VRK proteins were identified as two human EST sequences whose translation products have homology with the kinase domain of the unique vaccinia virus B1 kinase, from where its name is derived. During its evolution and adaptation to its host, the virus incorporated the cellular sequence required for viral life-cycle regulation. VRK1 is a chromatin kinase that regulates multiple processes, reflecting its late appearance in evolution as coordinator of preexisting functions in higher eukaryotes.
VRK1 Gene Structure and Expression
The human VRK1 gene is located on the chromosomal region 14q32.2, contains 12 exons and codes for a protein with 396 amino acids (Nichols and Traktman 2004). This gene has a polymorphism, marker rs722869, which is useful in the identification of population structure and genetic ancestry (Lao et al. 2006). Chromosomal translocations in this region do not affect VRK1. This kinase is not mutated in human cancer, although very infrequent point mutations have been detected scattered throughout the sequence. Recessive hereditary point mutations scattered throughout the sequence have been identified in very rare neurological diseases, and affected individuals are either homozygous or compound heterozygous. The murine VRK1 gene has 14 exons, coding for a protein with 440 aminoacids, and also express and alternatively splice message without exon 12. The human gene does not have any sequence corresponding to murine exons 12 and 13 (Nichols and Traktman 2004).
VRK1 is expressed in all types of human and murine cells tested (Nichols and Traktman 2004), particularly at very high levels if they are proliferating (Nezu et al. 1997; Santos et al. 2006). In murine embryonic hematopoietic development, the highest level of expression is achieved between days E11.5 and E13.5, a time of massive hematopoietic-liver expansion (Vega et al. 2003).
Depletion of VRK1 causes a block in cell cycle progression, since VRK1 is an early response gene following stimulation by growth factors and is necessary for G0 exit (Valbuena et al. 2008b) and G2/M transition (Kang et al. 2007). VRK1 gene-trap mice, which have a 15% residual level, are infertile because of loss of spermatogonia (Wiebe et al. 2010) and oocytes (Kim et al. 2012a).
VRK1 Protein Structure and Subcellular Localization
VRK (vaccinia-related kinases) form a branch of Ser-Thr kinases in the human kinome, which diverged very early from casein kinases (Manning et al. 2002). These kinases have homology to the catalytic domain of the B1R kinase of vaccinia virus that is required for viral DNA replication (Nichols and Traktman 2004). In yeast there is no orthologous gene, and in invertebrates D. melanogaster and C. elegans, there is only one VRK protein (Klerkx et al. 2009). In mammals there are three members, two of which (VRK1 and VRK2) are catalytically active (Lopez-Borges and Lazo 2000; Nichols and Traktman 2004), and a third one, VRK3, which is not active (Kang and Kim 2006), but can be activated by protein interactions (Park et al. 2015). The VRK catalytic domain has substitutions in several key residues making it an atypical kinase (Nichols and Traktman 2004). The relative conservation of the catalytic domain of VRK proteins indicates they can have an overlap of potential substrates in vitro, and the in vivo substrates are likely to be determined by subcellular localization.
The three dimensional structure of the VRK1 catalytic domain indicates it has a typical kinase domain, including the hinge region, catalytic loop, and DYG motif with some substitutions (PDB:3OP5). The structure of the C-terminus (PDV:2ALV), determined by NMR, has revealed that it has alternative conformations that are important for the structural stability and activity (Shin et al. 2011). The C-terminal region comprised between residues 300 and 355, up to the NLS (nuclear localization signal) can fold and inhibit the catalytic activity (Shin et al. 2011). The C-terminal region is modified by phosphorylation or protein interactions. In this way, some of the residues in the C-terminal region can interact with different ligands (Shin et al. 2011) and several proteins that interact with it can inhibit the VRK1 kinase activity, such as Ran-GDP (Sanz-Garcia et al. 2008) and macroH2A1.2 (Kim et al. 2012b).
Furthermore, VRK1 interacts differently with unrelated proteins. The N-terminus interacts with coilin (Cantarero et al. 2015) and Plk3 (Lopez-Sanchez et al. 2009); the C-terminus with p53 (Lopez-Sanchez et al. 2014); and both regions with NBS1 (Monsalve et al. 2016) and Sox2 (Moura et al. 2016).
