Vaccinia-related Kinase Research Articles

HnRNP A1 phosphorylated by VRK1 stimulates telomerase and its binding to telomeric DNA sequence

The telomere integrity is maintained via replication machinery, telomere associated proteins and telomerase. Many telomere associated proteins are regulated in a cell cycle-dependent manner. Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a single-stranded oligonucleotide binding protein, is thought to play a pivotal role in telomere maintenance. Here, we identified hnRNP A1 as a novel substrate for vaccinia-related kinase 1 (VRK1), a cell cycle regulating kinase. Phosphorylation by VRK1 potentiates the binding of hnRNP A1 to telomeric ssDNA and telomerase RNA in vitro and enhances its function for telomerase reaction. VRK1 deficiency induces a shortening of telomeres with an abnormal telomere arrangement and activation of DNA-damage signaling in mouse male germ cells. Together, our data suggest that VRK1 is required for telomere maintenance via phosphorylation of hnRNP A1, which regulates proteins associated with the telomere and telomerase RNA.

Defective folliculogenesis in female mice lacking Vaccinia-related kinase 1

The Vaccinia-related kinase 1(VRK1), which is generally implicated in modulating cell cycle, plays important roles in mammalian gametogenesis. Female infertility in VRK1-deficient mice was reported to be caused by defective meiotic progression in oocyte at postovulatory stage. VRK1 roles in folliculogenesis, however, remain largely unknown. Here, accurate quantification of folliculogenesis is performed by a direct visualization of ‘intact’ ovary in 3-dimensions (3-D) using a synchrotron X-ray microtomography. In VRK1-deficient ovaries, the numbers of pre-antral and antral follicles are significantly reduced by 38% and 46%, respectively, comparing to control. The oocytes volumes in antral and Graffian follicles also decrease by 42% and 37% in the mutants, respectively, indicating defects in oocyte quality at preovulatory stage. Genetic analysis shows that gene expressions related to folliculogenesis are down-regulated in VRK1-deficient ovaries, implying defects in folliculogenesis. We suggest that VRK1 is required for both follicle development and oocyte growth in mammalian female reproduction system.

Comparative kinome analysis to identify putative colon tumor biomarkers

Kinase domains are the type of protein domain most commonly found in genes associated with tumorigenesis. Because of this, the human kinome (the protein kinase component of the genome) represents a promising source of cancer biomarkers and potential targets for novel anti-cancer therapies. Alterations in the human colon kinome during the progression from normal colon (NC) through adenoma (AD) to adenocarcinoma (AC) were investigated using integrated transcriptomic and proteomic datasets. Two hundred thirty kinase genes and 42 kinase proteins showed differential expression patterns (fold change ≥ 1.5) in at least one tissue pair-wise comparison (AD vs. NC, AC vs. NC, and/or AC vs. AD). Kinases that exhibited similar trends in expression at both the mRNA and protein levels were further analyzed in individual samples of NC (n = 20), AD (n = 39), and AC (n = 24) by quantitative reverse transcriptase PCR. Individual samples of NC and tumor tissue were distinguishable based on the mRNA levels of a set of 20 kinases. Altered expression of several of these kinases, including chaperone activity of bc1 complex-like (CABC1) kinase, bromodomain adjacent to zinc finger domain protein 1B (BAZ1B) kinase, calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D), serine/threonine-protein kinase 24 (STK24), vaccinia-related kinase 3 (VRK3), and TAO kinase 3 (TAOK3), has not been previously reported in tumor tissue. These findings may have diagnostic potential and may lead to the development of novel targeted therapeutic interventions for colorectal cancer.Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-011-0831-6) contains supplementary material, which is available to authorized users.

