RESUMEN
During brain development, neural precursor cells (NPCs) expand initially, and then switch to generating stage-specific neurons while maintaining self-renewal ability. Because the NPC pool at the onset of neurogenesis crucially affects the final number of each type of neuron, tight regulation is necessary for the transitional timing from the expansion to the neurogenic phase in these cells. However, the molecular mechanisms underlying this transition are poorly understood. Here, we report that the telencephalon-specific loss of PAR3 before the start of neurogenesis leads to increased NPC proliferation at the expense of neurogenesis, resulting in disorganized tissue architecture. These NPCs demonstrate hyperactivation of hedgehog signaling in a smoothened-dependent manner, as well as defects in primary cilia. Furthermore, loss of PAR3 enhanced ligand-independent ciliary accumulation of smoothened and an inhibitor of smoothened ameliorated the hyperproliferation of NPCs in the telencephalon. Thus, these findings support the idea that PAR3 has a crucial role in the transition of NPCs from the expansion phase to the neurogenic phase by restricting hedgehog signaling through the establishment of ciliary integrity.
Asunto(s)
Proteínas Hedgehog , Células-Madre Neurales , Células-Madre Neurales/fisiología , Neuronas , Neurogénesis , Transducción de Señal/fisiologíaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is the cancer with the poorest prognosis. One of the major properties reflecting its poor prognosis is high-grade heterogeneity, which leads to insensitivity to anticancer treatments. Cancer stem cells (CSCs) acquire phenotypic heterogeneity, generating abnormally differentiated cells by asymmetric cell division. However, the detailed mechanism leading to phenotypic heterogeneity is largely unknown. Here, we showed that PDAC patients with co-upregulation of PKCλ and ALDH1A3 had the poorest clinical outcome. PKCλ knockdown by DsiRNA in the ALDH1high population of PDAC MIA-PaCa-2 cells attenuated the asymmetric distribution of the ALDH1A3 protein. To monitor asymmetric cell division of ALDH1A3-positive PDAC CSCs, we established stable Panc-1 PDAC clones expressing ALDH1A3-turboGFP (Panc-1-ALDH1A3-turboGFP cells). In addition to MIA-PaCa-2-ALDH1high cells, turboGFPhigh cells sorted from Panc-1-ALDH1A3-turboGFP cells showed asymmetric cell propagation of ALDH1A3 protein. PKCλ DsiRNA in Panc-1-ALDH1A3-turboGFP cells also attenuated the asymmetric distribution of ALDH1A3 protein. These results suggest that PKCλ regulates the asymmetric cell division of ALDH1A3-positive PDAC CSCs. Furthermore, Panc-1-ALDH1A3-turboGFP cells can be useful for the visualization and monitoring of CSC properties such as asymmetric cell division of ALDH1A3-positive PDAC CSCs in time-lapse imaging.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , División Celular Asimétrica , Línea Celular Tumoral , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/patología , Familia de Aldehído Deshidrogenasa 1/metabolismo , Células Madre Neoplásicas/patología , Neoplasias PancreáticasRESUMEN
Cell polarity is essential for various asymmetric cellular events, and the partitioning defective (PAR) protein PAR3 (encoded by PARD3 in mammals) plays a unique role as a cellular landmark to establish polarity. In epithelial cells, PAR3 localizes at the subapical border, such as the tight junction in vertebrates, and functions as an apical determinant. Although we know a great deal about the regulators of PAR3 localization, how PAR3 is concentrated and localized to a specific membrane domain remains an important question to be clarified. In this study, we demonstrate that ASPP2 (also known as TP53BP2), which controls PAR3 localization, links PAR3 and protein phosphatase 1 (PP1). The ASPP2-PP1 complex dephosphorylates a novel phosphorylation site, Ser852, of PAR3. Furthermore, Ser852- or Ser889-unphosphorylatable PAR3 mutants form protein clusters, and ectopically localize to the lateral membrane. Concomitance of clustering and ectopic localization suggests that PAR3 localization is a consequence of local clustering. We also demonstrate that unphosphorylatable forms of PAR3 exhibited a low molecular turnover and failed to coordinate rapid reconstruction of the tight junction, supporting that both the phosphorylated and dephosphorylated states are essential for the functional integrity of PAR3.