VRK1 Is a Nucleosomal Chromatin Kinase
VRK1 is a protein mostly located within the nucleus, and outside the nucleolus (Lopez-Borges and Lazo 2000; Vega et al. 2004), but the major fraction is interacting with chromatin (Kang et al. 2007; Salzano et al. 2015). In D. melanogaster, its ortholog is NHK-1 (nucleosomal histone kinase-1) (Cullen et al. 2005). In some cell types, particularly in epithelial cells, VRK1 is also present in the cytosol (Valbuena et al. 2007a), and there is a minor cytoplasmic subpopulation located in the Golgi apparatus (Lopez-Sanchez et al. 2009). However, the regulation of VRK1 subcellular localization is not known.
Within nuclei, VRK1 presents a granular pattern and is bound to chromatin. The VRK1 binding to chromatin presents two subpopulations, one associated with transcription factors and regulatory proteins (Valbuena et al. 2011b), and another forming stable complexes with histones (Salzano et al. 2015).
VRK1 participates in the regulation of telomere maintenance by phosphorylating hnRNPA1, a single-strand oligonucleotide binding protein, and facilitating its binding to telomeric ssDNA and telomerase RNA. Depletion of VRK1 causes a shortening of telomere in male germ cells and triggers a DNA-damage response (Choi et al. 2012).
VRK1 phosphorylates histone H3 in Thr3 and Ser10 inducing chromatin condensation in mitosis (Kang et al. 2007) (Sanz-Garcia et al. 2008). VRK1 protein level is higher in mitosis and is detected in chromatin, where it regulates the interaction of H3 with gamma heterochromatin protein 1 (HP1γ) (Kang et al. 2007). VRK1 is also able to form a stable and abundant complex with several histones (Salzano et al. 2015). Moreover, in interphase, VRK1 forms a complex with macroH2A1.2 that inhibits the kinase activity of VRK1 (Kim et al. 2012b) and promotes cell differentiation (Moura et al. 2016).
VRK1 Role in Nuclear Dynamics
VRK1 regulates several proteins implicated with nuclear processes, including chromatin organization, nuclear membrane assembly, and Cajal bodies formation, all of them dynamically reorganized during cell division.
VRK1 phosphorylates BAF in Ser4 and Thr2/Thr3 (Nichols et al. 2006). BAF is a protein regulating assembly and disassembly of the nuclear envelope (Nichols et al. 2006). Depletion of VRK1 causes the retention of BAF at the nuclear envelope and contributes to the aberrant structure of the nuclear envelope by preventing the role of binding partners that participate in NE disassembly (Molitor and Traktman 2014). The loss of VRK1 also prevents the interaction of BAF with chromosomes and alters BAF-chromosome interactions required for proper mitotic progression generating anaphase bridges and multipolar spindles (Molitor and Traktman 2014). Moreover, BAF phosphorylation by VRK1 can inhibit the preintegration complex of retroviruses in the chromosomes of host cells (Suzuki et al. 2010).
VRK1 interacts with RAN, the only nuclear small GTPase (Sanz-Garcia et al. 2008), which is implicated in nuclear transport and has an asymmetric distribution in the nucleus determined by its binding to either GDP or GTP. RAN is an allosteric regulator of VRK1 activity. RAN-GDP inhibits VRK1 kinase activity on substrates such as p53 and histone H3, and VRK1 is active when it is bound to RAN-GTP (Sanz-Garcia et al. 2008), which is localized proximal to the spindle in mitosis. This effect indicates that VRK1 activity in the nucleus is likely to be asymmetric despite its homogeneous distribution. In some cells, VRK1 is detected in the nucleolus, but its significance is unknown.
