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This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License., 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 gH2AX 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., DMM and M S-G were supported by JAE-CSIC-Fondo Social Europeo fellowships. DSM was supported by a Ministerio de Ciencia e Innovación-FPI predoctoral fellowship. This work was funded by grants from Ministerio de Econom ıa y Competitividad (SAF2010-14935, SAF2013-44810R, SAF2014-57791-REDC) and Consejerıa de Educacion de la Junta de Castilla y Leon (CSI002U14) to PAL.

VRK1 is a nuclear chromatin Ser-Thr kinase functionally associated with different processes that require chromatin remodeling. The VRK1 activity is regulated by entry in cell cycle, and this kinase is also required for nuclear envelope dynamics, chromatin condensation, and Golgi fragmentation. Among its substrates, there are several transcription factors: p53, c-Jun, ATF2, CREB1, and SOX2. VRK1 probably acts cooperating with other signaling pathways that also phosphorylate these transcription factors. VRK1 stabilizes p53 by a specific phosphorylation in Thr18 and after induction of p53 responses, there is a VRK1 downregulation in the lysosome, in which the autophagic pathway participates in a p53-dependent manner. VRK1 also contributes to chromatin condensation, nuclear envelop kinetics by phosphorylation of histone H3 and BAF, and the regulation of Cajal bodies by phosphorylation of coilin. In DNA-damage responses, VRK1 actively participates in chromatin remodeling and regulates H2AX, NBS1, and 53BP1. VRK1 is downstream of MEK1 and Plk3 in the induction of Golgi fragmentation during mitosis. Furthermore, VRK1 is expressed in most tissues, is necessary in the early G1 phase for entry in cell cycle, and is associated with proliferation markers.

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.

Under a Creative Commons license., 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., D.M.M, M. S-G and L. C. were supported by JAE-CSIC-Fondo Social Europeo fellowships. I. C-M. was supported by Ministerio de Economía y Competitividad-FPI-Fondo Social Europeo fellowship. This work was funded by grants from Ministerio de Economía y Competitividad (SAF2013-44810R; SAF2014-57791-REDC) and Consejería de Educación de la Junta de Castilla y León (CSI002U14 and UIC-017) to P.A.L.

Mice with thyroid-specific expression of oncogenic BRAF (Tg-Braf) develop papillary thyroid cancers (PTCs) that are locally invasive and have well-defined foci of poorly differentiated thyroid carcinoma (PDTC). To investigate the PTC-PDTC progression, we performed a microarray analysis using RNA from paired samples of PDTC and PTC collected from the same animals by laser capture microdissection. Analysis of eight paired samples revealed a profound deregulation of genes involved in cell adhesion and intracellular junctions, with changes consistent with an epithelial-mesenchymal transition (EMT). This was confirmed by immunohistochemistry, as vimentin expression was increased and E-cadherin lost in PDTC compared with adjacent PTC. Moreover, PDTC stained positively for phospho-Smad2, suggesting a role for transforming growth factor (TGF)β in mediating this process. Accordingly, TGFβ-induced EMT in primary cultures of thyroid cells from Tg-Braf mice, whereas wild-type thyroid cells retained their epithelial features. TGFβ-induced Smad2 phosphorylation, transcriptional activity and induction of EMT required mitogen-activated protein kinase (MAPK) pathway activation in Tg-Braf thyrocytes. Hence, tumor initiation by oncogenic BRAF renders thyroid cells susceptible to TGFβ-induced EMT, through a MAPK-dependent process.

Activating mutations in RAS genes and p21 Ras overactivation are common occurrences in a variety of human tumors. p21 Ras oncoproteins deregulate a number of signaling pathways, dedifferentiating the thyroid cell, and negatively regulating the expression of thyroid specific genes. In rat thyroid cells, Ras oncoproteins inhibit the TSH pathway by reducing PKA activity and thus the expression of thyroid specific genes, while in mouse melanocytes, Ras oncoproteins reduce the αMSH-stimulated cAMP signaling by increasing the expression of the phosphodiesterase-4B. Given these cell-dependent differences, we investigated if and how the TSH/CREB pathway is modulated by Ras oncoprotein in a human thyroid cell line. CREB phosphorylation was stimulated by TSH and forskolin in TAD-2 cells. Ras(V12) expression negatively regulated the TSH-stimulated CREB phosphorylation but was ineffective on forskolin-stimulated CREB phosphorylation. Phosphodiesterase inhibition by IBMX enhanced TSH-stimulated CREB phosphorylation, but did not restore TSH-stimulated CREB phosphorylation inhibited by Ras oncoprotein. These data indicate that Ras oncoprotein disrupts the TSH/CREB pathway, upstream adenylyl cyclase, and highlight the existence of mechanisms of interaction between Ras and the cAMP pathway different in human and in rat thyroid cells.