Asunto(s)
Polaridad Celular , Proteína Quinasa C , Animales , Proteínas de Ciclo Celular/metabolismo , Análisis por Conglomerados , Fosforilación , Proteína Quinasa C/metabolismo , Uniones Estrechas/metabolismoRESUMEN
Regnase-1-mediated mRNA decay (RMD), in which inflammatory mRNAs harboring specific stem-loop structures are degraded, is a critical part of proper immune homeostasis. Prior to initial translation, Regnase-1 associates with target stem-loops but does not carry out endoribonucleolytic cleavage. Single molecule imaging revealed that UPF1 is required to first unwind the stem-loops, thus licensing Regnase-1 to proceed with RNA degradation. Following translation, Regnase-1 physically associates with UPF1 using two distinct points of interaction: The Regnase-1 RNase domain binds to SMG1-phosphorylated residue T28 in UPF1; in addition, an intrinsically disordered segment in Regnase-1 binds to the UPF1 RecA domain, enhancing the helicase activity of UPF1. The SMG1-UPF1-Regnase-1 axis targets pioneer rounds of translation and is critical for rapid resolution of inflammation through restriction of the number of proteins translated by a given mRNA. Furthermore, small-molecule inhibition of SMG1 prevents RNA unwinding in dendritic cells, allowing post-transcriptional control of innate immune responses.
Asunto(s)
Macrófagos Peritoneales/inmunología , Degradación de ARNm Mediada por Codón sin Sentido/inmunología , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , Ribonucleasas/genética , Transactivadores/genética , Animales , Fibroblastos/citología , Fibroblastos/inmunología , Células HEK293 , Células HeLa , Homeostasis/genética , Homeostasis/inmunología , Humanos , Inmunidad Innata , Inflamación , Secuencias Invertidas Repetidas , Macrófagos/citología , Macrófagos/inmunología , Macrófagos Peritoneales/citología , Ratones , Ratones Noqueados , Mutación , Cultivo Primario de Células , Unión Proteica , Biosíntesis de Proteínas , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/inmunología , ARN Mensajero/metabolismo , Ribonucleasas/deficiencia , Ribonucleasas/inmunología , Imagen Individual de Molécula , Transactivadores/inmunologíaRESUMEN
Impaired cell polarity is a hallmark of diseased tissue. In the cardiovascular system, laminar blood flow induces endothelial planar cell polarity, represented by elongated cell shape and asymmetric distribution of intracellular organelles along the axis of blood flow. Disrupted endothelial planar polarity is considered to be pro-inflammatory, suggesting that the establishment of endothelial polarity elicits an anti-inflammatory response. However, a causative relationship between polarity and inflammatory responses has not been firmly established. Here, we find that a cell polarity protein, PAR-3, is an essential gatekeeper of GSK3ß activity in response to laminar blood flow. We show that flow-induced spatial distribution of PAR-3/aPKCλ and aPKCλ/GSK3ß complexes controls local GSK3ß activity and thereby regulates endothelial planar polarity. The spatial information for GSK3ß activation is essential for flow-dependent polarity to the flow axis, but is not necessary for flow-induced anti-inflammatory response. Our results shed light on a novel relationship between endothelial polarity and vascular homeostasis highlighting avenues for novel therapeutic strategies.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Proteínas de Ciclo Celular/fisiología , Polaridad Celular/fisiología , Endotelio Vascular/metabolismo , Inflamación/metabolismo , Proteínas de la Membrana/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Aorta/fisiopatología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Proteínas del Citoesqueleto/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Homeostasis/fisiología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas Nucleares/metabolismo , Proteína Quinasa C/metabolismo , Flujo Sanguíneo Regional , Proteínas Represoras/metabolismo , Transducción de SeñalRESUMEN
