Mammalian VRK (vaccinia-related kinases) were originally identified as two human EST, VRK1 and VRK2, expressed in proliferating cell lines and regenerating liver. These EST have homology to the catalytic region of the B1R kinase of vaccinia virus (Nezu et al. 1997) that is expressed early in viral infection and is required for viral DNA replication. These VRK proteins were classified as a new group of Ser-Thr kinases in the human kinome diverging very early from the same branch that led to casein kinase I (CK1) (Manning et al. 2002). This family has only one ortholog gene in invertebrates, such as C. elegans and Drosophila melanogaster, and is not present in yeast (Klerkx et al. 2009).
VRK2 Gene and Expression
The human VRK2 gene is located in chromosome 2p16.1 and generates two messages by alternative splicing. Their translation generates two protein isoforms, VRK2A (also known as VRK2) and VRK2B. The VRK2A corresponds to the full-length protein and is expressed from a message of 1833 nucleotides coding for a protein of 508 amino acids. The alternatively spliced variant includes a novel exon and generates a message of 1877 nucleotides. The additional exon has 44 nucleotides and is the new exon 13. The message for isoform B has an in-frame termination codon, generating a shorter protein of 397 amino acids that is truncated and lacks the C-terminal region that is present in isoform A (Nichols and Traktman 2004; Blanco et al. 2006). Isoform VRK2B detected is dispersed in cytoplasm and nuclei. VRK2B is likely to compensate nuclear VRK1 functions in those cells with low levels of VRK1 protein (Blanco et al. 2006), since both have similar substrate specificity (Sanz-Garcia et al. 2011). The murine VRK2 is similar to the human gene, but no alternatively spliced coding variant has been identified (Nichols and Traktman 2004).
Human VRK2 RNA is detected in all tissues, and the level is higher in those with high proliferation activity such as tumor cell lines. Fetal tissues express higher levels than adult organs (Nezu et al. 1997; Nichols and Traktman 2004).VRK2A has been detected in all cell types examined, whereas VRK2B has only been detected in cells in which VRK1 expressed at low levels or is cytoplasmic. Therefore, nuclear VRK2B might be functionally redundant with VRK1 in some roles not yet identified (Blanco et al. 2006).
During hematopoietic development in murine embryos, the highest level of VRK2 expression is achieved at days E11.5–E13.5, in thymus, spleen, and liver, at a time of massive hematopoietic-liver expansion (Vega et al. 2003). Expression increases again at day 17.5, just before delivery. In adult mice, high RNA levels are present in liver, kidney, and muscle (Vega et al. 2003).
VRK2 Protein Structure and Subcellular Localization
The human VRK2A protein is located in the endoplasmic reticulum (ER), partly in the perinuclear area (Nichols and Traktman 2004). The VRK2A isoform has a hydrophobic membrane-anchoring domain that is not present in isoform VRK2B. VRK2A localizes to endoplasmic reticulum (ER) membranes and colocalizes with calnexin and calreticulin, two ER markers, and is the isoform present in all cell types (Nichols and Traktman 2004; Blanco et al. 2006). VRK2A also colocalizes with mitochondria, as detected by its colocalization with the Mitotracker reagent (Blanco et al. 2006). VRK2B lacks the C-terminal region and its transmembrane hydrophobic tail, thus it is not bound to membranes. Thus VRK2B isoform is detected as granular deposits in both cytoplasm and nucleus, and its expression is restricted to some cells by unknown reasons, but it is a regulated expression (Blanco et al. 2006).
VRK2 Phosphorylation Substrates and Protein Interactions
The two VRK2 isoforms, which differ in their subcellular location, seem to have similar substrate specificity in vitro. VRK2A and VRK2 kinase activity have a similar specificity in the phosphorylation of peptides in vitro. The two VRK2 isoforms prefer basic sequences and their pattern of phosphorylation is similar to that of VRK1 (Sanz-Garcia et al. 2011). Therefore, in vivo specificity is likely to be determined by subcellular localization of the kinase and local substrate availability.
Both VRK2A and VRK2B phosphorylate human TP53 at Thr18 (equivalent to mouse Thr21) (Blanco et al. 2006). It is not known if the mitochondrial VRK2A phosphorylates mitochondrial p53.
VRK2A both murine and human as well as VRK1 are able to phosphorylate the N-terminal region of BAF, a small protein that interacts with DNA, and is required for nuclear envelope assembly. Phosphorylation of BAF by VRK1 results in loss of binding to DNA and inhibits nuclear envelope assembly (Nichols et al. 2006). VRK2, like VRK1, is also able to phosphorylate the amino terminus of BAF (barrier to autointegration factor) protein, which is required for assembly of the nuclear envelope, and also causes the loss of BAF binding to DNA (Nichols et al. 2006). The unique C. elegans ortholog for the VRK family, Vrk1–1, also phosphorylates Baf (Gorjanacz et al. 2008). This BAF phosphorylation impairs the formation of retroviral MoMLV (Moloney-murine leukemia virus) integration complexes (Suzuki et al. 2010).
