RESUMO
Inactivation of Rb is a major event in the development of hepatocellular carcinoma (HCC). The activity of CDK4, determined by T172 phosphorylation, correlates with the onset of RB phosphorylation and G1/S cell cycle transition. However, the regulation of CDK4 activation and of the Rb pathway in HCC remain unclear. Here, we report that cyclin Y, a novel member of the cyclin family, is a potential regulator of the Rb pathway. We demonstrate that the Cyclin Y protein was overexpressed in human HCC tissues and that it was associated with poor patient prognosis. Cyclin Y could regulate the G1/S phase transition in human HCC cell lines. We found that CDK4 can bind to Cyclin Y in vitro. Furthermore, the accumulation of Cyclin Y could activate CDK4 through T172 phosphorylation of CDK4, inactivate Rb with increasing Rb phosphorylation, and enable the expression of E2F target genes such as CDK2 and Cyclin A. Thus, our findings suggest that Cyclin Y plays a role in the G1/S phase transition of HCC cells via Cyclin Y/CDK4/Rb signaling and that Cyclin Y could be used as a potential prognostic biomarker in HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Ciclinas/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Neoplasias Hepáticas/metabolismo , Proteína do Retinoblastoma/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Pontos de Checagem do Ciclo Celular/genética , Ciclina A/genética , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Fosforilação , Prognóstico , Proteína do Retinoblastoma/antagonistas & inibidores , Transdução de Sinais , Análise Serial de TecidosRESUMO
Cyclin Y (CCNY) is a member of cyclin superfamily proteins involved in the regulation of the cell cycle in proliferating cells. Intriguingly, CCNY is highly expressed in terminally differentiated neuronal cells of multiple brain regions and acts as a postsynaptic protein, which plays an inhibitory role in long-term potentiation. However, the pathophysiological significance of CCNY in the nervous system remains largely unexplored. In this study, we revisited our RNA-sequencing (RNA-seq) data obtained from cultured hippocampal neurons virally overexpressing or depleting CCNY. Using RNA-seq-based bioinformatic disease analysis and synaptic gene ontology analysis, we identified that numerous genes associated with epilepsy (e.g. Chrna4, Gabrd, Nhlrc1, Reln, Samd12, Slc6a1, etc.) or neurodegenerative diseases (e.g. Psen1, Pdyn, Ndrg1, etc.) are affected by the level of CCNY expression. In agreement with the RNA-seq-based disease analysis, we found that Ccny knockout (KO) mice are more susceptible to kainic acid-induced epilepsy than wild-type mice. In addition, some epilepsy-associated genes that are regulated by CCNY levels were further validated in the brain of Ccny KO mice at the mRNA and protein levels. Collectively, our findings indicate that CCNY shifts the expression profile of epilepsy-associated genes and exerts a protective effect against kainic acid-induced epilepsy, suggesting CCNY as a potential pharmaceutical candidate for the treatment of epilepsy.
Assuntos
Ciclinas/genética , Epilepsia/induzido quimicamente , Epilepsia/genética , Agonistas de Aminoácidos Excitatórios , Ácido Caínico , Animais , Química Encefálica/genética , Células Cultivadas , Biologia Computacional , Feminino , Genótipo , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças Neurodegenerativas/genética , RNA-Seq , Proteína ReelinaRESUMO
Diabetes is associated with platelet hyper-reactivity and enhanced risk of thrombosis development. Here we compared protein expression in platelets from healthy donors and diabetic patients to identify differentially expressed proteins and their possible function in platelet activation. Mass spectrometry analyses identified cyclin Y (CCNY) in platelets and its reduced expression in platelets from diabetic patients, a phenomenon that could be attributed to the increased activity of calpains. To determine the role of CCNY in platelets, mice globally lacking the protein were studied. CCNY-/- mice demonstrated lower numbers of circulating platelets but platelet responsiveness to thrombin and a thromboxane A2 analogue were comparable with that of wild-type mice, as was agonist-induced α and dense granule secretion. CCNY-deficient platelets demonstrated enhanced adhesion to fibronectin and collagen as well as an attenuated spreading and clot retraction, indicating an alteration in "outside in" integrin signalling. This phenotype was accompanied by a significant reduction in the agonist-induced tyrosine phosphorylation of ß3 integrin. Taken together we have shown that CCNY is present in anucleated platelets where it is involved in the regulation of integrin-mediated outside in signalling associated with thrombin stimulation.
