Members of a novel family of mammalian protein kinases complement the DNA-negative phenotype of a vaccinia virus ts mutant defective in the B1 kinase

Temperature-sensitive (ts) mutants of vaccinia virus defective in the B1 kinase demonstrate a conditionally lethal defect in DNA synthesis. B1 is the prototypic member of a new family of protein kinases (vaccinia virus-related kinases, or VRK) that possess distinctive B1-like sequence features within their catalytic motifs (R. J. Nichols and P. Traktman, J. Biol. Chem., in press). Given the striking sequence similarity between B1 and the VRK enzymes, we proposed that they might share overlapping substrate specificity. We therefore sought to determine whether the human and mouse VRK1 enzymes (hVRK1 and mVRK1, respectively) could complement a B1 deficiency in vivo. Recombinant ts2 viruses expressing hVRK1, mVRK1, or wild-type B1 were able to synthesize viral DNA at high temperature, but those expressing the more distantly related human casein kinase 1 alpha 2 could not. Complementation required the enzymatic activity of hVRK1, since a catalytically inactive allele of hVRK1 was unable to confer a temperature-insensitive phenotype. Interestingly, rescue of viral DNA synthesis was not coupled to the ability to phosphorylate H5, the only virus-encoded protein shown to be a B1 substrate in vivo. Expression of hVRK1 during nonpermissive ts2 infections restored virus production and plaque formation, whereas expression of mVRK1 resulted in an intermediate level of rescue. Taken together, these observations indicate that enzymatically active cellular VRK1 kinases can perform the function(s) of B1 required for genome replication, most likely due to overlapping specificity for cellular and/or viral substrates.     

The vaccinia-related kinases phosphorylate the N' terminus of BAF, regulating its interaction with DNA and its retention in the nucleus

The vaccinia-related kinases (VRKs) comprise a branch of the casein kinase family whose members are characterized by homology to the vaccinia virus B1 kinase. The VRK orthologues encoded by Caenorhabditis elegans and Drosophila melanogaster play an essential role in cell division; however, substrates that mediate this role have yet to be elucidated. VRK1 can complement the temperature sensitivity of a vaccinia B1 mutant, implying that VRK1 and B1 have overlapping substrate specificity. Herein, we demonstrate that B1, VRK1, and VRK2 efficiently phosphorylate the extreme N' terminus of the BAF protein (Barrier to Autointegration Factor). BAF binds to both DNA and LEM domain-containing proteins of the inner nuclear membrane; in lower eukaryotes, BAF has been shown to play an important role during the reassembly of the nuclear envelope at the end of mitosis. We demonstrate that phosphorylation of ser4 and/or thr2/thr3 abrogates the interaction of BAF with DNA and reduces its interaction with the LEM domain. Coexpression of VRK1 and GFP-BAF greatly diminishes the association of BAF with the nuclear chromatin/matrix and leads to its dispersal throughout the cell. Cumulatively, our data suggest that the VRKs may modulate the association of BAF with nuclear components and hence play a role in maintaining appropriate nuclear architecture.     

The casein kinase 1 family: participation in multiple cellular processes in eukaryotes

Phosphorylation of serine, threonine and tyrosine residues by cellular protein kinases plays an important role in the regulation of various cellular processes. The serine/threonine specific casein kinase 1 and 2 protein kinase families--(CK1 and CK2)--were among the first protein kinases that had been described. In recent years our knowledge of the regulation and function of mammalian CK1 kinase family members has rapidly increased. Extracellular stimuli, the subcellular localization of CK1 isoforms, their interaction with various cellular structures and proteins, as well as autophosphorylation and proteolytic cleavage of their C-terminal regulatory domains influence CK1 kinase activity. Mammalian CK1 isoforms phosphorylate many different substrates among them key regulatory proteins involved in the control of cell differentiation, proliferation, chromosome segregation and circadian rhythms. Deregulation and/or the incidence of mutations in the coding sequence of CK1 isoforms have been linked to neurodegenerative diseases and cancer. This review will summarize our current knowledge about the function and regulation of mammalian CK1 isoforms.     

