RESUMO
Cyclin-dependent kinases (CDKs), in association with cyclins, control cell cycle progression by phosphorylating a large number of substrates. In animals, activation of CDKs regularly requires both the association with a cyclin and then phosphorylation of a highly conserved threonine residue in the CDK activation loop (the classical mechanism), mediated by a CDK-activating kinase (CAK). In addition to this typical mechanism of activation, some CDKs can also be activated by the association of a cyclin to a monomeric CDK previously phosphorylated by CAK although not all CDKs can be activated by this mechanism. In animals and yeast, cyclin, in addition to being required for CDK activation, provides substrate specificity to the cyclin/CDK complex; however, in plants both the mechanisms of CDKs activation and the relevance of the CDK-associated cyclin for substrate targeting have been poorly studied. In this work, by co-expressing proteins in E. coli, we studied maize CDKA2;1a and CDKB1;1, two of the main types of CDKs that control the cell cycle in plants. These kinases could be activated by the classical mechanism and by the association of CycD2;2a to a phosphorylated intermediate in its activation loop, a previously unproven mechanism for the activation of plant CDKs. Unlike CDKA2;1a, CDKB1;1 did not require CAK for its activation, since it autophosphorylated in its activation loop. Phosphorylation of CDKB1;1 and association of CycD2;2 was not enough for its full activation as association of maize CKS, a scaffolding protein, differentially stimulated substrate phosphorylation. Our results suggest that both CDKs participate in substrate recognition.
Assuntos
Proteínas Serina-Treonina Quinases , Zea mays , Animais , Proteínas Serina-Treonina Quinases/metabolismo , Zea mays/genética , Escherichia coli/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: Pituitary adenomas (PA) are the second most common intracranial tumors and are classified according to hormone they produce, and the transcription factors they express. The majority of PA occur sporadically, and their molecular pathogenesis is incompletely understood. METHODS: Here we performed transcriptome and proteome analysis of tumors derived from POU1F1 (GH-, TSH-, and PRL-tumors, N = 16), NR5A1 (gonadotropes and null cells adenomas, n = 17) and TBX19 (ACTH-tumors, n = 6) lineages as well as from silent ACTH-tumors (n = 3) to determine expression of kinases, cyclins, CDKs and CDK inhibitors. RESULTS: The expression profiles of genes encoding kinases were distinctive for each of the three PA lineage: NR5A1-derived tumors showed upregulation of ETNK2 and PIK3C2G and alterations in MAPK, ErbB and RAS signaling, POU1F1-derived adenomas showed upregulation of PIP5K1B and NEK10 and alterations in phosphatidylinositol, insulin and phospholipase D signaling pathways and TBX19-derived adenomas showed upregulation of MERTK and STK17B and alterations in VEGFA-VEGFR, EGF-EGFR and Insulin signaling pathways. In contrast, the expression of the different genes encoding cyclins, CDK and CDK inhibitors among NR5A1-, POU1F1- and TBX19-adenomas showed only subtle differences. CDK9 and CDK18 were upregulated in NR5A1-adenomas, whereas CDK4 and CDK7 were upregulated in POUF1-adenomas. CONCLUSIONS: The kinome of PA clusters these lesions into three distinct groups according to the transcription factor that drives their terminal differentiation. And these complexes could be harnessed as molecular therapy targets.
Assuntos
Adenoma , Neoplasias Hipofisárias , Adenoma/metabolismo , Hormônio Adrenocorticotrópico/genética , Proteínas Reguladoras de Apoptose/genética , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Humanos , Insulina , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/patologia , Proteínas Serina-Treonina Quinases , Fatores de Transcrição/genética , TranscriptomaRESUMO
BACKGROUND: Cancer is one of the main causes of death by disease; several alternative treatments have been developed to counteract this condition. Curcumin (diferuloylmethane), extracted from the rhizome of Curcuma longa, has antioxidant, anti-inflammatory, and anti-cancer properties; however, it has low water solubility and poor intestinal absorption. Carrier systems, such as nanoemulsions, can increase the bioavailability of lipophilic bioactive compounds. OBJECTIVE: To evaluate the effect of curcumin nanoemulsions prepared with lecithin modified with medium-chain fatty acids as an emulsifier, on the expression of the Cdk4, Ccne2, Casp8 and Cldn4 genes involved in the carcinogenesis process in K14E6 transgenic mice. METHODS: The emulsifier was prepared by interesterification of medium-chain fatty acids, pure lecithin, and immobilized phospholipase-1 on Duolite A568. An Ultraturrax homogenizer and a Branson Ultrasonic processor were used for the preparation of nano-emulsions, and a Zetasizer evaluated the particle size. qRT-PCR analysis was performed to quantify the cancer-related genes expressed in the K14E6 mice. The development and evolution of skin carcinogenesis were assessed through histological analysis to compare cell morphology. RESULTS: Ca 59% of the MCFA were incorporated via esterification into the PC within 12 hours of the reaction. An emulsifier yield used to formulate the NE of 86% was achieved. Nanoemulsions with a particle size of 44 nm were obtained. The curcumin nano-emulsion group had a 91.81% decrease in the tumorigenesis index and a reduction in tumor area of 89.95% compared to the sick group. Histological analysis showed that the group administered with free curcumin developed a microinvasive squamous cell carcinoma, as opposed to the group with nanoemulsion which presented only a slight inflammation. In gene expression, only a significant difference in Cdk4 was observed in the nanoemulsion group.
