RESUMEN
The resistance of cancer cells to therapy is responsible for the death of most patients with cancer1. Epithelial-to-mesenchymal transition (EMT) has been associated with resistance to therapy in different cancer cells2,3. However, the mechanisms by which EMT mediates resistance to therapy remain poorly understood. Here, using a mouse model of skin squamous cell carcinoma undergoing spontaneous EMT during tumorigenesis, we found that EMT tumour cells are highly resistant to a wide range of anti-cancer therapies both in vivo and in vitro. Using gain and loss of function studies in vitro and in vivo, we found that RHOJ-a small GTPase that is preferentially expressed in EMT cancer cells-controls resistance to therapy. Using genome-wide transcriptomic and proteomic profiling, we found that RHOJ regulates EMT-associated resistance to chemotherapy by enhancing the response to replicative stress and activating the DNA-damage response, enabling tumour cells to rapidly repair DNA lesions induced by chemotherapy. RHOJ interacts with proteins that regulate nuclear actin, and inhibition of actin polymerization sensitizes EMT tumour cells to chemotherapy-induced cell death in a RHOJ-dependent manner. Together, our study uncovers the role and the mechanisms through which RHOJ acts as a key regulator of EMT-associated resistance to chemotherapy.
Asunto(s)
Carcinoma de Células Escamosas , Resistencia a Antineoplásicos , Transición Epitelial-Mesenquimal , Neoplasias Cutáneas , Proteínas de Unión al GTP rho , Actinas/efectos de los fármacos , Actinas/metabolismo , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Transición Epitelial-Mesenquimal/efectos de los fármacos , Proteómica , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Animales , Ratones , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Perfilación de la Expresión Génica , GenomaRESUMEN
Activity-based protein profiling (ABPP) is a versatile strategy for identifying and characterizing functional protein sites and compounds for therapeutic development. However, the vast majority of ABPP methods for covalent drug discovery target highly nucleophilic amino acids such as cysteine or lysine. Here, we report a methionine-directed ABPP platform using Redox-Activated Chemical Tagging (ReACT), which leverages a biomimetic oxidative ligation strategy for selective methionine modification. Application of ReACT to oncoprotein cyclin-dependent kinase 4 (CDK4) as a representative high-value drug target identified three new ligandable methionine sites. We then synthesized a methionine-targeting covalent ligand library bearing a diverse array of heterocyclic, heteroatom, and stereochemically rich substituents. ABPP screening of this focused library identified 1oxF11 as a covalent modifier of CDK4 at an allosteric M169 site. This compound inhibited kinase activity in a dose-dependent manner on purified protein and in breast cancer cells. Further investigation of 1oxF11 found prominent cation-π and H-bonding interactions stabilizing the binding of this fragment at the M169 site. Quantitative mass-spectrometry studies validated 1oxF11 ligation of CDK4 in breast cancer cell lysates. Further biochemical analyses revealed cross-talk between M169 oxidation and T172 phosphorylation, where M169 oxidation prevented phosphorylation of the activating T172 site on CDK4 and blocked cell cycle progression. By identifying a new mechanism for allosteric methionine redox regulation on CDK4 and developing a unique modality for its therapeutic intervention, this work showcases a generalizable platform that provides a starting point for engaging in broader chemoproteomics and protein ligand discovery efforts to find and target previously undruggable methionine sites.
