RESUMEN
Malignant pleural mesothelioma (MPM) is an aggressive cancer with limited therapeutic options. We evaluated the impact of CDK4/6 inhibition by palbociclib in 28 MPM cell lines including 19 patient-derived ones, using various approaches including RNA-sequencing. Palbociclib strongly and durably inhibited the proliferation of 23 cell lines, indicating a unique sensitivity of MPM to CDK4/6 inhibition. When observed, insensitivity to palbociclib was mostly explained by the lack of active T172-phosphorylated CDK4. This was associated with high p16INK4A (CDKN2A) levels that accompany RB1 defects or inactivation, or (unexpectedly) CCNE1 overexpression in the presence of wild-type RB1. Prolonged palbociclib treatment irreversibly inhibited proliferation despite re-induction of cell cycle genes upon drug washout. A senescence-associated secretory phenotype including various potentially immunogenic components was irreversibly induced. Phosphorylated CDK4 was detected in 80% of 47 MPMs indicating their sensitivity to CDK4/6 inhibitors. Its absence in some highly proliferative MPMs was linked to very high p16 (CDKN2A) expression, which was also observed in public datasets in tumours from short-survival patients. Our study supports the evaluation of CDK4/6 inhibitors for MPM treatment, in monotherapy or combination therapy.
RESUMEN
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 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
CDK4 and CDK6 bound to D-type cyclins are master integrators of G1 phase cell cycle regulations by initiating the inactivating phosphorylation of the central oncosuppressor pRb. Because of their frequent deregulation in cancer, cyclin D-CDK4/6 complexes are emerging as especially promising therapeutic targets. The specific CDK4/6 inhibitor PD0332991 is currently tested in a growing number of phase II/III clinical trials against a variety of pRb-proficient chemotherapy-resistant cancers. We have previously shown that PD0332991 inhibits not only CDK4/6 activity but also the activation by phosphorylation of the bulk of cyclin D-CDK4 complexes stabilized by p21 binding. Here we show that PD0332991 has either a positive or a negative impact on the activation of cyclin D-CDK4/6 complexes, depending on their binding to p21. Indeed, whereas PD0332991 inhibits the phosphorylation and activity of p21-bound CDK4/6, it specifically stabilized activated cyclin D3-CDK4/6 complexes devoid of p21 and p27. After elimination of PD0332991, these activated cyclin D3-CDK4/6 complexes persisted for at least 24 h, resulting in paradoxical cell cycle entry in the absence of a mitogenic stimulation. This unsuspected positive effect of PD0332991 on cyclin D3-CDK4/6 activation should be carefully assessed in the clinical evaluation of PD0332991, which until now only involves discontinuous administration protocols.
Asunto(s)
Ciclina D3/metabolismo , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Complejos Multiproteicos/metabolismo , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Animales , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Medio de Cultivo Libre de Suero , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 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 , ADN/biosíntesis , Humanos , Fosforilación/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Proteína de Retinoblastoma/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
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
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
The homologous cyclin-dependent kinases (CDK) CDK4 and CDK6 integrate mitogenic and oncogenic signaling cascades with the cell cycle. Their activation requires binding to a D-type cyclin and then T-loop phosphorylation at T172 and T177 (respectively) by the only CDK-activating kinase identified in animal cells, cyclin H-CDK7. At odds with the existing data showing the constitutive activity of CDK7, we have recently identified the T172 phosphorylation of cyclin D-bound CDK4 as a crucial cell cycle regulatory target. Here we show that T172 phosphorylation of CDK4 is conditioned by its unique proline 173 residue. In contrast to CDK4, CDK6 does not contain such a proline and, unexpectedly, remained poorly phosphorylated and active in a variety of cells. Mutations of proline 173 did not adversely affect CDK4 activation by CDK7, but in cells they abolished CDK4 T172 phosphorylation and activity. Conversely, substituting a proline for the corresponding residue of CDK6 enforced its complete, apparently cyclin-independent T177 phosphorylation and dramatically increased its activity. These results lead us to propose that CDK4 might not be phosphorylated by CDK7 in intact cells but is more likely phosphorylated by another, presumably proline-directed kinase(s). Moreover, they provide a new model of a potentially oncogenic activating mutation of a CDK.
Asunto(s)
Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Mutación , Secuencia de Aminoácidos , Animales , Western Blotting , Células CHO , Línea Celular , Línea Celular Tumoral , Cricetinae , Cricetulus , Ciclina D3 , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/genética , Quinasas Ciclina-Dependientes/genética , Ciclinas/genética , Ciclinas/metabolismo , Electroforesis en Gel Bidimensional , Técnica del Anticuerpo Fluorescente Indirecta , Células HCT116 , Humanos , Inmunoprecipitación , Datos de Secuencia Molecular , Fosforilación , Prolina/genética , Prolina/metabolismo , Unión Proteica , Homología de Secuencia de Aminoácido , Treonina/genética , Treonina/metabolismo , Transfección , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
The Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) signaling pathways are aberrantly activated in many tumors, including highly proliferative glioblastomas, but how they are wired with the cell cycle remains imperfectly understood. Inhibitors of MEK/ERK and mTOR pathways are tested as anticancer agents. They are generally considered to induce a G(1) cell cycle arrest through down-regulation of D-type cyclins and up-regulation of p27(kip1). Here, we examined the effect of targeting mTOR by rapamycin and/or MEK by PD184352 in human glioblastoma cell lines. In combination, these drugs cooperatively and potently inhibited the G(1)-S transition and retinoblastoma protein phosphorylation. Their cooperation could not be explained by their partial and differential inhibitory effects on cyclin D1 or D3 but instead by their synergistic inhibition of the activating T172 phosphorylation of cyclin-dependent kinase (CDK) 4. This appeared independent of p27 and unrelated to weak modulations of the CDK-activating kinase activity. The T172 phosphorylation of CDK4 thus appears as a crucial node integrating the activity of both MEK/ERK and mTOR pathways. Combined inhibition of both pathways should be considered as a promising strategy for treatment of tumors harboring a deregulated CDK4 activity.
