RESUMEN
The imbalance in the phosphorylation and the dephosphorylation of proteins leads to various diseases. Therefore, in vivo, the functions of protein kinases and protein phosphatases are strictly regulated. Mg2+/Mn2+-dependent protein phosphatase PPM1M has been implicated in immunity and cancer; however, the regulation mechanism remains unknown. In this study, we show that PPM1M is regulated in different ways by multiple phosphorylation. PPM1M has four Ser/Thr-Pro motifs (Ser27, Ser43, Ser60, and Thr254) that are recognized by proline-directed kinases, and Ser60 was found to be phosphorylated by cyclin-dependent kinase 5 (CDK5) in the cell. The phospho-mimetic mutation of Ser27 and Ser43 in the N-terminal domain suppresses the nuclear localization of PPM1M and promotes its accumulation in the cytoplasm. The phospho-mimetic mutation of Ser60 decreases PPM1M activity; conversely, the phospho-mimetic mutation of Thr254 increases PPM1M activity. These results suggest that the subcellular localization and phosphatase activity of PPM1M are regulated by protein kinases, including CDK5, via phosphorylation at multiple sites. Thus, PPM1M is differentially regulated by proline-directed kinases, including CDK5.
Asunto(s)
Fosfoproteínas Fosfatasas , Proteínas , Fosforilación , Fosfoproteínas Fosfatasas/genética , ProlinaRESUMEN
Bone morphogenetic protein 2 (BMP2)-inducible kinase (BMP2K) is induced by the cytokine BMP2, which is also implicated in the production of bone differentiation. In addition to regulating bone differentiation, BMP2K is implicated in a variety of cancers. Therefore, understanding the variables that determine where in the cell this kinase functions may help in understanding malignancies linked to BMP2K. However, the mechanisms regulating the subcellular localization of BMP2K are mainly unknown. By liquid-liquid phase separation (LLPS), BMP2K forms droplets in the cytoplasm, but how the droplets are regulated remains unclear. The reason why BMP2K localizes to the cytoplasm irrespective of having a nuclear localization signal (NLS) is also unknown. Here we show the element that controls BMP2K's LLPS and cytoplasmic localization. A glutamine-rich area is necessary for BMP2K phase separation, and droplet formation is controlled by hyperosmolarity. Cytoplasmic localization of BMP2K is managed by inhibition of NLS function through phosphorylation of Ser-1010 and by a newly found cytoplasmic localization region that antagonizes the NLS. These results will provide an important biochemical foundation for the advancement of BMP2K-related cell biology, structural biology, and pathophysiology.
Asunto(s)
Proteína Morfogenética Ósea 2 , Señales de Localización Nuclear , Transporte Activo de Núcleo Celular , Proteína Morfogenética Ósea 2/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Señales de Localización Nuclear/metabolismo , Fosforilación , Espacio Intracelular/metabolismoRESUMEN
CaMK phosphatase (CaMKP/POPX2/PPM1F) is a Ser/Thr protein phosphatase that belongs to the PPM family. Accumulating evidence suggests that CaMKP is involved in the pathogenesis of various diseases, including cancer. To clarify the relationship between CaMKP activity and human breast cancer cell motility, we examined the phosphatase activity of CaMKP in cell extracts. CaMKP activity assays of the immunoprecipitates prepared from the cell extract revealed that cells exhibiting higher motility had higher CaMKP activity, with no significant differences in the specific activity being observed. Two CaMKP-specific inhibitors, 1-amino-8-naphthol-4-sulfonic acid (ANS) and 1-amino-8-naphthol-2,4-disulfonic acid (ANDS), inhibited the migration of highly invasive MDA-MB-231 breast cancer cells without significant cytotoxicity, while an inactive analog, naphthionic acid, did not. Furthermore, the cells lost their elongated morphology and assumed a rounded shape following treatment with ANS, whereas they retained their elongated morphology following treatment with naphthionic acid. Consistent with these findings, ANS and ANDS significantly enhanced the phosphorylation level of CaMKI, a cellular substrate of CaMKP, while naphthionic acid did not. The present data suggest that CaMKP could be a novel therapeutic target for cancer metastasis.
