Mechanism of regulation of Raf-1 by Ca2+/Calmodulin-dependent kinase II

The calcium-calmodulin dependent kinase II (CaMKII) is an ubiquitous serine/threonine protein kinase involved in multiple signalings and biological functions. It has been demonstrated that in epithelial and mesenchimal cells CaMKII participates with Ras to Raf-1 activation and that it is necessary for ERK activation by diverse factors. Raf-1 activation is complex. Maximal Raf-1 activation is reached by phosphorylation at Y341 by Src and at S338. Although early data proposed the involvement of p21-activated kinase 3 (Pak3), the kinase phosphorylating S338 is not definitively identified. Aim of my thesis is to go more insight into the molecular mechanisms of CaMKII/Raf-1 interaction and to verify the hypothesis that CaMKII phosphorylates Raf-1 at Ser338. To this purpose, I investigated the role of CaMKII in Raf-1 and ERK activation by oncogenic Ras and other factors, in COS-7 and NIH3T3 cells. Serum, SrcY527 and RasV12 activated CaMKII. CaMKII was necessary for Raf-1 and ERK activation by all these factors. CaMKII was necessary to the phosphorylation of S338 Raf-1 by serum, fibronectin or oncogenic Ras. Conversely, the inhibition of phosphatidylinositol 3-kinase, which in turn activates Pak3, was ineffective. The direct kinase activity of CaMKII on the serine 338 residue, was demonstrated in vitro by interaction of purified kinases. These results demonstrate that CaMKII phosphorylates Raf-1 at S338 and partecipates to ERK activation upon different physiologic and pathologic stimuli in the MAPK cascade. This kinase, might have a role in cancers harbouring oncogenic Ras and could represent a new therapeutic target for pharmacological intervention in these tumors

VRK1 phosphorylates and protects NBS1 from ubiquitination and proteasomal degradation in response to DNA damage

AbstractNBS1 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

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

Bilateral hypertrophy of masseteric and temporalis muscles, our fifteen patients and review of literature

OBJECTIVE: The association of bilateral hypertrophy of temporalis and masseteric muscles is a rare clinical entity. The origin of the condition is unclear, causing cosmetic problems, pain, and functional impairment. PATIENTS AND METHODS: In this paper we analyzed 15 patients treated at the Department of Maxillo-Facial Surgery of the University of Naples Federico II, from 2000 to 2013, for temporalis and/or masseteric muscle hypertrophy, and in particular, a rare case of a patient with a marked bilateral swelling of the temporalis and masseteric region, in conjunction with a review of the literature. RESULTS: Fourteen patients have not any kind of postoperatively problems. The last patient had been aware of the swelling for many years and complained of recurrent headaches. We adopted a new protocol fort these patients and the patient was very pleased with the treatment resul ts, and reported a reduct ion in headaches and a continuation of his well-being, in addition to greater self-confidence. The last follow-up was performed three years after the first treatment, and the patient showed a complete resolution of his symptoms, and just a small increase of the swelling. CONCLUSIONS: The treatment of temporalis and masseteric hypertrophy with Botulin toxin could be an effective option compared to conservative treatment or surgical intervention although the review of the literature shows that this is only a temporary treatment. In fact, surgery still remains the best option. The treatment must be repeated every 4/6 months for 2-3 consecutive years before having stable benefits. To overcome this problem, an association with a bite treatment allowed us to achieve more lasting and more stable results over time without a recurrence of symptoms between the treatments. Furthermore, this association has enabled us to obtain a more rapid reduction of the hypertrophy

PMCA-based detection of prions in the olfactory mucosa of patients with Sporadic Creutzfeldt-Jakob Disease

