VRK1 and AURKB form a complex that cross inhibit their kinase activity and the phosphorylation of histone H3 in the progression of mitosis.

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.

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

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.

VRK1 interacts with p53 forming a basal complex that is activated by UV-induced DNA damage.

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.

Preoperative serum CA 72.4 as prognostic factor of recurrence and death, especially at TNM stage II, for colorectal cancer.

BACKGROUND: Nowadays, evaluation of colorectal cancer prognosis and decision-making for treatment continues to be based primarily on TNM tumour stage. Administration of adjuvant chemotherapy is especially challenging for stage II patients that can have very different disease-related outcomes. Therefore, more reliable prognostic markers need to be developed to improve the selection of stage II patients at high risk for recurrence. Our purpose is to assess the prognostic value of preoperative serum CA 72.4 to improve the risk stratification of CRC patients. METHODS: Preoperative sera collected from 71 unselected patients between January 1994 and February 1997 was assayed for CA 72.4 and CEA levels. Patients were followed-up for at least 30 months or until relapse. Survival curves were estimated by the Kaplan-Meier method and the prognostic value was determined using Log-Rank test and Cox regression analysis. RESULTS: Preoperative CA 72.4 levels above 7 U/mL correlate with a worse prognosis, with associated recurrence and death percentages exceeding the displayed by CEA. In a multivariate analysis, its combination with CEA proved the most important independent factor predicting survival. Remarkably, at stage II CA 72.4 also discriminates better than CEA those patients that will relapse or die from those with a favourable prognosis; however, CEA has not a negligible effect on survival. CONCLUSIONS: The most outstanding finding of the present work is the correct classification of nearly every patient with bad prognosis (relapse or death) at TNM stage II when CEA and CA 72.4 are used altogether. This could improve the decision-making involved in the treatment of stage II colon cancer. Certainly further large-scale studies must be performed to determine whether CA 72.4 can be effectively used in the clinical setting.

VRK2 identifies a subgroup of primary high-grade astrocytomas with a better prognosis.

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.

Sensitivity of the kinase activity of human vaccinia-related kinase proteins to toxic metals.

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.

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.

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.

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.