Vaccinia-related kinase 1 (VRK1) confers resistance to DNA-damaging agents in human breast cancer by affecting DNA damage response

This is an open-access article distributed under the terms of the Creative Commons Attribution License.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.M. V-C and M. S-G have JAE-CSIC-Fondo Social Europeo predoctoral fellowships. This work was supported by grants from Ministerio de Educación, Ciencia e Innovación (SAF2010-14935 and CSD2007-0017) and Kutxa-Fundación Inbiomed to P.A.L.Peer Reviewe

Differential Inhibitor Sensitivity between Human Kinases VRK1 and VRK2

Human vaccinia-related kinases (VRK1 and VRK2) are atypical active Ser-Thr kinases implicated in control of cell cycle entry, apoptosis and autophagy, and affect signalling by mitogen activated protein kinases (MAPK). The specific structural differences in VRK catalytic sites make them suitable candidates for development of specific inhibitors. In this work we have determined the sensitivity of VRK1 and VRK2 to kinase inhibitors, currently used in biological assays or in preclinical studies, in order to discriminate between the two proteins as well as with respect to the vaccinia virus B1R kinase. Both VRK proteins and vaccinia B1R are poorly inhibited by inhibitors of different types targeting Src, MEK1, B-Raf, JNK, p38, CK1, ATM, CHK1/2 and DNA-PK, and most of them have no effect even at 100 µM. Despite their low sensitivity, some of these inhibitors in the low micromolar range are able to discriminate between VRK1, VRK2 and B1R. VRK1 is more sensitive to staurosporine, RO-31-8220 and TDZD8. VRK2 is more sensitive to roscovitine, RO 31–8220, Cdk1 inhibitor, AZD7762, and IC261. Vaccinia virus B1R is more sensitive to staurosporine, KU55933, and RO 31–8220, but not to IC261. Thus, the three kinases present a different pattern of sensitivity to kinase inhibitors. This differential response to known inhibitors can provide a structural framework for VRK1 or VRK2 specific inhibitors with low or no cross-inhibition. The development of highly specific VRK1 inhibitors might be of potential clinical use in those cancers where these kinases identify a clinical subtype with a poorer prognosis, as is the case of VRK1 in breast cancer

Sensibilidad a inhibidores de la familia de quinasas VRK y regulación de NFAT y COX-2 por la quinasa humana VRK2A

Memoria presentada por Marta Vázquez Cedeira para optar al grado de Doctor por la Universidad de Salamanca y realizada en el Instituto de Biología Molecular y Celular del Cancer de Salamanca.Esta memoria ha sido realizada siendo Marta Vázquez Cedeira beneficiaria de una beca de Iniciación a la Investigación y una beca predoctoral JAE del Consejo Superior de Investigaciones Científicas-Fondo Social Europeo para el desarrollo de la tesis doctoral (2006-2010). La investigación en el laboratorio ha sido financiada por los siguientes proyectos: • Ministerio de Educación y Ciencia (SAF2004-02900; SAF2007-60242; CDS-2007-0017 y SAF2010-14935) • Junta de Castilla y León, Consejería de Sanidad (SAN/673/SA05/08; BOCyL Nº 170) • Junta de Castilla y León, Consejería de Educación (CSI14A08; CSI006A11-2 y grupo de excelencia GR-15) • Kutxa-Fundación INBIOMEDPeer reviewe

Human kinase VRK2a regulates cell invasion through NFat activation and COX-2 expression

Resumen del trabajo presentado al 22nd Biennial Congress of the European Association for Cancer Research, celebrado en Barcelona (España) del 7 al 10 de julio de 2012.[Background]: Human VRK2A is a member of a new Ser/Thr kinase family that emerged late in evolution. VRK2A is poorly characterized, but its expression is higher in tumors and proliferative cells and it has been identified in a subgroup of breast carcinomas. The nuclear factor of activated T cells (NFAT) is a key factor regulating a variety of processes in cells and there is increasing evidence that NFAT plays a crucial role in cancer cells promoting invasion through cyclooxygenase-2 (COX-2) expression. NFAT proteins translocate to the nucleus upon phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore ionomycin (Io) stimulation. In addition, an inducible phosphorylation in NFAT transactivation domain (TAD) increases NFAT transcriptional activity and therefore COX-2 expression. To dilucidate what kinases are involved in this phosphorylation is essential to define NFAT modulation. The regulator of calcineurin (RCAN1) protein blocks NFAT activation by direct binding to calcineurin. However, RCAN1 phosphorylation relieves this inhibitory effect. [Methods]: We determined phosphorylation by in vitro kinase assays and measured transcriptional activity of NFAT and COX-2 promoter using luciferase reporter plasmids. Interactions were performing in pulldown assays. Matrigel transwell assay were used to measured cell invasion of breast cancer MDAMB-231 cell line and cell migration was observed in wound healing assays. [Results]: VRK2A phosphorylated NFAT N-terminal domain, enhancing NFAT transcriptional activity, and interaction between VRK2A catalytic domain and NFAT regulatory domain was detected. Also, VRK2A increased COX-2 expression through NFAT pathway since mutations in COX-2 promoter NFAT binding sites decreased induction of COX-2 by VRK2A. Furthermore, VRK2A down-regulation reduced cell invasion and migration upon PMA plus Io stimulation. In addition, we ruled out an indirect effect through the RCAN1 phosphorylation since VRK2A did not phosphorylate RCAN1 and it was not able to counteract RCAN1 inhibition on NFAT activity. [Conclusion]: These findings demonstrate the first reported role of VRK2A as an active kinase in a cell invasion context through NFAT-dependent COX-2 transcription.Peer Reviewe