Asunto(s)
Neoplasias de la Mama , Metionina , Humanos , Femenino , Quinasa 4 Dependiente de la Ciclina/metabolismo , Ligandos , Fosforilación , Oxidación-Reducción , Racemetionina/metabolismoRESUMEN
Cell cycle progression, including genome duplication, is orchestrated by cyclin-dependent kinases (CDKs). CDK activation depends on phosphorylation of their T-loop by a CDK-activating kinase (CAK). In animals, the only known CAK for CDK2 and CDK1 is cyclin H-CDK7, which is constitutively active. Therefore, the critical activation step is dephosphorylation of inhibitory sites by Cdc25 phosphatases rather than unrestricted T-loop phosphorylation. Homologous CDK4 and CDK6 bound to cyclins D are master integrators of mitogenic/oncogenic signaling cascades by initiating the inactivation of the central oncosuppressor pRb and cell cycle commitment at the restriction point. Unlike the situation in CDK1 and CDK2 cyclin complexes, and in contrast to the weak but constitutive T177 phosphorylation of CDK6, we have identified the T-loop phosphorylation at T172 as the highly regulated step determining CDK4 activity. Whether both CDK4 and CDK6 phosphorylations are catalyzed by CDK7 remains unclear. To answer this question, we took a chemical-genetics approach by using analogue-sensitive CDK7(as/as) mutant HCT116 cells, in which CDK7 can be specifically inhibited by bulky adenine analogs. Intriguingly, CDK7 inhibition prevented activating phosphorylations of CDK4/6, but for CDK4 this was at least partly dependent on its binding to p21 (cip1) . In response to CDK7 inhibition, p21-binding to CDK4 increased concomitantly with disappearance of the most abundant phosphorylation of p21, which we localized at S130 and found to be catalyzed by both CDK4 and CDK2. The S130A mutation of p21 prevented the activating CDK4 phosphorylation, and inhibition of CDK4/6 and CDK2 impaired phosphorylations of both p21 and p21-bound CDK4. Therefore, specific CDK7 inhibition revealed the following: a crucial but partly indirect CDK7 involvement in phosphorylation/activation of CDK4 and CDK6; existence of CDK4-activating kinase(s) other than CDK7; and novel CDK7-dependent positive feedbacks mediated by p21 phosphorylation by CDK4 and CDK2 to sustain CDK4 activation, pRb inactivation, and restriction point passage.
Asunto(s)
Puntos de Control del Ciclo Celular/genética , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/genética , Quinasas Ciclina-Dependientes/genética , Quinasas p21 Activadas/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Células HCT116 , Humanos , Mutación , Fosforilación , Unión Proteica , Fosfatasas cdc25/metabolismo , Quinasas p21 Activadas/genética , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
BACKGROUND: Transformation by the Tax oncoprotein of the human T cell leukemia virus type 1 (HTLV-1) is governed by actions on cellular regulatory signals, including modulation of specific cellular gene expression via activation of signaling pathways, acceleration of cell cycle progression via stimulation of cyclin-dependent kinase activity leading to retinoblastoma protein (pRb) hyperphosphorylation and perturbation of survival signals. These actions control early steps in T cell transformation and development of Adult T cell leukemia (ATL), an aggressive malignancy of HTLV-1 infected T lymphocytes. Post-translational modifications of Tax by phosphorylation, ubiquitination, sumoylation and acetylation have been implicated in Tax-mediated activation of the NF-κB pathway, a key function associated with Tax transforming potential. RESULTS: In this study, we demonstrate that acetylation at lysine K(346) in the carboxy-terminal domain of Tax is modulated in the Tax nuclear bodies by the acetyltransferase p300 and the deacetylases HDAC5/7 and controls phosphorylation of the tumor suppressor pRb by Tax-cyclin D3-CDK4-p21(CIP) complexes. This property correlates with the inability of the acetylation deficient K(346)R mutant, but not the acetylation mimetic K(346)Q mutant, to promote anchorage-independent growth of Rat-1 fibroblasts. By contrast, acetylation at lysine K(346) had no effects on the ability of Tax carboxy-terminal PDZ-binding domain to interact with the tumor suppressor hDLG. CONCLUSIONS: The identification of the acetyltransferase p300 and the deacetylase HDAC7 as enzymes modulating Tax acetylation points to new therapeutic targets for the treatment of HTLV-1 infected patients at risk of developing ATL.
Asunto(s)
Transformación Celular Viral , Productos del Gen tax/metabolismo , Histona Desacetilasas/metabolismo , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Lisina/metabolismo , Procesamiento Proteico-Postraduccional , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Animales , Línea Celular , Fibroblastos/virología , Humanos , RatasRESUMEN
Existing treatment strategies for pancreatobiliary malignancies are limited. Nowadays, surgery is the only path to cure these types of cancer, but only a small number of patients present with resectable tumors at the time of diagnosis. The notoriously poor prognosis, lack of diverse treatment options associated with pancreaticobiliary cancers, and their resistance to current therapies reflect the urge for the development of novel therapeutic targets. Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have emerged as an attractive therapeutic strategy in a number of cancers since their approval for treatment in patients with ER+/HER- breast cancer in combination with antiestrogens. In this article, we discuss the therapeutic potential of CDK4/6 inhibitors in pancreatobiliary cancers, notably cholangiocarcinoma and pancreatic ductal adenocarcinoma.
