulated PRL secretion was diminished, on the contrary, by the pharmacological PI3K/Akt inhibitors. Taken together, these findings indicate that the PI3K/Akt pathway exerts dual regulatory effects on both the Rap1/Raf-1/ERK-1/2 cascade and PRL release in pituitary cells, i.e. negative effects in unstimulated cells and positive ones in IGF-I-stimulated cells.


Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Prolactina/metabolismo , Proteínas Proto-Oncogênicas c-akt/fisiologia , Proteínas Proto-Oncogênicas c-raf/metabolismo , Somatotrofos/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Feminino , Hipófise/citologia , Hipófise/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Ratos , Ratos Wistar , Receptor Cross-Talk , Transdução de Sinais , Ativação Transcricional , Proteínas ras/metabolismo
14.
Neurochem Res ; 28(3-4): 557-64, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12675145

RESUMO

Involvement of different protein kinases regulated by cAMP and implication of muscarinic receptors in the regulation of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) mRNA levels and ChAT activity has been studied in NG108-15 cells. Dibutyryl cAMP enhanced both ChAT and VAChT mRNA levels and stimulated ChAT activity. Muscarinic stimulation or inhibition did not change ChAT activity or the receptor subtype mRNA pattern. MEK1/2 did not affect the regulation of ChAT and VAChT mRNA levels. However, PKA plays a major role in regulating ChAT and VAChT mRNA levels, because H89 decreased both. Strikingly, inhibition of PI3K by LY294002 had two opposite effects: ChAT mRNA level was decreased and VAChT mRNA level was increased. Such a result consolidates the observation that ChAT and VAChT genes, despite their unusual organization in a single "cholinergic locus," can be differentially or synergistically regulated, depending on the activated signaling pathways.


Assuntos
Proteínas de Transporte/genética , Colina O-Acetiltransferase/genética , Regulação da Expressão Gênica , Proteínas de Membrana Transportadoras , Sulfonamidas , Proteínas de Transporte Vesicular , Animais , Bucladesina/farmacologia , Colina O-Acetiltransferase/metabolismo , Cromonas/farmacologia , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Hibridomas , Isoquinolinas/farmacologia , Camundongos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , Ratos , Receptores Muscarínicos/fisiologia , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas , Proteínas Vesiculares de Transporte de Acetilcolina
15.
J Biol Chem ; 278(6): 3912-20, 2003 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-12464619

RESUMO

p21-activated kinases (PAK) are involved in the control of cytoskeleton dynamics and cell cycle progression. Here we report the characterization of a new mammalian PAK3 mRNA that contains a 45-bp alternatively spliced exon. This exon encodes for 15 amino acids that are inserted in the regulatory domain, inside the autoinhibitory domain but outside the Cdc42 and Rac interactive binding domain. The transcript of the 68-kDa new isoform named PAK3b is expressed in various areas of the adult mouse brain. In contrast to PAK3 without the exon b (PAK3a), whose basal kinase activity is weak in resting cells, PAK3b displays a high kinase activity in starved cells that is not further stimulated by active GTPases. Indeed, we demonstrate that the autoinhibitory domain of PAK3b no longer inhibits the kinase activity of PAK3. Moreover, we show that the 15-amino acid insertion within the autoinhibitory domain impedes the ability of PAK3b to bind to the GTPases Rac and Cdc42 and changes its specificity toward the GTPases. Altogether, our results show that the new PAK3b isoform has unique properties and would signal differently from PAK3a in neurons.


Assuntos
Encéfalo/enzimologia , GTP Fosfo-Hidrolases/metabolismo , Isoenzimas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , DNA Complementar , Éxons , Isoenzimas/genética , Camundongos , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por Substrato , Quinases Ativadas por p21
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