We previously reported that aberrant expression of atypical protein kinase C λ/ι (aPKCλ/ι) in low-grade squamous intraepithelial uterine cervix lesions was associated with an increased risk of progression to higher grade. This study aimed to investigate aPKCλ/ι expression patterns in cervical squamous cell carcinoma (SCC) and its association with disease progression. We immunohistochemically assessed aPKCλ/ι expression in 168 SCC samples and 13 normal uterine cervix samples. In 69.0% of SCC cases, aPKCλ/ι was expressed more abundantly than in normal epithelium, but there was no significant association between aPKCλ/ι intensity and disease progression (P=0.087, Cochran-Mantel-Haenszel test). aPKCλ/ι in normal cervical epithelium was confined to the cytoplasm or intercellular junctions. In contrast, aPKCλ/ι was predominantly localized within the nucleus in 36.9% of SCC samples (P<0.001, χ test), and the prevalence was significantly increased relative to advanced tumor stage (P<0.001, Cochran-Mantel-Haenszel test). Moreover, patients with SCC with aPKCλ/ι nuclear localization had worse prognoses than those with cytoplasmic localization (P<0.001, log-rank test). aPKCλ/ι localization differed between the intraepithelial lesion and adjacent invasive cancer in 40% of cases, while the expression pattern was similar between primary and matched metastatic tumors. In conclusion, aPKCλ/ι nuclear localization in cervical cancer is associated with tumor progression and worse prognosis. This is the first report to show aberrant nuclear aPKCλ/ι localization in a subgroup of cervical cancer patients and its association with worse prognosis.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Neoplasias del Cuello Uterino/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/patología , Progresión de la Enfermedad , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Pronóstico , Transporte de Proteínas , Neoplasias del Cuello Uterino/patología , Adulto JovenRESUMEN
Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that regulates the degradation of mRNAs harboring premature translation termination codons. NMD also influences the expression of many physiological transcripts. SMG-1 is a large kinase essential to NMD that phosphorylates Upf1, which seems to be the definitive signal triggering mRNA decay. However, the regulation of the kinase activity of SMG-1 remains poorly understood. Here, we reveal the three-dimensional architecture of SMG-1 in complex with SMG-8 and SMG-9, and the structural mechanisms regulating SMG-1 kinase. A bent arm comprising a long region of HEAT (huntington, elongation factor 3, a subunit of PP2A and TOR1) repeats at the N terminus of SMG-1 functions as a scaffold for SMG-8 and SMG-9, and projects from the C-terminal core containing the phosphatidylinositol 3-kinase domain. SMG-9 seems to control the activity of SMG-1 indirectly through the recruitment of SMG-8 to the N-terminal HEAT repeat region of SMG-1. Notably, SMG-8 binding to the SMG-1:SMG-9 complex specifically down-regulates the kinase activity of SMG-1 on Upf1 without contacting the catalytic domain. Assembly of the SMG-1:SMG-8:SMG-9 complex induces a significant motion of the HEAT repeats that is signaled to the kinase domain. Thus, large-scale conformational changes induced by SMG-8 after SMG-9-mediated recruitment tune SMG-1 kinase activity to modulate NMD.
Asunto(s)
Modelos Moleculares , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Fosfatidilinositol 3-Quinasas/genética , Proteínas Quinasas/genética , Multimerización de Proteína/fisiología , Proteínas Serina-Treonina Quinasas , Estructura Cuaternaria de Proteína , ARN Helicasas , Proteínas Recombinantes/metabolismo , Transactivadores/metabolismoRESUMEN
PKCι/λ has been proposed to be crucial in the early expression of long-term potentiation (LTP). Here, we further investigate the potential role of PKCι/λ in learning and memory by generating PKCι/λ conditional knockout mice specifically lacking PKCι/λ in the hippocampal CA1 pyramidal cells. Surprisingly, PKCι/λ cKO mice show normal hippocampal LTP and memory. Further close-up observation reveals compensation for PKCι/λ expression by PKMζ in PKCι/λ cKO mice. This compensation was not observed under basal conditions, but was detected either after LTP induction or learning-associated behavioral training. Accordingly, in the early stage of LTP expression, a switch from PKCι/λ- to PKMζ-dependent molecular mechanisms was detected in PKCι/λ cKO mice. Notably, when cKO mice were challenged with more difficult hippocampus-dependent learning tasks, moderate learning deficits were detected, suggesting a suboptimal compensation for PKCι/λ's function in PKCι/λ cKO mice. Thus, under physiological conditions, PKCι/λ is essential for hippocampal early-LTP and long-term memory (LTM).