Assuntos
Plaquetas/metabolismo , Ciclinas/genética , Integrinas/metabolismo , Adulto , Animais , Ciclinas/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Ativação Plaquetária/genética , Adesividade Plaquetária/genética , Agregação Plaquetária/genética , Transdução de Sinais/genética , Adulto JovemRESUMO
This study is designated to investigate the roles of cyclin Y (CCNY) and Wnt signaling pathway in regulating ovarian cancer (OC) cell proliferation, migration, and invasion. Quantitative real-time PCR (qRT-PCR), Western blot, MTT assay, cell scratch, and transwell test were used in our study, and transplanted tumor model was constructed on nude mice. C-Myc, cyclin D1, PFTK1, ki67, OGT, and ß-catenin protein expressions in tumor tissues were detected. CCNY was significantly upregulated in OC cell lines and tissues (both P < 0.05); significant association was observed between CCNY expression and clinicopathological stage, lymph node metastasis (LNM) (P < 0.05); and the CCNY expression in stages III to IV was higher than that in stages I to II, and patients with LNM had higher CCNY expression when compared with those in patients without LNM (P < 0.05); expressions of c-Myc, cyclin D, PFTK1, ki67, and OGT were upregulated in OC tissues compared with ovarian benign tissues, suggesting that these expressions were significantly different between the two groups (P < 0.05); CCNY significantly exacerbated proliferation, migration, and invasion of A2780 cells; c-Myc and cyclin D1 protein expressions increased as the expression of CCNY increased (P < 0.001); ß-catenin expressions in A2780 cells with over-expression of CCNY were significantly increased in the nucleus, but significantly decreased in the cytoplasm (both P < 0.05); high expressions of CCNY exacerbated the proliferation of A2780 cells in nude mice and significantly increased c-Myc, cyclin D1, PFTK1, ki67, and OGT protein expressions in tumor tissues which were transplanted into nude mice (P < 0.01). CCNY might exacerbate the proliferation, migration, and invasion of OC cells via activating the Wnt signaling pathway. Thus, this study provides a theoretical foundation for the development of therapeutic drugs that are able to cure OC by targeting CCNY.
Assuntos
Ciclinas/fisiologia , Proteínas de Neoplasias/fisiologia , Neoplasias Ovarianas/patologia , Via de Sinalização Wnt , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma/secundário , Adulto , Idoso , Animais , Diferenciação Celular , Divisão Celular , Linhagem Celular Tumoral , Movimento Celular , Núcleo Celular/metabolismo , Proliferação de Células , Ciclinas/biossíntese , Ciclinas/genética , Citoplasma/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Metástase Linfática , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Distribuição Aleatória , Proteínas Recombinantes de Fusão/metabolismoRESUMO
PFTK1 is a Cdc2-related protein kinase that is frequently upregulated in human hepatocellular carcinoma (HCC) where it correlates with metastatic features and motile phenotypes. To understand the modulated pathway underlining the PFTK1 action, here we show a physical interaction between PFTK1 and cyclin Y (CCNY) in promoting noncanonical Wnt signaling. In HCC cells, we found PFTK1 forms a direct complex with CCNY, and together readily upregulate key components of Wnt signaling (Dvl2 and Naked1). Exogenous expression of PFTK1 and CCNY activated Rho GTPases, which are known targets of the noncanonical path. In line with Rho GTPases activation, we also found marked actin polymerizations in cells with PFTK1-CCNY co-expressions. Our findings highlight a PFTK1-CCNY complex in activating noncanonical Wnt signaling in HCC cells.
