RESUMEN
Cytoplasmic intermediate filaments (cIFs) are found in all eumetazoans, except arthropods. To investigate the compatibility of cIFs in arthropods, we expressed human vimentin (hVim), a cIF with filament-forming capacity in vertebrate cells and tissues, transgenically in Drosophila Transgenic hVim could be recovered from whole-fly lysates by using a standard procedure for intermediate filament (IF) extraction. When this procedure was used to test for the possible presence of IF-like proteins in flies, only lamins and tropomyosin were observed in IF-enriched extracts, thereby providing biochemical reinforcement to the paradigm that arthropods lack cIFs. In Drosophila, transgenic hVim was unable to form filament networks in S2 cells and mesenchymal tissues; however, cage-like vimentin structures could be observed around the nuclei in internal epithelia, which suggests that Drosophila retains selective competence for filament formation. Taken together, our results imply that although the filament network formation competence is partially lost in Drosophila, a rudimentary filament network formation ability remains in epithelial cells. As a result of the observed selective competence for cIF assembly in Drosophila, we hypothesize that internal epithelial cIFs were the last cIFs to disappear from arthropods.-Gullmets, J., Torvaldson, E., Lindqvist, J., Imanishi, S. Y., Taimen, P., Meinander, A., Eriksson, J. E. Internal epithelia in Drosophila display rudimentary competence to form cytoplasmic networks of transgenic human vimentin.
Asunto(s)
Citoplasma/metabolismo , Drosophila/metabolismo , Epitelio/metabolismo , Vimentina/metabolismo , Animales , Animales Modificados Genéticamente , Western Blotting , Línea Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Filamentos Intermedios/enzimología , Filamentos Intermedios/metabolismo , Laminas/genética , Laminas/metabolismo , Tropomiosina/genética , Tropomiosina/metabolismo , Vimentina/genéticaRESUMEN
Nestin, a member of the cytoskeletal family of intermediate filaments, regulates the onset of myogenic differentiation through bidirectional signaling with the kinase Cdk5. Here, we show that these effects are also reflected at the organism level, as there is a loss of skeletal muscle mass in nestin-/- (NesKO) mice, reflected as reduced lean (muscle) mass in the mice. Further examination of muscles in male mice revealed that these effects stemmed from nestin-deficient muscles being more prone to spontaneous regeneration. When the regeneration capacity of the compromised NesKO muscle was tested by muscle injury experiments, a significant healing delay was observed. NesKO satellite cells showed delayed proliferation kinetics in conjunction with an elevation in p35 (encoded by Cdk5r1) levels and Cdk5 activity. These results reveal that nestin deficiency generates a spontaneous regenerative phenotype in skeletal muscle that relates to a disturbed proliferation cycle that is associated with uncontrolled Cdk5 activity.
Asunto(s)
Homeostasis , Músculo Esquelético/fisiología , Nestina/metabolismo , Regeneración , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Quinasa 5 Dependiente de la Ciclina/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Fibras Musculares Esqueléticas/fisiología , Mioblastos/metabolismo , Nestina/deficiencia , Tamaño de los Órganos , Fenotipo , Células Satélite del Músculo Esquelético/metabolismo , Cicatrización de HeridasRESUMEN
Contrary to cell cycle-associated cyclin-dependent kinases, CDK5 is best known for its regulation of signaling processes in differentiated cells and its destructive activation in Alzheimer's disease. Recently, CDK5 has been implicated in a number of different cancers, but how it is able to stimulate cancer-related signaling pathways remains enigmatic. Our goal was to study the cancer-promoting mechanisms of CDK5 in prostate cancer. We observed that CDK5 is necessary for proliferation of several prostate cancer cell lines. Correspondingly, there was considerable growth promotion when CDK5 was overexpressed. When examining the reasons for the altered proliferation effects, we observed that CDK5 phosphorylates S308 on the androgen receptor (AR), resulting in its stabilization and differential expression of AR target genes including several growth-priming transcription factors. However, the amplified cell growth was found to be separated from AR signaling, further corroborated by CDK5-dependent proliferation of AR null cells. Instead, we found that the key growth-promoting effect was due to specific CDK5-mediated AKT activation. Down-regulation of CDK5 repressed AKT phosphorylation by altering its intracellular localization, immediately followed by prominent cell cycle inhibition. Taken together, these results suggest that CDK5 acts as a crucial signaling hub in prostate cancer cells by controlling androgen responses through AR, maintaining and accelerating cell proliferation through AKT activation, and releasing cell cycle breaks.