Asunto(s)
Aprendizaje por Asociación/fisiología , Hipocampo/citología , Hipocampo/fisiología , Memoria a Largo Plazo/fisiología , Células Piramidales/fisiología , Animales , Condicionamiento Psicológico , Estimulación Eléctrica , Miedo , Femenino , Regulación de la Expresión Génica/genética , Potenciación a Largo Plazo/genética , Potenciación a Largo Plazo/fisiología , Masculino , Aprendizaje por Laberinto , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacologíaRESUMEN
BACKGROUND: Pancreatic cancer (PC) is the most lethal malignancy among solid tumors, and the most common risk factor for its development is cigarette smoking. Atypical protein kinase C (aPKC) isozymes function in cell polarity, proliferation, and survival, and have also been implicated in carcinogenesis. However, the involvement of aPKC in PC progression and the effect of nicotine, a major component of cigarette smoke, on the biological activities of aPKC remain to be fully elucidated. METHODS: We investigated the effects of nicotine on the proliferation, migration and invasion of the human PC cell lines Panc1 and BxPC3. We analyzed aPKC localization and activity by immunohistochemistry and in vitro kinase assays, respectively, to assess their involvement in the regulation of PC progression. Moreover, we examined the effect of nicotine on implanted peritoneal tumors of PC cells in mice. RESULTS: Nicotine enhanced cell proliferation, migration and invasion in Panc1 and BxPC3 cells. In nicotine-treated PC cells, the aPKC was significantly activated. We also found that nicotine induced phosphatidylinositol 3-kinase (PI3K) signal activation, and a specific inhibitor of the nicotine acetylcholine receptor (nAChR) as well as knockdown of nAChR prevented nicotine-mediated Akt phosphorylation and aPKC activation. In a peritoneal dissemination model of PC, nicotine-treated mice had larger tumors and increased numbers of nodules. Immunohistochemistry showed enhanced expression levels of aPKC and phosphorylated Akt in nodules from nicotine-treated mice. CONCLUSIONS AND GENERAL SIGNIFICANCE: Nicotine induces aberrant activation of aPKC via nAChR/PI3K signaling in PC cells, resulting in enhancement of cellular proliferation, migration and invasion.
Asunto(s)
Nicotina/farmacología , Neoplasias Pancreáticas/metabolismo , Proteína Quinasa C/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica , Nicotina/toxicidad , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Fumar/efectos adversosRESUMEN
Epithelial apicobasal polarity has fundamental roles in epithelial physiology and morphogenesis. The PAR complex, comprising PAR-3, PAR-6 and atypical protein kinase C (aPKC), is involved in determining cell polarity in various biological contexts, including in epithelial cells. However, it is not fully understood how the PAR complex induces apicobasal polarity. In this study, we found that PAR-3 regulates the protein expression of Girdin (also known as GIV or CCDC88A), a guanine-nucleotide-exchange factor (GEF) for heterotrimeric Gαi subunits, at the transcriptional level by cooperating with the AP-2 transcription factor. In addition, we confirmed that PAR-3 physically interacts with Girdin, and show that Girdin, together with the Gαi3 (also known as GNAI3), controls tight junction formation, apical domain development and actin organization downstream of PAR-3. Taken together, our findings suggest that transcriptional upregulation of Girdin expression and Girdin-Gαi3 signaling play crucial roles in regulating epithelial apicobasal polarity through the PAR complex.
Asunto(s)
Proteínas Portadoras/metabolismo , Polaridad Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas de Microfilamentos/genética , Transcripción Genética , Proteínas de Transporte Vesicular/genética , Animales , Secuencia de Bases , Comunicación Celular , Perros , Regulación de la Expresión Génica , Células HeLa , Células Hep G2 , Humanos , Células de Riñón Canino Madin Darby , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso , Unión Proteica , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Transducción de Señal , Uniones Estrechas/metabolismo , Factor de Transcripción AP-2/metabolismoRESUMEN