Assuntos
Carcinogênese/efeitos dos fármacos , Curcumina/farmacologia , Composição de Medicamentos/métodos , Fosfatidilcolinas/química , Neoplasias Cutâneas/tratamento farmacológico , Animais , Disponibilidade Biológica , Caspase 8/metabolismo , Claudina-4/metabolismo , Curcumina/administração & dosagem , Quinase 4 Dependente de Ciclina/metabolismo , Ciclinas/metabolismo , Emulsões/química , Lecitinas , Camundongos , Camundongos Transgênicos , Nanopartículas/química , Neoplasias Cutâneas/patologiaRESUMO
Cyclin dependent kinase A; 1 (CDKA; 1) is essential in G1/S transition of cell cycle and its oxidation has been implicated in cell cycle arrest during plant abiotic stress. In the present study, an evaluation at the molecular level was performed to find possible sites of protein oxidative modifications. In vivo studies demonstrated that carbonylation of maize CDKA,1 is associated with a decrease in complex formation with maize cyclin D (CycD). Control and in vitro oxidized recombinant CDKA; 1 were sequenced by mass spectrometry. Proline at the PSTAIRE cyclin-binding motif was identified as the most susceptible oxidation site by comparative analysis of the resulted peptides. The specific interaction between CDKA; 1 and CycD6; 1, measured by surface plasmon resonance (SPR), demonstrated that the affinity and the kinetic of the interaction depended on the reduced-oxidized state of the CDKA; 1. CDKA; 1 protein oxidative modification would be in part responsible for affecting cell cycle progression, and thus producing plant growth inhibition under oxidative stress.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Prolina/metabolismo , Zea mays/enzimologia , Sequência de Aminoácidos , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Ciclinas/química , Modelos Moleculares , Oxirredução , Prolina/química , Alinhamento de SequênciaRESUMO
The complex formed by the cyclin-dependent kinase A (CDKA) and cyclin D is responsible for the G1-S transition in the plant cell cycle. Maize (Zea mays L) CDKA; 1 and CycD6; 1 were cloned and expressed in E. coli. The present study describes the optimization of both proteins production using a statistical approach known as response surface methodology (RSM). The experimental design took into account the effects of four variables: optical density of the culture (OD600) before induction, isopropyl ß-d-1-thiogalactopyranoside (IPTG) concentration, post-induction temperature, and post-induction time. For each protein, a 24 full factorial central composite rotary design for these four independent variables (at five levels each) was employed to fit a polynomial model; which indicated that 30 experiments were required for this procedure. An optimization of CDKA; 1 and CycD6; 1 production levels in the soluble fraction was achieved. Protein conformation and stability were studied by circular dichroism and fluorescence spectroscopy. Finally, in vitro Cyc-CDK complex formation and its kinase activity were confirmed.