Common variants at VRK2 and TCF4 conferring risk of schizophrenia

Common sequence variants have recently joined rare structural polymorphisms as genetic factors with strong evidence for association with schizophrenia. Here we extend our previous genome-wide association study and meta-analysis (totalling 7 946 cases and 19 036 controls) by examining an expanded set of variants using an enlarged follow-up sample (up to 10 260 cases and 23 500 controls). In addition to previously reported alleles in the major histocompatibility complex region, near neurogranin (NRGN) and in an intron of transcription factor 4 (TCF4), we find two novel variants showing genome-wide significant association: rs2312147[C], upstream of vaccinia-related kinase 2 (VRK2) [odds ratio (OR) = 1.09, P = 1.9 × 10(-9)] and rs4309482[A], between coiled-coiled domain containing 68 (CCDC68) and TCF4, about 400 kb from the previously described risk allele, but not accounted for by its association (OR = 1.09, P = 7.8 × 10(-9)).

Classification, diagnosis and potential mechanisms in Pontocerebellar Hypoplasia

Pontocerebellar Hypoplasia (PCH) is group of very rare, inherited progressive neurodegenerative disorders with prenatal onset. Up to now seven different subtypes have been reported (PCH1-7). The incidence of each subtype is unknown. All subtypes share common characteristics, including hypoplasia/atrophy of cerebellum and pons, progressive microcephaly, and variable cerebral involvement. Patients have severe cognitive and motor handicaps and seizures are often reported. Treatment is only symptomatic and prognosis is poor, as most patients die during infancy or childhood. The genetic basis of different subtypes has been elucidated, which makes prenatal testing possible in families with mutations. Mutations in three tRNA splicing endonuclease subunit genes were found to be responsible for PCH2, PCH4 and PCH5. Mutations in the nuclear encoded mitochondrial arginyl- tRNA synthetase gene underlie PCH6. The tRNA splicing endonuclease, the mitochondrial arginyl- tRNA synthetase and the vaccinia related kinase1 are mutated in the minority of PCH1 cases. These genes are involved in essential processes in protein synthesis in general and tRNA processing in particular. In this review we describe the neuroradiological, neuropathological, clinical and genetic features of the different PCH subtypes and we report on in vitro and in vivo studies on the tRNA splicing endonuclease and mitochondrial arginyl-tRNA synthetase and discuss their relation to pontocerebellar hypoplasia.

Spotlight on the July 5 Issue

#### Notable in Neurology This issue features an article assessing the frequency of impulse control disorders in patients with restless legs syndrome with and without augmentation under dopaminergic therapy and another determining the steroid-sparing effect of methotrexate in patients with symptomatic generalized myasthenia gravis. A featured article described the phenotypes in 2 families with vaccinia-related kinase 1 mutations, including one novel mutation. ### A low-cost, tablet-based option for prehospital neurologic assessment: The iTREAT study![Graphic][1] The authors assessed the technical feasibility and reliability of a low-cost, tablet-based mobile telestroke option for ambulance transport. This platform reliably … [1]: /embed/inline-graphic-1.gif

NMR Solution Structure of Human Vaccinia-related Kinase 1 (VRK1) Reveals the C-terminal Tail Essential for Its Structural Stability and Autocatalytic Activity

Vaccinia-related kinase 1 (VRK1) is one of the mitotic kinases that play important roles in cell cycle, nuclear condensation, and transcription regulation. Kinase domain structures of two other VRK family members (VRK2 and VRK3) have been determined previously. However, the structure of VRK1, the most extensively studied and constitutively active VRK member, is yet to be characterized. Here, we present the nuclear magnetic resonance (NMR) solution structure of a catalytically active form of human VRK1 with its extended C-terminal tail (residues 1-361). The NMR structure of human VRK1 reveals that the C-terminal tail orients toward the catalytic site and forms a number of interactions that are critical for structural stability and catalysis. The role of this unique C-terminal tail was further investigated by deletion mutant studies where deletion of the terminal tail resulted in a dramatic reduction in the autocatalytic activity of VRK1. NMR titration studies carried out with ATP or an ATP analog confirm that ATP/ATP analogs interact with all of the crucial residues present in important motifs of the protein kinase such as the hinge region, catalytic loop, DYG motif, and thereby suggest that the catalytic domain of VRK1 is not atypical. In addition to the conventional interactions, some of the residues present on the extended C-terminal tail also interact with the ligands. These observations also substantiate the role of the extended C-terminal tail in the biological activity of VRK1.