RESUMEN
Background: CDK4/6 inhibitors (CDK4/6i) have been established as standard treatment against advanced Estrogen Receptor-positive breast cancers. These drugs are being tested against several cancers, including in combinations with other therapies. We identified the T172-phosphorylation of CDK4 as the step determining its activity, retinoblastoma protein (RB) inactivation, cell cycle commitment and sensitivity to CDK4/6i. Poorly differentiated (PDTC) and anaplastic (ATC) thyroid carcinomas, the latter considered one of the most lethal human malignancies, represent major clinical challenges. Several molecular evidence suggest that CDK4/6i could be considered for treating these advanced thyroid cancers. Methods: We analyzed by two-dimensional gel electrophoresis the CDK4 modification profile and the presence of T172-phosphorylated CDK4 in a collection of 98 fresh-frozen tissues and in 21 cell lines. A sub-cohort of samples was characterized by RNA sequencing and immunohistochemistry. Sensitivity to CDK4/6i (palbociclib and abemaciclib) was assessed by BrdU incorporation/viability assays. Treatment of cell lines with CDK4/6i and combination with BRAF/MEK inhibitors (dabrafenib/trametinib) was comprehensively evaluated by western blot, characterization of immunoprecipitated CDK4 and CDK2 complexes and clonogenic assays. Results: CDK4 phosphorylation was detected in all well-differentiated thyroid carcinomas (n=29), 19/20 PDTC, 16/23 ATC and 18/21 thyroid cancer cell lines, including 11 ATC-derived ones. Tumors and cell lines without phosphorylated CDK4 presented very high p16CDKN2A levels, which were associated with proliferative activity. Absence of CDK4 phosphorylation in cell lines was associated with CDK4/6i insensitivity. RB1 defects (the primary cause of intrinsic CDK4/6i resistance) were not found in 5/7 tumors without detectable phosphorylated CDK4. A previously developed 11-gene expression signature identified the likely unresponsive tumors, lacking CDK4 phosphorylation. In cell lines, palbociclib synergized with dabrafenib/trametinib by completely and permanently arresting proliferation. These combinations prevented resistance mechanisms induced by palbociclib, most notably Cyclin E1-CDK2 activation and a paradoxical stabilization of phosphorylated CDK4 complexes. Conclusion: Our study supports further clinical evaluation of CDK4/6i and their combination with anti-BRAF/MEK therapies as a novel effective treatment against advanced thyroid tumors. Moreover, the complementary use of our 11 genes predictor with p16/KI67 evaluation could represent a prompt tool for recognizing the intrinsically CDK4/6i insensitive patients, who are potentially better candidates to immediate chemotherapy.
Asunto(s)
Imidazoles , Oximas , Prolina/análogos & derivados , Tiocarbamatos , Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides , Humanos , Fosforilación , Proteínas Proto-Oncogénicas B-raf/genética , Línea Celular Tumoral , Neoplasias de la Tiroides/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Quinasa 4 Dependiente de la CiclinaRESUMEN
Cyclin-dependent kinase 4 (CDK4) is a master integrator that couples mitogenic/oncogenic signaling with the cell division cycle. It is deregulated in most cancers and inhibitors of CDK4 have become standard of care drugs for metastatic estrogen-receptor positive breast cancers and are being evaluated in a variety of other cancers. We previously characterized the T-loop phosphorylation at T172 of CDK4 as the highly regulated step that determines the activity of cyclin D-CDK4 complexes. Moreover we demonstrated that the highly variable detection of T172-phosphorylated CDK4 signals the presence or absence of the active CDK4 targeted by the CDK4/6 inhibitory drugs, which predicts the tumor cell sensitivity to these drugs including palbociclib. To date, the phosphorylation of CDK4 has been very poorly studied because only few biochemical techniques and reagents are available for it. In addition, the available ones including 2D-IEF separation of CDK4 modified forms are considered too tedious. The present report describes the generation, selection and characterization of the first monoclonal antibodies that specifically recognize the active CDK4 phosphorylated on its T172 residue. One key to this success was the immunization with a long phosphopeptide corresponding to the complete activation segment of CDK4. These monoclonal antibodies specifically recognize T172-phosphorylated CDK4 in a variety of assays, including western blotting, immunoprecipitation and, as a capture antibody, a sensitive ELISA from cell lysates. The specific immunoprecipitation of T172-phosphorylated CDK4 allowed to clarify the involvement of phosphorylations of co-immunoprecipitated p21 and p27, showing a privileged interaction of T172-phosphorylated CDK4 with S130-phosphorylated p21 and S10-phosphorylated p27. Abbreviations: 2D: two-dimensional; CAK: CDK-activating kinase; CDK: cyclin-dependent kinase; HAT: Hypoxanthine-Aminopterin-Thymidine; FBS: fetal bovine serum; IP: immunoprecipitation; ID: immunodetection; mAb: monoclonal antibody; PAGE: polyacrylamide gel electrophoresis; PBS: phosphate buffer saline; pRb: retinoblastoma susceptibility protein; SDS: sodium dodecyl sulfate; DTT: dithiotreitol; TET: tetracyclin repressor; Avi: Avi tag; TEV: tobacco etch virus cleavage site; EGFP: enhanced green fluorescent protein; BirA: bifunctional protein biotin ligase BirA; IRES: internal ribosome entry site; HIS: poly-HIS purification tag; DELFIA: dissociation-enhanced lanthanide fluorescent immunoassay; 3-MBPP1: 1-(1,1-dimethylethyl)-3[(3-methylphenyl) methyl]-1H-pyrazolo[3,4-d] pyrimidin-4-amine; BSA: bovine serum albumin; ECL: Enhanced chemiluminescence.