Assuntos
Proteína Quinase CDC2/genética , Ciclinas/genética , Escherichia coli/genética , Proteínas de Plantas/genética , Zea mays/genética , Sequência de Bases , Proteína Quinase CDC2/metabolismo , Ciclinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Isopropiltiogalactosídeo/metabolismo , Modelos Biológicos , Modelos Estatísticos , Proteínas de Plantas/metabolismo , Conformação Proteica , Solubilidade , Temperatura , TransfecçãoRESUMO
The Proliferating Cell Nuclear Antigen, PCNA, has roles in both G1 and S phases of the cell cycle. Here we show that maize PCNA can be found in cells in structures of a trimer or a dimer of trimer, in complexes of high molecular mass that change in size as germination proceeds, co-eluting with cell cycle proteins as CycD3;1 and CDKs (A/B1;1). Using different methodological strategies, we show that PCNA actually interacts with CycD3;1, CDKA, CDKB1;1, KRP1;1 and KRP4;1, all of which contain PIP or PIP-like motifs. Anti-PCNA immunoprecipitates show kinase activity that is inhibited by KRP1;1 and KRP4;2, indicating the formation of quaternary complexes PCNA-CycD/CDKs-KRPs in which PCNA would act as a platform. This inhibitory effect seems to be differential during the germination process, more pronounced as germination advances, suggesting a complex regulatory mechanism in which PCNA could bind different sets of cyclins/CDKs, some more susceptible to inhibition by KRPs than others.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Zea mays/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Ciclinas/metabolismo , Germinação , Fosforilação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Zea mays/enzimologia , Zea mays/fisiologiaRESUMO
We report an unanticipated system of joint regulation by cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK), involving collaborative multi-site phosphorylation of a single substrate. In budding yeast, the protein Ste5 controls signaling through a G1 arrest pathway. Upon cell-cycle entry, CDK inhibits Ste5 via multiple phosphorylation sites, disrupting its membrane association. Using quantitative time-lapse microscopy, we examined Ste5 membrane recruitment dynamics at different cell-cycle stages. Surprisingly, in S phase, where Ste5 recruitment should be blocked, we observed an initial recruitment followed by a steep drop-off. This delayed inhibition revealed a requirement for both CDK activity and negative feedback from the pathway MAPK Fus3. Mutagenesis, mass spectrometry, and electrophoretic analyses suggest that the CDK and MAPK modify shared sites, which are most extensively phosphorylated when both kinases are active and able to bind their docking sites on Ste5. Such collaborative phosphorylation can broaden regulatory inputs and diversify output dynamics of signaling pathways.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Sítios de Ligação , Pontos de Checagem do Ciclo Celular , Membrana Celular/enzimologia , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Ciclinas/metabolismo , Cinética , Proteínas Quinases Ativadas por Mitógeno/genética , Fosforilação , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por SubstratoRESUMO
Leishmania mexicana has a large family of cyclin-dependent kinases (CDKs) that reflect the complex interplay between cell cycle and life cycle progression. Evidence from previous studies indicated that Cdc2-related kinase 3 (CRK3) in complex with the cyclin CYC6 is a functional homologue of the major cell cycle regulator CDK1, yet definitive genetic evidence for an essential role in parasite proliferation is lacking. To address this, we have implemented an inducible gene deletion system based on a dimerised Cre recombinase (diCre) to target CRK3 and elucidate its role in the cell cycle of L. mexicana. Induction of diCre activity in promastigotes with rapamycin resulted in efficient deletion of floxed CRK3, resulting in G2/M growth arrest. Co-expression of a CRK3 transgene during rapamycin-induced deletion of CRK3 resulted in complementation of growth, whereas expression of an active site CRK3(T178E) mutant did not, showing that protein kinase activity is crucial for CRK3 function. Inducible deletion of CRK3 in stationary phase promastigotes resulted in attenuated growth in mice, thereby confirming CRK3 as a useful therapeutic target and diCre as a valuable new tool for analyzing essential genes in Leishmania.
Assuntos
Leishmania mexicana/citologia , Leishmania mexicana/genética , Proteínas Proto-Oncogênicas c-crk/genética , Proteínas Proto-Oncogênicas c-crk/metabolismo , Sequência de Aminoácidos , Animais , Proteína Quinase CDC2/metabolismo , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/genética , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Deleção de Genes , Integrases/genética , Integrases/metabolismo , Leishmania mexicana/enzimologia , Leishmaniose Cutânea/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Genética Reversa/métodos , Sirolimo/farmacologiaRESUMO
Trypanosoma cruzi, the etiologic agent of Chagas disease, is a protozoan parasite with a life cycle that alternates between replicative and non-replicative forms, but the components and mechanisms that regulate its cell cycle are poorly described. In higher eukaryotes, cyclins are proteins that activate cyclin-dependent kinases (CDKs), by associating with them along the different stages of the cell cycle. These cyclin-CDK complexes exert their role as major modulators of the cell cycle by phosphorylating specific substrates. For the correct progression of the cell cycle, the mechanisms that regulate the activity of cyclins and their associated CDKs are diverse and must be controlled precisely. Different types of cyclins are involved in specific phases of the eukaryotic cell cycle, preferentially activating certain CDKs. In this work, we characterized TcCYC6, a putative coding sequence of T. cruzi which encodes a protein with homology to mitotic cyclins. The overexpression of this sequence, fused to a tag of nine amino acids from influenza virus hemagglutinin (TcCYC6-HA), showed to be detrimental for the proliferation of epimastigotes in axenic culture and affected the cell cycle progression. In silico analysis revealed an N-terminal segment similar to the consensus sequence of the destruction box, a hallmark for the degradation of several mitotic cyclins. We experimentally determined that the TcCYC6-HA turnover decreased in the presence of proteasome inhibitors, suggesting that TcCYC6 degradation occurs via ubiquitin-proteasome pathway. The results obtained in this study provide first evidence that TcCYC6 expression and degradation are finely regulated in T. cruzi.