RESUMEN
Homozygous nonsense mutations in CEP55 are associated with several congenital malformations that lead to perinatal lethality suggesting that it plays a critical role in regulation of embryonic development. CEP55 has previously been studied as a crucial regulator of cytokinesis, predominantly in transformed cells, and its dysregulation is linked to carcinogenesis. However, its molecular functions during embryonic development in mammals require further investigation. We have generated a Cep55 knockout (Cep55-/-) mouse model which demonstrated preweaning lethality associated with a wide range of neural defects. Focusing our analysis on the neocortex, we show that Cep55-/- embryos exhibited depleted neural stem/progenitor cells in the ventricular zone as a result of significantly increased cellular apoptosis. Mechanistically, we demonstrated that Cep55-loss downregulates the pGsk3ß/ß-Catenin/Myc axis in an Akt-dependent manner. The elevated apoptosis of neural stem/progenitors was recapitulated using Cep55-deficient human cerebral organoids and we could rescue the phenotype by inhibiting active Gsk3ß. Additionally, we show that Cep55-loss leads to a significant reduction of ciliated cells, highlighting a novel role in regulating ciliogenesis. Collectively, our findings demonstrate a critical role of Cep55 during brain development and provide mechanistic insights that may have important implications for genetic syndromes associated with Cep55-loss.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Neocórtex/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Animales , Apoptosis/fisiología , Carcinogénesis/metabolismo , Células Cultivadas , Citocinesis/fisiología , Homocigoto , Humanos , Ratones , Ratones Noqueados , Células-Madre Neurales/metabolismo , FenotipoRESUMEN
WDR62 mutations that result in protein loss, truncation or single amino-acid substitutions are causative for human microcephaly, indicating critical roles in cell expansion required for brain development. WDR62 missense mutations that retain protein expression represent partial loss-of-function mutants that may therefore provide specific insights into radial glial cell processes critical for brain growth. Here we utilized CRISPR/Cas9 approaches to generate three strains of WDR62 mutant mice; WDR62 V66M/V66M and WDR62R439H/R439H mice recapitulate conserved missense mutations found in humans with microcephaly, with the third strain being a null allele (WDR62stop/stop). Each of these mutations resulted in embryonic lethality to varying degrees and gross morphological defects consistent with ciliopathies (dwarfism, anophthalmia and microcephaly). We find that WDR62 mutant proteins (V66M and R439H) localize to the basal body but fail to recruit CPAP. As a consequence, we observe deficient recruitment of IFT88, a protein that is required for cilia formation. This underpins the maintenance of radial glia as WDR62 mutations caused premature differentiation of radial glia resulting in reduced generation of neurons and cortical thinning. These findings highlight the important role of the primary cilium in neocortical expansion and implicate ciliary dysfunction as underlying the pathology of MCPH2 patients.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cilios/metabolismo , Ciliopatías/genética , Microcefalia/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Neocórtex/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Anoftalmos/embriología , Anoftalmos/genética , Anoftalmos/metabolismo , Apoptosis/genética , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/genética , Células Cultivadas , Cilios/genética , Cilios/patología , Ciliopatías/embriología , Ciliopatías/metabolismo , Ciliopatías/patología , Enanismo/embriología , Enanismo/genética , Enanismo/metabolismo , Células Ependimogliales/citología , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Fibroblastos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microcefalia/embriología , Microcefalia/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Mutación Missense , Neocórtex/embriología , Proteínas del Tejido Nervioso/genética , Neurogénesis/genética , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
Mutations of WD repeat domain 62 (WDR62) lead to autosomal recessive primary microcephaly (MCPH), and down-regulation of WDR62 expression causes the loss of neural progenitor cells (NPCs). However, how WDR62 is regulated and hence controls neurogenesis and brain size remains elusive. Here, we demonstrate that mitogen-activated protein kinase kinase kinase 3 (MEKK3) forms a complex with WDR62 to promote c-Jun N-terminal kinase (JNK) signaling synergistically in the control of neurogenesis. The deletion of Mekk3, Wdr62, or Jnk1 resulted in phenocopied defects, including premature NPC differentiation. We further showed that WDR62 protein is positively regulated by MEKK3 and JNK1 in the developing brain and that the defects of wdr62 deficiency can be rescued by the transgenic expression of JNK1. Meanwhile, WDR62 is also negatively regulated by T1053 phosphorylation, leading to the recruitment of F-box and WD repeat domain-containing protein 7 (FBW7) and proteasomal degradation. Our findings demonstrate that the coordinated reciprocal and bidirectional regulation among MEKK3, FBW7, WDR62, and JNK1, is required for fine-tuned JNK signaling for the control of balanced NPC self-renewal and differentiation during cortical development.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD/fisiología , MAP Quinasa Quinasa Quinasa 3/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Diferenciación Celular , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Femenino , Células HEK293 , Humanos , MAP Quinasa Quinasa Quinasa 3/genética , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Microcefalia/genética , Microcefalia/fisiopatología , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Fosforilación , Unión Proteica , Ratas , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
