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1.
Sci Rep ; 9(1): 13381, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31527692

RESUMO

Very rare polymorphisms in the human VRK1 (vaccinia-related kinase 1) gene have been identified in complex neuromotor phenotypes associated to spinal muscular atrophy (SMA), pontocerebellar hypoplasia (PCH), microcephaly, amyotrophic lateral sclerosis (ALS) and distal motor neuron dysfunctions. The mechanisms by which these VRK1 variant proteins contribute to the pathogenesis of these neurological syndromes are unknown. The syndromes are manifested when both of these rare VRK1 polymorphic alleles are implicated, either in homozygosis or compound heterozygosis. In this report, to identify the common underlying pathogenic mechanism of VRK1 polymorphisms, we have studied all human VRK1 variants identified in these neurological phenotypes from a biochemical point of view by molecular modeling, protein stability and kinase activity assays. Molecular modelling predicted that VRK1 variant proteins are either unstable or have an altered kinase activity. The stability and kinase activity of VRK1 pathogenic variants detected two groups. One composed by variants with a reduced protein stability: R133C, R358X, L195V, G135R and R321C. The other group includes VRK1variants with a reduced kinase activity tested on several substrates: histones H3 and H2AX, p53, c-Jun, coilin and 53BP1, a DNA repair protein. VRK1 variants with reduced kinase activity are H119R, R133C, G135R, V236M, R321C and R358X. The common underlying effect of VRK1 pathogenic variants with reduced protein stability or kinase activity is a functional insufficiency of VRK1 in patients with neuromotor developmental syndromes. The G135 variant cause a defective formation of 53BP1 foci in response to DNA damage, and loss Cajal bodies assembled on coilin.

2.
J Exp Clin Cancer Res ; 38(1): 203, 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101118

RESUMO

BACKGROUND: The VRK1 chromatin kinase regulates the organization of locally altered chromatin induced by DNA damage. The combination of ionizing radiation with inhibitors of DNA damage responses increases the accumulation of DNA damage in cancer cells, which facilitates their antitumor effect, a process regulated by VRK1. METHODS: Tumor cell lines with different genetic backgrounds were treated with olaparib to determine their effect on the activation of DNA repair pathways induced by ionizing radiation. The effect of combining olaparib with depletion of the chromatin kinase VRK1 was studied in the context of double-strand breaks repair pathway after treatment with ionizing radiation. The initiation and progression of DDR were studied by specific histone acetylation, as a marker of local chromatin relaxation, and formation of γH2AX and 53BP1 foci. RESULTS: In this work, we have studied the effect that VRK1 by itself or in collaboration with olaparib, an inhibitor of PARP, has on the DNA oxidative damage induced by irradiation in order to identify its potential as a new drug target. The combination of olaparib and ionizing radiation increases DNA damage permitting a significant reduction of their respective doses to achieve a similar amount of DNA damage detected by γH2AX and 53BP1 foci. Different treatment combinations of olaparib and ionizing radiation permitted to reach the maximum level of DNA damage at lower doses of both treatments. Furthermore, we have studied the effect that depletion of the VRK1 chromatin kinase, a regulator of DDR, has on this response. VRK1 knockdown impaired all steps in the DDR induced by these treatments, which were detected by a reduction of sequential markers such as H4K16 ac, γH2AX, NBS1 and 53BP1. Moreover, this effect of VRK1 is independent of TP53 or ATM, two genes frequently mutated in cancer. CONCLUSION: The protective DNA damage response induced by ionizing radiation is impaired by the combination of olaparib with depletion of VRK1, and can be used to reduce doses of radiation and their associated toxicity. Proteins implicated in DNA damage responses are suitable targets for development of new therapeutic strategies and their combination can be an alternative form of synthetic lethality.