Asunto(s)
Proliferación Celular , Quinasa 5 Dependiente de la Ciclina/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Androgénicos/metabolismo , Quinasa 5 Dependiente de la Ciclina/genética , Humanos , Masculino , Fosforilación , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/fisiopatología , Receptores Androgénicos/genética , Transducción de SeñalRESUMEN
Epithelial-mesenchymal transition (EMT) in cells is a developmental process adopted during tumorigenesis that promotes metastatic capacity. In this study, we advance understanding of EMT control in cancer cells with the description of a novel vimentin-ERK axis that regulates the transcriptional activity of Slug (SNAI2). Vimentin, ERK, and Slug exhibited overlapping subcellular localization in clinical specimens of triple-negative breast carcinoma. RNAi-mediated ablation of these gene products inhibited cancer cell migration and cell invasion through a laminin-rich matrix. Biochemical analyses demonstrated direct interaction of vimentin and ERK, which promoted ERK activation and enhanced vimentin transcription. Consistent with its role as an intermediate filament, vimentin acted as a scaffold to recruit Slug to ERK and promote Slug phosphorylation at serine-87. Site-directed mutagenesis established a requirement for ERK-mediated Slug phosphorylation in EMT initiation. Together, these findings identified a pivotal step in controlling the ability of Slug to organize hallmarks of EMT.
Asunto(s)
Proteína Quinasa 1 Activada por Mitógenos/biosíntesis , Factores de Transcripción/biosíntesis , Neoplasias de la Mama Triple Negativas/genética , Vimentina/biosíntesis , Animales , Carcinogénesis/genética , Movimiento Celular/genética , Proliferación Celular/genética , Embrión de Pollo , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Proteína Quinasa 1 Activada por Mitógenos/genética , Invasividad Neoplásica/genética , Metástasis de la Neoplasia , Fosforilación , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Neoplasias de la Mama Triple Negativas/patología , Vimentina/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Lamin A/C is part of the nuclear lamina, a meshwork of intermediate filaments underlying the inner nuclear membrane. The lamin network is anchoring a complex set of structural and linker proteins and is either directly or through partner proteins also associated or interacting with a number of signaling protein and transcription factors. During mitosis the nuclear lamina is dissociated by well established phosphorylation- dependent mechanisms. A-type lamins are, however, also phosphorylated during interphase. A recent study identified 20 interphase phosphorylation sites on lamin A/C and explored their functions related to lamin dynamics; movements, localization and solubility. Here we discuss these findings in the light of lamin functions in health and disease.
Asunto(s)
Interfase/genética , Lamina Tipo A/química , Mitosis , Distrofia Muscular de Emery-Dreifuss/genética , Transducción de Señal , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Filamentos Intermedios/química , Filamentos Intermedios/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Distrofia Muscular de Emery-Dreifuss/metabolismo , Distrofia Muscular de Emery-Dreifuss/patología , Mutación , Lámina Nuclear/química , Lámina Nuclear/metabolismo , Fosforilación , Transporte de Proteínas , SolubilidadRESUMEN
Nuclear lamins form the major structural elements that comprise the nuclear lamina. Loss of nuclear structural integrity has been implicated as a key factor in the lamin A/C gene mutations that cause laminopathies, whereas the normal regulation of lamin A assembly and organization in interphase cells is still undefined. We assumed phosphorylation to be a major determinant, identifying 20 prime interphase phosphorylation sites, of which eight were high-turnover sites. We examined the roles of these latter sites by site-directed mutagenesis, followed by detailed microscopic analysis - including fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and nuclear extraction techniques. The results reveal three phosphorylation regions, each with dominant sites, together controlling lamin A structure and dynamics. Interestingly, two of these interphase sites are hyper-phosphorylated in mitotic cells and one of these sites is within the sequence that is missing in progerin of the Hutchinson-Gilford progeria syndrome. We present a model where different phosphorylation combinations yield markedly different effects on the assembly, subunit turnover and the mobility of lamin A between, and within, the lamina, the nucleoplasm and the cytoplasm of interphase cells.
