(VRK) signaling in cell and tumor biology

VRK (vaccinia-related kinase) is a group of three proteins in the human kinome. These proteins, mainly VRK1 and VRK2, have been studied in the context of their substrates and interacting proteins in order to identify and characterize their signaling pathway, as well as their effect on other signaling pathways. VRK1 is mostly a nuclear kinase that specifically phosphorylates p53, c-Jun, ATF2, CREB, BAF and histone H3. VRK1 is an early response gene and is implicated in regulation of cell cycle progression. VRK1 is activated in response to DNA damage phosphorylating p53, which is stabilized and activated; this active p53 induces a downregulatory mechanism of VRK1 that permits the reversal of p53 induced effects. The activity of nuclear VRK1 is regulated by its interaction with the Ran small GTPase. Also, VRK1 is a downstream component of the signaling pathway of MEK-Plk3 that induces Golgi fragmentation in mitosis. VRK2 Correspondence/Reprint request: Dr. P.A. Lazo, IBMCC-Centro de Investigación del Cáncer, CSICUniversidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain E-mail: [email protected]; Marta Sanz-García et al. 136 has two isoforms; VRK2A is cytosolic and bound to endoplasmic reticulum and mitochondrial membranes. VRK2B is a shorter isoform free in cytosol and nucleus. VRK2A affects cellular signaling by interaction with scaffold proteins, as JIP1. The JIP1-VRK2A signalosome blocks the incorporation of JNK, preventing its activation, and thus reducing the stress response to inflammatory cytokines as interleukin-1β and to hypoxia. 1. The vaccinia-related kinase (VRK) family The completion of the human genome project has led to the identification of 518 proteins that constitute the human kinome [1]. Approximately half of them are not well characterized regarding the signaling pathways in which they are integrated, and their biological functions. But in most likelihood they can probably account for the specificity and regulation of many biological functions. VRK (vaccinia-related kinases) were classified as a new group of Ser-Thr kinases in the human kinome [1], and originally were identified as two human EST, VRK1 and VRK2, which have homology to catalytic region of the B1R kinase of vaccinia virus [2] that is expressed early in viral infection since it is required for viral DNA replication [3-5]. The VRK gene family appeared late in evolution. In lower eukaryotes, D. melanogaster (NHK-1) and C. elegans (Vrk1), there is only one member, and no ortholog has been identified in unicellular eukaryotes, like yeasts S. cerevisiae or S. pombe. In mammals there are three members, two of which (VRK1 and VRK2) are catalytically active [6-8], and a third one, VRK3, is not active and might function as a scaffold protein [9]. The VRK catalytic domain is distantly related to the casein kinase group, forming an early divergent branch in the human kinome [1], but the conservation of the kinase domain is weak and has substitutions in several key residues in this domain [10]. These proteins maintain the structure of the kinase domain, but the rest of the protein is very divergent among them, suggesting that the CK1-like catalytic domain recombined early in evolution with other proteins to form a divergent protein family with new regulatory properties, affecting interactions, regulation, and subcellular localization. In one of these recombination events the kinase domain was picked up by pox viruses (vaccinia, variola, smallpox) and generated the B1R protein, which is the only kinase in poxviruses genome; B1R is required for poxvirus replication and its inactivating mutations can be partially recovered by overexpression of mammalian VRK1, or VRK2 proteins, indicating that in poxvirus there was an additional functional divergence due to viral life requirement [11]. Structurally, the three VRK proteins differ in their regulatory region located C-terminal for VRK1 and VRK2 and N-terminal for VRK3. These regulatory regions are unrelated Vaccinia-related kinase (VRK) signaling in cell and tumor biology 137 among them and have no homology to any known feature identified in other proteins. The length of the regulatory region is variable. It is approximately one hundred amino acids in VRK1 and two hundred aminoacids in VRK2, thus their subcellular localization and regulation is likely to be different among mammalian VRK proteins and those of lower eukaryotes. The relative conservation of catalytic domain of VRK proteins indicates they can have an overlap of potential substrates. The catalytic domain of these kinases have some key substitutions which make them differ from other closely related kinases, nevertheless two of them VRK1 and VRK2 are catalytically active and have a 56 % sequence identity [6, 8, 12]. These differences might be exploited for the design of VRK specific inhibitors [13], which may be achieved by determination of their crystal structure. The crystal structure of the catalytic domain of VRK2 and VRK3, with 38 % sequence identity and without any significant difference, has been determined providing the framework for future inhibitor development [10]. Human VRK1 gene is located on the chromosomal region 14q32.2. This gene has a polymorphism, marker rs722869, that in combination with other nine polymorphisms in other genes has proven very useful and is included in the most informative set to study the identification of human continental population structure and genetic ancestry [14]. VRK1 gene expression has been studied in hematopoietic murine development, and its highest expression coincides with