Protein kinase Cδ regulates vaccinia-related kinase 1 in DNA damage-induced apoptosis.

Vaccinia-related kinase 1 (VRK1) is a novel serine/threonine kinase that plays an important role in cell proliferation. However, little is known about the upstream regulators of VRK1 activity. Here we provide evidence for a role of protein kinase Cδ (PKCδ) in the regulation of murine VRK1. We show that PKCδ interacts with VRK1, phosphorylates the Ser-355 residue in the putative regulatory region, and negatively regulates its kinase activity in vitro. Intriguingly, PKCδ-induced cell death was facilitated by phosphorylation of VRK1 when cells were exposed to a DNA-damaging agent. In addition, p53 played a critical role in the regulation of DNA damage-induced cell death accompanied by PKCδ-mediated modulation of VRK1. In p53-deficient cells, PKCδ-mediated phosphorylation of VRK1 had no effect on cell viability. However, cells overexpressing p53 exhibited significant reduction of cell viability when cotransfected with both VRK1 and PKCδ. Taken together, these results indicate that PKCδ regulates phosphorylation and down-regulation of VRK1, thereby contributing to cell cycle arrest and apoptotic cell death in a p53-dependent manner.

Vaccinia-Related Kinase 1 Is Required for the Maintenance of Undifferentiated Spermatogonia in Mouse Male Germ Cells

Vaccinia-related kinase 1 (VRK1) is a crucial protein kinase for mitotic regulation. VRK1 is known to play a role in germ cell development, and its deficiency results in sterility. Here we describe that VRK1 is essential for the maintenance of spermatogonial stem cells. To determine whether VRK1 plays a role in these cells, we assessed the population size of undifferentiated spermatogonia. Flow cytometry analyses showed that the number of undifferentiated spermatogonia was markedly reduced in VRK1-deficient testes. VRK1 was highly expressed in spermatogonial populations, and approximately 66% of undifferentiated spermatogonia that were sorted as an Ep-CAM+/c-kit−/alpha-6-integrin+ population showed a positive signal for VRK1. Undifferentiated stem cells expressing Plzf and Oct4 but not c-kit also expressed VRK1, suggesting that VRK1 is an intrinsic factor for the maintenance of spermatogonial stem cells. Microarray analyses of the global testicular transcriptome and quantitative RT-PCR of VRK1-deficient testes revealed significantly reduced expression levels of undifferentiated spermatogonial marker genes in early postnatal mice. Together, these results suggest that VRK1 is required for the proliferation and differentiation of undifferentiated spermatogonia, which are essential for spermatogenic cell maintenance.

VRK2 Inhibits Mitogen-Activated Protein Kinase Signaling and Inversely Correlates with ErbB2 in Human Breast Cancer

The epidermal growth factor (EGF)-ErbB-mitogen-activated protein kinase (MAPK) transcription signaling pathway is altered in many types of carcinomas, and this pathway can be regulated by new protein-protein interactions. Vaccinia-related kinase (VRK) proteins are Ser-Thr kinases that regulate several signal transduction pathways. In this work, we study the effect of VRK2 on MAPK signaling using breast cancer as a model. High levels of VRK2 inhibit EGF and ErbB2 activation of transcription by the serum response element (SRE). This effect is also detected in response to H-Ras(G12V) or B-Raf(V600E) oncogenes and is accompanied by a reduction in phosphorylated extracellular signal-regulated kinase (ERK) levels, p90RSK levels, and SRE-dependent transcription. Furthermore, VRK2 knockdown has the opposite effect, increasing the transcriptional response to stimulation with EGF and leading to increased levels of ERK phosphorylation. The molecular mechanism lies between MAPK/ERK kinase (MEK) and ERK, since MEK remains phosphorylated while ERK phosphorylation is blocked by VRK2A. This inhibition of the ERK signaling pathway is a consequence of a direct protein-protein interaction between VRK2A, MEK, and kinase suppressor of Ras 1 (KSR1). Identification of new correlations in human cancer can lead to a better understanding of the biology of individual tumors. ErbB2 and VRK2 protein levels were inversely correlated in 136 cases of human breast carcinoma. In ErbB2(+) tumors, there is a significant reduction in the VRK2 level, suggesting a role for VRK2A in ErbB2-MAPK signaling. Thus, VRK2 downregulation in carcinomas permits signal transmission through the MEK-ERK pathway without affecting AKT signaling, causing a signal imbalance among pathways that contributes to the phenotype of breast cancer.