Asunto(s)
Anticuerpos Monoclonales , Neoplasias , Ciclo Celular , Quinasa 4 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Humanos , Neoplasias/metabolismo , Fosforilación , Proteína de Retinoblastoma/metabolismoRESUMEN
Cyclin-dependent kinase 4 (CDK4) is a master integrator of mitogenic and antimitogenic extracellular signals. It is also crucial for many oncogenic transformation processes. Various molecular features of CDK4 activation remain poorly known or debated, including the regulation of its association with D-type cyclins, its activating Thr172 phosphorylation, and the roles of Cip/Kip CDK "inhibitors" in these processes. Thr172 phosphorylation of CDK4 was reinvestigated using two-dimensional gel electrophoresis in various experimental systems, including human fibroblasts, canine thyroid epithelial cells stimulated by thyrotropin, and transfected mammalian and insect cells. Thr172 phosphorylation of CDK4 depended on prior D-type cyclin binding, but Thr172 phosphorylation was also found in p16-bound CDK4. Opposite effects of p27 on cyclin D3-CDK4 activity observed in different systems depended on its stoichiometry in this complex. Thr172-phosphorylated CDK4 was enriched in complexes containing p21 or p27, even at inhibitory levels of p27 that precluded CDK4 activity. Deletion of the p27 nuclear localization signal sequence relocalized cyclin D3-CDK4 in the cytoplasm but did not affect CDK4 phosphorylation. Within cyclin D3 complexes, T-loop phosphorylation of CDK4, but not of CDK6, was directly regulated, identifying it as a determining target for cell cycle control by extracellular factors. Collectively, these unexpected observations indicate that CDK4-activating kinase(s) should be reconsidered.
Asunto(s)
Quinasa 4 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclinas/metabolismo , Señales de Localización Nuclear/metabolismo , Animales , Células Cultivadas , Quinasa 4 Dependiente de la Ciclina/análisis , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Citoplasma/enzimología , Perros , Activación Enzimática , Humanos , Señales de Localización Nuclear/genética , Fosforilación , Proteínas Tirosina Quinasas Receptoras/metabolismo , Serina/metabolismo , Treonina/metabolismoRESUMEN
Cyclin D-CDK4/6 are the first CDK complexes to be activated in the G1 phase in response to oncogenic pathways. The specific CDK4/6 inhibitor PD0332991 (palbociclib) was recently approved by the FDA and EMA for treatment of advanced ER-positive breast tumors. Unfortunately, no reliable predictive tools are available for identifying potentially responsive or insensitive tumors. We had shown that the activating T172 phosphorylation of CDK4 is the central rate-limiting event that initiates the cell cycle decision and signals the presence of active CDK4. Here, we report that the profile of post-translational modification including T172 phosphorylation of CDK4 differs among breast tumors and associates with their subtypes and risk. A gene expression signature faithfully predicted CDK4 modification profiles in tumors and cell lines. Moreover, in breast cancer cell lines, the CDK4 T172 phosphorylation best correlated with sensitivity to PD0332991. This gene expression signature identifies tumors that are unlikely to respond to CDK4/6 inhibitors and could help to select a subset of patients with HER2-positive and basal-like tumors for clinical studies on this class of drugs.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/patología , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/química , Piperazinas/farmacología , Procesamiento Proteico-Postraduccional , Piridinas/farmacología , Transcriptoma , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Análisis por Micromatrices , Fosforilación , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