Assuntos
Histonas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Células A549 , Acetilação/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/genética , Cromatina/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/radioterapia , Fosforilação/efeitos dos fármacos , Ftalazinas/farmacologia , Piperazinas/farmacologia , Interferência de RNA , Radiação Ionizante , Mutações Sintéticas Letais/genética
3.
Cell Mol Life Sci ; 75(13): 2375-2388, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29679095

RESUMO

DNA damage causes a local distortion of chromatin that triggers the sequential processes that participate in specific DNA repair mechanisms. This initiation of the repair response requires the involvement of a protein whose activity can be regulated by histones. Kinases are candidates to regulate and coordinate the connection between a locally altered chromatin and the response initiating signals that lead to identification of the type of lesion and the sequential steps required in specific DNA damage responses (DDR). This initiating kinase must be located in chromatin, and be activated independently of the type of DNA damage. We review the contribution of the Ser-Thr vaccinia-related kinase 1 (VRK1) chromatin kinase as a new player in the signaling of DNA damage responses, at chromatin and cellular levels, and its potential as a new therapeutic target in oncology. VRK1 is involved in the regulation of histone modifications, such as histone phosphorylation and acetylation, and in the formation of γH2AX, NBS1 and 53BP1 foci induced in DDR. Induction of DNA damage by chemotherapy or radiation is a mainstay of cancer treatment. Therefore, novel treatments can be targeted to proteins implicated in the regulation of DDR, rather than by directly causing DNA damage.


Assuntos
Cromatina/metabolismo , Dano ao DNA/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Histonas/metabolismo , Humanos
4.
Cell Mol Life Sci ; 75(14): 2591-2611, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29340707

RESUMO

Regulation of cell division requires the integration of signals implicated in chromatin reorganization and coordination of its sequential changes in mitosis. Vaccinia-related kinase 1 (VRK1) and Aurora B (AURKB) are two nuclear kinases involved in different steps of cell division. We have studied whether there is any functional connection between these two nuclear kinases, which phosphorylate histone H3 in Thr3 and Ser10, respectively. VRK1 and AURKB are able to form a stable protein complex, which represents only a minor subpopulation of each kinase within the cell and is detected following nocodazole release. Each kinase is able to inhibit the kinase activity of the other kinase, as well as inhibit their specific phosphorylation of histone H3. In locations where the two kinases interact, there is a different pattern of histone modifications, indicating that there is a local difference in chromatin during mitosis because of the local complexes formed by these kinases and their asymmetric intracellular distribution. Depletion of VRK1 downregulates the gene expression of BIRC5 (survivin) that recognizes H3-T3ph, both are dependent on the activity of VRK1, and is recovered with kinase active murine VRK1, but not with a kinase-dead protein. The H3-Thr3ph-survivin complex is required for AURB recruitment, and their loss prevents the localization of ACA and AURKB in centromeres. The cross inhibition of the kinases at the end of mitosis might facilitate the formation of daughter cells. A sequential role for VRK1, AURKB, and haspin in the progression of mitosis is proposed.


Assuntos
Aurora Quinase B/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Células Cultivadas , Ativação Enzimática , Retroalimentação Fisiológica/fisiologia , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional/fisiologia , Receptor Cross-Talk/fisiologia
5.
Cell Signal ; 33: 49-58, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28189587

RESUMO

The activation of p53 in response to different types of cellular stress induces several protective reactions including cell cycle arrest, senescence or cell death. These protective effects are a consequence of the activation of p53 by specific phosphorylation performed by several kinases. The reversion of the cell cycle arrest, induced by p53, is a consequence of the phosphorylated and activated p53, which triggers its own downregulation and that of its positive regulators. The different down-regulatory processes have a sequential and temporal order of events. The mechanisms implicated in p53 down-regulation include phosphatases, deacetylases, and protein degradation by the proteasome or autophagy, which also affect different p53 protein targets and functions. The necessary first step is the dephosphorylation of p53 to make it available for interaction with mdm2 ubiquitin-ligase, which requires the activation of phosphatases targeting both p53 and p53-activating kinases. In addition, deacetylation of p53 is required to make lysine residues accessible to ubiquitin ligases. The combined action of these downregulatory mechanisms brings p53 protein back to its basal levels, and cell cycle progression can resume if cells have overcome the stress or damage situation. The specific targeting of these down-regulatory mechanisms can be exploited for therapeutic purposes in cancers harbouring wild-type p53.