Asunto(s)
Neoplasias de la Mama , Naftoles , Humanos , Femenino , Células MDA-MB-231 , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina , Movimiento Celular , Línea Celular TumoralRESUMEN
CaMK phosphatase (CaMKP/PPM1F/POPX2) is a Mn2+-dependent, calyculin A/okadaic acid-insensitive Ser/Thr protein phosphatase that belongs to the PPM family. CaMKP is thought to be involved in regulation of not only various protein kinases, such as CaM kinases and p21-activated protein kinase, but also of cellular proteins regulated by phosphorylation. A large-scale screening of a chemical library identified gallic acid and some of its alkyl esters as novel CaMKP inhibitors highly specific to CaMKP. Surprisingly, they caused specific carbonylation of CaMKP, leading to its inactivation. Under the same conditions, no carbonylation nor inactivation was observed when PPM1A, which is affiliated with the same family as CaMKP, and λ-phosphatase were used. The carbonylation reaction was inhibited by SH compounds such as cysteamine in a dose-dependent manner with a concomitant decrease in CaMKP inhibition by ethyl gallate. The pyrogallol structure of gallate was necessary for the gallate-mediated carbonylation of CaMKP. Point mutations of CaMKP leading to impairment of phosphatase activity did not significantly affect the gallate-mediated carbonylation. Ethyl gallate resulted in almost complete inhibition of CaMKP under the conditions where the carbonylation level was nearly identical to that of CaMKP carbonylation via metal-catalyzed oxidation with ascorbic acid/FeSO4, which resulted in only a partial inhibition of CaMKP. The gallate-mediated carbonylation of CaMKP absolutely required divalent cations such as Mn2+, Cu2+, Co2+ and Fe2+, and was markedly enhanced by a phosphopeptide substrate. When MDA-MB-231 cells transiently expressing CaM kinase I, a CaMKP substrate, were treated by ethyl gallate, significant enhancement of phosphorylation of CaM kinase I was observed, suggesting that ethyl gallate can penetrate into cells to inactivate cellular CaMKP. All the presented data strongly support the hypothesis that CaMKP undergoes carbonylation of its specific amino acid residues by incubation with alkyl gallates and the divalent metal cations, leading to inactivation specific to CaMKP.
Asunto(s)
Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina , Fosfoproteínas Fosfatasas , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/química , Oxidación-Reducción , Fosfoproteínas Fosfatasas/química , Fosforilación , Carbonilación Proteica , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 2/metabolismoRESUMEN
Ca2+/calmodulin (CaM)-dependent protein kinase Iδ (CaMKIδ) is a Ser/Thr kinase that plays pivotal roles in Ca2+ signalling. CaMKIδ is activated by Ca2+/CaM-binding and phosphorylation at Thr180 by CaMK kinase (CaMKK). In this study, we characterized four splice variants of mouse CaMKIδ (mCaMKIδs: a, b, c and d) found by in silico analysis. Recombinant mCaMKIδs expressed in Escherichia coli were phosphorylated by CaMKK; however, only mCaMKIδ-a and c showed protein kinase activities towards myelin basic protein in vitro, with mCaMKIδ-b and mCaMKIδ-d being inactive. Although mCaMKIδ-a and mCaMKIδ-c underwent autophosphorylation in vitro, only mCaMKIδ-c underwent autophosphorylation in 293T cells. Site-directed mutagenesis showed that the autophosphorylation site is Ser349, which is found in the C-terminal region of only variants c and b (Ser324). Furthermore, phosphorylation of these sites (Ser324 and Ser349) in mCaMKIδ-b and c was more efficiently catalyzed by cAMP-dependent protein kinase in vitro and in cellulo as compared to the autophosphorylation of mCaMKIδ-c. Thus, variants of mCaMKIδ possess distinct properties in terms of kinase activities, autophosphorylation and phosphorylation by another kinase, suggesting that they play physiologically different roles in murine cells.