UNLABELLED: Although thyroid gland function is mainly under the control of pituitary TSH, other factors, such as iodine, play a role in this process. The thyroid is capable of producing different iodolipids such as 6-iodo-deltalactone and 2-iodohexadecanal (2-IHDA). It was shown that these iodolipids mimic some of the inhibitory effects of excess iodide on several thyroid parameters. OBJECTIVES: To study the effect of 2-IHDA on cell proliferation and apoptosis in FRTL-5 cells. RESULTS: FRTL-5 cells were grown in the presence of TSH and treated with increasing concentrations of KI and 2-IHDA (0.5, 5, 10 and 33 µM) for 24, 48 and 72 h. Whereas KI inhibited cell proliferation only at 33 µM after 72 h of treatment, 2-IHDA inhibited in a time and concentration dependent manner. Analysis of cell cycle by flow cytometric DNA analysis revealed an accumulation of cells in G1 phase induced by 2-IHDA. The expression of cyclin A, cyclin D1 and cyclin D3 were reduced after treatment with 2-IHDA whereas CDK4 and CDK6 proteins were not modified. 2-IHDA induced a dynamic change in cytoplasmic to nuclear accumulation of p21 and p27 causing these proteins to be accumulated mostly in the nucleus. We also observed evidence of a pro-apoptotic effect of 2-IHDA at highest concentrations. No significant effect of KI was observed. CONCLUSION: These results suggest that the inhibitory effects of 2-IHDA on FRTL-5 thyroid cell proliferation are mediated by cell cycle arrest in G1/S phase and cell death by apoptosis.
Asunto(s)
Aldehídos/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Glándula Tiroides/citología , Tirotropina/farmacología , Animales , Apoptosis , Línea Celular , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Ciclinas/metabolismo , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Ratas , Glándula Tiroides/efectos de los fármacosRESUMEN
We have investigated the role of the different classes of MAPKs, i.e. ERKs, c-Jun N-terminal kinases (JNKs), and p38 MAPK in the proliferation of dog and human thyroid epithelial cells (thyrocytes) in primary cultures. In these cells, TSH, acting through cAMP, epidermal growth factor, hepatocyte growth factor (HGF), and phorbol 12-myristate 13-acetate induce DNA synthesis. With the exception of HGF, all of these factors require the presence of insulin for mitogenic effects to be expressed. We found that TSH and forskolin are without effect on the phosphorylation and activity of the different classes of MAPKs. In contrast, all the cAMP-independent growth factors, whereas without effect on the phosphorylation and activity of JNKs and p38 MAPK, stimulated the ERKs. This effect was strong and sustained in response to HGF, epidermal growth factor and 12-myristate 13-acetate but weak and transient in response to insulin. Moreover, whereas in stimulated cells DNA synthesis was inhibited by PD 098059, an inhibitor of MAPK kinase 1 and consequently of ERKs, it was not modified by SB 203580, an inhibitor of p38 MAPK. Taken together, these data 1) exclude a role of JNKs and p38 MAPK in the proliferation of dog and human thyrocytes; 2) suggest that the mitogenic action of the cAMP-independent agents requires a strong and sustained activation of both ERKs and phosphatidylinositol 3-kinase/protein kinase B as realized by HGF alone or by the other agents together with insulin; and 3) show that TSH and cAMP do not activate ERKs but that the weak activation of ERKs by insulin is nevertheless necessary for DNA synthesis to occur.