During an immune response against a microbial pathogen, activated naive T lymphocytes give rise to effector cells that provide acute host defense and memory cells that provide long-lived immunity. It has been shown that T lymphocytes can undergo asymmetric division, enabling the daughter cells to inherit unequal amounts of fate-determining proteins and thereby acquire distinct fates from their inception. In this study, we show that the absence of the atypical protein kinase C (PKC) isoforms, PKCζ and PKCλ/ι, disrupts asymmetric CD8(+) T lymphocyte division. These alterations were associated with aberrant acquisition of a pre-effector transcriptional program, detected by single-cell gene expression analyses, in lymphocytes that had undergone their first division in vivo and enhanced differentiation toward effector fates at the expense of memory fates. Together, these results demonstrate a role for atypical PKC in regulating asymmetric division and the specification of divergent CD8(+) T lymphocyte fates early during an immune response.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , División Celular/inmunología , Inmunidad Innata , Isoenzimas/inmunología , Listeriosis/inmunología , Proteína Quinasa C/inmunología , Animales , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/microbiología , Linfocitos T CD8-positivos/patología , Diferenciación Celular , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Células Dendríticas/patología , Regulación de la Expresión Génica , Memoria Inmunológica , Isoenzimas/genética , Isoenzimas/metabolismo , Listeria monocytogenes/inmunología , Listeriosis/enzimología , Listeriosis/microbiología , Listeriosis/patología , Ratones , Ratones Noqueados , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Transducción de Señal , Análisis de la Célula Individual , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/microbiología , Subgrupos de Linfocitos T/patologíaRESUMEN
Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades mRNAs containing premature translation termination codons (PTCs). SMG-1 and Upf1 transiently form a surveillance complex termed "SURF" that includes eRF1 and eRF3 on post-spliced mRNAs during recognition of PTC. If an exon junction complex (EJC) exists downstream from the SURF complex, SMG-1 phosphorylates Upf1, the step that is a rate-limiting for NMD. We provide evidence of an association between the SURF complex and the ribosome in association with mRNPs, and we suggest that the SURF complex functions as a translation termination complex during NMD. We identified SMG-8 and SMG-9 as novel subunits of the SMG-1 complex. SMG-8 and SMG-9 suppress SMG-1 kinase activity in the isolated SMG-1 complex and are involved in NMD in both mammals and nematodes. SMG-8 recruits SMG-1 to the mRNA surveillance complex, and inactivation of SMG-8 induces accumulation of a ribosome:Upf1:eRF1:eRF3:EJC complex on mRNP, which physically bridges the ribosome and EJC through eRF1, eRF3, and Upf1. These results not only reveal the regulatory mechanism of SMG-1 kinase but also reveal the sequential remodeling of the ribosome:SURF complex to the predicted DECID (DECay InDucing) complex, a ribosome:SURF:EJC complex, as a mechanism of in vivo PTC discrimination.
Asunto(s)
Codón sin Sentido/metabolismo , Regulación Enzimológica de la Expresión Génica , Complejos Multienzimáticos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Subunidades de Proteína/metabolismo , Estabilidad del ARN/fisiología , Animales , Caenorhabditis elegans/enzimología , Proteínas de Caenorhabditis elegans/metabolismo , Glutatión/análogos & derivados , Glutatión/metabolismo , Células HeLa , Humanos , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Ribosomas/metabolismoRESUMEN
Polarized hepatocytes contain tight junctions (TJs), which are among the most important junctions for sealing the bile canalicular lumen from the sinusoidal space. Alterations in TJs are implicated in chronic cholestatic liver diseases, such as primary biliary cirrhosis and primary sclerosing cholangitis, which have lipid peroxidation marker elevations or antioxidant vitamin decreases. However, the effect of oxidative stress on hepatocyte polarity or liver morphology is unknown. We found that carbon tetrachloride (CCl4)-induced oxidative stress resulted in disassembly of TJs. Ultrastructural analysis revealed disruption in TJs, Golgi morphology, and expansion of the bile canalicular lumen size in CCl4-treated hepatocytes. The Par complex [Par-3-atypical protein kinase C (aPKC) and Par-6 ternary complex] regulates TJs and lumen formation, and the Par-3-aPKC complex formation was inhibited by CCl4 treatment. Moreover, the antioxidant compound vitamin E prohibited a CCl4-induced disturbance in TJs and Par-3-aPKC complex formation. aPKC phosphorylates Par-3 and down-regulates its own affinity with Par-3. Importantly, aPKC kinase activity and Par-3 phosphorylation were significantly increased in CCl4-treated rat livers. These results indicate that the Par-3-aPKC complex plays a crucial role in the maintenance of hepatocyte polarity and sealing of the bile canalicular lumen. Our findings suggest that bile canalicular lumen expansion might explain the presence of cholestasis in patients with primary biliary cirrhosis and primary sclerosing cholangitis.