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder caused by the conformational conversion of the prion protein (PrPC) into an abnormally folded form, named prion (or PrPSc). The combination of the polymorphism at codon 129 of the PrP gene (coding either methionine or valine) with the biochemical feature of the proteinase-K resistant PrP (generating either PrPSc type 1 or 2) gives rise to different PrPSc strains, which cause variable phenotypes of sCJD. The definitive diagnosis of sCJD and its classification can be achieved only post-mortem after PrPSc identification and characterization in the brain. By exploiting the Real-Time Quaking-Induced Conversion (RT-QuIC) assay, traces of PrPSc were found in the olfactory mucosa (OM) of sCJD patients, thus demonstrating that PrPSc is not confined to the brain. Here, we have optimized another technique, named protein misfolding cyclic amplification (PMCA) for detecting PrPSc in OM samples of sCJD patients. OM samples were collected from 27 sCJD and 2 genetic CJD patients (E200K). Samples from 34 patients with other neurodegenerative disorders were included as controls. Brains were collected from 26 sCJD patients and 16 of them underwent OM collection. Brain and OM samples were subjected to PMCA using the brains of transgenic mice expressing human PrPC with methionine at codon 129 as reaction substrates. The amplified products were analyzed by Western blot after proteinase K digestion. Quantitative PMCA was performed to estimate PrPSc concentration in OM. PMCA enabled the detection of prions in OM samples with 79.3% sensitivity and 100% specificity. Except for a few cases, a predominant type 1 PrPSc was generated, regardless of the tissues analyzed. Notably, all amplified PrPSc were less resistant to PK compared to the original strain. In conclusion, although the optimized PMCA did not consent to recognize sCJD subtypes from the analysis of OM collected from living patients, it enabled us to estimate for the first time the amount of prions accumulating in this biological tissue. Further assay optimizations are needed to faithfully amplify peripheral prions whose recognition could lead to a better diagnosis and selection of patients for future clinical trials

VRK1 phosphorylates Tip60/KAT5 and is required for H4K16 acetylation in response to DNA damage

Dynamic remodeling of chromatin requires acetylation and methylation of histones, frequently affecting the same lysine residue. These alternative epigenetic modifications require the coordination of enzymes, writers and erasers, mediating them such as acetylases and deacetylases. In cells in G0/G1, DNA damage induced by doxorubicin causes an increase in histone H4K16ac, a marker of chromatin relaxation. In this context, we studied the role that VRK1, a chromatin kinase activated by DNA damage, plays in this early step. VRK1 depletion or MG149, a Tip60/KAT5 inhibitor, cause a loss of H4K16ac. DNA damage induces the phosphorylation of Tip60 mediated by VRK1 in the chromatin fraction. VRK1 directly interacts with and phosphorylates Tip60. Furthermore, the phosphorylation of Tip60 induced by doxorubicin is lost by depletion of VRK1 in both ATM +/+ and ATM−/− cells. Kinase-active VRK1, but not kinase-dead VRK1, rescues Tip60 phosphorylation induced by DNA damage independently of ATM. The Tip60 phosphorylation by VRK1 is necessary for the activating acetylation of ATM, and subsequent ATM autophosphorylation, and both are lost by VRK1 depletion. These results support that the VRK1 chromatin kinase is an upstream regulator of the initial acetylation of histones, and an early step in DNA damage responses (DDR).This work was supported by grants from Agencia Estatal de Investigación-Ministerio de Economía y Competitividad-FEDER [SAF2016-75744-R; RED2018-102801-T, PID2019-105610RB-I00] Consejería de Educación de la Junta de Castilla y León-ERDF [CLC-2017-01, and UIC-258] to P.A.L., R.G.-G, P.M.-G. and I.C.-M. were supported by Consejería de Educación-Junta de Castilla y León-Fondo Social Europeo (ESF), Ministerio de Educación y Universidades [FPU 16-01883] and MINECO-FPI [BES-2014-067729] predoctoral fellowships respectively

Novel role of the human kinase VRK1 in response to DNA Damage induced by oxidative stress