ZO-1 and ZONAB Interact to Regulate Proximal Tubular Cell Differentiation

<h3>Abstract</h3> Collateral lethality occurs when loss of one paralog renders cancer cells dependent on the remaining paralog. Combining genome scale CRISPR/Cas9 screens coupled with RNA-sequencing in over 900 cancer cell lines, we found that cancers of nervous system lineage, including adult and pediatric gliomas and neuroblastomas, required the nuclear kinase Vaccinia-Related Kinase 1 (VRK1) for their survival. <i>VRK1</i> dependency was inversely correlated with expression of its paralog VRK2. <i>VRK2</i> knockout (KO) sensitized cells to <i>VRK1</i> suppression, and conversely, VRK2 overexpression increased cell fitness in the setting of VRK1 suppression. DNA methylation of the <i>VRK2</i> promoter was associated with low VRK2 expression in human neuroblastomas, and adult and pediatric gliomas. Mechanistically, depletion of <i>VRK1</i> reduced Barrier-to-Autointegration Factor (BAF) phosphorylation during mitosis, resulting in DNA damage and apoptosis. Together, these studies identify VRK1 as a synthetic lethal target in <i>VRK2</i> promoter-methylated adult and pediatric gliomas and neuroblastomas. <h3>Statement of Significance</h3> We credential VRK1 as a target in adult and pediatric gliomas, and neuroblastomas with <i>VRK2</i> promoter methylation. This demonstrates the utility of paralog-driven synthetic lethal interactions for biomarker-linked, targeted therapeutics.

SNP-Arrays Provide New Insights Into the Pathogenesis of Post-Transplant Diffuse Large B-Cell Lymphoma (PT-DLBCL).

Abstract 444Post transplant lymphoproliferative disorders (PTLD) are important complications of solid organ transplantation associated with severe morbidity and mortality. DLBCL represents the most common form of monomorphic PTLD. Since knowledge of the pathogenesis of monoclonal PTLD is limited, we studied a series 44 cases of PT-DLBCL with high-density genome wide SNP-based arrays, and compared them with 105 cases immunocompetent DLBCL (IC-DLBCL) and 28 cases of HIV-DLBCL. Methods. DNA was extracted from frozen biopsies. All 177 DNA profiles were obtained using the Affymetrix GeneChip Human Mapping 250K NspI arrays. Recurrent minimal common regions (MCR) were defined using the algorithm by Lenz et al. (PNAS, 2008). Differences in frequencies between subgroups were evaluated using Fisher's exact test followed by multiple test correction. Results. The most common MCR were losses at 2p16.1 (29%) and 17p11.2-p13.3 (22%, TP53), and gains at 11q14.2-q24.2 (22%) and 7q (22%). Amplifications occurred in 7% at 9p23-p24 (PTPRD, JAK2, JMJD2C). MCRs more frequently deleted in PT-DLBCL when compared to IC-DLBCL included a series of small interstitial deletions at 1p32.2, 2p16.1, 3p14.2, 4p14, 14q13.2, 20p12.3, 20q13.32. Some of these aberrations targeted known fragile sites (FRA3B, FRA1B and FRA2E). FRA2E deletion showed the most significant difference with IC-DLBCL (PT-DLBCL 30% vs IC-DLBCL 4%, p<0.0001, adj.p<0.0005) and contained two genes, Vaccinia Related Kinase 2 (VRK2) and Fanconi anemia, complementation group L (FANCL). VRK2 is a negative regulator of MAPK pathway. FANCL is an E3 ubiquitin ligase, that is part of the FA nuclear protein complex, mainly involved in DNA repair, but also in telomere stability, chromatin remodelling, cell cycle regulation and apoptosis. No deletions of WW domain-containing Oxidoreductase (WWOX) were observed in PT-DLBCL, as opposed to HIV-DLBCL. Aside from the small interstitial deletions, PT-DLBCL overall showed a profile similar to IC-DLBCL, but PT-DLBCL contained no 13q14.3 deletions (0% vs 15%; p=0.003, adj.p = 0.04), which among other genes contains MIR15 and MIR16, and had fewer copy neutral LOH at 6p21.32-p21.33 (HLA-DR locus; 4% vs 24%; p=0.005, adj.p = 0.04). By immunophenotype, PT-DLBCL resembles most closely activated B cell like (ABC) / non germinal center (GC) B-cells. However, when compared with gene expression-classified ABC DLBCL, the MCRs characteristic of this subtype were under-represented in PT-DLBCL: gains of 18q (BCL2; 5% vs 32%; p=0.007, adj.p=0.07) and 3q (0% vs 32%; p=0.02; p=0.006, adj.p=0.07) and 6q losses (PRDM1 and TNFAIP3; 9% vs 23%; p=0.06, adj.p=0.3). Also, MCRs in GC such as 2p14-p16.1 (REL) gains were less common in PT-DLBCL compared to GC-DLBCL (11% vs 37%, p=0.019, adj.p = 0.13). In conclusion, PT-DLBCL shared similarities with IC-DLBCL, but presented unique features, such as the lack of copy neutral LOH at 6p (HLA-DR locus) and frequent breakage at fragile sites. The latter appeared partially different from what observed in HIV-DLBCL, suggesting different pathogenetic mechanisms in the context of the two conditions of immunodeficiency. The comparison with IC GC- and ABC-DLBCL subtypes indicated discrepancies between the PT-DLBCL phenotype and their genomic profile. Disclosures:No relevant conflicts of interest to declare.