Asunto(s)
Canalículos Biliares/enzimología , Canalículos Biliares/patología , Tetracloruro de Carbono/toxicidad , Polaridad Celular/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Proteína Quinasa C/metabolismo , Animales , Canalículos Biliares/efectos de los fármacos , Proteínas Portadoras/metabolismo , Activación Enzimática/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Hepatocitos/ultraestructura , Humanos , Hígado/efectos de los fármacos , Hígado/patología , Hígado/ultraestructura , Masculino , Modelos Biológicos , Proteínas del Tejido Nervioso , Ratas Wistar , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Vitamina E/farmacologíaRESUMEN
Atypical protein kinase C λ/ι (aPKCλ/ι) is a regulator of epithelial cellular polarity. It is also overexpressed in several cancers and functions in cell proliferation and invasion. Therefore, we hypothesized that aPKCλ/ι may be involved in development and progression of cervical intraepithelial neoplasia (CIN), the precancerous disease of cervical cancer induced by human papillomavirus. To do this, we investigated the relationship between aPKCλ/ι expression and CIN. aPKCλ/ι expression level and subcellular localization were assessed in 192 CIN biopsy samples and 13 normal epithelial samples using immunohistochemistry. aPKCλ/ι overexpression (normal epithelium, 7.7%; CIN1, 41.7%; CIN2/3, 76.4%) and aPKCλ/ι nuclear localization (normal epithelium, 0.0%; CIN1, 36.9%; CIN2/3, 78.7%) were higher in CIN samples than normal samples (P<0.05), suggesting that CIN grade is related to aPKCλ/ι overexpression and nuclear localization. Then, 140 CIN cases were retrospectively analyzed for 4-yr cumulative disease progression and regression rates using the Cox proportional hazards model. CIN1 cases with aPKCλ/ι overexpression or aPKCλ/ι nuclear localization had a higher progression rate than CIN1 cases with normal aPKCλ/ι expression levels or cytoplasmic localization (62.5% vs. 9.7% and 63.1% vs. 9.4%, respectively; P<0.001). Multivariate analysis indicated that human papillomavirus types 16 and 18, aPKCλ/ι overexpression (hazard ratio=4.26; 95% confidence interval, 1.50-12.1; P=0.007), and aPKCλ/ι nuclear localization (hazard ratio=3.59; 95% confidence interval, 1.24-10.4; P=0.019) were independent risk factors for CIN1 progression. In conclusion, aPKCλ/ι could be useful for the therapeutic management of patients with CIN, particularly those with non-human papillomavirus 16/18 types.
Asunto(s)
Biomarcadores de Tumor/análisis , Proteína Quinasa C/biosíntesis , Displasia del Cuello del Útero/patología , Neoplasias del Cuello Uterino/patología , Adulto , Progresión de la Enfermedad , Femenino , Humanos , Immunoblotting , Inmunohistoquímica , Estimación de Kaplan-Meier , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/epidemiología , Pronóstico , Proteína Quinasa C/análisis , Estudios Retrospectivos , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/metabolismo , Displasia del Cuello del Útero/virologíaRESUMEN
Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance mechanism that eliminates aberrant mRNAs containing premature termination codons (PTCs). NMD inhibits the production of aberrant proteins that still retain, at least in part, wild-type function as well as dominant-negative peptides. Therefore, the selective inhibition of NMD has the potential to ameliorate NMD-exacerbated mutant phenotypes. However, we do not have sufficient knowledge of how to effectively suppress NMD with minimum cytotoxic effects. In this study, we aimed to identify NMD-related factors that can be targeted to efficiently inhibit NMD without causing significant cytotoxicity to restore the levels of truncated but partially functional proteins. We evaluated the knockdown of 15 NMD components in Ullrich congenital muscular dystrophy fibroblasts, which have a homozygous frameshift mutation causing a PTC in the collagen type VI α 2 gene. Of the 15 NMD factors tested, knockdown of SMG-8 produced the best effect for restoring defective mRNA and protein levels without affecting cell growth, cell-cycle progression, or endoplasmic reticulum stress. The efficacy of SMG-8 knockdown to improve the mutant phenotype was confirmed using another cell line, from a cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy patient who carries a PTC-containing mutation in HtrA serine peptidase 1. Our results suggest that SMG-8 is an appropriate target for inhibiting NMD to improve NMD-exacerbated mutant phenotypes. NMD inhibition by knockdown of SMG-8 may also be useful to induce synergy in combining the use of read-through drugs for patients with nonsense mutation-associated diseases.
