RESUMEN
The Ser/Thr protein phosphatase 2 A (PP2A) regulates the dephosphorylation of many phosphoproteins. Substrate recognition are mediated by B regulatory subunits. Here, we report the identification of a substrate conserved motif [RK]-V-x-x-[VI]-R in FAM122A, an inhibitor of B55α/PP2A. This motif is necessary for FAM122A binding to B55α, and computational structure prediction suggests the motif, which is helical, blocks substrate docking to the same site. In this model, FAM122A also spatially constrains substrate access by occluding the catalytic subunit. Consistently, FAM122A functions as a competitive inhibitor as it prevents substrate binding and dephosphorylation of CDK substrates by B55α/PP2A in cell lysates. FAM122A deficiency in human cell lines reduces the proliferation rate, cell cycle progression, and hinders G1/S and intra-S phase cell cycle checkpoints. FAM122A-KO in HEK293 cells attenuates CHK1 and CHK2 activation in response to replication stress. Overall, these data strongly suggest that FAM122A is a short helical motif (SHeM)-dependent, substrate-competitive inhibitor of B55α/PP2A that suppresses multiple functions of B55α in the DNA damage response and in timely progression through the cell cycle interphase.
Asunto(s)
Secuencias de Aminoácidos , Interfase , Proteína Fosfatasa 2 , Humanos , Puntos de Control del Ciclo Celular/genética , Proliferación Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa de Punto de Control 2/metabolismo , Quinasa de Punto de Control 2/genética , Células HEK293 , Fosforilación , Unión Proteica , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/genéticaRESUMEN
Prostate cancer (PCa) is the most common cancer diagnosed in men worldwide and the second leading cause of cancer-related deaths in US males in 2022. Prostate cancer also represents the second highest cancer mortality disparity between non-Hispanic blacks and whites. However, there is a relatively small number of prostate normal and cancer cell lines compared to other cancers. To identify the molecular basis of PCa progression, it is important to have prostate epithelial cell (PrEC) lines as karyotypically normal as possible. Our lab recently developed a novel methodology for the rapid and efficient immortalization of normal human PrEC that combines simultaneous CRISPR-directed inactivation of CDKN2A exon 2 (which directs expression of p16INK4A and p14ARF) and ectopic expression of an hTERT transgene. To optimize this methodology to generate immortalized lines with minimal genetic alterations, we sought to target exon 1α of the CDKN2A locus so that p16INK4A expression is ablated while p14ARF expression remains unaltered. Here we describe the establishment of two cell lines: one with the above-mentioned p16INK4A only loss, and a second line targeting both products in the CDKN2A locus. We characterize the potential lineage origin of these new cell lines along with our previously obtained clones, revealing distinct gene expression signatures. Based on the analyses of protein markers and RNA expression signatures, these cell lines are most closely related to a subpopulation of basal prostatic cells. Given the simplicity of this one-step methodology and the fact that it uses only the minimal genetic alterations necessary for immortalization, it should also be suitable for the establishment of cell lines from primary prostate tumor samples, an urgent need given the limited number of available prostate cancer cell lines.
RESUMEN
BACKGROUND & AIMS: Autophagy plays roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelial homeostasis. METHODS: We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histologic and biochemical analyses. We fluorescence-activated cell sorted esophageal basal cells based on fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID and then subjected these cells to transmission electron microscopy, image flow cytometry, three-dimensional organoid assays, RNA sequencing, and cell cycle analysis. Three-dimensional organoids were subjected to passaging, single-cell RNA sequencing, cell cycle analysis, and immunostaining. RESULTS: Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells under homeostatic conditions and also was associated with significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Esophageal basal cells with high AV level (Cyto-IDHigh) displayed limited organoid formation capability on initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-IDLow). RNA sequencing suggested increased autophagy in Cyto-IDHigh esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. Single-cell RNA sequencing of three-dimensional organoids generated by Cyto-IDLow and Cyto-IDHigh cells identified expansion of 3 cell populations and enrichment of G2/M-associated genes in the Cyto-IDHigh group. Ki67 expression was also increased in organoids generated by Cyto-IDHigh cells, including in basal cells localized beyond the outermost cell layer. CONCLUSIONS: Autophagy contributes to maintenance of the esophageal proliferation-differentiation gradient. Esophageal basal cells with high AV level exhibit limited proliferation and generate three-dimensional organoids with enhanced self-renewal capacity.