Helicobacter pylori-induced modification of the histone H3 phosphorylation status in gastric epithelial cells reflects its impact on cell cycle regulation

Post-translational modifications of core histones are important components of the epigenetic landscape. Recent investigations of bacterial or toxin-induced effects on histone phosphorylation and acetylation in host cells have linked the changes to transcriptional alterations of key cellular response pathways. However, these changes may have other reasons and functional consequences. Here, we show that infection of gastric epithelial cell lines with the carcinogenic bacterium Helicobacter pylori leads to changes in histone H3 phosphorylation: type IV secretion system (T4SS)-dependent decreases of H3 phosphorylation levels at serine 10 (pH3Ser10) and threonine 3 (pH3Thr3) were observed. Immunofluorescence experiments with pH3Ser10 and cyclin B1 revealed that a H. pylori-induced transient pre-mitotic arrest was responsible for the observed reduction. This causal link was substantiated further by showing that H. pylori causes a strong decrease of the cell division cycle 25 (CDC25C) phosphatase. As a consequence, mitotic histone H3 kinases such as vaccinia-related kinase 1 (VRK1) and Aurora B were not fully activated in infected cells. We show that VRK1 activity, measured using a kinase activity assay, was reduced after H. pylori infection by approximately 40%. Moreover, over–expression of VRK1, but not Aurora B, compensated for the H. pylori-induced decrease of pH3Ser10. Rephosphorylation of H3Ser10 was IκB kinase α (IKKα)-dependent and occurred at later time points of infection. Taken together, our work highlights the impact of bacterial pathogens on host cell chromatin; this modulation reflects the subversion of key cellular processes such as cell cycle progression.

Spinal Muscular Atrophy with Pontocerebellar Hypoplasia Is Caused by a Mutation in the VRK1 Gene

The spinal muscular atrophies (SMAs) are a genetically and clinically heterogeneous group of disorders characterized by degeneration and loss of anterior horn cells in the spinal cord, leading to muscle weakness and atrophy. Spinal muscular atrophy with pontocerebellar hypoplasia (SMA-PCH, also known as pontocerebellar hypoplasia type 1 [PCH1]) is one of the rare infantile SMA variants that include additional clinical manifestations, and its genetic basis is unknown. We used a homozygosity mapping and positional cloning approach in a consanguineous family of Ashkenazi Jewish origin and identified a nonsense mutation in the vaccinia-related kinase 1 gene (VRK1) as a cause of SMA-PCH. VRK1, one of three members of the mammalian VRK family, is a serine/threonine kinase that phosphorylates p53 and CREB and is essential for nuclear envelope formation. Its identification as a gene involved in SMA-PCH implies new roles for the VRK proteins in neuronal development and maintenance and suggests the VRK genes as candidates for related phenotypes.