Primary cilia are non-motile, cell cycle-associated organelles that can be found on most vertebrate cell types. Comprised of microtubule bundles organised into an axoneme and anchored by a mature centriole or basal body, primary cilia are dynamic signalling platforms that are intimately involved in cellular responses to their extracellular milieu. Defects in ciliogenesis or dysfunction in cilia signalling underlie a host of developmental disorders collectively referred to as ciliopathies, reinforcing important roles for cilia in human health. Whilst primary cilia have long been recognised to be present in striated muscle, their role in muscle is not well understood. However, recent studies indicate important contributions, particularly in skeletal muscle, that have to date remained underappreciated. Here, we explore recent revelations that the sensory and signalling functions of cilia on muscle progenitors regulate cell cycle progression, trigger differentiation and maintain a commitment to myogenesis. Cilia disassembly is initiated during myoblast fusion. However, the remnants of primary cilia persist in multi-nucleated myotubes, and we discuss their potential role in late-stage differentiation and myofiber formation. Reciprocal interactions between cilia and the extracellular matrix (ECM) microenvironment described for other tissues may also inform on parallel interactions in skeletal muscle. We also discuss emerging evidence that cilia on fibroblasts/fibro-adipogenic progenitors and myofibroblasts may influence cell fate in both a cell autonomous and non-autonomous manner with critical consequences for skeletal muscle ageing and repair in response to injury and disease. This review addresses the enigmatic but emerging role of primary cilia in satellite cells in myoblasts and myofibers during myogenesis, as well as the wider tissue microenvironment required for skeletal muscle formation and homeostasis.
Asunto(s)
Centrosoma/metabolismo , Cilios/fisiología , Músculo Esquelético/fisiología , Animales , Axonema , Ciclo Celular/fisiología , Diferenciación Celular/fisiología , Cilios/metabolismo , Citoesqueleto , Matriz Extracelular , Humanos , Desarrollo de Músculos/fisiología , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Orgánulos , Transducción de SeñalRESUMEN
Mutations of the microtubule (MT)-associated protein Doublecortin X (DCX) gene disrupt cortical layering in brain development. Whilst many of these pathogenic DCX mutations are within the doublecortin domains (DC1 and DC2) that mediate direct DCX-MT association, a pathogenic mutation DCX E2K that causes cognitive impairment and pachygyria in human patients lies within the regulatory DCX N-terminus (DCX-N) preceding the DC1 domain. Here, we characterise the impact of DCX E2K on cytoskeletal association and regulation in neuronal cells. We show that the DCX E2K mutant protein retains the ability to interact with and bundle MTs, but these MTs show a reduced sensitivity to nocodazole-induced depolymerisation as well as slower α-tubulin exchange rates. Furthermore, we showed increased association of DCX E2K mutant with the actin filament (F-ACT) network. These results highlight the importance of the N-terminus of DCX in regulating association and co-ordination of MT and F-ACT networks.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Mutación , Neuropéptidos/genética , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Neuropéptidos/metabolismo , Nocodazol/farmacología , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/farmacologíaRESUMEN
WD40-repeat protein 62 (WDR62) is a spindle pole protein required for normal cell division and neuroprogenitor differentiation during brain development. Microcephaly-associated mutations in WDR62 lead to mitotic mislocalization, highlighting a crucial requirement for precise WDR62 spatiotemporal distribution, although the regulatory mechanisms are unknown. Here, we demonstrate that the WD40-repeat region of WDR62 is required for microtubule association, whereas the disordered C-terminal region regulates cell-cycle-dependent compartmentalization. In agreement with a functional requirement for the WDR62JNK1 complex during neurogenesis, WDR62 specifically recruits JNK1 (also known as MAPK8), but not JNK2 (also known as MAPK9), to the spindle pole. However, JNK-mediated phosphorylation of WDR62 T1053 negatively regulated microtubule association, and loss of JNK signaling resulted in constitutive WDR62 localization to microtubules irrespective of cell cycle stage. In contrast, we identified that Aurora A kinase (AURKA) and WDR62 were in complex and that AURKA-mediated phosphorylation was required for the spindle localization of WDR62 during mitosis. Our studies highlight complex regulation of WDR62 localization, with opposing roles for JNK and AURKA in determining its spindle association.