Purpose: Calcium/calmodulin-dependent kinase II (CaMKII) is involved in the regulation of cell proliferation. Its endogenous inhibitor (hCaKIINα) is expressed in some cell types. We determined the role of CaMKII in RET-stimulated proliferation and hCaMKIINα in medullary thyroid carcinoma (MTC). Experimental Design: We analyzed the role of RET mutants on CaMKII activation in NIH3T3 and in MTC cell lines, and determined the effect of CaMKII inhibition on RET/ERK pathway and cell proliferation. Then the expression of hCaKIINα mRNA was determined by real-time PCR in primary MTC and it was correlated with some clinicopathologic parameters. Results: RET C634Y and RETM918T mutants expressed in NIH3T3 cells induced CaMKII activation. CaMKII was activated in unstimulated MTC cells carrying the same RET mutants and it was inhibited by RET inhibition. Inhibition of CaMKII in these cells induced a reduction of Raf-1, MEK, and ERK phosphorylation, cyclin D expression, and cell proliferation. hCaKIINα mRNA expression in primary MTC was very variable and did not correlate with gender and age at diagnosis. Serum calcitonin, (R2 = 0.032; P = 0.017), tumor volume (P = 0.0079), lymph node metastasis (P = 0.033), and staging (P = 0.0652) were negatively correlated with the hCaKIINα mRNA expression. Conclusions: CaMKII is activated by RET mutants and is activated at baseline in MTC cells where it mediates the oncogenic pathway leading to cell proliferation. The mRNA expression of its endogenous inhibitor hCaKIINα inversely correlates with the severity of MTC. CaMKII might represent a new target for MTC therapy and hCaKIINα is a marker of disease extension. Clin Cancer Res; 20(6); 1513–20. ©2014 AACR.

The calcium/calmodulin-dependent kinase II (CaMKII) participates with Ras to Raf-1 activation, and it is necessary for activation of the extracellular signal-regulated kinase (ERK) by different factors in epithelial and mesenchimal cells. Raf-1 activation is a complex multistep process, and its maximal activation is achieved by phosphorylation at Y341 by Src and at S338 by other kinase/s. Although early data proposed the involvement of p21-activated kinase 3 (Pak3), the kinase phosphorylating S338 remains to be definitively identified. In this study, we verified the hypothesis that CaMKII phosphorylates Raf-1 at Ser338. To do so, we determined the role of CaMKII in Raf-1 and ERK activation by oncogenic Ras and other factors. Serum, fibronectin, Src (Y527) and Ras (V12) activated CaMKII and ERK, at different extents. The inhibition of CaMKII attenuated Raf-1 and ERK activation by all these factors. CaMKII was also necessary for the phosphorylation of Raf-1 at S338 by serum, fibronectin and Ras. Conversely, inhibition of Pak3 activation by blocking phosphatidylinositol 3-kinase was ineffective. The direct phosphorylation of S338 Raf-1 by CaMKII was demonstrated in vitro by interaction of purified kinases. These results demonstrate that Ras activates CaMKII, which, in turn, phosphorylates Raf-1 at S338 and participates in ERK activation upon different stimuli.

Hashimoto's thyroiditis is the most frequent autoimmune disorder, characterized by the presence of a large lymphocytic infiltration and secretion of inflammatory cytokines in the thyroid. Infiltrating lymphocytes and cytokines play a pivotal role in the progression of HT, characterized by the progressive destruction of the normal follicular architecture of the gland and death of follicular cells, ending with loss of thyroid function. Integrins are plasma membrane receptors for the cell–extra-cellular matrix components, with both structural and signaling functions. Integrin-mediated fibronectin (FN) binding is necessary for the correct function and survival of thyroid follicular cells. The purpose of this study was to determine the effect of interferon-γ (IFN-γ) stimulation on integrin expression and signaling in the thyroid cell. Cytotoxicity, integrin expression, cell adhesion to FN, and FN-stimulated ERK and AKT phosphorylation were determined in a normal human thyroid cell line treated with IFN-γ. IFN-γ induced apoptosis and reduced the expression of the integrin αvβ3. Integrin-mediated cell adhesion to FN was strongly impaired. Similarly, FN-stimulated ERK and AKT phosphorylation were inhibited. In conclusion, our study in a thyroid cell model demonstrates that IFN-γ induces apoptosis and inhibits the expression of the integrin αvβ3, reducing cell adhesion to FN and the succeeding outside-in signaling. These results suggest that integrins mediate the cytotoxic effect of IFN-γ and are involved in the destructive mechanism of autoimmune thyroiditis.

RET/papillary thyroid carcinoma (PTC), TRK-T, or activating mutations of Ras and BRaf are frequent genetic alterations in PTC, all leading to the activation of the extracellular-regulated kinase (Erk) cascade. The aim of this study was to investigate the role of calmodulin-dependent kinase II (CaMKII) in the signal transduction leading to Erk activation in PTC cells. In normal thyroid cells, CaMKII and Erk were in the inactive form in the absence of stimulation. In primary PTC cultures and in PTC cell lines harboring the oncogenes RET/PTC-1 or BRafV600E, CaMKII was active also in the absence of any stimulation. Inhibition of calmodulin or phospholipase C (PLC) attenuated the level of CaMKII activation. Expression of recombinant RET/PTC-3, BRafV600E, or RasV12 induced CaMKII activation. Inhibition of CaMKII attenuated Erk activation and DNA synthesis in thyroid papillary carcinoma (TPC-1), a cell line harboring RET/PTC-1, suggesting that CaMKII is a component of the Erk signal cascade in this cell line. In conclusion, PTCs contain an active PLC/Ca2+/calmodulin-dependent signal inducing constitutive activation of CaMKII. This kinase is activated by BRafV600E, oncogenic Ras, and by RET/PTC. CaMKII participates to the activation of the Erk pathway by oncogenic Ras and RET/PTC and contributes to their signal output, thus modulating tumor cell proliferation.