Asunto(s)
AMP Cíclico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Factor de Crecimiento de Hepatocito/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , Glándula Tiroides/citología , Animales , Arsenitos/farmacología , Carcinógenos/farmacología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Colforsina/farmacología , Perros , Inhibidores Enzimáticos/farmacología , Células Epiteliales/citología , Células Epiteliales/enzimología , Flavonoides/farmacología , Humanos , Hipoglucemiantes/farmacología , Imidazoles/farmacología , Insulina/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Piridinas/farmacología , Compuestos de Sodio/farmacología , Acetato de Tetradecanoilforbol/farmacología , Tirotropina/farmacología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Mitosis is triggered by the abrupt dephosphorylation of inhibitory Y15 and T14 residues of cyclin B1-bound cyclin-dependent kinase (CDK)1 that is also phosphorylated at T161 in its activation loop. The sequence of events leading to the accumulation of fully phosphorylated cyclin B1-CDK1 complexes remains unclear. Two-dimensional gel electrophoresis allowed us to determine whether T14, Y15, and T161 phosphorylations occur on same CDK1 molecules and to characterize the physiological occurrence of their seven phosphorylation combinations. Intriguingly, in cyclin B1-CDK1, the activating T161 phosphorylation never occurred without the T14 phosphorylation. This strict association could not be uncoupled by a substantial reduction of T14 phosphorylation in response to Myt1 knockdown, suggesting some causal relationship. However, T14 phosphorylation was not directly required for T161 phosphorylation, because Myt1 knockdown did uncouple these phosphorylations when leptomycin B prevented cyclin B1-CDK1 complexes from accumulating in cytoplasm. The coupling mechanism therefore depended on unperturbed cyclin B1-CDK1 traffic. The unexpected observation that the activating phosphorylation of cyclin B1-CDK1 was tightly coupled to its T14 phosphorylation, but not Y15 phosphorylation, suggests a mechanism that prevents premature activation by constitutively active CDK-activating kinase. This explained the opposite effects of reduced expression of Myt1 and Wee1, with only the latter inducing catastrophic mitoses.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Ciclina B1/metabolismo , Activación Enzimática , Procesamiento Proteico-Postraduccional , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Ciclina A/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel Bidimensional , Ácidos Grasos Insaturados/farmacología , Fase G2 , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Carioferinas/antagonistas & inhibidores , Mitosis , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Unión Proteica , Transporte de Proteínas , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Interferencia de ARN , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Treonina/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína Exportina 1RESUMEN
The study of normal signal transduction pathways regulating the proliferation and differentiation of a cell type allows to predict and to understand the perversions of these pathways which lead to tumorigenesis. In the case of the human thyroid cell, three cascades are mostly involved in tumorigenesis: The pathways and genetic events affecting them are described. Caveats in the use of models and the interpretation of results are formulated and the still pending questions are outlined.
Asunto(s)
Transducción de Señal , Glándula Tiroides/metabolismo , Glándula Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Animales , Genes Relacionados con las Neoplasias/genética , Humanos , Transducción de Señal/genética , Neoplasias de la Tiroides/genéticaRESUMEN
Cyclin-dependent kinase (CDK) 4 is a master integrator that couples mitogenic/oncogenic signalling cascades with the inactivation of the central oncosuppressor Rb and the cell cycle. Its activation requires binding to a D-type cyclin and then T-loop phosphorylation at T172 by the only identified CDK-activating kinase in animal cells, cyclin H-CDK7. In contrast with the observed constitutive activity of cyclin H-CDK7, we have recently identified the T172-phosphorylation of cyclin D-bound CDK4 as a crucial cell cycle regulatory target. Intriguingly, the homologous T177-phosphorylation of CDK6 is weak in several systems and does not present this regulation. In this Perspective, we review the recent advances and debates on the multistep mechanism leading to activation of D-type cyclin-CDK4 complexes. This involves a re-evaluation of the implication of Cip/Kip CDK "inhibitors" and CDK7 in this process.
Asunto(s)
Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Proteína de Retinoblastoma/metabolismo , Animales , Ciclo Celular , Ciclina H/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Neoplasias/metabolismo , Fosforilación , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
How cAMP-dependent protein kinases [protein kinase A (PKA)] transduce the mitogenic stimulus elicited by TSH in thyroid cells to late activation of cyclin D3-cyclin-dependent kinase 4 (CDK4) remains enigmatic. Here we show in PC Cl3 rat thyroid cells that TSH/cAMP, like insulin, activates the mammalian target of rapamycin (mTOR)-raptor complex (mTORC1) leading to phosphorylation of S6K1 and 4E-BP1. mTORC1-dependent S6K1 phosphorylation in response to both insulin and cAMP required amino acids, whereas inhibition of AMP-activated protein kinase and glycogen synthase kinase 3 enhanced insulin but not cAMP effects. Unlike insulin, TSH/cAMP did not activate protein kinase B or induce tuberous sclerosis complex 2 phosphorylation at T1462 and Y1571. However, like insulin, TSH/cAMP produced a stable increase in mTORC1 kinase activity that was associated with augmented 4E-BP1 binding to raptor. This could be caused in part by T246 phosphorylation of PRAS40, which was found as an in vitro substrate of PKA. Both in PC Cl3 cells and primary dog thyrocytes, rapamycin inhibited DNA synthesis and retinoblastoma protein phosphorylation induced by TSH and insulin. Although rapamycin reduced cyclin D3 accumulation, the abundance of cyclin D3-CDK4 complexes was not affected. However, rapamycin inhibited the activity of these complexes by decreasing the TSH and insulin-mediated stimulation of activating T172 phosphorylation of CDK4. We propose that mTORC1 activation by TSH, at least in part through PKA-dependent phosphorylation of PRAS40, crucially contributes to mediate cAMP-dependent mitogenesis by regulating CDK4 T172-phosphorylation.