Asunto(s)
Aurora Quinasa A/metabolismo , Microtúbulos/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Huso Acromático/metabolismo , Proteínas de Ciclo Celular , Línea Celular , Células HeLa , Humanos , Sistema de Señalización de MAP Quinasas/genética , Proteína Quinasa 8 Activada por Mitógenos/genética , Mitosis/genética , Neurogénesis/genética , Fosforilación , Estructura Terciaria de ProteínaRESUMEN
Dynamic microtubule changes after a cell stress challenge are required for cell survival and adaptation. Stathmin (STMN), a cytoplasmic microtubule-destabilizing phosphoprotein, regulates interphase microtubules during cell stress, but the signaling mechanisms involved are poorly defined. In this study ectopic expression of single alanine-substituted phospho-resistant mutants demonstrated that STMN Ser-38 and Ser-63 phosphorylation were specifically required to maintain interphase microtubules during hyperosmotic stress. STMN was phosphorylated on Ser-38 and Ser-63 in response to hyperosmolarity, heat shock, and arsenite treatment but rapidly dephosphorylated after oxidative stress treatment. Two-dimensional PAGE and Phos-tag gel analysis of stress-stimulated STMN phospho-isoforms revealed rapid STMN Ser-38 phosphorylation followed by subsequent Ser-25 and Ser-63 phosphorylation. Previously, we delineated stress-stimulated JNK targeting of STMN. Here, we identified cAMP-dependent protein kinase (PKA) signaling as responsible for stress-induced STMN Ser-63 phosphorylation. Increased cAMP levels induced by cholera toxin triggered potent STMN Ser-63 phosphorylation. Osmotic stress stimulated an increase in PKA activity and elevated STMN Ser-63 and CREB (cAMP-response element-binding protein) Ser-133 phosphorylation that was substantially attenuated by pretreatment with H-89, a PKA inhibitor. Interestingly, PKA activity and subsequent phosphorylation of STMN were augmented in the absence of JNK activation, indicating JNK and PKA pathway cross-talk during stress regulation of STMN. Taken together our study indicates that JNK- and PKA-mediated STMN Ser-38 and Ser-63 phosphorylation are required to preserve interphase microtubules in response to hyperosmotic stress.
Asunto(s)
Interfase/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Microtúbulos/metabolismo , Presión Osmótica/fisiología , Transducción de Señal/fisiología , Estatmina/metabolismo , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Microtúbulos/genética , Células PC12 , Fosforilación/fisiología , Ratas , Estatmina/genéticaRESUMEN
The c-Jun N-terminal kinases (JNKs) are a group of stress-activated protein kinases that regulate gene expression changes through specific phosphorylation of nuclear transcription factor substrates. To address the mechanisms underlying JNK nuclear entry, we employed a semi-intact cell system to demonstrate for the first time that JNK1 nuclear entry is dependent on the importin α2/ß1 heterodimer and independent of importins α3, α4, ß2, ß3, 7 and 13. However, quantitative image analysis of JNK1 localization following exposure of cells to either arsenite or hyperosmotic stress did not indicate its nuclear accumulation. Extending our analyses to define the dynamics of nuclear trafficking of JNK1, we combined live cell imaging analyses with fluorescence recovery after photobleaching (FRAP) protocols. Subnuclear and subcytoplasmic bleaching protocols revealed the slowed movement of JNK1 in both regions in response to hyperosmotic stress. Strikingly, while movement into the nucleus of green fluorescent protein (GFP) or transport of a GFP-T-antigen fusion protein as estimated by initial rates and time to reach half-maximal recovery (t1/2) measures remained unaltered, hyperosmotic stress slowed the nuclear entry of GFP-JNK1. In contrast, arsenite exposure which did not alter the initial rates of nuclear accumulation of GFP, GFP-T-antigen or GFP-JNK1, decreased the t1/2 for nuclear accumulation of both GFP and GFP-JNK1. Thus, our results challenge the paradigm of increased nuclear localization of JNK broadly in response to all forms of stress-activation and are consistent with enhanced interactions of stress-activated JNK1 with scaffold and substrate proteins throughout the nucleus and the cytosol under conditions of hyperosmotic stress.