Assuntos
Carcinoma Hepatocelular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Neoplasias Hepáticas/metabolismo , Via de Sinalização Wnt , Linhagem Celular Tumoral , Humanos , Ligação Proteica , Mapeamento de Interação de ProteínasRESUMO
Cyclin Y is a highly conserved cyclin among eumetazoans, yet its function and regulation are poorly understood. To search for Cyclin Y-interacting proteins, we screened a yeast two-hybrid library using human Cyclin Y (CCNY) as a bait and identified the following interactors: CDK14 and four members of the 14-3-3 family (ε, ß, η, τ). The interaction between CCNY and 14-3-3 proteins was confirmed both in vitro and in vivo. The results showed that Ser-100 and Ser-326 residues in CCNY were crucial for 14-3-3 binding. Interestingly, binding of CCNY to 14-3-3 significantly enhanced the association between CCNY and CDK14. Our findings may add a new layer of regulation of CCNY binding to its kinase partner.
Assuntos
Proteínas 14-3-3/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Sequência de Aminoácidos , Animais , Quinases Ciclina-Dependentes/química , Células HEK293 , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Serina/metabolismo , Frações Subcelulares/metabolismoRESUMO
The family protein of cyclins, as well as cyclin-dependent kinases (CDKs) cooperating with them, are broadly researched, as a matter of their dysfunction may lead to tumor transformation. Cyclins are defined as key regulators that have a controlling function of the mammalian nuclear cell divides. Cyclin Y (CCNY) is a recently characterized member of the cyclin family and was first identified from the human testis cDNA library. It is an actin-binding protein acting through decreased actin dynamics at a steady state and during glycine-induced long-term potentiation (LTP) and involves the inhibition of cofilin activation. What is more, CCNY is a positive regulatory subunit of the CDK14/PFTK1 complexes affected by the activation of the Wnt signaling pathway in the G2/M phase by recruiting CDK14/PFTK1 to the plasma membrane and promoting phosphorylation of LRP6. The expression of CCNY has been significantly mentioned within the cell migration and invasion activity both in vivo and in vitro. The aim of this review is evaluation of the expression of CCNY in the physiology processes and compare the expression of this protein in cancer cells, taking into account the impact of the level of expression on tumor progression.
Assuntos
Quinases Ciclina-Dependentes , Testículo , Animais , Humanos , Masculino , Testículo/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Núcleo Celular/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Fosforilação , Mamíferos/metabolismoRESUMO
Many psychiatric disorders accompany deficits in cognitive functions and synaptic plasticity, and abnormal lipid modifications of neuronal proteins are associated with their pathophysiology. Lipid modifications, including palmitoylation and myristoylation, play crucial roles in the subcellular localization and trafficking of proteins. Cyclin Y (CCNY), enriched in the postsynaptic compartment, acts as an inhibitory modulator of functional and structural long-term potentiation (LTP) in the hippocampal neurons. However, cellular and molecular mechanisms underlying CCNY-mediated inhibitory functions in the synapse remain largely unknown. Here, we report that myristoylation located CCNY to the trans-Golgi network (TGN), and subsequent palmitoylation directed the myristoylated CCNY from the TGN to the synaptic cell surface. This myristoylation-dependent palmitoylation of CCNY was required for the inhibitory role of CCNY in excitatory synaptic transmission, activity-induced dynamics of AMPA receptors and PSD-95, LTP, and spatial learning. Furthermore, spatial learning significantly reduced palmitoyl- and myristoyl-CCNY levels, indicating that spatial learning lowers the synaptic abundance of CCNY. Our findings provide mechanistic insight into how CCNY is clustered adjacent to postsynaptic sites where it could play its inhibitory roles in synaptic plasticity and spatial learning.
Assuntos
Potenciação de Longa Duração , Receptores de AMPA , Ciclinas/metabolismo , Hipocampo/fisiologia , Humanos , Lipídeos , Lipoilação/fisiologia , Potenciação de Longa Duração/fisiologia , Plasticidade Neuronal/fisiologia , Receptores de AMPA/metabolismo , Aprendizagem Espacial , Sinapses/metabolismoRESUMO
Cyclin Y (CCNY) is a novel cyclin and highly conserved in metazoan species. Previous studies from our and other laboratory indicate that CCNY play a crucial role in tumor progression. There are two CCNY isoform which has different subcellular distributions, with cytoplasmic isoform (CCNYc) and membrane distribution isoform (CCNYm). However, the expression and function of CCNY isoforms is still unclear. We firstly found CCNYc was expressed in natural lung cancer tissue and cells through the subcellular distribution. Co-IP and immunofluorescence showed that both CCNYm and CCNYc could interact with PFTK1. Further studies illustrated that CCNYc but not CCNYm enhanced cell migration and invasion activity both in vivo and vitro. The function of CCNYc could be inhibited by suppression of PFTK1 expression. In addition, our data indicated that tropomyosin 4 (TPM4), a kind of actin-binding proteins, was down-regulated by suppression of CCNY. F-actin assembly could be controlled by CCNYc as well as PFTK1 and TPM4. As a result, CCNY was mainly expressed in lung cancer. CCNYc could promote cell motility and invasion. It indicated that CCNYc/PFTK1 complex could promote cell metastasis by regulating the formation of F-actin via TPM4.