Asunto(s)
AMP Cíclico/farmacología , Quinasa 4 Dependiente de la Ciclina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Glándula Tiroides/metabolismo , Animales , Western Blotting , Células Cultivadas , Ciclina D3/metabolismo , Perros , Electroforesis en Gel Bidimensional , Inmunoprecipitación , Fosforilación/efectos de los fármacos , Unión Proteica , Ratas , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Glándula Tiroides/citología , Tirotropina/farmacologíaRESUMEN
How cyclic AMP (cAMP) could positively or negatively regulate G1 phase progression in different cell types or in cancer cells versus normal differentiated counterparts has remained an intriguing question for decades. At variance with the cAMP-dependent mitogenesis of normal thyroid epithelial cells, we show here that cAMP and cAMP-dependent protein kinase activation inhibit S-phase entry in four thyroid carcinoma cell lines that harbor a permanent activation of the Raf/ERK pathway by different oncogenes. Only in Ret/PTC1-positive TPC-1 cells did cAMP markedly inhibit the Raf/ERK cascade, leading to mTOR pathway inhibition, repression of cyclin D1 and p21 and p27 accumulation. p27 knockdown did not prevent the DNA synthesis inhibition. In the other cells, cAMP little affected these signaling cascades and levels of cyclins D or CDK inhibitors. However, cAMP differentially inhibited the pRb-kinase activity and T172-phosphorylation of CDK4 complexed to cyclin D1 or cyclin D3, whereas CDK-activating kinase activity remained unaffected. At variance with current conceptions, our studies in thyroid carcinoma cell lines and previously in normal thyrocytes identify the activating phosphorylation of CDK4 as a common target of opposite cell cycle regulations by cAMP, irrespective of its impact on classical mitogenic signaling cascades and expression of CDK4 regulatory partners.
Asunto(s)
AMP Cíclico/farmacología , Quinasa 4 Dependiente de la Ciclina/metabolismo , Neoplasias de la Tiroides/enzimología , Neoplasias de la Tiroides/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclina A/metabolismo , Ciclina D , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , ADN de Neoplasias/biosíntesis , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Mutación/genética , Fosforilación/efectos de los fármacos , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteína de Retinoblastoma/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Cyclin-dependent kinase (CDK)4 is a master integrator that couples mitogenic and antimitogenic extracellular signals with the cell cycle. It is also crucial for many oncogenic transformation processes. In this overview, we address various molecular features of CDK4 activation that are critical but remain poorly known or debated, including the regulation of its association with D-type cyclins, its subcellular location, its activating Thr172-phosphorylation and the roles of Cip/Kip CDK "inhibitors" in these processes. We have recently identified the T-loop phosphorylation of CDK4, but not of CDK6, as a determining target for cell cycle control by extracellular factors, indicating that CDK4-activating kinase(s) might have to be reconsidered.
RESUMEN
Two distinct mitogenic modes coexist in the physiologically relevant model of primary cultures of dog thyroid epithelial cells. The differentiation-associated mitogenic stimulation by TSH and cAMP specifically requires the assembly and activation of cyclin D3-cyclin-dependent kinase (CDK)4 associated to p27(kip1), while the dedifferentiating proliferation induced by growth factors is associated with induction of cyclin D1. Here, we suggest that the related CDK "inhibitors" p21(cip1) and p27 are differentially utilized as positive CDK4 regulators in these mitogenic stimulations. p21 was induced by EGF + serum, but repressed by TSH, which, as previously shown, upregulates p27. In response to EGF + serum, p21 supported the nuclear localization, phosphorylation and pRb-kinase activity of CDK4. Unexpectedly, partly different site-specificities of pRb-kinase activity, leading to similar differences in the phosphorylation pattern of pRb in intact cells, were associated with cyclin D3-CDK4 bound to p27 in TSH-stimulated cells, or with CDK4 bound to p21 in growth factor-stimulated cells. These differences were ascribed to the predominant association of the latter complex to cyclin D1. Indeed, in different cell types and species, cyclin D1 varied from cyclin D3 by more efficiently driving the phosphorylation of pRb at sites (Ser807/811 and Thr826) required for its electrophoretic mobility shift. Therefore, different D-type cyclins could differently impact some pRb functions, which should be considered not only in the understanding of the relationships between cell cycle and differentiation expression in the distinct mitogenic modes of thyroid cells, but also in various development or differentiation models associated with dramatic switches in the expression of individual D-type cyclins.