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Autofagia , Proliferación Celular , Homeostasis , Ratones Noqueados , Organoides , Animales , Ratones , Organoides/metabolismo , Esófago/patología , Esófago/citología , Esófago/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Proteína 7 Relacionada con la Autofagia/metabolismo , Proteína 7 Relacionada con la Autofagia/genética , 4-Nitroquinolina-1-Óxido , Autorrenovación de las Células , Mucosa Esofágica/patología , Mucosa Esofágica/metabolismo , Mucosa Esofágica/citología , Análisis de la Célula IndividualRESUMEN
Anticancer nucleosides are effective against solid tumors and hematological malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4'-ethynyl-2'-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (DCK) phosphorylation for its activity and induced replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å co-crystal structure of DCK bound to EdC and UDP reveals how the rigid 4'-alkyne of EdC fits within the active site of DCK. Remarkably, EdC was resistant to cytidine deamination and SAMHD1 metabolism mechanisms and exhibited higher potency against ALL compared to FDA approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a pre-clinical nucleoside prodrug candidate for DLBCL and ALL.
RESUMEN
Serine/threonine protein phosphatase 2 (PP2A) forms heterotrimeric holoenzymes, where a scaffold subunit bridges the PP2A catalytic subunit to a B regulatory subunit, e.g., B55α. The PP2A/B55α holoenzyme plays key roles in signaling and cell-cycle control targeting multiple substrates. Here, we describe semiquantitative approaches to determine PP2A/B55α substrate specificity. Parts I and II detail approaches to assess PP2A/B55α-mediated dephosphorylation of immobilized substrate peptide variants. Parts III and IV detail methods to assess PP2A/B55α-substrate-binding specificity. These approaches are adaptable to other serine/threonine phosphatases. For complete details on the use and execution of this protocol, please refer to Fowle et al..1.
RESUMEN
The Ser/Thr protein phosphatase 2A (PP2A) is a highly conserved collection of heterotrimeric holoenzymes responsible for the dephosphorylation of many regulated phosphoproteins. Substrate recognition and the integration of regulatory cues are mediated by B regulatory subunits that are complexed to the catalytic subunit (C) by a scaffold protein (A). PP2A/B55 substrate recruitment was thought to be mediated by charge-charge interactions between the surface of B55α and its substrates. Challenging this view, we recently discovered a conserved SLiM [ RK ]- V -x-x-[ VI ]- R in a range of proteins, including substrates such as the retinoblastoma-related protein p107 and TAU (Fowle et al. eLife 2021;10:e63181). Here we report the identification of this SLiM in FAM122A, an inhibitor of B55α/PP2A. This conserved SLiM is necessary for FAM122A binding to B55α in vitro and in cells. Computational structure prediction with AlphaFold2 predicts an interaction consistent with the mutational and biochemical data and supports a mechanism whereby FAM122A uses the 'SLiM' in the form of a short α-helix to dock to the B55α top groove. In this model, FAM122A spatially constrains substrate access by occluding the catalytic subunit with a second α-helix immediately adjacent to helix 1. Consistently, FAM122A functions as a competitive inhibitor as it prevents binding of substrates in in vitro competition assays and the dephosphorylation of CDK substrates by B55α/PP2A in cell lysates. Ablation of FAM122A in human cell lines reduces the rate of proliferation, progression through cell cycle transitions and abrogates G1/S and intra-S phase cell cycle checkpoints. FAM122A-KO in HEK293 cells results in attenuation of CHK1 and CHK2 activation in response to replication stress. Overall, these data strongly suggest that FAM122A is a 'SLiM'-dependent, substrate-competitive inhibitor of B55α/PP2A that suppresses multiple functions of B55α in the DNA damage response and in timely progression through the cell cycle interphase.