Asunto(s)
Núcleo Celular/metabolismo , Espacio Intracelular/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Presión Osmótica , Sorbitol/farmacología , Estrés Fisiológico , Animales , Antígenos Transformadores de Poliomavirus/metabolismo , Arsenitos/farmacología , Núcleo Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Recuperación de Fluorescencia tras Fotoblanqueo , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Espacio Intracelular/efectos de los fármacos , Carioferinas/metabolismo , Cinética , Ratones , Presión Osmótica/efectos de los fármacos , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ratas , Estrés Fisiológico/efectos de los fármacos , Fracciones Subcelulares/enzimologíaRESUMEN
We applied pulse-shape analysis (PulSA) to monitor protein localization changes in mammalian cells by flow cytometry. PulSA enabled high-throughput tracking of protein aggregation, translocation from the cytoplasm to the nucleus and trafficking from the plasma membrane to the Golgi as well as stress-granule formation. Combining PulSA with tetracysteine-based oligomer sensors in a cell model of Huntington's disease enabled further separation of cells enriched with monomers, oligomers and inclusion bodies.
Asunto(s)
Citometría de Flujo/métodos , Enfermedad de Huntington/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Aparato de Golgi/metabolismo , Humanos , Proteína Huntingtina , Cuerpos de Inclusión/metabolismo , Transporte de ProteínasRESUMEN
Excitotoxicity resulting from overstimulation of glutamate receptors is a major cause of neuronal death in cerebral ischemic stroke. The overstimulated ionotropic glutamate receptors exert their neurotoxic effects in part by overactivation of calpains, which induce neuronal death by catalyzing limited proteolysis of specific cellular proteins. Here, we report that in cultured cortical neurons and in vivo in a rat model of focal ischemic stroke, the tyrosine kinase Src is cleaved by calpains at a site in the N-terminal unique domain. This generates a truncated Src fragment of ~52 kDa, which we localized predominantly to the cytosol. A cell membrane-permeable fusion peptide derived from the unique domain of Src prevents calpain from cleaving Src in neurons and protects against excitotoxic neuronal death. To explore the role of the truncated Src fragment in neuronal death, we expressed a recombinant truncated Src fragment in cultured neurons and examined how it affects neuronal survival. Expression of this fragment, which lacks the myristoylation motif and unique domain, was sufficient to induce neuronal death. Furthermore, inactivation of the prosurvival kinase Akt is a key step in its neurotoxic signaling pathway. Because Src maintains neuronal survival, our results implicate calpain cleavage as a molecular switch converting Src from a promoter of cell survival to a mediator of neuronal death in excitotoxicity. Besides unveiling a new pathological action of Src, our discovery of the neurotoxic action of the truncated Src fragment suggests new therapeutic strategies with the potential to minimize brain damage in ischemic stroke.