Assuntos
Ciclo Celular , Estresse Fisiológico , Proteína Supressora de Tumor p53/metabolismo , Animais , Autofagia , Humanos , Fosforilação , Proteólise
7.
Sci Rep ; 6: 28532, 2016 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-27334688

RESUMO

Sox2 is a pluripotency transcription factor that as an oncogene can also regulate cell proliferation. Therefore, genes implicated in several different aspects of cell proliferation, such as the VRK1 chromatin-kinase, are candidates to be targets of Sox2. Sox 2 and VRK1 colocalize in nuclei of proliferating cells forming a stable complex. Sox2 knockdown abrogates VRK1 gene expression. Depletion of either Sox2 or VRK1 caused a reduction of cell proliferation. Sox2 up-regulates VRK1 expression and both proteins cooperate in the activation of CCND1. The accumulation of VRK1 protein downregulates SOX2 expression and both proteins are lost in terminally differentiated cells. Induction of neural differentiation with retinoic acid resulted in downregulation of Sox2 and VRK1 that inversely correlated with the expression of differentiation markers such as N-cadherin, Pax6, mH2A1.2 and mH2A2. Differentiation-associated macro histones mH2A1.2and mH2A2 inhibit CCND1 and VRK1 expression and also block the activation of the VRK1 promoter by Sox2. VRK1 is a downstream target of Sox2 and both form an autoregulatory loop in epithelial cell differentiation.


Assuntos
Ciclo Celular/genética , Diferenciação Celular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Oncogenes/genética , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição SOXB1/genética , Biomarcadores/metabolismo , Caderinas/genética , Carcinogênese/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Ciclina D1/genética , Regulação para Baixo/genética , Epitélio/metabolismo , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Fator de Transcrição PAX6/genética , Regiões Promotoras Genéticas/genética , Regulação para Cima/genética
8.
Biochim Biophys Acta ; 1863(4): 760-9, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26869104

RESUMO

NBS1 is an early component in DNA-Damage Response (DDR) that participates in the initiation of the responses aiming to repair double-strand breaks caused by different mechanisms. Early steps in DDR have to react to local alterations in chromatin that are induced by DNA damage. NBS1 participates in the early detection of DNA damage and functions as a platform for the recruitment and assembly of components that are sequentially required for the repair process. In this work we have studied whether the VRK1 chromatin kinase can affect the activation of NBS1 in response to DNA damage induced by ionizing radiation. VRK1 is forming a basal preassembled complex with NBS1 in non-damaged cells. Knockdown of VRK1 resulted in the loss of NBS1 foci induced by ionizing radiation, an effect that was also detected in cell-cycle arrested cells and in ATM (-/-) cells. The phosphorylation of NBS1 in Ser343 by VRK1 is induced by either doxorubicin or IR in ATM (-/-) cells. Phosphorylated NBS1 is also complexed with VRK1. NBS1 phosphorylation by VRK1 cooperates with ATM. This phosphorylation of NBS1 by VRK1 contributes to the stability of NBS1 in ATM (-/-) cells, and the consequence of its loss can be prevented by treatment with the MG132 proteasome inhibitor of RNF8. We conclude that VRK1 regulation of NBS1 contributes to the stability of the repair complex and permits the sequential steps in DDR.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteólise , Ubiquitinação , Proteínas Mutadas de Ataxia Telangiectasia/genética , Dano ao DNA/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Células Tumorais Cultivadas
9.
Sci Rep ; 5: 10543, 2015 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-26068304