Emerging biological functions of the vaccinia-related kinase (VRK) family.

The vaccinia-related kinases (VRKs) branched off early from the family of casein kinase (CK) I and compose a relatively uncharacterized family of the kinome. The VRKs were discovered due to their close sequence relation to the vaccinia virus B1R serine/threonine kinase. They were first described in phosphorylation of transcription factors that led to the discovery of an autoregulatory mechanism between VRK and the tumor suppressor transcription factor p53. The relevance of VRKs has broadened recently by introduction of its members as essential regulators in cell signaling, nuclear envelope dynamics, chromatin modifications, apoptosis and cellular stress response. Several phosphorylation substrates have been described, as well as the first positive and negative regulators of VRK. We provide an overview of the VRKs across species and discuss the wide diversity of cellular and organismal requirements for this kinase family.

Proteomics Identification of Nuclear Ran GTPase as an Inhibitor of Human VRK1 and VRK2 (Vaccinia-related Kinase) Activities

Human vaccinia-related kinase (VRK) 1 is a novel serine-threonine kinase that regulates several transcription factors, nuclear envelope assembly, and chromatin condensation and is also required for cell cycle progression. The regulation of this kinase family is unknown. Mass spectrometry has permitted the identification of Ran as an interacting and regulatory protein of the VRK serine-threonine kinase activities. The stable interaction has been validated by pulldown of endogenous proteins as well as by reciprocal immunoprecipitations. The three members of the VRK family stably interact with Ran, and the interaction was not affected by the bound nucleotide, GDP or GTP. The interaction was stronger with the RanT24N that is locked in its inactive conformation and cannot bind nucleotides. None of the kinases phosphorylated Ran or RCC1. VRK1 does not directly interact with RCC1, but if Ran is present they can be isolated as a complex. The main effect of the interaction of inactive RanGDP with VRK1 is the inhibition of its kinase activity, which was detected by a reduction in VRK1 autophosphorylation and a reduction in phosphorylation of histone H3 in residues Thr-3 and Ser-10. The kinase activity inhibition can be relieved by the interaction with the constitutively active RanGTP or RanL43E, which locks Ran in its GTP-bound active conformation. In this complex, the interaction with VRK proteins does not alter the effect of its guanine exchange factor, RCC1. Ran is a novel negative regulator of nuclear VRK1 and VRK2 kinase activity, which may vary in different subcellular localizations generating an asymmetric intracellular distribution of kinase activity depending on local protein interactions.

A Central Role for CK1 in Catalyzing Phosphorylation of the p53 Transactivation Domain at Serine 20 after HHV-6B Viral Infection

The tumor suppressor protein p53 is activated by distinct cellular stresses including radiation, hypoxia, type I interferon, and DNA/RNA virus infection. The transactivation domain of p53 contains a phosphorylation site at Ser20 whose modification stabilizes the binding of the transcriptional co-activator p300 and whose mutation in murine transgenics induces B-cell lymphoma. Although the checkpoint kinase CHK2 is implicated in promoting Ser20 site phosphorylation after irradiation, the enzyme that triggers this phosphorylation after DNA viral infection is undefined. Using human herpesvirus 6B (HHV-6B) as a virus that induces Ser20 site phosphorylation of p53 in T-cells, we sought to identify the kinase responsible for this virus-induced p53 modification. The p53 Ser20 kinase was fractionated and purified using cation, anion, and dye-ligand exchange chromatography. Mass spectrometry identified casein kinase 1 (CK1) and vaccinia-related kinase 1 (VRK1) as enzymes that coeluted with virus-induced Ser20 site kinase activity. Immunodepletion of CK1 but not VRK1 removed the kinase activity from the peak fraction, and bacterially expressed CK1 exhibited Ser20 site kinase activity equivalent to that of the virus-induced native CK1. CK1 modified p53 in a docking-dependent manner, which is similar to other known Ser20 site p53 kinases. Low levels of the CK1 inhibitor D4476 selectively inhibited HHV-6B-induced Ser20 site phosphorylation of p53. However, x-ray-induced Ser20 site phosphorylation of p53 was not blocked by D4476. These data highlight a central role for CK1 as the Ser20 site kinase for p53 in DNA virus-infected cells but also suggest that distinct stresses may selectively trigger different protein kinases to modify the transactivation domain of p53 at Ser20.