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Prostate cell lines from diverse backgrounds are important to addressing disparities in prostate cancer (PCa) incidence and mortality rates among Black men. ACRJ-PC28 was developed from a transrectal needle biopsy and established via inactivation of the CDKN2A locus and simultaneous expression of human telomerase. Characterization assays included growth curve analysis, immunoblots, IHC, 3D cultures, immunofluorescence imaging, confocal microscopy, flow cytometry, WGS, and RNA-Seq. ACRJ-PC28 has been passaged more than 40 times in vitro over 10 months with a doubling time of 45 hours. STR profiling confirmed the novelty and human origin of the cell line. RNA-Seq confirmed the expression of prostate specific genes alpha-methylacyl-CoA racemase (AMACR) and NKX3.1 and Neuroendocrine specific markers synaptophysin (SYP) and enolase 2 (ENO2) and IHC confirmed the presence of AMACR. Immunoblots indicated the cell line is of basal-luminal type; expresses p53 and pRB and is AR negative. WGS confirmed the absence of exonic mutations and the presence of intronic variants that appear to not affect function of AR, p53, and pRB. RNA-Seq data revealed numerous TP53 and RB1 mRNA splice variants and the lack of AR mRNA expression. This is consistent with retention of p53 function in response to DNA damage and pRB function in response to contact inhibition. Soft agar anchorage-independent analysis indicated that the cells are transformed, confirmed by principal component analysis (PCA) where ACRJ-PC28 cells cluster alongside other PCa tumor tissues, yet was distinct. The novel methodology described should advance prostate cell line development, addressing the disparity in PCa among Black men.
Asunto(s)
Células Neuroendocrinas , Neoplasias de la Próstata , Masculino , Humanos , Proteína p53 Supresora de Tumor/genética , Células Neuroendocrinas/metabolismo , Neoplasias de la Próstata/genética , Línea Celular , ARN Mensajero , Región del CaribeRESUMEN
Protein phosphorylation is a reversible post-translation modification essential in cell signaling. This study addresses a long-standing question as to how the most abundant serine/threonine protein phosphatase 2 (PP2A) holoenzyme, PP2A/B55α, specifically recognizes substrates and presents them to the enzyme active site. Here, we show how the PP2A regulatory subunit B55α recruits p107, a pRB-related tumor suppressor and B55α substrate. Using molecular and cellular approaches, we identified a conserved region 1 (R1, residues 615-626) encompassing the strongest p107 binding site. This enabled us to identify an 'HxRVxxV619-625' short linear motif (SLiM) in p107 as necessary for B55α binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous B55α/PP2A substrates, including TAU, contain a related SLiM C-terminal from a proximal phosphosite, 'p[ST]-P-x(4,10)-[RK]-V-x-x-[VI]-R.' Mutation of conserved SLiM residues in TAU dramatically inhibits dephosphorylation by PP2A/B55α, validating its generality. A data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55α groove, and phosphosite presentation. Altogether, these data provide key insights into PP2A/B55α's mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55α's role in multiple cellular processes.
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Proteína Fosfatasa 2/genética , Proteína p107 Similar a la del Retinoblastoma/genética , Células HEK293 , Holoenzimas/metabolismo , Humanos , Fosforilación , Proteína Fosfatasa 2/metabolismo , Proteína p107 Similar a la del Retinoblastoma/metabolismoRESUMEN
BACKGROUND: Immortalization of primary prostate epithelial cells (PrEC) with just hTERT expression is particularly inefficient in the absence of DNA tumor viral proteins or p16INK4A knockdown. MATERIALS AND METHODS: Here, we describe the establishment of immortalized normal prostate epithelial cell line models using CRISPR technology to inactivate the CDKN2A locus concomitantly with ectopic expression of an hTERT transgene. RESULTS: Using this approach, we have obtained immortal cell clones that exhibit fundamental characteristics of normal cells, including diploid genomes, near normal karyotypes, normal p53 and pRB cell responses, the ability to form non-invasive spheroids, and a non-transformed phenotype. Based on marker expression, these clones are of basal cell origin. CONCLUSIONS: Use of this approach resulted in the immortalization of independent clones of PrEC that retained normal characteristics, were stable, and non-transformed. Thus, this approach could be used for the immortalization of normal primary prostate cells. This technique could also be useful for establishing cell lines from prostate tumor tissues of different tumor grades and/or from patients of diverse ethnicities to generate cell line models that facilitate the study of the molecular basis of disease disparity.