RESUMO

Cajal bodies (CBs) are nuclear organelles associated with ribonucleoprotein functions and RNA maturation. CBs are assembled on coilin, its main scaffold protein, in a cell cycle dependent manner. The Ser-Thr VRK1 (vaccinia-related kinase 1) kinase, whose activity is also cell cycle regulated, interacts with and phosphorylates coilin regulating assembly of CBs. Coilin phosphorylation is not necessary for its interaction with VRK1, but it occurs in mitosis and regulates coilin stability. Knockdown of VRK1 or VRK1 inactivation by serum deprivation causes a loss of coilin phosphorylation in Ser184 and of CBs formation, which are rescued with an active VRK1, but not by kinase-dead VRK1. The phosphorylation of coilin in Ser184 occurs during mitosis before assembly of CBs. Loss of coilin phosphorylation results in disintegration of CBs, and of coilin degradation that is prevented by proteasome inhibitors. After depletion of VRK1, coilin is ubiquitinated in nuclei, which is partly mediated by mdm2, but its proteasomal degradation occurs in cytosol and is prevented by blocking its nuclear export. We conclude that VRK1 is a novel regulator of CBs dynamics and stability in cell cycle by protecting coilin from ubiquitination and degradation in the proteasome, and propose a model of CB dynamics.


Assuntos
Ciclo Celular/fisiologia , Corpos Enovelados/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Corpos Enovelados/genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células MCF-7 , Proteínas Nucleares/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Serina-Treonina Quinases/genética
10.
Epigenetics ; 10(5): 373-83, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25923214

RESUMO

All types of DNA damage cause a local alteration and relaxation of chromatin structure. Sensing and reacting to this initial chromatin alteration is a necessary trigger for any type of DNA damage response (DDR). In this context, chromatin kinases are likely candidates to participate in detection and reaction to a locally altered chromatin as a consequence of DNA damage and, thus, initiate the appropriate cellular response. In this work, we demonstrate that VRK1 is a nucleosomal chromatin kinase and that its depletion causes loss of histones H3 and H4 acetylation, which are required for chromatin relaxation, both in basal conditions and after DNA damage, independently of ATM. Moreover, VRK1 directly and stably interacts with histones H2AX and H3 in basal conditions. In response to DNA damage induced by ionizing radiation, histone H2AX is phosphorylated in Ser139 by VRK1. The phosphorylation of H2AX and the formation of γH2AX foci induced by ionizing radiation (IR), are prevented by VRK1 depletion and are rescued by kinase-active, but not kinase-dead, VRK1. In conclusion, we found that VRK1 is a novel chromatin component that reacts to its alterations and participates very early in DDR, functioning by itself or in cooperation with ATM.


Assuntos
Acetilação/efeitos da radiação , Cromatina/metabolismo , Reparo do DNA/efeitos da radiação , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Acetilação/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Linhagem Celular , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Humanos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Inibidores de Proteínas Quinases/farmacologia , Pironas/farmacologia , Radiação Ionizante , Serina/metabolismo
11.
J Neurosci ; 35(3): 936-42, 2015 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-25609612