Asunto(s)
Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina , Animales , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/metabolismo , Línea Celular , AMP Cíclico/genética , AMP Cíclico/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Fosforilación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The CCDC26 gene is considered to encode a functional noncoding RNA associated with acute myeloid leukemia and other cancers. However, investigations into the physiological roles of CCDC26 are rare. Previously, we reported that CCDC26 regulated proliferation and cell death of leukemia cells through KIT, a receptor tyrosine kinase, by using K562 leukemia cells and their derivative CCDC26-knockdown (KD) cells. Here we propose a new role of CCDC26 in the differentiation of erythroid cells. We showed that expression of embryonic (ε- and ζ-) globins was markedly upregulated in CCDC26-KD cells compared with K562 control cells during hemin-induced differentiation. In contrast, expression of fetal-type γ-globin, a major globin expressed in original K562 cells, was decreased. These changes in the expression of globin genes mainly took place at the transcriptional level, with significant suppression of transcription of adult (ß-, δ-) globins in CCDC26-KD cells. Re-introduction of exogenous CCDC26 into the CCDC26-KD cells recovered low-level expression of the embryonal globins. These results suggest CCDC26 has a role in switching transcription of globin genes in the differentiation of erythroid cells. The expression profile of the CCDC26-KD cells and control cells suggests FOG-2, a transcriptional modulator, as a candidate for a mediator of the CCDC26-associated regulation. We showed that both embryonic globins were transcriptionally activated in FOG-2-KD K562 cells. The KIT inhibitor ISCK03 suppressed the production of hemoglobin in K562 cells but did not affect transcription of globin genes. To summarize, FOG-2, but not KIT, is responsible for globin transcriptional regulation by CCDC26.
Asunto(s)
Proteínas de Unión al ADN/genética , Células Eritroides/citología , Globinas/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , ARN Largo no Codificante/genética , Factores de Transcripción/genética , Diferenciación Celular/efectos de los fármacos , Células Eritroides/efectos de los fármacos , Células Eritroides/metabolismo , Regulación del Desarrollo de la Expresión Génica , Globinas/metabolismo , Hemoglobinas/genética , Hemoglobinas/metabolismo , Humanos , Imidazoles/farmacología , Células K562 , Sulfonamidas/farmacologíaRESUMEN
Docosahexaenoic acid (DHA) has been shown to have neuroprotective effects in Parkinson's disease, but the underlying mechanism has not been fully elucidated. DHA is metabolized to DHA epoxides (EDPs) and hydroxides by cytochrome P450s (P450s), and EDPs are further hydroxylated to the corresponding diols, dihydroxydocosapentaenoic acids (DHDPs) by soluble epoxide hydrolase (sEH). In the present study, we investigated the roles of these DHA metabolites in the beneficial effects of DHA supplementation on a rotenone-induced rat model of Parkinson's disease. Metabolite analysis by LC-MS revealed that CYP2A1, 2C11, 2C13, 2C23, and 2E1 contributed to the formation of EDPs, and these P450s and sEH were expressed in the rat brain. We found that DHA supplementation in rats improved the motor dysfunction induced by rotenone. In addition, DHA reversed the decrease in tyrosine hydroxylase and the increase in lipid peroxidation generated by rotenone in the striatum. DHA supplementation also induced mRNA expression of antioxidant genes, such as sod1 and catalase, and Nrf2 protein expression in the striatum. However, these effects of DHA supplementation were eliminated by cosupplementation with the sEH inhibitor TPPU. Supplementation with DHA increased the amount of 19,20-DHDP in the rat brain, while the amount of EDPs was not significantly increased. In addition, TPPU suppressed the increase in DHDPs and increased EDPs in the brain. In PC12 cells, 19,20-DHDP increased the mRNA levels of sod1 and catalase along with Nrf2 induction. This study suggests that DHA metabolites-DHDPs generated by P450s and sEH-have an important role in improving rotenone-induced Parkinson's disease.