VRK1 Regulates Cajal Bodies in Cell Proliferation
Cajal bodies (CBs) are nuclear organelles associated with ribonucleoprotein functions and RNA maturation. CBs are assembled on coilin, its main scaffold protein, in a cell cycle-dependent manner. VRK1 interacts and phosphorylates coilin in at least eight residues regulating assembly of CBs (Sanz-Garcia et al. 2011). Coilin phosphorylation is not necessary for its interaction with VRK1, but it occurs in mitosis and regulates coilin stability (Cantarero et al. 2015). Knockdown of VRK1 or VRK1 inactivation by serum deprivation causes a loss of coilin phosphorylation in Ser184 and impairs CBs formation. The phosphorylation of coilin in Ser184 occurs during mitosis before the assembly of CBs. Loss of coilin phosphorylation in Ser184 results in disintegration of CBs, and of coilin degradation (Cantarero et al. 2015). Knockdown of VRK1 facilitates coilin ubiquitination in nuclei, which is partly mediated by hdm2/MDM2, but its proteasomal degradation occurs in cytosol and is prevented by blocking its nuclear export. VRK1 is a novel regulator of CBs dynamics and stability in cell cycle by protecting coilin from ubiquitination and degradation in the proteasome (Cantarero et al. 2015).
VRK1 Phosphorylation of Transcription Factors
The transcription factor ATF2 is phosphorylated in residues Ser62 and Thr73 by VRK1 (Sevilla et al. 2004b). These residues are in the same region, but are different from the Thr69 and Thr71 targeted by JNK. Thus they can have an additive effect even at maximum stimulation of VRK1 and JNK signaling pathways (Sevilla et al. 2004a). Furthermore, residues Ser62 and Thr72 are activated in response to cAMP signals mediated by PKA, which can cooperate with VRK1 in case of suboptimal stimulation. VRK1 phosphorylates CREB1 in Ser133 and is integrated in the transcriptional protein complex required for cyclin D1 (CCND1) gene expression (Kang et al. 2008), consistent with a VRK1 role in cell cycle entry and progression (Valbuena et al. 2008b).
VRK1 also forms a stable basal complex with p53, which upon DNA damage induces activation of the kinase and immediate phosphorylation of p53 in Thr18 (Lopez-Sanchez et al. 2014).
Sox2 is a pluripotency transcription factor that also forms a basal complex with VRK1 in different cell types and both colocalize in dividing cells within a stratified epithelium (Moura et al. 2016).
In the context of transcriptional regulation, VRK1 has been detected forming a complex with the p300 acetyl transferase, a transcriptional cofactor (Guermah et al. 2006), which is able to contribute to the protection of VRK1 by a p53-dependent degradation through DRAM (Valbuena et al. 2008a).
VRK1 Regulates Cell Cycle and p53
VRK1 phosphorylates p53 uniquely in Thr18 (Lopez-Borges and Lazo 2000). This phosphorylation prevents p53 interaction with HDM2 and favors its interaction with transcriptional cofactors. Thus, VRK1 induces a stabilization and accumulation of p53 that exerts its known biological effects (Vega et al. 2004). A persistent accumulation of p53 is deleterious for the cell. Therefore, it is necessary to downregulate p53, but this requires also removing its VRK1 stabilizer. The accumulation of p53 can be reverted by its downregulation, which requires the inactivation of its stabilizers mechanisms, such as phosphorylating kinases. P53 induces the expression of DRAM (damage-regulated autophagy modulator) that targets VRK1 for proteolytic degradation by autophagy (Valbuena et al. 2006; Valbuena et al. 2011a). Depletion of beclin1 (BECN1) also prevents VRK1 downregulation by autophagy (Valbuena et al. 2011a). By this mechanism, VRK1 protein is removed, and dephosphorylated p53 becomes available for proteasomal degradation. In tumors with p53 mutations, this autoregulatory loop is not induced and there is an accumulation of VRK1 (Valbuena et al. 2007b; Valbuena et al. 2011a). Thus, VRK1 appears to play a fundamental role allowing cells to respond to DNA damage and initiate protective responses (Vega et al. 2004).