the time of maximum cellular expansion in days E11.5 to E13.5 [15]. Endogenous VRK1 protein is detected in different Figure 1. Structure of the three human VRK proteins. ATP: ATP binding site; KD: kinase domain; Plk: consensus target for Plk3. NLS: nuclear localization signal. BAB: Basic-acid-basic region. TM: transmembrane region. Marta Sanz-García et al. 138 intracellular compartments, suggesting that localization is regulated and that it might play different functional roles depending on its localization. VRK1 has a canonical nuclear localization signal (KKRKK) in residues 356 – 360, which accounts for its nuclear localization [6, 8]. In interphase the endogenous VRK1 has a granular aspect, probably forming aggregates, dispersed within the nucleus, and in mitosis it is dispersed throughout the cell and does not bind to chromatin. In some cell types the endogenous VRK1 protein is mostly cytosolic presenting a particulate appearance, and a minor fraction is nuclear [16], but the underlying cause is unknown. Also there is a subpopulation in the Golgi apparatus where VRK1 colocalizes with giantin where it participates in the control of Golgi fragmentation, and perhaps in VRK1 recycling [17]. This differential distribution is tissue specific [16]. These different locations of human VRK1 are also detected in human normal tissue biopsies. For example in testes and esophagus VRK1 is detected as nuclear with all antibodies. However in small intestine or kidney, a nuclear and a cytosolic subpopulation are detected depending on the antibody used [16]. The factors determining, and regulating, these different subcellular localizations are unknown. The human VRK2 gene has 14 exons and can generate two different transcripts by alternative splicing, one lacking exon 13 that contains a termination codon, encoding two proteins of 508 (VRK2A) or 397 (VRK2B) aminoacids respectively. The largest protein is called VRK2A, and the other is VRK2B [12], both are identical up to residue 393 and have a common kinase domain. The VRK2A protein has, at the end of its carboxy terminus, a hydrophobic region of 17 aminoacids that anchors the protein to membranes in endoplasmic reticulum and mitochondria. VRK2A colocalizes with calreticulin and calnexin indicating its presence in the endoplasmic reticulum [12]; it also colocalizes with mitotracker, a lipidic marker of mitochondria [12]. VRK2B, lacking its hydrophobic tail, is free in the cytosol and the nucleus of the cell. The shorter isoform B is more similar in size to VRK1, lacking the membrane anchor sequence, and detected in those cells where VRK1 is mostly cytosolic, suggesting they might have some functional redundancy, but how their expression is coordinated is not known. Therefore, although both proteins might have similar or identical kinase properties, their natural substrates may be different due to their different subcellular localization. Additional variants of VRK2 have been identified by RT-PCR, but the expected corresponding proteins have not been detected and may represent splicing intermediates. Many cell types have been studied for the expression of both isoforms, such as normal B-cells, several lymphoma cell lines and several frequently used carcinoma cell lines such as HeLa, A549, H1299, MCF7, among others. All of them expressed both messages [12]. All Vaccinia-related kinase (VRK) signaling in cell and tumor biology 139 of them express VRK2A protein, but the level of the VRK2B protein was variable, and in some cell lines was not detectable [12]. The human VRK3 gene is located on 19q13.33, and codes for a protein with a degenerate kinase domain located in the carboxy terminal region that has no kinase activity due to critical aminoacid substitutions [8]. Its crystal structure has revealed important structural differences that make it unable to bind ATP [10]. VRK3 non-functional kinase-dead domain maintains its overall structure so that it can function as scaffold for other proteins [10]. The VRK3 protein is detected in the nucleus and has a bipartite nuclear localization signal [8, 9]. In humans the expression of VRK proteins has been detected in all tissues studied; but not all cells in a tissue express these proteins. The reason for this heterogeneous expression is not yet known, but it may be associated to the proliferation and differentiation state of individual cells. In normal epithelium higher levels are detected near the basal layer, and the intensity of the signal decreases as the cell matures [18]. 2. Biological roles of VRK proteins The biological information available on the three VRK proteins is very limited, and most of it was obtained by studying the human proteins in two main directions. One of them implies the characterization of the signaling pathway in which VRK proteins are implicated, either by substrate identification or by interaction with other proteins. The other is directed at their effect on some biological response, such as hypoxia, interleukin1β or DNA damage. In both situations it is expected that information will expand as these proteins acquire more relevance in the context of tumor biology, and start to attract more attention. In the case of VRK1 some of its targets have been identified, including several transcription factors (Fig. 2), which can serve as starting points to characterize the VRK pathway by itself, or their interaction with other pathways. In this review we will summarized the different biological processes where VRK proteins have been shown to participate. 3. VRK proteins and cell signaling 3.1. VRK2 downregulates the response to hypoxia or interleukin-1β