Asunto(s)
Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Ciclinas/metabolismo , Células Epiteliales/metabolismo , Proteína de Retinoblastoma/metabolismo , Glándula Tiroides/citología , Glándula Tiroides/metabolismo , Animales , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Ciclina D3 , Perros , Activación Enzimática , Células Epiteliales/citología , Humanos , Mitosis , Modelos Biológicos , Fosforilación , Transporte de Proteínas , Especificidad por SustratoRESUMEN
To control the G1/S transition and the progression through the S phase, the activation of the cyclin-dependent kinase (CDK) 2 involves the binding of cyclin E then cyclin A, the activating Thr-160 phosphorylation within the T-loop by CDK-activating kinase (CAK), inhibitory phosphorylations within the ATP binding region at Tyr-15 and Thr-14, dephosphorylation of these sites by cdc25A, and release from Cip/Kip family (p27kip1 and p21cip1) CDK inhibitors. To re-assess the precise relationship between the different phosphorylations of CDK2, and the influence of cyclins and CDK inhibitors upon them, we introduce here the use of the high resolution power of two-dimensional gel electrophoresis, combined to Tyr-15- or Thr-160-phosphospecific antibodies. The relative proportions of the potentially active forms of CDK2 (phosphorylated at Thr-160 but not Tyr-15) and inactive forms (non-phosphorylated, phosphorylated only at Tyr-15, or at both Tyr-15 and Thr-160), and their respective association with cyclin E, cyclin A, p21, and p27, were demonstrated during the mitogenic stimulation of normal human fibroblasts. Novel observations modify the current model of the sequential CDK2 activation process: (i) Tyr-15 phosphorylation induced by serum was not restricted to cyclin-bound CDK2; (ii) Thr-160 phosphorylation engaged the entirety of Tyr-15-phosphorylated CDK2 associated not only with a cyclin but also with p27 and p21, suggesting that Cip/Kip proteins do not prevent CDK2 activity by impairing its phosphorylation by CAK; (iii) the potentially active CDK2 phosphorylated at Thr-160 but not Tyr-15 represented a tiny fraction of total CDK2 and a minor fraction of cyclin A-bound CDK2, underscoring the rate-limiting role of Tyr-15 dephosphorylation by cdc25A.
Asunto(s)
Quinasas CDC2-CDC28/metabolismo , Electroforesis en Gel Bidimensional/métodos , Adenosina Trifosfato/metabolismo , Western Blotting , Ciclo Celular , Línea Celular , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Fase G1 , Humanos , Mitógenos/química , Mitógenos/metabolismo , Modelos Biológicos , Fosforilación , Pruebas de Precipitina , Unión Proteica , Fase S , Treonina/química , Factores de Tiempo , Tirosina/química , Fosfatasas cdc25/metabolismoRESUMEN
According to current concepts, the cell cycle commitment after restriction (R) point passage requires the sustained stimulation by mitogens of the synthesis of labile d-type cyclins, which associate with cyclin-dependent kinase (CDK) 4/6 to phosphorylate pRb family proteins and sequester the CDK inhibitor p27kip1. In primary cultures of dog thyroid epithelial cells, the cAMP-dependent cell cycle induced by a sustained stimulation by thyrotropin or forskolin differs from growth factor mitogenic pathways, as cAMP does not upregulate d-type cyclins but increases p27 levels. Instead, cAMP induces the assembly of required cyclin D3-CDK4 complexes, which associate with nuclear p27. In this study, the arrest of forskolin stimulation rapidly slowed down the entry of dog thyrocytes into S phase and the phosphorylation of pRb family proteins. The pRb kinase activity, but not the formation, of the cyclin D3-CDK4-p27 complex was strongly reduced. Using two-dimensional gel electrophoresis, a phosphorylated form of CDK4 was separated. It appeared in response to forskolin and was bound to both cyclin D3 and p27, presumably reflecting the activating Thr-172 phosphorylation of CDK4. Upon forskolin withdrawal or after cycloheximide addition, this CDK4 phosphoform unexpectedly persisted in p27 complexes devoid of cyclin D3 but it disappeared from the more labile cyclin D3 complexes. These data demonstrate that the assembly of the cyclin D3-CDK4-p27 holoenzyme and the subsequent phosphorylation and activation of CDK4 depend on distinct cAMP actions. This provides a first example of a crucial regulation of CDK4 phosphorylation by a mitogenic cascade and a novel mechanism of cell cycle control at the R point.