Asunto(s)
Degradación de ARNm Mediada por Codón sin Sentido , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Codón sin Sentido , Colágeno Tipo VI/química , Colágeno Tipo VI/genética , ADN Complementario/genética , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Mutación , Proteínas Nucleares/genética , Fenotipo , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/genética , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Subunidades de Proteína , ARN Interferente Pequeño/genética , Esclerosis/genética , Esclerosis/metabolismo , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
The establishment of epithelial polarity is tightly linked to the dramatic reorganization of microtubules (MTs) from a radial array to a vertical alignment of non-centrosomal MT bundles along the lateral membrane, and a meshwork under the apical and basal membranes. However, little is known about the underlying molecular mechanism of this polarity-dependent MT remodeling. The evolutionarily conserved cell polarity-regulating kinase PAR-1 (known as MARK in mammals), whose activity is essential for maintaining the dynamic state of MTs, has indispensable roles in promoting this process. Here, we identify a novel PAR-1-binding protein, which we call microtubule crosslinking factor 1 (MTCL1), that crosslinks MTs through its N-terminal MT-binding region and subsequent coiled-coil motifs. MTCL1 colocalized with the apicobasal MT bundles in epithelial cells, and its knockdown impaired the development of these MT bundles and the epithelial-cell-specific columnar shape. Rescue experiments revealed that the N-terminal MT-binding region was indispensable for restoring these defects of the knockdown cells. MT regrowth assays indicated that MTCL1 was not required for the initial radial growth of MTs from the apical centrosome but was essential for the accumulation of non-centrosomal MTs to the sublateral regions. Interestingly, MTCL1 recruited a subpopulation of PAR-1b (known as MARK2 in mammals) to the apicobasal MT bundles, and its interaction with PAR-1b was required for MTCL1-dependent development of the apicobasal MT bundles. These results suggest that MTCL1 mediates the epithelial-cell-specific reorganization of non-centrosomal MTs through its MT-crosslinking activity, and cooperates with PAR-1b to maintain the correct temporal balance between dynamic and stable MTs within the apicobasal MT bundles.
Asunto(s)
Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Polaridad Celular/fisiología , Células Cultivadas , Perros , Células Epiteliales/enzimología , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación , Células de Riñón Canino Madin Darby , Ratones , Morfogénesis , Unión Proteica , TransfecciónRESUMEN
BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein that mediates the assembly and release of virus-like particles (VLPs) from an infected cell membrane. The Gag C-terminal p6 domain contains short sequence motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. Gag p6 has also been found to be phosphorylated during HIV-1 infection and this event may affect virus replication. However, the kinase that directs the phosphorylation of Gag p6 toward virus replication remains to be identified. In our present study, we identified this kinase using a proteomic approach and further delineate its role in HIV-1 replication. RESULTS: A proteomic approach was designed to systematically identify human protein kinases that potently interact with HIV-1 Gag and successfully identified 22 candidates. Among this panel, atypical protein kinase C (aPKC) was found to phosphorylate HIV-1 Gag p6. Subsequent LC-MS/MS and immunoblotting analysis with a phospho-specific antibody confirmed both in vitro and in vivo that aPKC phosphorylates HIV-1 Gag at Ser487. Computer-assisted structural modeling and a subsequent cell-based assay revealed that this phosphorylation event is necessary for the interaction between Gag and Vpr and results in the incorporation of Vpr into virions. Moreover, the inhibition of aPKC activity reduced the Vpr levels in virions and impaired HIV-1 infectivity of human primary macrophages. CONCLUSION: Our current results indicate for the first time that HIV-1 Gag phosphorylation on Ser487 is mediated by aPKC and that this kinase may regulate the incorporation of Vpr into HIV-1 virions and thereby supports virus infectivity. Furthermore, aPKC inhibition efficiently suppresses HIV-1 infectivity in macrophages. aPKC may therefore be an intriguing therapeutic target for HIV-1 infection.
Asunto(s)
VIH-1/fisiología , Interacciones Huésped-Patógeno , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional , Ensamble de Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Fosforilación , Virión/metabolismoRESUMEN
Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades mRNAs containing premature termination codons (PTCs). SMG-1-mediated Upf1 phosphorylation takes place in the decay inducing complex (DECID), which contains a ribosome, release factors, Upf1, SMG-1, an exon junction complex (EJC) and a PTC-mRNA. However, the significance and the consequence of Upf1 phosphorylation remain to be clarified. Here, we demonstrate that SMG-6 binds to a newly identified phosphorylation site in Upf1 at N-terminal threonine 28, whereas the SMG-5:SMG-7 complex binds to phosphorylated serine 1096 of Upf1. In addition, the binding of the SMG-5:SMG-7 complex to Upf1 resulted in the dissociation of the ribosome and release factors from the DECID complex. Importantly, the simultaneous binding of both the SMG-5:SMG-7 complex and SMG-6 to phospho-Upf1 are required for both NMD and Upf1 dissociation from mRNA. Thus, the SMG-1-mediated phosphorylation of Upf1 creates a binding platforms for the SMG-5:SMG-7 complex and for SMG-6, and triggers sequential remodeling of the mRNA surveillance complex for NMD induction and recycling of the ribosome, release factors and NMD factors.