VRK1 colocalizes with p63, Ki67, and Sox2 in proliferating cells in stratified squamous epithelium (Santos et al. 2006; Moura et al. 2016). VRK1 gene expression is regulated by entry in cell cycle after stimulation with serum and is expressed at the same time as MYC and FOS, two early genes and positively correlated with phospho-RB. Serum withdrawal results in loss of VRK1 gene expression (Valbuena et al. 2008b). VRK1 gene expression is activated by Sox2 and both positively cooperate in the regulation of cyclinD1 (CCND1) expression (Moura et al. 2016). VRK1 gene expression precedes that of CCND1 gene before the restriction point in G1 (Valbuena et al. 2008b). VRK1 protein forms part of the transcriptional complex of the CCND1 gene promoter (Kang et al. 2008). VRK1 knockdown prevents cell division (Vega et al. 2004), causes loss of RB phosphorylation, and induces accumulation of p27, suggesting a cell cycle blockade (Valbuena et al. 2008b). Depletion of VRK1 also reduces proliferation in human breast cancer cells resulting in a reduction of metastases formation in orthotopic xenograph models (Molitor and Traktman 2013).
In mitosis there is a fragmentation of the Golgi apparatus that is required for its distribution into daughter cells; this process is mainly induced by MEK1 and PLK3, VRK1 being a downstream step. The VRK1 subpopulation located in Golgi apparatus is an essential component of the MEK1-PLK3 signaling pathway required for Golgi fragmentation in mitosis, in which Plk3 directly phosphorylates VRK1 in Ser342 (Lopez-Sanchez et al. 2009).
VRK1 in DNA Damage Responses
VRK1 is a chromatin kinase that reacts to alterations in chromatin structure as a consequence of DNA damage. VRK1 kinase activity is induced tenfold by different types of DNA damage, such as pyrimidine dimers, single- or double-strand DNA breaks (Sanz-Garcia et al. 2012). The VRK1 activity is also regulated by histone interactions, and its activation induced by a locally altered chromatin, consequence of DNA damage. VRK1, as early participant in DDR, facilitates histones H3 and H4 acetylation and thus chromatin relaxation to permit the DNA repair processes (Salzano et al. 2015).
VRK1 participates as a very early component in DNA damage response by its direct interaction and phosphorylation of histone H2AX in Ser-139 (γH2AX) in response to ionizing radiation or doxorubicin (Salzano et al. 2015). γH2AX protects the damage site before the specific type of DNA damage and serves as platform for the recruitment of repair mechanisms. This activation of γH2AX by VRK1 is independent of the cell cycle, because it also occurs in arrested and nondividing cells. VRK1 is critical because under normal conditions DNA damage occurs independently of the individual cell situation regarding cell division and proliferations status, differentiation stage, or cell interactions that are heterogeneous within a tissue. This is particularly relevant for stem cells that are nondividing when DNA damage occurs, and therefore repair and cell division are not associated in time.
In cases of DNA double-strand breaks, there is a sensor mechanism that requires the participation of NBS1. NBS1 forms a preassembled basal complex with VRK1, and after DNA damage, NBS1 is phosphorylated by VRK1 in Ser343. This phosphorylation protects NBS1 from ubiquitination mediated by RNF8 and subsequent proteasomal degradation that facilitates the sequential steps in DNA repair processes (Monsalve et al. 2016). One of these steps in nondividing cells is the repair mediated by nonhomologous end joining (NHEJ), the main double-strand break repair system, which is mediated by 53BP1. After induction of double-strand breaks by gamma irradiation, VRK1 binds to 53BP1 and phosphorylates it in Ser25 (Sanz-Garcia et al. 2012). The phosphorylation and activation by VRK1 of these sequentially acting proteins in DDR, H2AX, NBS1, and 53BP1 is independent of ATM activation. VRK1 is functional in p53-/- or ATM-/- cell lines, and in arrested or nondividing cells. Therefore, VRK1 is a very early initiating kinase in DNA repair processes, and VRK1 depletion prevents the activating phosphorylation of ATM in Ser1981 and DNA-PK in Ser2056 (Sanz-Garcia et al. 2012; Salzano et al. 2015). Because of the role of VRK1 in DDR, the knockdown of VRK1 hypersensitizes tumor cells to treatment with either ionizing radiation or doxorubicin, and similar effects are achieved with significant reduction in treatment doses (Salzano et al. 2014). Moreover, high levels of VRK1 confer resistance to treatment, which is partly a reason for its association with a poorer prognosis in breast cancer (Finetti et al. 2008).