Clustered Charge-to-Alanine Mutagenesis of the Vaccinia Virus A20 Gene: Temperature-Sensitive Mutants Have a DNA-Minus Phenotype and Are Defective in the Production of Processive DNA Polymerase Activity

Although the vaccinia virus DNA polymerase is inherently distributive, a highly processive form of the enzyme exists within the cytoplasm of infected cells (W. F. McDonald, N. Klemperer, and P. Traktman, Virology 234:168-175, 1997). In the accompanying report we outline the purification of the 49-kDa A20 protein as a stoichiometric component of the processive polymerase complex (N. Klemperer, W. McDonald, K. Boyle, B. Unger, and P. Traktman, J. Virol. 75:12298-12307, 2001). To complement this biochemical analysis, we undertook a genetic approach to the analysis of the structure and function of the A20 protein. Here we report the application of clustered charge-to-alanine mutagenesis of the A20 gene. Eight mutant viruses containing altered A20 alleles were isolated using this approach; two of these, tsA20-6 and tsA20-ER5, have tight temperature-sensitive phenotypes. At the nonpermissive temperature, neither virus forms macroscopic plaques and the yield of infectious virus is <1% of that obtained at the permissive temperature. Both viruses show a profound defect in the accumulation of viral DNA at the nonpermissive temperature, although both the A20 protein and DNA polymerase accumulate to wild-type levels. Cytoplasmic extracts prepared from cells infected with the tsA20 viruses show a defect in processive polymerase activity; they are unable to direct the formation of RFII product using a singly primed M13 template. In sum, these data indicate that the A20 protein plays an essential role in the viral life cycle and that viruses with A20 lesions exhibit a DNA(-) phenotype that is correlated with a loss in processive polymerase activity as assayed in vitro. The vaccinia virus A20 protein can, therefore, be considered a new member of the family of proteins (E9, B1, D4, and D5) with essential roles in vaccinia virus DNA replication.     

Mitotic histone H3 phosphorylation by vaccinia-related kinase 1 in mammalian cells

Mitotic chromatin condensation is essential for cell division in eukaryotes. Posttranslational modification of the N-terminal tail of histone proteins, particularly by phosphorylation by mitotic histone kinases, may facilitate this process. In mammals, aurora B is believed to be the mitotic histone H3 Ser10 kinase; however, it is not sufficient to phosphorylate H3 Ser10 with aurora B alone. We show that histone H3 is phosphorylated by vaccinia-related kinase 1 (VRK1). Direct phosphorylation of Thr3 and Ser10 in H3 by VRK1 both in vitro and in vivo was observed. Loss of VRK1 activity was associated with a marked decrease in H3 phosphorylation during mitosis. Phosphorylation of Ser10 by VRK1 is similar to that by aurora B. Moreover, expression and chromatin localization of VRK1 depended on the cell cycle phase. Overexpression of VRK1 resulted in a dramatic condensation of nuclei. Our findings collectively support a role of VRK1 as a novel mitotic histone H3 kinase in mammals.     

BAFfled by Poxviruses?

Successful viruses engage in a dynamic interplay with their hosts, where both utilize diverse strategies to impose their supremacy. In this issue of Cell Host & Microbe, Wiebe and Traktman describe a novel interaction between vaccinia virus and mammalian cells. A host protein called BAF can bind ectopic cytoplasmic DNA and block viral DNA replication, whereas vaccinia in turn counteracts this inhibition with a virus-encoded serine threonine kinase that inactivates BAF.     