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Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata/genética , ARN Neoplásico/genética , Telomerasa/genética , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Telomerasa/biosíntesisRESUMEN
The PPP2R2A gene encodes the B55α regulatory subunit of PP2A. Here, we report that PPP2R2A is hemizygously lost in ~42% of prostate adenocarcinomas, correlating with reduced expression, poorer prognosis, and an increased incidence of hemizygous loss (>75%) in metastatic disease. Of note, PPP2R2A homozygous loss is less common (5%) and not increased at later tumor stages. Reduced expression of B55α is also seen in prostate tumor tissue and cell lines. Consistent with the possibility that complete loss of PPP2R2A is detrimental in prostate tumors, PPP2R2A deletion in cells with reduced but present B55α reduces cell proliferation by slowing progression through the cell cycle. Remarkably, B55α-low cells also appear addicted to lower B55α expression, as even moderate increases in B55α expression are toxic. Reconstitution of B55α expression in prostate cancer (PCa) cell lines with low B55α expression reduces proliferation, inhibits transformation and blocks xenograft tumorigenicity. Mechanistically, we show B55α reconstitution reduces phosphorylation of proteins essential for centrosomal maintenance, and induces centrosome collapse and chromosome segregation failure; a first reported link between B55α/PP2A and the vertebrate centrosome. These effects are dependent on a prolonged metaphase/anaphase checkpoint and are lethal to PCa cells addicted to low levels of B55α. Thus, we propose the reduction in B55α levels associated with hemizygous loss is necessary for centrosomal integrity in PCa cells, leading to selective lethality of B55α reconstitution. Such a vulnerability could be targeted therapeutically in the large pool of patients with hemizygous PPP2R2A deletions, using pharmacologic approaches that enhance PP2A/B55α activity.
RESUMEN
The process of tumor cell invasion and metastasis includes assembly of invadopodia, protrusions capable of degrading the extracellular matrix (ECM). The effect of cell cycle progression on invadopodia has not been elucidated. In this study, by using invadopodia and cell cycle fluorescent markers, we show in 2D and 3D cultures, as well as in vivo, that breast carcinoma cells assemble invadopodia and invade into the surrounding ECM preferentially during the G1 phase. The expression (MT1-MMP, also known as MMP14, and cortactin) and localization (Tks5; also known as SH3PXD2A) of invadopodia components are elevated in G1 phase, and cells synchronized in G1 phase exhibit significantly higher ECM degradation compared to the cells synchronized in S phase. The cyclin-dependent kinase inhibitor (CKI) p27kip1 (also known as CDKN1B) localizes to the sites of invadopodia assembly. Overexpression and stable knockdown of p27kip1 lead to contrasting effects on invadopodia turnover and ECM degradation. Taken together, these findings suggest that expression of invadopodia components, as well as invadopodia function, are linked to cell cycle progression, and that invadopodia are controlled by cell cycle regulators. Our results caution that this coordination between invasion and cell cycle must be considered when designing effective chemotherapies.
Asunto(s)
Matriz Extracelular/metabolismo , Fase G1 , Podosomas/metabolismo , Animales , Línea Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Matriz Extracelular/genética , Técnicas de Inactivación de Genes , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Ratones , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismo , Podosomas/genética , Fase SRESUMEN
PP2A is a highly conserved eukaryotic serine/threonine protein phosphatase of the PPP family of phosphatases with fundamental cellular functions. In cells, PP2A targets specific subcellular locations and substrates by forming heterotrimeric holoenzymes, where a core dimer consisting of scaffold (A) and catalytic (C) subunits complexes with one of many B regulatory subunits. PP2A plays a key role in positively and negatively regulating a myriad of cellular processes, as it targets a very sizable fraction of the cellular substrates phosphorylated on Ser/Thr residues. This review focuses on insights made toward the understanding on how the subunit composition and structure of PP2A holoenzymes mediates substrate specificity, the role of substrate modulation in the signaling of cellular division, growth, and differentiation, and its deregulation in cancer.