RESUMO

Spinal muscular atrophy with pontocerebellar hypoplasia (SMA-PCH) is an infantile SMA variant with additional manifestations, particularly severe microcephaly. We previously identified a nonsense mutation in Vaccinia-related kinase 1 (VRK1), R358X, as a cause of SMA-PCH. VRK1-R358X is a rare founder mutation in Ashkenazi Jews, and additional mutations in patients of different origins have recently been identified. VRK1 is a nuclear serine/threonine protein kinase known to play multiple roles in cellular proliferation, cell cycle regulation, and carcinogenesis. However, VRK1 was not known to have neuronal functions before its identification as a gene mutated in SMA-PCH. Here we show that VRK1-R358X homozygosity results in lack of VRK1 protein, and demonstrate a role for VRK1 in neuronal migration and neuronal stem cell proliferation. Using shRNA in utero electroporation in mice, we show that Vrk1 knockdown significantly impairs cortical neuronal migration, and affects the cell cycle of neuronal progenitors. Expression of wild-type human VRK1 rescues both proliferation and migration phenotypes. However, kinase-dead human VRK1 rescues only the migration impairment, suggesting the role of VRK1 in neuronal migration is partly noncatalytic. Furthermore, we found that VRK1 deficiency in human and mouse leads to downregulation of amyloid-ß precursor protein (APP), a known neuronal migration gene. APP overexpression rescues the phenotype caused by Vrk1 knockdown, suggesting that VRK1 affects neuronal migration through an APP-dependent mechanism.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Movimento Celular/genética , Cerebelo/anormalidades , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Malformações do Sistema Nervoso/metabolismo , Neurônios/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Atrofias Musculares Espinais da Infância/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Doenças Cerebelares/genética , Doenças Cerebelares/metabolismo , Doenças Cerebelares/patologia , Cerebelo/metabolismo , Cerebelo/patologia , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/metabolismo , Deficiências do Desenvolvimento/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Imagem por Ressonância Magnética , Camundongos , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/patologia , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/genética , Atrofias Musculares Espinais da Infância/genética , Atrofias Musculares Espinais da Infância/patologia
12.
Oncotarget ; 5(7): 1770-8, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24731990

RESUMO

Vaccinia-related kinase 1 (VRK1) belongs to a group of sixteen kinases associated to a poorer prognosis in human breast carcinomas, particularly in estrogen receptor positive cases based on gene expression arrays. In this work we have studied the potential molecular mechanism by which the VRK1 protein can contribute to a poorer prognosis in this disease. For this aim it was first analyzed by immunohistochemistry the VRK1 protein level in normal breast and in one hundred and thirty six cases of human breast cancer. The effect of VRK1 to protect against DNA damage was determined by studying the effect of its knockdown on the formation of DNA repair foci assembled on 53BP1 in response to treatment with ionizing radiation or doxorubicin in two breast cancer cell lines. VRK1 protein was detected in normal breast and in breast carcinomas at high levels in ER and PR positive tumors. VRK1 protein level was significantly lower in ERBB2 positive cases. Next, to identify a mechanism that can link VRK1 to poorer prognosis, VRK1 was knocked-down in two breast cancer cell lines that were treated with ionizing radiation or doxorubicin, both inducing DNA damage. Loss of VRK1 resulted in reduced formation of DNA-damage repair foci complexes assembled on the 53BP1 scaffold protein, and this effect was independent of damaging agent or cell type. This observation is consistent with detection of high VRK1 protein levels in ER and PR positive breast cancers. We conclude that VRK1 can contribute to make these tumors more resistant to DNA damage-based therapies, such as ionizing radiation or doxorubicin, which is consistent with its association to a poor prognosis in ER positive breast cancer. VRK1 is potential target kinase for development of new specific inhibitors which can facilitate sensitization to other treatments in combination therapies; or alternatively be used as a new cancer drugs.


Assuntos
Neoplasias da Mama/enzimologia , Carcinoma/enzimologia , Dano ao DNA , Reparo do DNA , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/metabolismo , Mama/enzimologia , Neoplasias da Mama/química , Carcinoma/química , Linhagem Celular Tumoral , Reparo do DNA/genética , Doxorrubicina , Feminino , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Prognóstico , Proteínas Serina-Treonina Quinases/genética , Radiação Ionizante , Receptor ErbB-2/análise , Receptores Estrogênicos/análise , Receptores de Progesterona/análise , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
13.
FEBS Lett ; 588(5): 692-700, 2014 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-24492002

RESUMO

DNA damage immediate cellular response requires the activation of p53 by kinases. We found that p53 forms a basal stable complex with VRK1, a Ser-Thr kinase that responds to UV-induced DNA damage by specifically phosphorylating p53. This interaction takes place through the p53 DNA binding domain, and frequent DNA-contact mutants of p53, such as R273H, R248H or R280K, do not disrupt the complex. UV-induced DNA damage activates VRK1, and is accompanied by phosphorylation of p53 at Thr-18 before it accumulates. We propose that the VRK1-p53 basal complex is an early-warning system for immediate cellular responses to DNA damage.