RESUMO
While positive regulators of hippocampal long-term potentiation (LTP) have extensively been investigated, relatively little is known about the inhibitory regulators of LTP. We previously reported that Cyclin Y (CCNY), a member of cyclin family generally known to function in proliferating cells, is a novel postsynaptic protein that serves as a negative regulator of functional LTP. However, whether CCNY plays a role in structural LTP, which is mechanistically linked to functional LTP, and which mechanisms are involved in the CCNY-mediated suppression of LTP at the molecular level remain elusive. Here, we report that CCNY negatively regulates the plasticity-induced changes in spine morphology through the control of actin dynamics. We observed that CCNY directly binds to filamentous actin and interferes with LTP-induced actin polymerization as well as depolymerization by blocking the activation of cofilin, an actin-depolymerizing factor, thus resulting in less plastic spines and the impairment of structural LTP. These data suggest that CCNY acts as an inhibitory regulator for both structural and functional LTP by modulating actin dynamics through the cofilin-actin pathway. Collectively, our findings provide a mechanistic insight into the inhibitory modulation of hippocampal LTP by CCNY, highlighting a novel function of a cyclin family protein in non-proliferating neuronal cells.
Assuntos
Plasticidade Neuronal , Fatores de Despolimerização de Actina , Actinas , Ciclinas , Proteínas dos Microfilamentos , SinapsesRESUMO
AMPK is one of the main regulators of energy homeostasis in the cell, achieving this role in part by upregulating autophagy in times of nutrient deprivation. Previous reports have described several AMPK substrates involved in autophagy regulation; however, there are still undiscovered AMPK downstream effectors that could play an important role in autophagy. In a new article, Dohmen et al. discovered that the CCNY-CDK16 complex is a novel AMPK substrate involved in autophagy activation.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Animais , Autofagia/fisiologia , Metabolismo Energético , Humanos , Fosforilação , Serina-Treonina Quinases TOR/metabolismoRESUMO
Anaplastic lymphoma kinase (ALK) rearrangement, one of the common oncogene rearrangements in the mutational history of lung adenocarcinoma, occurs in approximately 5% of non-small cell lung cancer (NSCLC) patients who could be effectively treated with ALK tyrosine kinase inhibitors (TKIs). The earlier phase III PROFILE 1014 study has shown that crizotinib, a first-generation ALK-TKI, significantly improved progression-free survival (PFS) compared with platinum-based chemotherapy in patients with previously untreated advanced ALK-positive NSCLC. Thus, clinicians must screen potential candidates for this driver alteration to guide ALK inhibitor therapy with a molecular testing platform capable of capturing all ALK fusions. Echinoderm microtubule-associated proteins, including the EML4 gene, are the most common ALK rearrangement partner. With the widespread use of the next-generation sequencing (NGS) techniques, which could approach enable the simultaneous screening of multiple genetic alterations, increasingly ALK rearrangement partners have been documented. However, the concurrent two ALK rearrangements within the same patient have rarely previously been reported. Here, we describe a novel CCNY-ALK (C1:A20) and ATIC-ALK (A7:A20), coexisting in the same case with poorly differentiated NSCLC and providing evidence of its sensitivity to ALK inhibitors. The newly identified rearrangement partners can be added to the list of ALK rearrangements that occurred in ALK-positive NSCLC, as it could lead to prolonged disease control. Also, while different ALK rearrangement variants might bring differing clinical outcomes, we discuss the impact of the co-mutations of these two ALK rearrangements on the sensitivity to ALK inhibitors. However, the impact of co-mutations on the pathogenesis of NSCLC should be further studied to supply more theoretical insight that co-mutations present for personalized anti-cancer therapy.