Resumen del trabajo presentado al 15th ASEICA International Congress, celebrado en Sevilla (España) del 21 al 23 de octubre de 2015.[Introduction]: In basal conditions, cells are exposed to exogenous and endogenous agents, such as ionizing radiation (IR) or oxidative stress, among others, which are responsible for double- and single-strand breaks (DSBs and SSBs, respectively) in the DNA. As a consequence of this, cells can activate different DNA repair mechanisms based on the type of DNA lesions. All these mechanisms take part in a global process called DNA Damage Response (DDR), essential to maintain the integrity and stability of genetic information. It is widely known that oxidative stress induces both SSBs and DSBs, particularly in cells with high metabolic rates, like cancer or neural cells. For this reason, it is particularly relevant to know the proteins which detect and repair the damage, returning to homeostatic equilibrium afterwards. Recently, VRK1 has been described to be required for the assembly of 53BP1 foci and participates in the recruitment and formation of γ H2AX foci in response to ionizing radiation. Furthermore, VRK1 interacts with p53 and is activated by UV-induced DNA damage. Overall, these processes could be an indicator of the crucial role this kinase may play in several steps of DDR. [Objectives]: Our aim is to determine whether VRK1 is activated and participates in the assembly of 53BP1 foci after inducing oxidative stress in human cancer cell lines.[Methods]: To study VRK1 activation by oxidative stress, cell lines were starved and treated with 10 mM of hydrogen peroxide (H2O2). Next, endogenous p53 was immunoprecipitated at different points in time and the level of threonine 18 phosphorylation, which depends on VRK1 activation, was determined with a phosphospecific antibody. Moreover, in vitro assays were performed, using purified protein GST-p53 (1-85) as substrate and analyzing its phosphorylation in threonine 18. Along this same line, the assembly of 53BP1 foci was assessed by immunofluorescence in the presence or absence of VRK1. [Results]: In this work analysis, we can observe that VRK1 is activated by oxidative stress in the absence of serum, which reduces VRK1 activation mediated by growth factors. Exclusively under H2O2 exposition, this kinase is able to activate and phosphorylate p53. In turn, what it is shown is that VRK1 plays an essential role in DNA repair after oxidative stress induction. When cell lines were treated with H2O2, the number of 53BP1 foci increased considerably for the following 30-60 minutes, beginning to decrease at that point. On the contrary, VRK1 depletion was accompanied by a huge reduction of the number of 53BP1 foci, despite the fact that cells had been previously treated with hydrogen peroxide. [Conclusions]: Based on these results, we conclude that VRK1 participates in DNA repair process after H2O2 exposure, because it is activated and required for the assembly of 53BP1 foci under these conditions. Furthermore, it could also be possible that this kinase plays a crucial role in the early steps of DDR, mainly indicated by its hypothetical involvement in the recruitment and formation of γ H2AX foc.Peer Reviewe

The Vaccinia-Related Kinase I (VRK1) in epigenetic regulation of chromatin

Resumen del trabajo presentado al 15th ASEICA International Congress, celebrado en Sevilla (España) del 21 al 23 de octubre de 2015.[Introduction]: The differences in chromatin compaction are determined by the interaction of DNA with histones and other non histone chromosomal proteins. Chromatin needs to relax or condense to regulate gene transcription or DNA damage response, among other processes. These key reversible mechanisms depend on histone post translational modifications, such as lysine acetylation or methylation, which are regulated by ATP-dependent enzymes: histone acetyltransferases (HAT) and methyltransferases (KMT), called “writers”, and histone deacetylases (HDACs) and demethylases (KDMT), called “erasers”. Epigenetics deregulation is gradually gaining importance as potential player in aging and neurodegeneration. VRK1 is a nuclear Ser-Thr kinase implicated in many cellular processes, such as proliferation, cell cycle or DNA Damage Repair (DDR). Previously we observed that VRK1 is present in the chromatin fraction and is able to interact and phosphorylate H2AX in Ser139 after DNA Damage induced by irradiation. Besides, it is necessary for H4 acetylation in an ATM-independent manner. [Objectives]: We aim to study if VRK1regulates epigenetic mechanisms and has a role in chromatin condensation/relaxation by regulating histone writers or erasers. [Methods]: We used pharmacological inhibitors of HATs (C646 and MG149); HDACs (Entinostat and SAHA); KMTs (Chaetocin); and KDMTs (JMJD2 inhibitor). Some of them are advancing in preclinical regulatory studies. Serum-deprived U2OS cells were silenced for VRK1 and immunofluorescence experiments for lysine-specific H3 methylation and H4 acetylation were performed. [Results]: First, as we knew that VRK1 was necessary for 53BP1 and γ H2AX foci formation after irradiation and to confirm that VRK1 has a role in relaxing chromatin after DNA damage, we tested the effect of histone acetylation/deacetylation and methylation/demethylation inhibition on foci formation. The depletion of VRK1 by siRNA in U2OS, A549 and ATM-/- cells affected the acetylation of H3 in Lys14 and Lys9 and also the H4 acetylation in Lys16, important markers of transcriptional activation. In contrast, siVRK1 enhanced the H3 methylation in Lys9 (a gene repressor marker) but not in Lys4 (transcriptional activator residue), confirming the role of VRK1 in positively mediating gene transcription. As expected, the pharmacological treatment of C646 and of MG149 reduced H3K9 and H4K16 acetylation, while the inhibition of HATs and VRK1 depletion didn’t rescue both acetylations, showing that siVRK1 works as a HATs inhibitor. Moreover, the H3K9 trimethylation enhanced by siVRK1 or JMJD2 inhibitor had a further increase with the presence of the both inhibitions, suggesting that siVRK1 is also a strong KDMT inhibitor. [Conclusions]: In conclusion, we suppose that VRK1 regulates epigenetic mechanisms and supports the chromatin relaxation through two ways: activating HATs and also activating KDMTs. These results could be useful to further studies that will expand the roles of VRK1 in chromatin remodeling and also in the number of target proteins regulated by VRK1. Futurperspectives could be focus on the activation of HATs p300 and Tip60 or some of the members of the Jumonji family by VRK1.Peer Reviewe