Assuntos
Dano ao DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Reparo do DNA , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética , Raios Ultravioleta
14.
BMC Clin Pathol ; 13(1): 23, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-24079673

RESUMO

BACKGROUND: Malignant astrocytomas are the most common primary brain tumors and one of the most lethal among human cancers despite optimal treatment. Therefore, the characterization of molecular alterations underlying the aggressive behavior of these tumors and the identification of new markers are thus an important step towards a better patient stratification and management. METHODS AND RESULTS: VRK1 and VRK2 (Vaccinia-related kinase-1, -2) expression, as well as proliferation markers, were determined in a tissue microarray containing 105 primary astrocytoma biopsies. Kaplan Meier and Cox models were used to find clinical and/or molecular parameters related to overall survival. The effects of VRK protein levels on proliferation were determined in astrocytoma cell lines. High levels of both protein kinases, VRK1 or VRK2, correlated with proliferation markers, p63 or ki67. There was no correlation with p53, reflecting the disruption of the VRK-p53-DRAM autoregulatory loop as a consequence of p53 mutations. High VRK2 protein levels identified a subgroup of astrocytomas that had a significant improvement in survival. The potential effect of VRK2 was studied by analyzing the growth characteristics of astrocytoma cell lines with different EGFR/VRK2 protein ratios. CONCLUSION: High levels of VRK2 resulted in a lower growth rate suggesting these cells are more indolent. In high-grade astrocytomas, VRK2 expression constitutes a good prognostic marker for patient survival.

15.
J Med Virol ; 85(12): 2119-27, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24009184

RESUMO

John Cunningham virus (JCV) infects chronically human populations worldwide and probably might confer a higher risk for colorectal cancer (CRC). The prevalence of JCV DNA has been determined in normal colon mucosa and compared it with different degrees of colorectal lesions, as well as viral presence in the urine of the individuals in the study. JCV DNA was detected by a nested-PCR approach targeting the JCV small-t antigen in 100 healthy controls, and 100 patients undergoing biopsy for diagnosis of colorectal disorders. JCV DNA was detected in 40% of normal mucosa from controls and patients. JCV DNA presence in urine was also similar in controls and patients (37-41% range). JCV DNA detection in normal mucosa and urine reflects the infected population in Portugal. However, in cases with colorectal tumor lesions, JCV DNA was detected in 90% cases, independently of histological type or grade, and this increase was significantly higher with respect to its normal surrounding mucosa. This higher detection of JCV DNA in tumor lesions with respect to its own normal mucosa suggested that a selection for virus containing cells has occurred at some early stage in tumor initiation or progression. JCV may have a specific tropism for colon epithelial cells with some inherent predisposition that makes them more prone to oncogenic transformation, with selection of infected cells. Several p53 polymorphisms in intron 2, common to both groups, were more frequently detected in colorectal pathology cases. A novel p53 mutation in the 3' untranslated region (exon 11) was identified in 10 patients.


Assuntos
Adenocarcinoma/virologia , Adenoma/virologia , Pólipos do Colo/virologia , Neoplasias Colorretais/virologia , Mucosa Intestinal/virologia , Vírus JC/genética , Infecções por Polyomavirus/virologia , Infecções Tumorais por Vírus/virologia , Adenocarcinoma/genética , Adenoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Pólipos do Colo/genética , Neoplasias Colorretais/genética , Análise Mutacional de DNA , DNA Viral/genética , DNA Viral/urina , Feminino , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Razão de Chances , Infecções por Polyomavirus/genética , Proteína Supressora de Tumor p53/genética , Infecções Tumorais por Vírus/genética , Adulto Jovem
16.
J Biol Inorg Chem ; 18(4): 473-82, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23483238