Asunto(s)
Calpaína/química , Regulación Enzimológica de la Expresión Génica , Neuronas/metabolismo , Familia-src Quinasas/química , Animales , Isquemia Encefálica/patología , Calpaína/metabolismo , Muerte Celular , Membrana Celular/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Masculino , Modelos Biológicos , Mutación , Péptidos/química , Ratas , Ratas Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transducción de Señal , Accidente Cerebrovascular/enzimología , Accidente Cerebrovascular/patología , Familia-src Quinasas/metabolismoRESUMEN
Based on bioinformatics interrogation of the genome, >500 mammalian protein kinases can be clustered within seven different groups. Of these kinases, the mitogen-activated protein kinase (MAPK) family forms part of the CMGC group of serine/threonine kinases that includes extracellular signal regulated kinases (ERKs), cJun N-terminal kinases (JNKs), and p38 MAPKs. With the JNKs considered attractive targets in the treatment of pathologies including diabetes and stroke, efforts have been directed to the discovery of new JNK inhibitory molecules that can be further developed as new therapeutics. Capitalizing on our biochemical understanding of JNK, we performed in silico screens of commercially available chemical databases to identify JNK1-interacting compounds and tested their in vitro JNK inhibitory activity. With in vitro and cell culture studies, we showed that the compound, 4'-methyl-N(2)-3-pyridinyl-4,5'-bi-1,3-thiazole-2,2'-diamine (JNK Docking (JD) compound 123, but not the related compound (4'-methyl-N~2~-(6-methyl-2-pyridinyl)-4,5'-bi-1,3-thiazole-2,2'-diamine (JD124), inhibited JNK1 activity towards a range of substrates. Molecular docking, saturation transfer difference NMR experiments and enzyme kinetic analyses revealed both ATP- and substrate-competitive inhibition of JNK by JD123. In characterizing JD123 further, we noted its ATP-competitive inhibition of the related p38-γ MAPK, but not ERK1, ERK2, or p38-α, p38-ß or p38-δ. Further screening of a broad panel of kinases using 10µM JD123, identified inhibition of kinases including protein kinase Bß (PKBß/Aktß). Appropriately modified thiazole diamines, as typified by JD123, thus provide a new chemical scaffold for development of inhibitors for the JNK and p38-γ MAPKs as well as other kinases that are also potential therapeutic targets such as PKBß/Aktß.
Asunto(s)
Diaminas/química , Diaminas/farmacología , Proteína Quinasa 8 Activada por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Tiazoles/química , Tiazoles/farmacología , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Línea Celular , Células Cultivadas , Propuestas de Licitación , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Proteína Quinasa 8 Activada por Mitógenos/química , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular/métodos , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
The impact of aberrant centrosomes and/or spindles on asymmetric cell division in embryonic development indicates the tight regulation of bipolar spindle formation and positioning that is required for mitotic progression and cell fate determination. WD40-repeat protein 62 (WDR62) was recently identified as a spindle pole protein linked to the neurodevelopmental defect of microcephaly but its roles in mitosis have not been defined. We report here that the in utero electroporation of neuroprogenitor cells with WDR62 siRNAs induced their cell cycle exit and reduced their proliferative capacity. In cultured cells, we demonstrated cell-cycle-dependent accumulation of WDR62 at the spindle pole during mitotic entry that persisted until metaphase-anaphase transition. Utilizing siRNA depletion, we revealed WDR62 function in stabilizing the mitotic spindle specifically during metaphase. WDR62 loss resulted in spindle orientation defects, decreased the integrity of centrosomes displaced from the spindle pole and delayed mitotic progression. Additionally, we revealed JNK phosphorylation of WDR62 is required for maintaining metaphase spindle organization during mitosis. Our study provides the first functional characterization of WDR62 and has revealed requirements for JNK/WDR62 signaling in mitotic spindle regulation that may be involved in coordinating neurogenesis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Metafase , Proteínas Asociadas a Microtúbulos/metabolismo , Procesamiento Proteico-Postraduccional , Huso Acromático/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proliferación Celular , Centrosoma/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Femenino , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Microcefalia , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Proteínas del Tejido Nervioso , Células-Madre Neurales/metabolismo , Células-Madre Neurales/fisiología , Fosforilación , Profase , Transporte de Proteínas , ARN Interferente Pequeño/genéticaRESUMEN