VRK2A phosphorylates the JIP1 scaffold protein, but its significance is unknown since its effects on MAPK signaling are independent of phosphorylation and only requires the protein interaction (Blanco et al. 2007; Blanco et al. 2008).
The autophosphorylation activity of both VRK2 proteins can be inhibited in vitro by a stable interaction between the small nuclear GTPase Ran in a nucleotide-free or GDP-bound conformation. The GTP-bound form of Ran has no apparent effect on VRK2 kinase activity (Sanz-Garcia et al. 2008). The asymmetric distribution of RAN-GDP and RAN-GTP in cells might thus reflect a differential kinase activity depending on their localization. VRK2A has a membrane targeting domain that allows VRK2A to localize to intracellular membranes in the endoplasmic reticulum and mitochondria. VRK2B lacks this domain.
VRK2A also directly interacts with the scaffold KSR1, implicated in signal transmission from EGF, ERBB2, KRAS, and BRAF signals that activate AP1-dependent transcription (Fernandez et al. 2010). In this complex VRK2 also directly interacts with MEK1 (Fernandez et al. 2010).
The C-terminal region of VRK2A interacts with an Epstein-Barr Virus (EBV) protein, BHRF1, which is a homolog of BCL2 and enhances cell survival, but no interaction with human BCL2 has been detected (Li et al. 2006).
VRK2 Modulates MAPK Signaling
VRK2A isoform modulates signaling mediated by MAPK pathways. This action is mediated by a direct interaction between a scaffold protein and VRK2A, and does not depend on its kinase activity. Stress responses, such as hypoxia (Blanco et al. 2007), and response to inflammatory cytokines, such as interleukin-1β (IL-1β) (Blanco et al. 2008), mediate signals by the activation of Jun dependent transcription. These responses are inhibited by VRK2A which forms a macromolecular complex, signalosome, which is anchored to endoplasmic reticulum membranes. In this complex VRK2A interacts with TAK1, the first MAP kinase (MAPKKK) and the JIP1 scaffold protein (Blanco et al. 2007; Blanco et al. 2008). This complex prevents the incorporation of JNK (c-Jun N-terminal kinase), which cannot be activated by phosphorylation, inhibiting c-Jun dependent transcription. The amount of VRK2A in the cells seems to control the fraction of JIP1 free and bound, thus creating a compartmentalization with two complexes, one active without VRK2A, and another inactive with VRK2 (Fig. 2).
Many proliferative responses mediated by oncogenes are transmitted by MAPK complexes resulting in the activation of RAF-MEK-ERK pathway, which is assembled on the KSR1 scaffold protein (Fig. 2). Among the activated oncogenes that can be modulated by VRK2A are ERBB2, RAS, and RAF. VRK2A directly interacts with MEK1 and KSR1 preventing the activation of ERK1 and p90RSK (Fernandez et al. 2010). VRK2A does not affect signals that partly use this pathway, such as AKT. In this way, VRK2A inhibits at a downstream level the signal initiated in ERBB2, which is amplified in many tumors types. Also reduced level of P-ERK was observed in the presence of VRK2, and VRK3, in the analysis of the EGFR response pathway (Komurov et al. 2010).
VRK2 Regulates Cell Invasion by Controlling Cox2 Expression
VRK2 can control tumor cell invasion in part by regulating the gene expression of COX2 (cyclooxygenase-2). This effect of VRK2 on cell invasiveness is mediated by a direct phosphorylation of the NFAT1 (nuclear factor of activated T cells) transcription factor in Ser-32 by VRK2. The COX2 gene proximal promoter ha an NFAT1 target sequence (Vazquez-Cedeira and Lazo 2012). The treatment of tumor cells with phorbol esters that induce a similar phosphorylation of NFAT1 and activation of COX2 gene expression that facilitates cell invasion (Vazquez-Cedeira and Lazo 2012). Knockdown of vrk2 reduces invasion by MDA-MB-231 and MDA-MB-435 tumors cells induced by phorbol esters and ionomycin treatment (Vazquez-Cedeira and Lazo 2012).
VRK2 Regulates the Intrinsic Pathway of Apoptosis
Epstein-Barr virus (EBV) expresses the BHRF1 protein that is a homolog of mammalian Bcl2, which has a protective role and favors cell survival. BHRF1 interacts with the C-terminal region of VRK2A, and if VRK2A levels are high, cells are protected against apoptosis, and this protection depends on the interaction and not on the kinase activity of VRK2 (Li et al. 2006).