Human VRK2 (vaccinia-related kinase 2) modulates tumor cell invasion by hyperactivation of NFAT1 and expression of cyclooxygenase-2

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.Supported by a Junta para Ampliación de Estudios e Investigaciones Científicas-Consejo Superior de Investigaciones Científicas-Fondo Social Europeo fellowship. This work was supported by grants from Ministerio de Educación, Ciencia e Innovación Grant SAF2010-14935, Consolider-Ingenio 2010 Grant CSD07-0017, Junta de Castilla y León (Consejería de Educación, Grant CSI-006A11-2), Fundación Sandra Ibarra, and Kutxa-Fundación Inbiomed.Peer Reviewe

Substrate profiling of human vaccinia-related kinases identifies coilin, a Cajal body nuclear protein, as a phosphorylation target with neurological implications

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. © 2011 Elsevier B.V.M. S-G., and M. V-C have JAE/CSIC predoctoral fellowships. This work was supported by grants from Ministerio de Educación, Ciencia e Innovación (SAF2010-14935, and CSD2007-0017), Junta de Castilla y León-Consejería de Educación (GR15, CSI006A11-2), CSIC (PA1003032) and Kutxa-Fundación Inbiomed to P.A.L; and by the Legacy Heritage Bio-Medical Programof the Israel Science Foundation (grant no. 1872/2008) to P.R. and E.L.L.Peer Reviewe

VRK1 y AurKB interaccionan en mitosis regulando la fosforilación específica de la histona 3 y la correcta progresión del ciclo celular

Resumen del trabajo presentado al XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Granada (España) del 9 al 12 de septiembre de 2014.El ciclo celular es un proceso altamente regulado en el que participan diversas proteínas quinasa, como la Aurora B y la VRK1. Estas dos quinasas controlan varios procesos para una correcta progresión del ciclo celular, entre los cuales se destaca la condensación de la cromatina. Durante la mitosis, las dos serina-treonina quinasas contribuyen significativamente en la condensación de la cromatina en varios mecanismos, entre los que se destaca la fosforilación de las histonas, más precisamente de la histona 3. VRK1 fosforila específicamente la treonina 3 (T3) de la histona 3 y la Aurora B fosforila específicamente la histona 3 en la serina 10 (S10). En este trabajo, hemos demostrado que VRK1 interacciona con Aurora B y que esta interacción se produce durante la mitosis, concretamente en las fases más tardías. Además, analizamos el efecto de la interacción VRK1-Aurora B en la capacidad de fosforilar a la histona 3. Así, confirmamos que cantidades crecientes de Aurora B, afectaban a la fosforilación de la histona 3, en el residuo T3, por VRK1 y que este efecto era independiente de la actividad quinasa de la Aurora B. De la misma manera, cantidades crecientes de VRK1, activa o inactiva, afectaban a la fosforilación de la histona 3, en el residuo S10, por la Aurora B. El mismo efecto sobre la histona 3 fue verificado en ensayos quinasa con (32P[ATP]). Posteriormente, hemos analizado durante el ciclo celular los niveles de fosforilación de la histona 3, en los dos residuos T3 y S10, y hemos demostrado que la disminución de la fosforilación de la histona 3 en los dos residuos coincide con la interacción VRK1-Aurora B. Con estos resultados concluimos que la interacción VRK1-Aurora B afecta negativamente la fosforilación de la histona 3 por las dos quinasas, y que este puede ser un mecanismo regulatorio para una correcta salida de la mitosis.Peer Reviewe