The stathmin (STMN) family of tubulin-binding phosphoproteins are critical regulators of interphase microtubule dynamics and organization in a broad range of cellular processes. c-Jun N-terminal kinase (JNK) signalling to STMN family proteins has been implicated specifically in neuronal maturation, degeneration and cell stress responses more broadly. Previously, we characterized mechanisms underlying JNK phosphorylation of STMN at proline-flanked serine residues (Ser25 and Ser38) that are conserved across STMN-like proteins. In this study, we demonstrated using in vitro kinase assays and alanine replacement of serine residues that JNK phosphorylated the STMN-like domain (SLD) of SCG10 on Ser73, consistent with our previous finding that STMN Ser38 was the primary JNK target site. In addition, we confirmed that a JNK binding motif ((41)KKKDLSL(47)) that facilitates JNK targeting of STMN is conserved in SCG10. In contrast, SCLIP was phosphorylated by JNK primarily on Ser60 which corresponds to Ser25 on STMN. Moreover, although the JNK-binding motif identified in STMN and SCG10 was not conserved in SCLIP, JNK phosphorylation of SCLIP was inhibited by a substrate competitive peptide (TI-JIP) highlighting kinase-substrate interaction as required for JNK targeting. Thus, STMN and SCG10 are similarly targeted by JNK but there are clear differences in JNK recognition and phosphorylation of the closely related family member, SCLIP.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Estatmina/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia Conservada , Humanos , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteína Quinasa 8 Activada por Mitógenos/genética , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Estatmina/química , Estatmina/genética , Especificidad por SustratoRESUMEN
DiOHF (3',4'-dihydroxyflavonol) is cardioprotective against I/R (ischaemia/reperfusion) injury. The biological activities of flavonols are associated with kinase modulation to alter cell signalling. We thus investigated the effects of DiOHF on the activation of MAPKs (mitogen-activated protein kinases) that regulate the cardiac stress response. In an ovine model of I/R, JNK (c-Jun N-terminal kinase), p38(MAPK), ERK (extracellular-signal-regulated kinase) and Akt were activated, and NP202, a pro-drug of DiOHF, reduced infarct size and inhibited JNK and p38(MAPK) activation, whereas ERK and Akt phosphorylation were unaltered. Similarly, in cultured myoblasts, DiOHF pre-treatment preserved viability and inhibited activation of JNK and p38(MAPK), but not ERK in response to acute oxidative and chemotoxic stress. Furthermore, DiOHF prevented stress-activation of the direct upstream regulators MKK4/7 (MAPK kinase 4/7) and MKK3/6 respectively. We utilized small-molecule affinity purification and identified CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) as a kinase targeted by DiOHF and demonstrated potent CaMKII inhibition by DiOHF in vitro. Moreover, the specific inhibition of CaMKII with KN-93, but not KN-92, prevented oxidative stress-induced activation of JNK and p38(MAPK). The present study indicates DiOHF inhibition of CaMKII and attenuation of MKK3/6âp38(MAPK) and MKK4/7âJNK signalling as a requirement for the protective effects of DiOHF against stress stimuli and myocardial I/R injury.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Cardiotónicos/farmacología , Flavonoles/farmacología , Sistema de Señalización de MAP Quinasas , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Animales , Arsenitos/farmacología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Línea Celular , Peróxido de Hidrógeno/farmacología , MAP Quinasa Quinasa 4/metabolismo , Ratones , Daño por Reperfusión Miocárdica/enzimología , Daño por Reperfusión Miocárdica/patología , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Fosforilación , Procesamiento Proteico-Postraduccional , Ratas , Oveja Doméstica , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Signal processing by intracellular kinases controls near all biological processes but how signal pathway functions evolve with changed cellular context is poorly understood. Functional specificity of c-Jun N-terminal Kinases (JNK) are partly encoded by signal strength. Here we reveal that intracellular pH (pHi) is a significant component of the JNK network and defines signal response to specific stimuli. We show pHi regulates JNK activity in response to cell stress, with the relationship between pHi and JNK activity dependent on specific stimuli and upstream kinases activated. Using the optogenetic clustering tag CRY2, we show that an increase in pHi promotes the light-induced phase transition of ASK1 to augment JNK activation. While increased pHi similarly promoted CRY2-tagged JNK2 to form light-induced condensates, this attenuated JNK activity. Mathematical modelling of feedback signalling incorporating pHi and differential contributions by ASK1 and JNK2 condensates was sufficient to delineate signal responses to specific stimuli. Taking pHi and ASK1/JNK2 signal contributions into consideration may delineate oncogenic versus tumour suppressive JNK functions and cancer cell drug responses.