Asunto(s)
Ácidos Docosahexaenoicos/administración & dosificación , Ácidos Grasos Insaturados/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Enfermedad de Parkinson Secundaria/tratamiento farmacológico , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Catalasa/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Modelos Animales de Enfermedad , Ácidos Docosahexaenoicos/metabolismo , Epóxido Hidrolasas/antagonistas & inhibidores , Epóxido Hidrolasas/metabolismo , Humanos , Masculino , Factor 2 Relacionado con NF-E2/metabolismo , Fármacos Neuroprotectores/metabolismo , Oxidación-Reducción/efectos de los fármacos , Enfermedad de Parkinson Secundaria/inducido químicamente , Enfermedad de Parkinson Secundaria/patología , Ratas , Rotenona/toxicidad , Superóxido Dismutasa-1/metabolismoRESUMEN
Protein phosphatase PPM1H is known to participate in various biological or pathophysiological mechanisms. However, little is known about the molecular mechanisms of its regulation. In this study, we investigated the protein kinases that directly phosphorylate PPM1H, identifying them as cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase I (CaMKI). In vitro and in silico analyses showed that the phosphorylation sites of PPM1H by PKA and CaMKI were Ser-123 and Ser-210, respectively. The phosphorylation state of PPM1H in cells exhibited the kinase activator- and inhibitor-dependent changes. In mouse neuroblastoma Neuro2a cells, phosphorylation of Ser-210 was much higher in the phospho-mimetic mutant (S123D) than in the non-phosphorylatable mutant (S123A) when they were treated with ionomycin. This suggests that a hierarchical phosphorylation, with initial phosphorylation of Ser-123 promoting subsequent phosphorylation of Ser-210, occurs in these neuron-like cells. Moreover, in cell-based assay a PPM1H(S123A/S210A) double mutant barely dephosphorylated Smad1, a transcription factor known as an endogenous substrate of PPM1H. These results suggest that cAMP and Ca2+/calmodulin regulate dephosphorylation of Smad1 through the dual phosphorylation of PPM1H at Ser-123 and Ser-210.
Asunto(s)
Proteína Smad1/metabolismo , Animales , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/metabolismo , Línea Celular Tumoral , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células HEK293 , Humanos , Ratones , FosforilaciónRESUMEN
Ca2+/calmodulin-dependent protein kinase I isoforms (CaMKIα, ß, γ, and δ) play important roles in Ca2+ signaling in eukaryotic cells by being activated by CaMK kinase (CaMKK) through phosphorylation at a Thr residue in the activation loop. However, we have recently found that, unlike rat CaMKIα (rCaMKIα), C-terminally truncated fragments of zebrafish and mouse CaMKIδ [zCaMKIδ(1-299) and mCaMKIδ(1-297)] produced by Escherichia coli exhibit almost full activity in the absence of CaMKK. To address the CaMKK-independent activation mechanism of CaMKIδ in E. coli cells, here we performed comparative analyses between recombinant zCaMKIδ(1-299) and rCaMKIα(1-294) in vitro. By using a kinase-dead mutant of zCaMKIδ(1-299) and λ phosphatase coexpression method, we elucidated that zCaMKIδ(1-299) was highly autophosphorylated and activated in E. coli during cell culture, but rCaMKIα(1-294) was not. The major autophosphorylation site leading to activation of the kinase was Ser296, determined using mass spectrometry analysis in conjunction with site-directed mutagenesis. Furthermore, mimicking phosphorylation at Ser296 in full-length zCaMKIδ resulted in additional activation of the kinase compared with CaMKI fully activated by CaMKK. Our results provide the first evidence that CaMKIδ is activated through CaMKK-independent phosphorylation at Ser296, which might be a clue to understand the physiological regulation of CaMKIδ isoform.
Asunto(s)
Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/metabolismo , Activación Enzimática/fisiología , Proteínas de Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/genética , Escherichia coli/enzimología , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones Endogámicos BALB C , Mutagénesis Sitio-Dirigida , Mutación , Fosforilación , Procesamiento Proteico-Postraduccional , Ratas , Alineación de Secuencia , Serina/química , Pez Cebra , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
Protein phosphorylation plays important roles in regulating a variety of biological processes in animals, plants, and microorganisms. Therefore, it is important to use appropriate techniques to detect and analyze protein kinases and protein phosphatases. In this chapter, we describe the method to detect protein phosphatase activities using fluorogenic substrates such as 4-methylumbelliferyl phosphate (MUP) after separating proteins by one-dimensional or two-dimensional polyacrylamide gel electrophoresis.