Asunto(s)
Proteínas Portadoras/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido , Telomerasa/metabolismo , Transactivadores/metabolismo , Proteínas 14-3-3/química , Sitios de Unión , Células HEK293 , Células HeLa , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas , Estructura Terciaria de Proteína , ARN Helicasas , Telomerasa/química , Treonina/fisiología , Transactivadores/químicaRESUMEN
BACKGROUND & AIMS: The apical-basal polarity of pancreatic acinar cells is essential for maintaining tissue architecture. However, the mechanisms by which polarity proteins regulate acinar pancreas injury and regeneration are poorly understood. METHODS: Cerulein-induced pancreatitis was induced in mice with conditional deletion of the polarity protein Par3 in the pancreas. The impact of Par3 loss on pancreas injury and regeneration was assessed by histologic analyses and transcriptional profiling by RNA sequencing. Mice were pretreated with the bromodomain and extraterminal domain (BET) inhibitor JQ1 before cotreatment with cerulein to determine the effect of BET inhibition on pancreas injury and regeneration. RESULTS: Initially, we show that Par3 is increased in acinar-ductal metaplasia (ADM) lesions present in human and mouse chronic pancreatitis specimens. Although Par3 loss disrupts tight junctions, Par3 is dispensable for pancreatogenesis. However, with aging, Par3 loss results in low-grade inflammation, acinar degeneration, and pancreatic lipomatosis. Par3 loss exacerbates acute pancreatitis-induced injury and chronic pancreatitis-induced acinar cell loss, promotes pancreatic lipomatosis, and prevents regeneration. Par3 loss also results in suppression of chronic pancreatitis-induced ADM and primary ciliogenesis. Notably, targeting BET proteins attenuates chronic pancreatitis-induced loss of primary cilia and promotes ADM in mice lacking pancreatic Par3. Targeting BET proteins also attenuates cerulein-induced acinar cell loss and enhances recovery of acinar cell mass and body weight of mice lacking pancreatic Par3. CONCLUSIONS: Combined, this study demonstrates how Par3 restrains chronic pancreatitis-induced changes in the pancreas and identifies a potential role for BET inhibitors to attenuate pancreas injury and facilitate regeneration.
RESUMEN
Solute carrier family 20 member 1 (SLC20A1) is a sodium/inorganic phosphate symporter, which has been identified as a prognostic marker in several types of cancer, including pancreatic cancer. However, to the best of our knowledge, the association between SLC20A1 expression and cancer stem cell (CSC) markers, such as aldehyde dehydrogenase 1 (ALDH1), in pancreatic ductal adenocarcinoma (PDAC), and the role of SLC20A1 in PDAC CSCs remains unclear. In the present study, a genomic dataset of primary pancreatic cancer (The Cancer Genome Atlas, Pan-Cancer Atlas) was downloaded and analyzed. Kaplan-Meier analysis and multivariate Cox regression analysis were performed to evaluate the overall survival, disease-specific survival (DSS), disease-free interval (DFI) and progression-free interval (PFI). Subsequently, SLC20A1 small interfering RNA (siRNA) knockdown (KD) was induced in the PANC-1 and MIA-PaCa-2 PDAC cell lines, and in sorted high ALDH1 activity (ALDH1high) cells, after which, cell viability, in vitro tumor sphere formation, cell death and caspase-3 activity were examined. The results revealed that patients with high expression of SLC20A1 (SLC20A1 high) at tumor stage I had a poor prognosis compared with patients with low expression of SLC20A1 (SLC20A1 low) in terms of DSS, DFI and PFI. In addition, patients with high expression of SLC20A1 and ALDH1A3 (SLC20A1 high ALDH1A3 high) exhibited poorer clinical outcomes than patients with high expression of SLC20A1 and low expression of ALDH1A3 (SLC20A1 high ALDH1A3 low), low expression of SLC20A1 and high expression of ALDH1A3 (SLC20A1 low ALDH1A3 high) and SLC20A1 low ALDH1A3 low. SLC20A1 siRNA KD in ALDH1high cells isolated from PANC-1 and MIA-PaCa-2 cell lines resulted in suppression of in vitro tumorsphere formation, and enhancement of cell death and caspase-3 activity. These results suggested that SLC20A1 was involved in cell survival via the suppression of caspase-3-dependent apoptosis, and contributed to cancer progression and poor clinical outcomes in PDAC. In conclusion, SLC20A1 may be used as a prognostic marker and novel therapeutic target of ALDH1-positive pancreatic CSCs.