VRK2 Controls Chaperonins
Chaperonins are proteins controlling the correct folding of proteins and thus prevent pathological protein aggregation that causes neurodegenerative diseases. The chaperonin TRiC is an important regulator in the elimination of misfolded proteins. TRiC protein level is downregulated by VRK2 through the proteasome by recruiting the E3 ligase COP1 (Kim et al. 2014). In this context VRK2 phosphorylates the USP25, a deubiquitinase that interacts with TRiC, in Thr680, Thr727, and Ser745 residues causing the inactivation of USP25 deubiquitinase activity (Kim et al. 2015) and thus permitting protein degradation. By this mechanism, VRK2 can regulate the accumulation of neurpathogenic proteins with poly-Q expansions (>40) that form intracellular aggregates. High levels of VRK2 facilitate degradation of proteins such as huntingtin and thus reduces the formation of its poly-Q aggregates; and VRK2 depletion facilitates formation of huntingtin poly-Q aggregates (Kim et al. 2015). It is possible that a reduction of intracellular poly-Q aggregates in cells might be achieved if specific VRK2 inhibitors are designed. This will represent a leap forward in the control of neurodegenerative diseases associated to intracellular poly-Q aggregation.
VRK2 in Human Cancer: Breast Cancer and Glioblastomas
VRK2 levels in human breast cancer correlated positively with estrogen and progesterone receptors. VRK2 was inversely correlated with ErbB2, thus Erb2+ breast cancer have low levels of VRK2. VRK2 can block the signal mediated by MAPK in response to EGF or its receptors (ERBB family), as well as signals from mutated RAS or BRAF. By reducing VRK2 levels, the inhibition of MAPK pathway transcriptional signaling is partly removed in these tumors (Fernandez et al. 2010). This downstream inhibitory effect of VRK2A on MAPK can also be occurring in other tumors with mutations along this signaling pathway, but this needs to be demonstrated.
Expression of VRK2 has been studied in human primary astrocytomas (Rodriguez-Hernandez et al. 2013). High expression of VRK2, as well as VRK1, correlated with proliferation markers p63 or Ki67, but there was no correlation with p53. High levels of VRK2 identified a subgroup of high-grade astrocytomas that presented an improvement in survival using Kaplan-Meier and Cox models (Rodriguez-Hernandez et al. 2013). Overexpression of VRK2 in astrocytomas cell lines resulted in a reduction of its growth (Rodriguez-Hernandez et al. 2013). Thus the better prognosis of some astrocytomas might be a consequence of the downregulation of MAPK signaling by VRK2. Also high levels of VRK2 have been reported in colorectal adenocarcinomas (Hennig et al. 2012).
VRK2 in Viral Infections
VRK2 received its name from the homology of its catalytic domain and that of the unique vaccinia virus kinase B1, which is required for Vaccinia virus DNA replication early in infection. Despite their divergence, VRK2 and VRK1 are able to partially rescue the replication of vaccinia virus strains with defective B1 kinase (Boyle and Traktman 2004).
Targeting VRK2 with Kinase Inhibitors
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 signaling by mitogen activated protein kinases (MAPK). The specific structural differences in VRK catalytic sites make them suitable candidates for 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 the low sensitivity, some of these inhibitors in the low micromolar range are able to discriminate between VRK1 and VRK2. VRK2 is more sensitive to roscovitine, RO 31-8220, Cdk1 inhibitor, AZD7762, and IC261. VRK1 is more sensitive to staurosporine, RO 31-8220 and TDZD8 (Vazquez-Cedeira et al. 2011). 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).
VRK2 in Neurological and Psychiatric Diseases
Several polymorphisms with the VRK2 loci on chromosome 2p16.1, but outside the coding region, have been identified in several studies as associated to predisposition several neurological and psychiatric disorders such as epilepsy (Epilepsies. ILARCoC 2014), bipolar disorders (Kerner et al. 2013), and schizophrenia (Irish Schizophrenia Genomics Consortium and the Wellcome Trust Case Control Consortium 2012; Tesli et al. 2016). The role of VRK2 as regulator MAPK signaling pathways might explain this VRK2 role, since MAPK pathways are targets of drugs used in these diseases such as valproic acid, lithium, or antidepressants.
VRK2 is a Ser-Thr kinase and has two isoforms, cytosolic and nuclear. The full-length VRK2 cytosolic isoform (A) inhibits MAPK signaling by a direct interaction with the corresponding JIP1 or KSR1 scaffold proteins. As a consequence high levels of VRK2A inhibits in a dose dependent manner signals mediated by the TAK1-MKK7-JNK in response to hypoxia or interleukin, and the signal of the ERBB2-RAS-RAF-MEK-ERK pathway. Downregulation of VRK2A relieves this inhibition and allows signal transmission in response to stimuli initiated in Receptor-tyrosine kinases (RTK), such as ERBB2. Thus, in breast cancer, VRK2A and ERBB2 present an inverse correlation. VRK2 controls protein quality by regulating degradation of misfolded proteins. Mitochondrial VRK2 controls the association of Bax to mitochondrial membranes and thus modulates apoptosis. Allelic noncoding polymorphisms of the VRK2 gene have been linked to several neurological diseases.
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