Asunto(s)
Pruebas de Enzimas , Colorantes Fluorescentes , Fosfoproteínas Fosfatasas , Animales , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Pruebas de Enzimas/métodos , Colorantes Fluorescentes/química , Fosfoproteínas Fosfatasas/análisis , Fosfoproteínas Fosfatasas/química , Ratas , Especificidad por SustratoRESUMEN
We surveyed genome sequences from the basidiomycetous mushroom Coprinopsis cinerea and isolated a cDNA homologous to CMKA, a calmodulin-dependent protein kinase (CaMK) in Aspergillus nidulans. We designated this sequence, encoding 580 amino acids with a molecular weight of 63,987, as CoPK02. CoPK02 possessed twelve subdomains specific to protein kinases and exhibited 43, 35, 40% identity with rat CaMKI, CaMKII, CaMKIV, respectively, and 40% identity with CoPK12, one of the CaMK orthologs in C. cinerea. CoPK02 showed significant autophosphorylation activity and phosphorylated exogenous proteins in the presence of Ca2+/CaM. By the CaM-overlay assay we confirmed that the C-terminal sequence (Trp346-Arg358) was the calmodulin-binding site, and that the binding of Ca2+/CaM to CoPK02 was reduced by the autophosphorylation of CoPK02. Since CoPK02 evolved in a different clade from CoPK12, and showed different gene expression compared to that of CoPK32, which is homologous to mitogen-activated protein kinase-activated protein kinase, CoPK02 and CoPK12 might cooperatively regulate Ca2+-signaling in C. cinerea.
Asunto(s)
Basidiomycota/enzimología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Secuencia de Aminoácidos , Animales , Basidiomycota/genética , Basidiomycota/crecimiento & desarrollo , Sitios de Unión , Señalización del Calcio , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Calmodulina/metabolismo , Catálisis , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Perfilación de la Expresión Génica , Genes Fúngicos , Fosforilación , Filogenia , Ratas , Homología de Secuencia de AminoácidoRESUMEN
Casein kinase 1 (CK1) is a widely expressed Ser/Thr kinase in eukaryotic organisms that is involved in various cellular processes (e.g., circadian rhythm and apoptosis). Therefore, preparing highly active CK1 and investigating its properties in vitro have important implications for understanding the biological roles of the kinase. However, recombinant CK1 undergoes autoinactivation via autophosphorylation in Escherichia coli cells and thus is undesirably prepared as a phosphorylated and inactivated kinase. To circumvent this problem, we established a protein expression system using E. coli strain BL21(DE3)pλPP in which λ protein phosphatase (λPPase) is constitutively expressed. Using this system, recombinant CK1 isoforms (α, δ and ε) were readily prepared as unphosphorylated forms. Furthermore, we found that CK1s prepared using BL21(DE3)pλPP showed markedly higher activity than those prepared by the conventional BL21(DE3). Finally, we demonstrated that the kinase activity of CK1δ from BL21(DE3)pλPP was higher than that prepared by a conventional method consisting of troublesome steps such as in vitro λPPase treatment. Thus, this simple method using BL21(DE3)pλPP is valuable for preparing highly active CK1s. It may also be applicable to other kinases that are difficult to prepare because of phosphorylation in E. coli cells.
Asunto(s)
Bacteriófago lambda/enzimología , Quinasa de la Caseína I , Escherichia coli , Expresión Génica , Fosfoproteínas Fosfatasas/biosíntesis , Proteínas Virales/biosíntesis , Bacteriófago lambda/genética , Quinasa de la Caseína I/biosíntesis , Quinasa de la Caseína I/química , Quinasa de la Caseína I/genética , Quinasa de la Caseína I/aislamiento & purificación , Escherichia coli/química , Escherichia coli/enzimología , Escherichia coli/genética , Humanos , Fosfoproteínas Fosfatasas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Virales/genéticaRESUMEN
Intracellular signal transduction is built on the basis of the subtle balance between phosphorylation and dephosphorylation. Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F/POPX2) and CaMKP-N (PPM1E/POPX1) are Ser/Thr phosphatases that belong to the PPM (protein phosphatase, Mg2+/Mn2+-dependent) family. The former was discovered in rat brain as a novel protein phosphatase regulating Ca2+/calmodulin-dependent protein kinases (CaMKs), whereas the latter was first identified in human cDNA databases using the rat CaMKP sequence. Subsequent studies have revealed that they are involved in various cellular functions through regulation of not only CaMKs but also other protein kinases such as AMP-activated protein kinase. Furthermore, accumulating evidence shows possible involvement of CaMKP and CaMKP-N in the pathogenesis of various diseases including cancer. Therefore, the biochemistry of CaMKP and CaMKP-N largely contributes to molecular medicine targeting these phosphatases. In this review, we summarized recent progress in the enzymology and biology of CaMKP and CaMKP-N. We also focused on etiology studies in which CaMKP and CaMKP-N are involved. Based on the emerging evidence, future perspectives of studies on these phosphatases and related issues to be elucidated are discussed.