VRK1 regulates AurKB activity and localization in early mitosis

Resumen del trabajo presentado al 15th ASEICA International Congress, celebrado en Sevilla (España) del 21 al 23 de octubre de 2015.[Introduction]: The regulation of cell cycle relies on the activity of several protein kinases. These kinases control the correct transmission of genetic information to the newly-formed cells, DNA replication, chromosome segregation, and chromatin condensation, between innumerous other mechanisms. Between these kinases, we can found two different serine-threonine kinases, the Aurora Kinase B (AurKB) and the Vaccinia-related Kinase 1 (VRK1). VRK1 is a nuclear kinase implicated in p53 regulation and DNA damage repair, as well as in the chromatin condensation through histone H3 phosphorylation, mainly, in Thr3. Besides, VRK1 regulates CCND1 gene and correlates positively with proliferation markers such as the Ki67. In turn, AurKB is a component of the Chromosomal Passenger Complex (CPC) necessary for accurate spindle-kinetochore attachments during chromosome segregation and cytokinesis. This kinase contributes also to chromatin condensation, phosphorylating the histone H3Ser10. [Objectives]: We hypothesize that VRK1 and Aurora B might form a complex, and that the establishment of this interaction affects the activity of AurKB. Moreover, we hypothesize that VRK1 may affect the centromeric localization of AurKB, through modulation of histone H3T3 phosphorylation. [Methods]: VRK1 and Aurora B interaction was assessed by reciprocal immunoprecipitations in HEK 293T. The cell cycle-dependent interaction, between both kinases was analyzed performing the arrested of HeLa or U2OS cells in G1/S, after double Thymidine block or in G2/M, after Thymidine-Nocodazole block. The same protocol was used to assess the co-localization of VRK1 and AurKB by immunofluorescence. In vitro kinase assay, in presence of cold ATP or Kinase assay (32P[ATP]) was performed to evaluate histone H3Ser10 phosphorylation by GST-AurKB, after incubation with active or inactive GST-VRK1. Finally, sub-cellular localization of AurKB was assessed by immunofluorescence after VRK1 knockdown. [Results]: Herein, we showed that VRK1 interact with AurKB. This interaction was observed, between transfected VRK1 and AurKB, and also, among endogenous proteins. The interaction seemed to occur mainly in mitosis, when VRK1 and AurKB levels were increased. However, we could not find a possible co-localization between VRK1 and AurKB during mitosis. The effect of this interaction results in the inhibition of AurKB activity. On the other hand, VRK1 downregulation affects the centromeric localization of AurKB-dependent on the H3T3 phosphorylation levels. [Conclusions]: VRK1 interacts with AurKB during mitosis, and this interaction impairs the kinase activity of AurKB. VRK1 regulates AurKB localization in early phases of mitosis.Peer Reviewe

Vaccinia-Related Kinase-3

VRK3 is the most divergent member of the VRK family. It has no kinase activity due to several substitutions in key residues within its catalytic domain. VRK3 is mostly located within the nucleus and appears to play a scaffold role, where it downregulates MAPK signaling and reduces the level of p-ERK by recruiting the VHR phosphatasePeer Reviewe

Vaccinia-related kinase (VRK) signaling

22 páginas, 5 figuras.VRK (vaccinia-related kinase) is a group of three proteins in the human kinome. These proteins, mainly VRK1 and VRK2, have been studied in the context of their substrates and interacting proteins in order to identify and characterize their signaling pathway, as well as their effect on other signaling pathways. VRK1 is mostly a nuclear kinase that specifically phosphorylates p53, c-Jun, ATF2, CREB, BAF and histone H3. VRK1 is an early response gene and is implicated in regulation of cell cycle progression. VRK1 is activated in response to DNA damage phosphorylating p53, which is stabilized and activated; this active p53 induces a downregulatory mechanism of VRK1 that permits the reversal of p53 induced effects. The activity of nuclear VRK1 is regulated by its interaction with the Ran small GTPase. Also, VRK1 is a downstream component of the signaling pathway of MEK-Plk3 that induces Golgi fragmentation in mitosis. VRK2 has two isoforms; VRK2A is cytosolic and bound to endoplasmic reticulum and mitochondrial membranes. VRK2B is a shorter isoform free in cytosol and nucleus. VRK2A affects cellular signaling by interaction with scaffold proteins, as JIP1. The JIP1-VRK2A signalosome blocks the incorporation of JNK, preventing its activation,and thus reducing the stress response to inflammatory cytokines as interleukin-1β and to hypoxia.This work was supported by grants from Ministerio de Educación y Ciencia e Innovación (SAF2007-60242 and CSD2007-0017), Junta de Castilla y León (Consejería de Educación, CSI-14A08 and GR15; Consejería de Sanidad) and Fundación Sandra Ibarra.Peer reviewe