Asunto(s)
Interfase , Lamina Tipo A/metabolismo , Procesamiento Proteico-Postraduccional , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Células HeLa , Humanos , Datos de Secuencia Molecular , Lámina Nuclear/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas , Transporte de Proteínas , Factores de TranscripciónRESUMEN
The AP-1 transcription factor c-Jun has been shown to be essential for stress-induced apoptosis in several models. However, the molecular mechanisms underlying the proapoptotic activity of c-Jun are poorly understood. We identify the apoptosis-antagonizing transcription factor (AATF) as a novel nucleolar stress sensor, which is required as a cofactor for c-Jun-mediated apoptosis. Overexpression or down-regulation of AATF expression levels led to a respective increase or decrease in the amount of activated and phosphorylated c-Jun with a proportional alteration in the induction levels of the proapoptotic c-Jun target genes FasL and TNF-α. Accordingly, AATF promoted commitment of ultraviolet (UV)-irradiated cells to c-Jun-dependent apoptosis. Whereas AATF overexpression potentiated UV-induced apoptosis in wild-type cells, c-Jun-deficient mouse embryonic fibroblasts were resistant to AATF-mediated apoptosis induction. Furthermore, AATF mutants defective in c-Jun binding were also defective in inducing AP-1 activity and c-Jun-mediated apoptosis. UV irradiation induced a translocation of AATF from the nucleolus to the nucleus, thereby enabling its physical association to c-Jun. Analysis of AATF deletion mutants revealed that the AATF domains required for compartmentalization, c-Jun binding, and enhancement of c-Jun transcriptional activity were all also required to induce c-Jun-dependent apoptosis. These results identify AATF as a nucleolar-confined c-Jun cofactor whose expression levels and spatial distribution determine the stress-induced activity of c-Jun and the levels of c-Jun-mediated apoptosis.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Represoras/metabolismo , Animales , Apoptosis/efectos de la radiación , Proteínas Reguladoras de la Apoptosis/química , Nucléolo Celular/efectos de la radiación , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Proteínas Nucleares/química , Unión Proteica/efectos de la radiación , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de la radiación , Proteínas Represoras/química , Rayos UltravioletaRESUMEN
Intermediate filament (IF) proteins comprise a large family with more than 70 members. Initially, IFs were assumed to provide only structural reinforcement for the cell. However, IFs are now known to be dynamic structures that are involved in a wide range of cellular processes during all stages of life, from development to ageing, and during homeostasis and stress. This Commentary discusses some lesser-known functional and regulatory aspects of IFs. We specifically address the emerging roles of nestin in myogenesis and cancer cell migration, and examine exciting evidence on the regulation of nestin and lamin A by the notch signalling pathway, which could have repercussions for our understanding of the roles of IF proteins in development and ageing. In addition, we discuss the modulation of the post-translational modifications of neuronally expressed IFs and their protein-protein interactions, as well as IF glycosylation, which not only has a role in stress and ageing, but might also regulate IFs during development. Although many of these recent findings are still preliminary, they nevertheless open new doors to explore the functionality of the IF family of proteins.
Asunto(s)
Envejecimiento/metabolismo , Filamentos Intermedios/metabolismo , Envejecimiento/genética , Animales , Humanos , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Filamentos Intermedios/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Nestina , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Many types of progenitor cells are distinguished by the expression of the intermediate filament protein nestin, a frequently used stem cell marker, the physiological roles of which are still unknown. Whereas myogenesis is characterized by dynamically regulated nestin levels, we studied how altering nestin levels affects myoblast differentiation. Nestin determined both the onset and pace of differentiation. Whereas depletion of nestin by RNAi strikingly accelerated the process, overexpression of nestin completely inhibited differentiation. Nestin down-regulation augmented the early stages of differentiation, at the level of cell-cycle withdrawal and expression of myogenic markers, but did not affect proliferation of undifferentiated dividing myoblasts. Nestin regulated the cleavage of the Cdk5 activator protein p35 to its degradation-resistant form, p25. In this way, nestin has the capacity to halt myoblast differentiation by inhibiting sustained activation of Cdk5 by p25, which is critical for the progress of differentiation. Our results imply that nestin regulates the early stages of myogenesis rather than maintains the undifferentiated state of progenitor cells. In the bidirectional interrelationship between nestin and Cdk5, Cdk5 regulates the organization and stability of its own nestin scaffold, which in turn controls the effects of Cdk5. This nestin-Cdk5 cross-talk sets the pace of muscle differentiation.