Asunto(s)
Calcio/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteína Fosfatasa 2C/metabolismo , Secuencia de Aminoácidos , Animales , Dominio Catalítico , ADN Complementario/genética , Enfermedad , Humanos , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 2C/química , Proteína Fosfatasa 2C/genética , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
Brain edema is a severe complication that accompanies ischemic stroke. Increasing evidence shows that inflammatory cytokines impair tight junctions of the blood-brain barrier, suggesting the involvement of microglia in brain edema. In this study, we examined the role of microglia in the progression of ischemic brain edema using mice with permanent middle cerebral artery occlusion. The intensity of T2-weighted imaging (T2WI) in the cerebral cortex and the striatum was elevated 3â¯h after occlusion and spread to peripheral regions of the ischemic hemisphere. Merged images of 2,3,5-triphenyl tetrazolium chloride staining and T2WI revealed the exact vasogenic edema region, which spread from the ischemic core to outside the ischemic region. Microglia were strongly activated in the ischemic region 3â¯h after occlusion and, notably, activated microglia were observed in the non-ischemic region 24â¯h after occlusion. Pretreatment with minocycline, an inhibitor of microglial activation clearly suppressed not only vasogenic edema but also infarct formation. We demonstrated in this study that vasogenic edema spreads from the ischemic core to the peripheral region, which can be elicited, at least in part, by microglial activation induced by ischemia.
Asunto(s)
Edema Encefálico/etiología , Encéfalo/patología , Infarto de la Arteria Cerebral Media/complicaciones , Microglía/patología , Animales , Edema Encefálico/patología , Progresión de la Enfermedad , Infarto de la Arteria Cerebral Media/patología , Masculino , Ratones Endogámicos ICR , Agua/análisisRESUMEN
Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) is a Ser/Thr phosphatase that belongs to the PPM family. Growing evidence suggests that PPM phosphatases including CaMKP act as a complex with other proteins to regulate cellular functions. In this study, using the two-dimensional far-western blotting technique with digoxigenin-labeled CaMKP as a probe, in conjunction with peptide mass fingerprinting analysis, we identified neurofilament L (NFL) as a CaMKP-binding protein in a Triton-insoluble fraction of rat brain. We confirmed binding of fluorescein-labeled CaMKP (F-CaMKP) to NFL in solution by fluorescence polarization. The analysis showed that the dissociation constant of F-CaMKP for NFL is 73 ± 17 nM (n = 3). Co-immunoprecipitation assay using a cytosolic fraction of NGF-differentiated PC12 cells showed that endogenous CaMKP and NFL form a complex in cells. Furthermore, the effect of CaMKP on self-assembly of NFL was examined. Electron microscopy revealed that CaMKP markedly prevented NFL from forming large filamentous aggregates, suggesting that CaMKP-binding to NFL inhibits its filament association. These findings may provide new insights into a novel mechanism for regulating network formation of neurofilaments during neuronal differentiation.