RESUMO
Cyclin-dependent kinases (CDKs) are perceived as the engine that drives cell cycle progression whereas cyclins are considered to be the gears that are changed to aid the transition between cycle phases. CDK14 is a cdc2-related serine/threonine protein kinase and plays an important role in normal cell cycle progression. However, its distribution and function in the central nervous system (CNS) lesion remain unclear. In this study, we mainly investigated the protein expression and cellular localization of CDK14 during spinal cord injury (SCI). Western blot analysis revealed that the expression of CDK14 was gradually increased and reached a peak at 3 days after SCI. The expression of CDK14 was further analyzed by immunohistochemistry. Double immunofluorescence staining showed that CDK14 was co-expressed prominent in astrocytes. Co-localization CDK14/proliferating cell nuclear antigen (PCNA) were detected in glial cells. cyclin Y, which can interact with CDK14, was detected that had same expression trend was consistent with CDK14 Western blot results in SCI. Double-immunofluorescence staining indicated that CDK14 co-expressed with cyclin Y in some cells. Co-immunoprecipitation had been showed that CDK14 could interact with cyclin Y after acute SCI. Taken together, these data suggested that both CDK14 and cyclin Y may play important roles in spinal cord pathophysiology.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Gliose , Traumatismos da Medula Espinal/metabolismo , Animais , Células Cultivadas , Masculino , Neuroglia/metabolismo , Ligação Proteica , Transporte Proteico , Ratos , Ratos Sprague-DawleyRESUMO
In this study, DNA arrays have been employed to monitor gene expression patterns in testis of mice exposed to tobacco smoke for 24 weeks and compared to control animals. The results of the analysis revealed significant changes in expression of several genes that may have a role in spermatogenesis. Cdk14 was chosen for further characterization because of a suggested role in the testis and in regulation of Wnt signaling. RT-PCR analysis confirmed down regulation of Cdk14 in mice exposed to cigarette smoke (CS). Cdk14 is expressed in all testicular cells; spermatogonia- and Sertoli-derived cell lines treated with cigarette smoke extract (CSE) in vitro showed down-regulation of CDK14 mRNA and protein levels as well as down-regulation of ß-catenin levels. CS-induced down-regulation of CDK14 mRNA and protein levels was also observed in several lung epithelium-derived cell lines including primary normal human bronchial epithelial cells (NHBE), suggesting that the effect is not restricted to the testis. Similar to testicular cells, CS-induced down-regulation of CDK14 in lung cells correlated with decreased levels of ß-catenin, a finding suggesting impaired Wnt signaling. In the lungs, CDK14 was localized to the alveolar and bronchial epithelium.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Proteínas Quinases/metabolismo , Fumaça/efeitos adversos , Fumar/efeitos adversos , Testículo/efeitos dos fármacos , Animais , Quinases Ciclina-Dependentes/genética , Regulação para Baixo , Perfilação da Expressão Gênica , Humanos , Exposição por Inalação/efeitos adversos , Masculino , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Quinases/genética , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/enzimologia , Fumar/genética , Fumar/metabolismo , Espermatogônias/efeitos dos fármacos , Espermatogônias/enzimologia , Testículo/enzimologia , Fatores de Tempo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Cyclin Y, a membrane associated cyclin, is capable of binding and activating CDK14. Here we report that human cyclin Y (CCNY) is a phosphoprotein in vivo and that phosphorylation of CCNY by CDK14 triggers its ubiquitination and degradation. Inactivation of either CDK14 or Cul1 results in accumulation of CCNY. An in vivo and in vitro mapping of CCNY phosphorylation sites by mass spectrometry revealed that the flanking regions of the conserved cyclin box are heavily phosphorylated. Phosphorylation of CCNY at Serines 71 and 73 creates a putative phospho-degron that controls its association with an SCF complex. Mutation of serine to alanine at these two sites stabilized CCNY and enhanced the activity of CCNY/CDK14 on phosphorylation of LRP6. Our results provide insight into autoregulation of the cyclin Y/CDK14 pair in CDK14 activation and cyclin Y turnover which is a process that is involved in membrane proximal signaling.