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Neoplasias/enzimología , Neoplasias/patología , Proteína Fosfatasa 2/metabolismo , Animales , Ciclo Celular/fisiología , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Holoenzimas/química , Holoenzimas/metabolismo , Humanos , Fosforilación , Proteína Fosfatasa 2/química , Transducción de Señal , Especificidad por SustratoRESUMEN
Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.
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Quinasa 9 Dependiente de la Ciclina/metabolismo , Animales , Línea Celular Tumoral , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , Metilación de ADN , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
In their active hypophosphorylated state, members of the retinoblastoma family of pocket proteins negatively regulate cell cycle progression at least in part by repressing expression of E2F-dependent genes. Mitogen-dependent activation of G1 and G1/S Cyclin Dependent Kinases (CDKs) results in coordinated hyperphosphorylation and inactivation of these proteins, which no longer bind and repress E2Fs. S and G2/M CDKs maintain pocket protein hyperphosphorylated through the end of mitosis. The inactivating action of inducible CDKs is opposed by the Ser/Thr protein phosphatases PP2A and PP1. Various trimeric PP2A holoenzymes have been implicated in dephosphorylation of pocket proteins in response to specific cellular signals and stresses or as part of an equilibrium with CDKs throughout the cell cycle. PP1 has specifically been implicated in dephosphorylation of pRB in late mitosis and early G1. This review is particularly focused on the emerging role of PP2A as a major hub for integration of growth suppressor signals that require rapid inactivation of pocket proteins. Of note, activation of particular PP2A holoenzymes triggers differential activation of pocket proteins in the presence of active CDKs.
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Proteína Fosfatasa 2/metabolismo , Proteína de Retinoblastoma/metabolismo , Animales , Quinasas Ciclina-Dependientes/metabolismo , Factores de Transcripción E2F/metabolismo , Interfase , Estrés Oxidativo , Fosforilación , Proteína Fosfatasa 2/química , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteína de Retinoblastoma/química , Transducción de SeñalRESUMEN
BACKGROUND: CDK9 is the catalytic subunit of the Positive Transcription Elongation Factor b (P-TEFb), which phosphorylates the CTD of RNAPII and negative elongation factors enabling for productive elongation after initiation. CDK9 associates with T-type cyclins and cyclin K and its activity is tightly regulated in cells at different levels. CDK9 is also the catalytic subunit of TAK (Tat activating Kinase), essential for HIV1 replication. Because of CDK9's potential as a therapeutic target in AIDS, cancer, inflammation, and cardiomyophathy it is important to understand the consequences of CDK9 inhibition. A previous gene expression profiling study performed with human glioblastoma T98G cells in which CDK9 activity was inhibited either with a dominant negative mutant form of CDK9 (dnCDK9) or the pharmacological inhibitor Flavopiridol unveiled striking differences in gene expression effects. In the present report we extended these studies by (1) using both immortalized normal human fibroblasts and primary human astrocytes, (2) eliminating potential experimental variability due to transduction methodology and (3) also modulating CDK9 activity with siRNA. FINDINGS: Striking differences in the effects on gene expression resulting from the strategy used to inhibit CDK9 activity (dnCDK9 or FVP) remain even when potential variability due to viral transduction is eliminated. siRNA mediated CDK9 knockdown in human fibroblasts and astrocytes efficiently reduced CDK9 expression and led to potent changes in gene expression that exhibit little correlation with the effects of dnCDK9 or FVP. Interestingly, HEXIM1 a validated CDK9 target gene, was found to be potently downregulated by dnCDK9, FVP and siCDK9, but the cluster of genes with expression profiles similar to HEXIM1 was small. Finally, cluster analysis of all treatments revealed higher correlation between treatments than cell type origin. CONCLUSION: The nature of the strategy used to inhibit CDK9 profoundly affects the patterns of gene expression resulting from CDK9 inhibition. These results suggest multiple variables that affect outcome, including kinetics of inhibition, potency, off-target effects, and selectivity issues. This is particularly important when considering CDK9 as a potential target for therapeutic intervention.