Asunto(s)
MAP Quinasa Quinasa Quinasa 5 , Concentración de Iones de Hidrógeno , MAP Quinasa Quinasa Quinasa 5/metabolismo , MAP Quinasa Quinasa Quinasa 5/genética , Humanos , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/genética , Estrés Fisiológico , Transducción de Señal , Animales , Optogenética , Sistema de Señalización de MAP QuinasasRESUMEN
Phosphorylation of STAT3 (signal transducer and activator of transcription 3) is critical for its nuclear import and transcriptional activity. Although a shorter STAT3ß spliceform was initially described as a negative regulator of STAT3α, gene knockout studies have revealed that both forms play critical roles. We have expressed STAT3α and STAT3ß at comparable levels to facilitate a direct comparison of their functional effects, and have shown their different cytokine-stimulated kinetics of phosphorylation and nuclear translocation. Notably, the sustained nuclear translocation and phosphorylation of STAT3ß following cytokine exposure contrasted with a transient nuclear translocation and phosphorylation of STAT3α. Importantly, co-expression of the spliceforms revealed that STAT3ß enhanced and prolonged the phosphorylation and nuclear retention of STAT3α, but a STAT3ß R609L mutant, with a disrupted SH2 (Src homology 2) domain, was not tyrosine phosphorylated following cytokine stimulation and could not cross-regulate STAT3α. The physiological importance of prolonged phosphorylation and nuclear retention was indicated by transcriptome profiling of STAT3(-/-) cells expressing either STAT3α or STAT3ß, revealing the complexity of genes that are up- and down-regulated by the STAT3 spliceforms, including a distinct set of STAT3ß-specific genes regulated under basal conditions and after cytokine stimulation. These results highlight STAT3ß as a significant transcriptional regulator in its own right, with additional actions to cross-regulate STAT3α phosphorylation and nuclear retention after cytokine stimulation.
Asunto(s)
Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Empalme Alternativo , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Citocinas/farmacología , Cartilla de ADN/genética , Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Cinética , Ratones , Mutagénesis Sitio-Dirigida , Fosforilación/efectos de los fármacos , Factor de Transcripción STAT3/química , Factor de Transcripción STAT3/deficiencia , Tirosina/química , Dominios Homologos srcRESUMEN
Maintenance of cardiac structure and Z-disc signaling are key factors responsible for protecting the heart in a setting of stress, but how these processes are regulated is not well defined. We recently demonstrated that PI3K(p110α) protects the heart against myocardial infarction. The aim of this study was to determine whether PI3K(p110α) directly regulates components of the Z-disc and cardiac structure. To address this question, a unique three-dimensional virtual muscle model was applied to gene expression data from transgenic mice with increased or decreased PI3K(p110α) activity under basal conditions (sham) and in a setting of myocardial infarction to display the location of structural proteins. Key findings from this analysis were then validated experimentally. The three-dimensional virtual muscle model visually highlighted reciprocally regulated transcripts associated with PI3K activation that encoded key components of the Z-disc and costamere, including melusin. Studies were performed to assess whether PI3K and melusin interact in the heart. Here, we identify a novel melusin-PI3K interaction that generates lipid kinase activity. The direct impact of PI3K(p110α) on myocyte structure was assessed by treating neonatal rat ventricular myocytes with PI3K(p110α) inhibitors and examining the myofiber morphology of hearts from PI3K transgenic mice. Results demonstrate that PI3K is critical for myofiber maturation and Z-disc alignment. In summary, PI3K regulates the expression of genes essential for cardiac structure and Z-disc signaling, interacts with melusin, and is critical for Z-disc alignment.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Regulación Enzimológica de la Expresión Génica , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Costameras/metabolismo , Proteínas del Citoesqueleto/química , Insuficiencia Cardíaca/metabolismo , Inmunoprecipitación , Proteínas Sustrato del Receptor de Insulina/metabolismo , Ratones , Ratones Transgénicos , Microscopía Confocal/métodos , Células Musculares/citología , Proteínas Musculares/química , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
Excessive proliferation and stabilization of the microtubule (MT) array in cardiac myocytes can accompany pathological cardiac hypertrophy, but the molecular control of these changes remains poorly characterized. In this study, we examined MT stabilization in two independent murine models of heart failure and revealed increases in the levels of post-translationally modified stable MTs, which were closely associated with STAT3 activation. To explore the molecular signaling events contributing to control of the cardiac MT network, we stimulated cardiac myocytes with an α-adrenergic agonist phenylephrine (PE), and observed increased tubulin content without changes in detyrosinated (glu-tubulin) stable MTs. In contrast, the hypertrophic interleukin-6 (IL6) family cytokines increased both the glu-tubulin content and glu-MT density. When we examined a role for ERK in regulating cardiac MTs, we showed that the MEK/ERK-inhibitor U0126 increased glu-MT density in either control cardiac myocytes or following exposure to hypertrophic agents. Conversely, expression of an activated MEK1 mutant reduced glu-tubulin levels. Thus, ERK signaling antagonizes stabilization of the cardiac MT array. In contrast, inhibiting either JAK2 with AG490, or STAT3 signaling with Stattic or siRNA knockdown, blocked cytokine-stimulated increases in glu-MT density. Furthermore, the expression of a constitutively active STAT3 mutant triggered increased glu-MT density in the absence of hypertrophic stimulation. Thus, STAT3 activation contributes substantially to cytokine-stimulated glu-MT changes. Taken together, our results highlight the opposing actions of STAT3 and ERK pathways in the regulation of MT changes associated with cardiac myocyte hypertrophy.