Asunto(s)
Encéfalo/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/química , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Proteínas de Neurofilamentos/química , Proteínas de Neurofilamentos/metabolismo , Neuronas/metabolismo , Animales , Sitios de Unión , Química Encefálica , Células PC12 , Unión Proteica , Ratas , Distribución TisularRESUMEN
In this study, protective actions of the sex steroid hormones, progesterone, testosterone, and 17ß-estradiol, against oxygen-glucose deprivation (OGD)/reoxygenation-induced neuronal cell death were examined using rat organotypic hippocampal slice cultures. Progesterone, testosterone, and 17ß-estradiol significantly attenuated neuronal cell death elicited by OGD/reoxygenation. While the neuroprotection conferred by progesterone was not affected by SU-10603, an inhibitor of cytochrome P45017α, finasteride, a 5α-reductase inhibitor that blocks the conversion of progesterone to allopregnanolone, partially reversed the neuroprotection induced by progesterone. The progesterone metabolite, allopregnanolone attenuated neuronal injury induced by OGD/reoxygenation. Pretreatment with letrozole, a cytochrome P450 aromatase inhibitor or 4-hydroxyphenyl-1-naphthol, a 17ß-hydroxysteroid dehydrogenase 2 inhibitor showed no effect on testosterone-mediated neuroprotection, while finasteride completely abolished the protective action of testosterone. Treatment with 5α-dihydrotestosterone significantly suppressed neuronal injury. Pretreatment with mifepristone, a progesterone receptor antagonist and hydroxyflutamid, an androgen receptor antagonist significantly diminished the neuroprotective effects of progesterone and testosterone, respectively. ICI182,780, an estrogen receptor antagonist, showed no effect on neuroprotection mediated by 17ß-estradiol. Pretreatment with actinomycin D or cycloheximide clearly abolished the neuroprotective effects of progesterone and testosterone, while actinomycin D and cycloheximide did not show any effect on neuroprotection mediated by 17ß-estradiol. Taken together, progesterone protects neurons via progesterone receptor-dependent genomic pathway, and allopregnanolone is involved in progesterone-mediated neuroprotection. Testosterone and its metabolite 5α-dihydrotestosterone protect neurons via the genomic pathway of the androgen receptor. Metabolism of sex steroid hormones in the brain might complicate their protective actions in the brain.
Asunto(s)
Glucosa/metabolismo , Hipocampo/metabolismo , Hipoxia/metabolismo , Oxígeno/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Dihidrotestosterona/farmacología , Estradiol/farmacología , Finasterida/farmacología , Glucosa/deficiencia , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Fármacos Neuroprotectores , Pregnanolona/farmacología , Progesterona/farmacología , Ratas , Receptores Androgénicos/metabolismo , Testosterona/farmacología , Tetrahidronaftalenos/farmacologíaRESUMEN
We describe here the expression and characterization of a constitutively active fragment of zebrafish Ca(2+)/calmodulin-dependent protein kinase (CaMK) Iδ designated zCaMKIδ(1-299) that lacks an autoinhibitory domain. We used a simple one-step purification method to isolate the recombinant enzyme at high yield (220 mg/l of the culture medium) from the soluble fraction of lysates prepared from Escherichia coli. Unlike the corresponding fragment of CaMKIα (CaMKΙα(1-294)), the kinase activity of zCaMKIδ(1-299), without activation procedures, was comparable to that of wild-type zCaMKIδ activated by CaMK kinase. zCaMKIδ(1-299) exhibited broad substrate specificity highly similar to that of wild-type zCaMKIδ, and complementary to that of the cAMP-dependent protein kinase catalytic subunit (PKAc). The protein kinase activity of zCaMKIδ(1-299) was higher compared with that of PKAc as well as CX-30K-CaMKII that comprises a constitutively active fragment of CaMKII fused to the N-terminal region of Xenopus CaMKI. Furthermore, kinase activity was highly stable against thermal inactivation and repeated freezing-thawing. Thus, zCaMKIδ(1-299) represents a readily available alternative that can be used as a "High-performance phosphorylating reagent" alone or in combination with PKAc in diverse experiments on protein phosphorylation and dephosphorylation.