Asunto(s)
Benzamidas/farmacología , Proliferación Celular/efectos de los fármacos , Quinasa 4 Dependiente de la Ciclina/metabolismo , Glioblastoma/patología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Sirolimus/farmacología , Factores de Transcripción/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales , Benzamidas/administración & dosificación , Línea Celular Tumoral , Replicación del ADN/efectos de los fármacos , Combinación de Medicamentos , Evaluación Preclínica de Medicamentos , Glioblastoma/metabolismo , Humanos , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Proteína Reguladora Asociada a mTOR , Proteína de Retinoblastoma/metabolismo , Sirolimus/administración & dosificación , Serina-Treonina Quinasas TOR , Células Tumorales CultivadasRESUMEN
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 thyroid epithelial cells. TSH via cAMP induces proliferation and differentiation expression, whereas growth factors including epidermal growth factor (EGF) induce proliferation and dedifferentiation. Divergent models of TSH/cAMP-dependent mitogenesis have emerged from different thyroid cell culture systems. In the FRTL-5 rat cell line, cAMP cross-signals with transduction pathways of growth factors to induce cyclin D1 and p21(cip1) and down-regulate p27(kip1). By contrast, in canine primary cultures, mitogenic pathways of cAMP and growth factors are fully distinct. cAMP does not induce D-type cyclins and p21, it up-regulates p27, and it stimulates the formation and activity of cyclin D3-cyclin-dependent kinase (CDK) 4 complexes. In primary cultures of normal human thyrocytes, EGF + serum increased cyclin D1 and p21 accumulation, and it stimulated the assembly and activity of cyclin D1-CDK4-p21 complexes. By contrast, TSH repressed or did not induce cyclin D1 and p21, and it rather up-regulated p27. TSH did not increase cyclin D1-CDK4 activity, but it stimulated the activating phosphorylation of CDK4 and the pRb-kinase activity of preexisting cyclin D3-CDK4 complexes. As recently demonstrated in dog thyrocytes and other systems, cyclin D1 and cyclin D3 differently oriented the site specificity of CDK4 pRb-kinase activity, which might differently impact some pRb functions. Cyclin D1 or cyclin D3 are thus differentially used in the distinct mitogenic stimulations by growth factors or TSH, and potentially in hyperproliferative diseases generated by the overactivation of their respective signaling pathways. At variance with dog thyroid primary cultures, rat thyroid cell lines might not be valid models of TSH-dependent mitogenesis of human thyrocytes.
Asunto(s)
Ciclina D1/metabolismo , Ciclinas/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Mitosis , Glándula Tiroides/efectos de los fármacos , Tirotropina/farmacología , Animales , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Ciclina D3 , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , 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/antagonistas & inhibidores , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Perros , Humanos , Mitosis/efectos de los fármacos , Fosforilación , Ratas , Suero , Glándula Tiroides/citología , Glándula Tiroides/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
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
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
The cAMP-dependent mitogenic stimulation elicited by thyroid-stimulating hormone (TSH) in primary cultures of canine thyroid epithelial cells is unique as it upregulates the cyclin-dependent kinase (CDK) inhibitor p27kip1 but not D-type cyclins. TSH and cAMP promote the assembly of required cyclin D3-CDK4 complexes and their nuclear import. Here, the nuclear translocation of these complexes strictly correlated in individual cells with the enhanced presence of nuclear p27. p27, like cyclin D3, supported the TSH-stimulated pRb-kinase activity of the CDK4 complex and, as demonstrated using the high-resolution power of the two-dimensional (2D) gel electrophoresis, the phosphorylation of CDK4, presumably by the nuclear CDK-activating kinase. In the presence of TSH, transforming growth factor beta (TGFbeta) did not affect the assembly of cyclin D3-CDK4, but it strongly inhibited the pRb-kinase activity associated with both cyclin D3 and p27, not only by preventing the nuclear import of cyclin D3-CDK4 and its binding to p27, but also by inhibiting CDK4 phosphorylation within residual p27-bound cyclin D3-CDK4 complexes. No alterations of the relative abundance of multiple (un)phosphorylated forms of cyclin D3 and p27 demonstrated by 2D-gel electrophoresis were associated with these processes. This study suggests a crucial positive role of p27 in the TSH-stimulated nuclear import, phosphorylation, and catalytic activity of cyclin D3-bound CDK4. Moreover, it demonstrates a technique to directly assess the in vivo phosphorylation of endogenous CDK4, which might appear as a last regulated step targeted by the antagonistic cell cycle effects of TSH and TGFbeta.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Células Epiteliales/enzimología , Proteínas Proto-Oncogénicas , Tirotropina/farmacología , Factor de Crecimiento Transformador beta/farmacología , Proteínas Supresoras de Tumor/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/genética , Animales , Dominio Catalítico/efectos de los fármacos , Dominio Catalítico/genética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular/efectos de los fármacos , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclina D3 , Quinasa 4 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/efectos de los fármacos , Ciclinas/efectos de los fármacos , Perros , Electroforesis en Gel Bidimensional/métodos , Células Epiteliales/efectos de los fármacos , Holoenzimas/efectos de los fármacos , Holoenzimas/metabolismo , Fosforilación/efectos de los fármacos , Subunidades de Proteína/efectos de los fármacos , Subunidades de Proteína/metabolismo , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/enzimología , Tirotropina/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Supresoras de Tumor/efectos de los fármacosRESUMEN
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.