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Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Terapia Molecular Dirigida , Inhibidores de Proteínas Quinasas/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Línea Celular Transformada , Análisis por Conglomerados , Quinasa 9 Dependiente de la Ciclina/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Flavonoides/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación/efectos de los fármacos , Piperidinas/farmacología , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
The positive transcription elongation factor b (P-TEFb) is involved in physiological and pathological events including inflammation, cancer, AIDS, and cardiac hypertrophy. The balance between its active and inactive form is tightly controlled to ensure cellular integrity. We report that the transcriptional repressor CTIP2 is a major modulator of P-TEFb activity. CTIP2 copurifies and interacts with an inactive P-TEFb complex containing the 7SK snRNA and HEXIM1. CTIP2 associates directly with HEXIM1 and, via the loop 2 of the 7SK snRNA, with P-TEFb. In this nucleoprotein complex, CTIP2 significantly represses the Cdk9 kinase activity of P-TEFb. Accordingly, we show that CTIP2 inhibits large sets of P-TEFb- and 7SK snRNA-sensitive genes. In hearts of hypertrophic cardiomyopathic mice, CTIP2 controls P-TEFb-sensitive pathways involved in the establishment of this pathology. Overexpression of the ß-myosin heavy chain protein contributes to the pathological cardiac wall thickening. The inactive P-TEFb complex associates with CTIP2 at the MYH7 gene promoter to repress its activity. Taken together, our results strongly suggest that CTIP2 controls P-TEFb function in physiological and pathological conditions.
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Cardiomegalia/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Cardiomegalia/genética , Cardiomegalia/patología , Quinasa 9 Dependiente de la Ciclina/genética , Quinasa 9 Dependiente de la Ciclina/metabolismo , Células HEK293 , Humanos , Ratones , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Factor B de Elongación Transcripcional Positiva/genética , Estructura Secundaria de Proteína , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
The phosphorylation state of pocket proteins during the cell cycle is determined at least in part by an equilibrium between inducible cyclin-dependent kinases (CDKs) and serine/threonine protein phosphatase 2A (PP2A). Two trimeric holoenzymes consisting of the core PP2A catalytic/scaffold dimer and either the B55α or PR70 regulatory subunit have been implicated in the activation of p107/p130 and pRB, respectively. While the phosphorylation state of p107 is very sensitive to forced changes of B55α levels in human cell lines, regulation of p107 in response to physiological modulation of PP2A/B55α has not been elucidated. Here we show that fibroblast growth factor 1 (FGF1), which induces maturation and cell cycle exit in chondrocytes, triggers rapid accumulation of p107-PP2A/B55α complexes coinciding with p107 dephosphorylation. Reciprocal solution-based mass spectrometric analysis identified the PP2A/B55α complex as a major component in p107 complexes, which also contain E2F/DPs, DREAM subunits, and/or cyclin/CDK complexes. Of note, p107 is one of the preferred partners of B55α, which also associates with pRB in RCS cells. FGF1-induced dephosphorylation of p107 results in its rapid accumulation in the nucleus and formation of larger complexes containing p107 and enhances its interaction with E2F4 and other p107 partners. Consistent with a key role of B55α in the rapid activation of p107 in chondrocytes, limited ectopic expression of B55α results in marked dephosphorylation of p107 while B55α knockdown results in hyperphosphorylation. More importantly, knockdown of B55α dramatically delays FGF1-induced dephosphorylation of p107 and slows down cell cycle exit. Moreover, dephosphorylation of p107 in response to FGF1 treatment results in early recruitment of p107 to the MYC promoter, an FGF1/E2F-regulated gene. Our results suggest a model in which FGF1 mediates rapid dephosphorylation and activation of p107 independently of the CDK activities that maintain p130 and pRB hyperphosphorylation for several hours after p107 dephosphorylation in maturing chondrocytes.