Cisplatin‐mediated activation of extracellular signal‐regulated kinases 1/2 (ERK1/2) by inhibition of ERK1/2 phosphatases

The mechanism(s) underlying neurodegeneration-associated activation of ERK1/2 remain poorly understood. We report that in cultured rat cortical neurons, whose basal ERK1/2 phosphorylation required NMDA receptors (NMDAR), the neurotoxic DNA intercalating drug cisplatin increased ERK1/2 phosphorylation via NMDAR despite reducing their activity. The rate of ERK1/2 dephosphorylation was lowered by cisplatin. Cisplatin-treated neurons showed general transcription inhibition likely accounting for the reduced expression of the ERK1/2-selective phosphatases including the dual specificity phosphatase-6 (DUSP6) and the DUSP3 activator vaccinia-related kinase-3 (VRK3). Hence, cisplatin effects on ERK1/2 may be due to the deficient ERK1/2 inhibition by the transcription-regulated phosphatases. Indeed, the transcription inhibitor actinomycin D reduced expression of DUSP6 and VRK3 while inducing the NMDAR-dependent activation of ERK1/2 and the impairment of ERK1/2 dephosphorylation. Thus, cisplatin-mediated transcriptional inhibition of ERK1/2 phosphatases contributed to delayed and long lasting accumulation of phospho-ERK1/2 that was driven by the basal NMDAR activity. Our results provide the first direct evidence for transcriptionally-regulated inactivation of neuronal ERK1/2. Its disruption likely contributes to neurodegeneration-associated activation of ERK1/2.

The C/H3 Domain of p300 Is Required to Protect VRK1 and VRK2 from their Downregulation Induced by p53

BackgroundThe vaccinia-related kinase 1 (VRK1) protein, an activator of p53, can be proteolytically downregulated by an indirect mechanism, which requires p53-dependent transcription.Principal FindingsIn this work we have biochemically characterized the contribution of several p53 transcriptional cofactors with acetyl transferase activity to the induction of VRK1 downregulation that was used as a functional assay. Downregulation of VRK1 induced by p53 is prevented in a dose dependent manner by either p300 or CBP, but not by PCAF, used as transcriptional co-activators, suggesting that p53 has a different specificity depending on the relative level of these transcriptional cofactors. This inhibition does not require p53 acetylation, since a p53 acetylation mutant also induces VRK1 downregulation. PCAF can not revert the VRK1 protection effect of p300, indicating that these two proteins do not compete for a common factor needed to induce VRK1 downregulation. The protective effect is also induced by the C/H3 domain of p300, a region implicated in binding to several transcription factors and SV40 large T antigen; but the protective effect is lost when a mutant C/H3Del33 is used. The protective effect is a consequence of direct binding of the C/H3 domain to the transactivation domain of p53. A similar downregulatory effect can also be detected with VRK2 protein.Conclusions/SignificanceSpecific p53-dependent effects are determined by the availability and ratios of its transcriptional cofactors. Specifically, the downregulation of VRK1/VRK2 protein levels, as a consequence of p53 accumulation, is thus dependent on the levels of the p300/CBP protein available for transcriptional complexes, since in this context this cofactor functions as a repressor of the effect. These observations point to the relevance of knowing the cofactor levels in order to determine one effect or another.

VRK1 (Vaccinia-related kinase 1)

Review on VRK1 (Vaccinia-related kinase 1), with data on DNA, on the protein encoded, and where the gene is implicated.