The cellular protection of cells in response to DNA damage is also mediated by the regulation of p53 by VRK1. In non-damaged cells, p53 forms a stable complex with VRK1 that after DNA damage is immediately phosphorylated in Thr18 and stabilized triggering p53-dependent cell-cycle arrest (Lopez-Sanchez et al. 2014).
VRK1 in Human Cancer
VRK1 is expressed at high level in most tumors, probably reflecting its association with proliferation, because this kinase positively correlates with cell cycle markers cdk2, p63, and survivin, as shown in head and neck squamous cell carcinomas (Santos et al. 2006). In lung cancer with p53 mutations, there are also very high levels of VRK1, reflecting the disruption of its downregulatory loop (Valbuena et al. 2007b). In breast cancer, VRK1 overexpression identifies a subgroup with poorer prognosis (Finetti et al. 2008). VRK1 is also overexpressed in colorectal adenocarcinomas (Hennig et al. 2012) and breast cancer (Finetti et al. 2008).
VRK1 in Human Neurological Diseases
Patients with rare diseases and complex clinical presentations represent a challenge for clinical diagnostics. Genomic approaches are allowing the identification of novel variants in genes for very rare disorders. Several human mutations indicate that VRK1 plays a complex role in neurological diseases.
Spinal muscular atrophy with pontocerebellar hypoplasia (SMA-PCH) is an infantile SMA variant with additional manifestations, particularly severe microcephaly. The homozygous mutation R358X, detected in two siblings from an Ashkenazi Jewish family, causes a syndrome characterized by pontocerebellar hypoplasia, spinal-muscular atrophy, mild mental retardation, and death in childhood (Renbaum et al. 2009). Heterozygous carriers of this mutation have no pathology. VRK1-R358X homozygosity results in lack of VRK1 protein and reduces neuronal migration in part by a reduction in levels of APP. This migration phenotype can be rescued by VRK1 or APP, suggesting VRK1 regulates neuronal migration by an APP-dependent mechanism (Vinograd-Byk et al. 2015).
Three patients from two unrelated families presented a complex neurological phenotype characterized by axonal sensorimotor neuropathy and microcephaly (Gonzaga-Jauregui et al. 2013). Whole exome sequencing detected compound heterozygous (V236M and R89Q) alleles in two affected siblings from one family. In an unrelated patient, a homozygous nonsense variant (R358X) in VRK1 was also detected in an Ashkenazi Jewish family, but the phenotype was partially different from the unrelated cases with the same mutation. An additional VRK1 mutation, R133C, was identified in a patient with cognitive disorder and pontocerebellar hypoplasia (Najmabadi et al. 2011).
Two different VRK1 mutations, H119R and R321C, were detected as the only genetic in a case of amyotrophic lateral sclerosis (ALS) that mainly affects lower limbs and without any known origin (Nguyen et al. 2015). Two additional mutations, G135R and L195V, have been identified in patients with new motor phenotypes (Stoll et al. 2016).
The VRK1 Signaling Network
Targeting VRK1 with Kinase Inhibitors
Human vaccinia-related kinases (VRK1 and VRK2) are atypical active Ser-Thr kinases implicated in control of cell cycle, proliferation, apoptosis, and autophagy. The structural differences in VRK catalytic sites make them suitable candidates for the development of specific inhibitors. These two kinases have been tested for their sensitivity to inhibitors targeting other kinases such as Src, MEK1, B-Raf, JNK, p38, CK1, ATM, CHK1/2, and DNA-PK and most of them have no effect even at 100 micromolar. But despite their low sensitivity, some of these inhibitors in the low micromolar range are able to discriminate between VRK1 and VRK2. VRK1 is more sensitive to staurosporine, RO-31-8220 and TDZD8, while VRK2 is more sensitive to roscovitine, RO 31-8220, Cdk1 inhibitor, AZD7762, and IC261. In both cases, some inhibition was achieved at doses unsuitable for clinical use. Thus, the two kinases present a different pattern of sensitivity to kinase inhibitors (Vazquez-Cedeira et al. 2011). This differential response to known inhibitors can provide a structural framework for VRK1 or VRK2 specific inhibitors with low or no cross-inhibition and high specificity (Fedorov et al. 2007). 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 VRK1 overexpression in breast cancer.
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.
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