Chromatin dynamics in response to DNA damage

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.[Introduction]: Genome integrity is continuously challenged by exogenous and endogenous agents causing DNA damage, both in euchromatin and heterocromatin. The repair of these lesions involves changes in the histone organization, such as recruitment and/or incorporation of histone variants or covalent modifications of histones, in order to promote the formation of open, relaxed chromatin structues. Recently, a growing number of chromatin components, remodelers and modifications has been identificated as key players in DNA repair, emphasizing the complex role of chromatin in this process. On the other hand, defects in DNA repair pathways enable cancer cells to survive DNA damage. For this reason, the combination of DNA-damaging agents with specific inhibitors of these pathways could be used in cancer treatment. Currently, specific inhibitors against chromatin remodelers have been developed, but it is still unclear how they act in tumor cells. Our aim ls to detemine the effect of histone acetylation/deacetylation and methylation/demethylation inhibition on DNA repair foci. [Results and conclusion]: We use pharmacological inhibitors of HAIs (C646 and MGl49); HDACs (Entinostat and SAHA); KMTs (Chaetocin); and KDMs (JMJD2 inhibitor). Some of them are being employed in preclinical regulatory studies. In this analysis, we can observe that closed chromatin induced by HAIs inhibitors seems to affect the formation of yH2AX and 53BP1 foci, and the dynamics offoci formation corresponds with the increase of fluorescence level of H4 acetylation after inducing DNA damage. Based on these results, we conclude that chromatin relaxation is an essential early step in DNA repair, which can be blocked by specific inhibitors against HAIs, sensibilizing cells to treatmentsPeer Reviewe

Regulation of epigenetic modifications of chromatin by the VRK1 kinase

Resumen del póster presentado a la European Association for Cancer Research (EACR) Conference on Basic Epigenetic Mechanisms in Cancer, celebrada en Berlin (Alemania) del 8 al 11 de noviembre de 2015.Chromatin organization is regulated by covalent modifications of histones such as acetylation or methylation in specific residues, which can be regulated by phosphorylations. VRK1 is a nuclear Ser-Thr kinase implicated in many nuclear processes, such as proliferation, cell cycle or DNA Damage Repair (DDR). VRK1 is present in the chromatin fraction and is able to interact and phosphorylate H2AX in Ser139 after DNA Damage induced by irradiation, and also phosphorylates histone H3 in Thr3. In this work we have studied the role of human VRK1 Ser-Thr in the control of DNA damage response to IR and the role of specific acetylation and methylation of histones. We have shown that VRK1 is necessary for a proper formation of 53BP1 and γH2AX foci formation at double strand-breaks induced by ionizing radiation in and ATM-independent manner. DSB imply a local relaxation of chromatin and therefore it is likely that it might alter the modifications of histones. Fist we tested if acetylation is required for formation of DSB foci, and whether these acetylations require VRK1. The depletion of VRK1 by siRNA i affected the acetylation of H3 in Lys14 and Lys9 and also the H4 acetylation in Lys16, important markers of transcriptional activation. In contrast, siVRK1 enhanced the H3 methylation in Lys9 (a gene repressor marker) but not in Lys4 (transcriptional activator residue), confirming the role of VRK1 in positively mediating gene transcription. Also we have determined if manipulation of VRK1 levels can alter the tumour cell sensitivity to drugs currently used in clinical trials that target histone acetylation or methylation. Pharmacological inhibition of histone acetyl transferases (HATs) or VRK1 depletion did not rescue H3K9 and H4K16 acetylations, indicating that VRK1 is a regulator of HATs. We conclude that VRK1 regulates epigenetic mechanisms and has a role in chromatin condensation.Peer Reviewe