Asunto(s)
Cardiomegalia/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Microtúbulos/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Cardiomegalia/patología , Cardiomiopatía Hipertrófica/metabolismo , Cardiomiopatía Hipertrófica/patología , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Interleucina-6/metabolismo , Masculino , Ratones , Ratones Endogámicos , Ratones Transgénicos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , ARN Interferente Pequeño , Ratas , Ratas Sprague-Dawley , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
An improved understanding of the roles of protein kinases in intracellular signalling and disease progression has driven significant advances in protein kinase inhibitor discovery. Peptide inhibitors that target the kinase protein substrate-binding site have continued to attract attention. In the present paper, we describe a novel JNK (c-Jun N-terminal kinase) inhibitory peptide PYC71N, which inhibits JNK activity in vitro towards a range of recombinant protein substrates including the transcription factors c-Jun, ATF2 (activating trancription factor 2) and Elk1, and the microtubule regulatory protein DCX (doublecortin). Analysis of cell culture studies confirmed the actions of a cell-permeable version of PYC71 to inhibit c-Jun phosphorylation during acute hyperosmotic stress. The analysis of the in vitro data for the kinetics of this inhibition indicated a substrate-inhibitor complex-mediated inhibition of JNK by PYC71N. Alanine-scanning replacement studies revealed the importance of two residues (PYC71N Phe9 or Phe11 within an FXF motif) for JNK inhibition. The importance of these residues was confirmed through interaction studies showing that each change decreased interaction of the peptide with c-Jun. Furthermore, PYC71N interacted with both non-phosphorylated (inactive) JNK1 and the substrate c-Jun, but did not recognize active JNK1. In contrast, a previously characterized JNK-inhibitory peptide TIJIP [truncated inhibitory region of JIP (JNK-interacting protein)], showed stronger interaction with active JNK1. Competition binding analysis confirmed that PYC71N inhibited the interaction of c-Jun with JNK1. Taken together, the results of the present study define novel properties of the PYC71N peptide as well as differences from the characterized TIJIP, and highlight the value of these peptides to probe the biochemistry of JNK-mediated substrate interactions and phosphorylation.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/química , Fragmentos de Péptidos/química , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Proteína Doblecortina , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Cinética , Proteína Quinasa 8 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 8 Activada por Mitógenos/química , Proteína Quinasa 9 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 9 Activada por Mitógenos/química , Células PC12 , Fragmentos de Péptidos/farmacología , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ratas , Proteínas Recombinantes/químicaRESUMEN
A central event in the pathogenesis of motor neuron disease (MND) is the loss of neuromuscular junctions (NMJs), yet the mechanisms that lead to this event in MND remain to be fully elucidated. Maintenance of the NMJ relies upon neural agrin (n-agrin) which, when released from the nerve terminal, activates the postsynaptic Muscle Specific Kinase (MuSK) signaling complex to stabilize clusters of acetylcholine receptors. Here, we report that muscle from MND patients has an increased proportion of slow fibers and muscle fibers with smaller diameter. Muscle cells cultured from MND biopsies failed to form large clusters of acetylcholine receptors in response to either non-MND human motor axons or n-agrin. Furthermore, levels of expression of MuSK, and MuSK-complex components: LRP4, Caveolin-3, and Dok7 differed between muscle cells cultured from MND patients compared to those from non-MND controls. To our knowledge, this is the first time a fault in the n-agrin-LRP4-MuSK signaling pathway has been identified in muscle from MND patients. Our results highlight the n-agrin-LRP4-MuSK signaling pathway as a potential therapeutic target to prolong muscle function in MND.