RESUMO

The human vaccinia-related kinase (VRK) proteins VRK1 and VRK2 regulate different processes, such as the cell cycle, DNA damage response, and signaling by mitogen-activated protein kinases in response to growth factors or cellular stress. Alterations in expression levels of these Ser-Thr kinases are associated with cancer and neurodegenerative diseases. These functions suggest that they might also be targets of toxic metals, and thus contribute to the pathogenic effects associated with metal intoxication. VRK1 is inhibited by cadmium, copper, and mercury, and VRK2 is more sensitive to cadmium and much less sensitive to copper and mercury. Both kinases are insensitive to lead and cobalt. VRK1 is in general more sensitive than VRK2 in the low micromolar range. This inhibitory effect induced by these metals was detected in an autophosphorylation assay, as well as in phosphorylation assays using p53 and histone H3 as substrates. The accumulation of these three metals in cells can contribute, by inhibition of VRKs, to their toxic pathogenic effects, particularly their neurological manifestations. In this context copper has not generally been associated with any intoxication syndrome, except Wilson's syndrome, but it might be implicated in some alterations with which it has not yet been associated.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metais Pesados/toxicidade , Proteínas Serina-Treonina Quinases/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração Inibidora 50
17.
J Biol Chem ; 287(51): 42739-50, 2012 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-23105117

RESUMO

Human VRK2 (vaccinia-related kinase 2), a kinase that emerged late in evolution, affects different signaling pathways, and some carcinomas express high levels of VRK2. Invasion by cancer cells has been associated with NFAT1 (nuclear factor of activated T cells) activation and expression of the COX-2 (cyclooxygenase 2) gene. We hypothesized that VRK proteins might play a regulatory role in NFAT1 activation in tumor cells. We demonstrate that VRK2 directly interacts and phosphorylates NFAT1 in Ser-32 within its N-terminal transactivation domain. VRK2 increases NFAT1-dependent transcription by phosphorylation, and this effect is only detected following cell phorbol 12-myristate 13-acetate and ionomycin stimulation and calcineurin activation. This NFAT1 hyperactivation by VRK2 increases COX-2 gene expression through the proximal NFAT1 binding site in the COX-2 gene promoter. Furthermore, VRK2A down-regulation by RNA interference reduces COX-2 expression at transcriptional and protein levels. Therefore, VRK2 down-regulation reduces cell invasion by tumor cells, such as MDA-MB-231 and MDA-MB-435, upon stimulation with phorbol 12-myristate 13-acetate plus ionomycin. These findings identify the first reported target and function of human VRK2 as an active kinase playing a role in regulation of cancer cell invasion through the NFAT pathway and COX-2 expression.


Assuntos
Ciclo-Oxigenase 2/genética , Fatores de Transcrição NFATC/metabolismo , Neoplasias/enzimologia , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Proteínas de Ligação a DNA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ionomicina/farmacologia , Isoenzimas/metabolismo , Camundongos , Modelos Biológicos , Proteínas Musculares/metabolismo , Fatores de Transcrição NFATC/química , Fatores de Transcrição NFATC/genética , Invasividade Neoplásica , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genética , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Genética/efeitos dos fármacos
18.
Cell Mol Life Sci ; 69(22): 3881-93, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22752157