Asunto(s)
Condrocitos/citología , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteína p107 Similar a la del Retinoblastoma/metabolismo , Animales , Ciclo Celular , Línea Celular Tumoral , Condrocitos/metabolismo , Condrosarcoma/metabolismo , Humanos , Fosforilación , RatasRESUMEN
Cell cycle progression is negatively regulated by the retinoblastoma family of pocket proteins and CDK inhibitors (CKIs). In contrast, CDKs promote progression through multiple phases of the cell cycle. One prominent way by which CDKs promote cell cycle progression is by inactivation of pocket proteins via hyperphosphorylation. Reactivation of pocket proteins to halt cell cycle progression requires dephosphorylation of multiple CDK-phosphorylated sites and is accomplished by PP2A and PP1 serine/threonine protein phosphatases. The same phosphatases are also implicated in dephosphorylation of multiple CDK substrates as cells exit mitosis and reenter the G1 phase of the cell cycle. This review is primarily focused on the role of PP2A and PP1 in the activation of pocket proteins during the cell cycle and in response to signaling cues that trigger cell cycle exit. Other functions of PP2A during the cell cycle will be discussed in brief, as comprehensive reviews on this topic have been published recently (De Wulf et al., 2009; Wurzenberger and Gerlich, 2011).
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/genética , Quinasas Ciclina-Dependientes/metabolismo , Proteína Fosfatasa 2/fisiología , Animales , Holoenzimas/genética , Holoenzimas/metabolismo , Holoenzimas/fisiología , Humanos , Modelos Biológicos , Fosforilación/genética , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Especificidad por Sustrato/genéticaRESUMEN
BACKGROUND: The Positive Transcription Elongation Factor b (P-TEFb) is a complex of Cyclin Dependent Kinase 9 (CDK9) with either cyclins T1, T2 or K. The complex phosphorylates the C-Terminal Domain of RNA polymerase II (RNAPII) and negative elongation factors, stimulating productive elongation by RNAPII, which is paused after initiation. P-TEFb is recruited downstream of the promoters of many genes, including primary response genes, upon certain stimuli. Flavopiridol (FVP) is a potent pharmacological inhibitor of CDK9 and has been used extensively in cells as a means to inhibit CDK9 activity. Inhibition of P-TEFb complexes has potential therapeutic applications. RESULTS: It has been shown that Lipopolysaccharide (LPS) stimulates the recruitment of P-TEFb to Primary Response Genes (PRGs) and proposed that P-TEFb activity is required for their expression, as the CDK9 inhibitor DRB prevents localization of RNAPII in the body of these genes. We have previously determined the effects of FVP in global gene expression in a variety of cells and surprisingly observed that FVP results in potent upregulation of a number of PRGs in treatments lasting 4-24 h. Because inhibition of CDK9 activity is being evaluated in pre-clinical and clinical studies for the treatment of several pathologies, it is important to fully understand the short and long term effects of its inhibition. To this end, we determined the immediate and long-term effect of FVP in the expression of several PRGs. In exponentially growing normal human fibroblasts, the expression of several PRGs including FOS, JUNB, EGR1 and GADD45B, was rapidly and potently downregulated before they were upregulated following FVP treatment. In serum starved cells re-stimulated with serum, FVP also inhibited the expression of these genes, but subsequently, JUNB, GADD45B and EGR1 were upregulated in the presence of FVP. Chromatin Immunoprecipitation of RNAPII revealed that EGR1 and GADD45B are transcribed at the FVP-treatment time points where their corresponding mRNAs accumulate. These results suggest a possible stress response triggered by CDK9 inhibition than ensues transcription of certain PRGs. CONCLUSIONS: We have shown that certain PRGs are transcribed in the presence of FVP in a manner that might be independent of CDK9, suggesting a possible alternative mechanism for their transcription when P-TEFb kinase activity is pharmacologically inhibited. These results also show that the sensitivity to FVP is quite variable, even among PRGs.