Asunto(s)
Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/genética , Dominio Catalítico , Clonación Molecular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Estabilidad de Enzimas , Escherichia coli/genética , Fosforilación , Especificidad por Sustrato , Pez Cebra/genética , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) is a Ser/Thr protein phosphatase that belongs to the PPM family. It is important to identify an endogenous regulator of CaMKP. Using an Escherichia coli two-hybrid screening method, we identified the C-terminal cytoplasmic fragment of protocadherin γ subfamily C5 (Pcdh-γC5), which was generated by intracellular processing, as a CaMKP-binding protein. Dephosphorylation of phosphorylated Ca(2+)/calmodulin-dependent protein kinase I (CaMKI) by CaMKP was significantly activated by the C-terminal cytoplasmic fragment, Pcdh-γC5(715-944), both in vitro and in cells, suggesting that the C-terminal fragment functions as an endogenous activator of CaMKP. The nuclear translocation of the fragment was blocked by its binding to cytoplasmic CaMKP to form a ternary complex with CaMKI. Taken together, these results strongly suggest that the C-terminal cytoplasmic fragment of Pcdh-γC5 acts as a scaffold for CaMKP and CaMKI to regulate CaMKP activity. These findings may provide new insights into the reversible regulation of CaMKP in cells.
Asunto(s)
Cadherinas/metabolismo , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/metabolismo , Regulación de la Expresión Génica , Neuronas/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Transporte Activo de Núcleo Celular/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Proteínas Relacionadas con las Cadherinas , Cadherinas/química , Cadherinas/genética , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/genética , Línea Celular Tumoral , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citosol/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Neuronas/citología , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Unión Proteica , Ratas , Proteínas Recombinantes de Fusión , Transducción de SeñalRESUMEN
Microglia are activated quickly in response to external pathogens or cell debris and clear these substances via the inflammatory response. However, excessive activation of microglia can be harmful to host cells due to the increased production of reactive oxygen species and proinflammatory cytokines. Superoxide dismutase 2 (SOD2) is reportedly induced under various inflammatory conditions in the central nervous system. We herein demonstrated that activated microglia strongly express SOD2 and examined the role of SOD2, focusing on regulation of the microglial activity and the susceptibility of microglia to oxidative stress. When rat primary microglia were treated with LPS, poly(I:C), peptidoglycan, or CpG oligodeoxynucleotide, respectively, the mRNA and protein levels of SOD2 largely increased. However, an increased expression of SOD2 was not detected in the primary neurons or astrocytes, indicating that SOD2 is specifically induced in microglia under inflammatory conditions. The activated microglia showed high tolerance to oxidative stress, whereas SOD2 knockdown conferred vulnerability to oxidative stress. Interestingly, the production of proinflammatory cytokines was increased in the activated microglia treated with SOD2 siRNA compared with that observed in the control siRNA-treated cells. Pretreatment with NADPH oxidase inhibitors, diphenylene iodonium and apocynin, decreased in not only reactive oxygen species generation but also the proinflammatory cytokine expression. Notably, SOD2 knockdown largely potentiated the nuclear factor κB activity in the activated microglia. Taken together, increased SOD2 conferred tolerance to oxidative stress in the microglia and decreased proinflammatory cytokine production by attenuating the nuclear factor κB activity. Therefore, SOD2 might regulate neuroinflammation by controlling the microglial activities.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Microglía/enzimología , Estrés Oxidativo , Superóxido Dismutasa/biosíntesis , Animales , Citocinas/biosíntesis , Citocinas/genética , Técnicas de Silenciamiento del Gen , Inflamación/inducido químicamente , Inflamación/enzimología , Inflamación/genética , Inflamación/patología , Lipopolisacáridos/farmacología , Microglía/patología , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Oligodesoxirribonucleótidos/farmacología , Poli I-C/farmacología , Ratas , Ratas Wistar , Superóxido Dismutasa/genéticaRESUMEN
The protective roles of astrocytes in neurotoxicity induced by environmental chemicals, such as methylmercury (MeHg), are largely unknown. We found that conditioned medium of MeHg-treated astrocytes (MCM) attenuated neuronal cell death induced by MeHg, suggesting that astrocytes-released factors can protect neuronal cells. The increased expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) was observed in MeHg-treated astrocytes. NGF and BDNF were detected in culture media as homodimers, which are able to bind specific tyrosine kinase receptors, tropomyosin related kinase (Trk) A and TrkB, respectively. The TrkA antagonist and TrkB antagonist abolished the protective effects of MCM in neuronal cell death induced by MeHg. Taken together, astrocytes synthesize and release NGF and BDNF in response to MeHg to protect neurons from MeHg toxicity. This study is considered to show a novel defense mechanism against MeHg-induced neurotoxicity.