RESUMO

The spatial and temporal regulation of intracellular signaling is determined by the spatial and temporal organization of complexes assembled on scaffold proteins, which can be modulated by their interactions with additional proteins as well as subcellular localization. The scaffold KSR1 protein interacts with MAPK forming a complex that conveys a differential signaling in response to growth factors. The aim of this work is to determine the unknown mechanism by which VRK2A downregulates MAPK signaling. We have characterized the multiprotein complex formed by KSR1 and the Ser-Thr kinase VRK2A. VRK2A is a protein bound to the endoplasmic reticulum (ER) and retains a fraction of KSR1 complexes on the surface of this organelle. Both proteins, VRK2A and KSR1, directly interact by their respective C-terminal regions. In addition, MEK1 is also incorporated in the basal complex. MEK1 independently interacts with the CA5 region of KSR1 and with the N-terminus of VRK2A. Thus, VRK2A can form a high molecular size (600-1,000 kDa) stable complex with both MEK1 and KSR1. Knockdown of VRK2A resulted in disassembly of these high molecular size complexes. Overexpression of VRK2A increased the amount of KSR1 in the particulate fraction and prevented the incorporation of ERK1/2 into the complex after stimulation with EGF. Neither VRK2A nor KSR1 interact with the VHR, MKP1, MKP2, or MKP3 phosphatases. The KSR1 complex assembled and retained by VRK2A in the ER can have a modulatory effect on the signal mediated by MAPK, thus locally affecting the magnitude of its responses, and can explain differential responses depending on cell type.


Assuntos
Retículo Endoplasmático/metabolismo , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Regulação para Baixo , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Complexos Multiproteicos/metabolismo , Ligação Proteica , Proteínas Quinases/genética , Estrutura Quaternária de Proteína , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , RNA Interferente Pequeno
19.
J Biol Chem ; 287(28): 23757-68, 2012 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-22621922

RESUMO

Cellular responses to DNA damage require the formation of protein complexes in a highly organized fashion. The complete molecular components that participate in the sequential signaling response to DNA damage remain unknown. Here we demonstrate that vaccinia-related kinase 1 (VRK1) in resting cells plays an important role in the formation of ionizing radiation-induced foci that assemble on the 53BP1 scaffold protein during the DNA damage response. The kinase VRK1 is activated by DNA double strand breaks induced by ionizing radiation (IR) and specifically phosphorylates 53BP1 in serum-starved cells. VRK1 knockdown resulted in the defective formation of 53BP1 foci in response to IR both in number and size. This observed effect on 53BP1 foci is p53- and ataxia-telangiectasia mutated (ATM)-independent and can be rescued with VRK1 mutants resistant to siRNA. VRK1 knockdown also prevented the activating phosphorylation of ATM, CHK2, and DNA-dependent protein kinase in response to IR. VRK1 activation in response to DNA damage is a novel and early step in the signaling of mammalian DNA damage responses.


Assuntos
Dano ao DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quinase do Ponto de Checagem 2 , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Microscopia Confocal , Mutação , Nucleossomos/enzimologia , Nucleossomos/genética , Nucleossomos/efeitos da radiação , Fosforilação/efeitos da radiação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Radiação Ionizante , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
20.
J Proteomics ; 75(2): 548-60, 2011 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-21920476

RESUMO

Protein phosphorylation by kinases plays a central role in the regulation and coordination of multiple biological processes. In general, knowledge on kinase specificity is restricted to substrates identified in the context of specific cellular responses, but kinases are likely to have multiple additional substrates and be integrated in signaling networks that might be spatially and temporally different, and in which protein complexes and subcellular localization can play an important role. In this report the substrate specificity of atypical human vaccinia-related kinases (VRK1 and VRK2) using a human peptide-array containing 1080 sequences phosphorylated in known signaling pathways has been studied. The two kinases identify a subset of potential peptide targets, all of them result in a consensus sequence composed of at least four basic residues in peptide targets. Linear peptide arrays are therefore a useful approach in the characterization of kinases and substrate identification, which can contribute to delineate the signaling network in which VRK proteins participate. One of these target proteins is coilin; a basic protein located in nuclear Cajal bodies. Coilin is phosphorylated in Ser184 by both VRK1 and VRK2. Coilin colocalizes and interacts with VRK1 in Cajal bodies, but not with the mutant VRK1 (R358X). VRK1 (R358X) is less active than VRK1. Altered regulation of coilin might be implicated in several neurological diseases such as ataxias and spinal muscular atrophies.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Especificidade por Substrato , Transfecção , Vírus Vaccinia/genética
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