PIKfyve lipid kinase is a protein kinase: downregulation of 5'-phosphoinositide product formation by autophosphorylation

A subset of phosphoinositide 3-kinase family members are dual specificity enzymes; their protein kinase activity is thought to bring about an additional level to their intracellular regulation. Here we have examined whether the 5'-phosphoinositide kinase PIKfyve, reported previously to catalyze the formation of PtdIns 5-P and PtdIns 3,5-P(2) in vitro [Sbrissa et al. (1999) J. Biol. Chem. 274, 21589-21597], displays dual specificity. We now report that PIKfyve possesses an intrinsic protein kinase activity inseparable from its lipid kinase activity and, besides itself, can phosphorylate exogenous proteins in a substrate-specific manner. Both the autophosphorylation and transphosphorylation were demonstrated with PIKfyve immunopurified or affinity-purified from heterologously transfected COS cells, infected Sf9 cells, or native 3T3-L1 adipocytes. Conversely, no protein kinase activity was associated with immunopurified lipid kinase dead point (K1831E) or truncated (Delta1812-2052) PIKfyve mutants. PIKfyve autophosphorylation or transphosphorylation engaged Ser but not Thr or Tyr residues. PIKfyve autophosphorylation was largely abrogated upon pretreatment with PIKfyve lipid substrates or phosphatases. The impact of autophosphorylation on the PIKfyve lipid kinase activity was further examined with purified PIKfyve preparations. A decrease of 70% in the lipid product formation was associated with PIKfyve autophosphorylation, which was reversed upon treatment with phosphatases. In the cellular context, PIKfyve, or a fraction of it, was found in a phosphorylated form. Collectively, these results indicate that PIKfyve is a dual specificity kinase, which can generate and relay protein phosphorylation signals to regulate the formation of its lipid products, and possibly other events, in the context of living cells.     

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.     

Multivalent DNA and nucleosome acidic patch interactions specify VRK1 mitotic localization and activity

A key role of chromatin kinases is to phosphorylate histone tails during mitosis to spatiotemporally regulate cell division. Vaccinia-related kinase 1 (VRK1) is a serine–threonine kinase that phosphorylates histone H3 threonine 3 (H3T3) along with other chromatin-based targets. While structural studies have defined how several classes of histone-modifying enzymes bind to and function on nucleosomes, the mechanism of chromatin engagement by kinases is largely unclear. Here, we paired cryo-electron microscopy with biochemical and cellular assays to demonstrate that VRK1 interacts with both linker DNA and the nucleosome acidic patch to phosphorylate H3T3. Acidic patch binding by VRK1 is mediated by an arginine-rich flexible C-terminal tail. Homozygous missense and nonsense mutations of this acidic patch recognition motif in VRK1 are causative in rare adult-onset distal spinal muscular atrophy. We show that these VRK1 mutations interfere with nucleosome acidic patch binding, leading to mislocalization of VRK1 during mitosis, thus providing a potential new molecular mechanism for pathogenesis.     

Functional Disruption of the Moloney Murine Leukemia Virus Preintegration Complex by Vaccinia-related Kinases

Retroviral integration is executed by the preintegration complex (PIC), which contains viral DNA together with a number of proteins. Barrier-to-autointegration factor (BAF), a cellular component of Moloney murine leukemia virus (MMLV) PICs, has been demonstrated to protect viral DNA from autointegration and stimulate the intermolecular integration activity of the PIC by its DNA binding activity. Recent studies reveal that the functions of BAF are regulated by phosphorylation via a family of cellular serine/threonine kinases called vaccinia-related kinases (VRK), and VRK-mediated phosphorylation causes a loss of the DNA binding activity of BAF. These results raise the possibility that BAF phosphorylation may influence the integration activities of the PIC through removal of BAF from viral DNA. In the present study, we report that VRK1 was able to abolish the intermolecular integration activity of MMLV PICs in vitro. This was accompanied by an enhancement of autointegration activity and dissociation of BAF from the PICs. In addition, in vitro phosphorylation of BAF by VRK1 abrogated the activity of BAF in PIC function. Among the VRK family members, VRK1 as well as VRK2, which catalyze hyperphosphorylation of BAF, could abolish PIC function. We also found that treatment of PICs with certain nucleotides such as ATP resulted in the inhibition of the intermolecular integration activity of PICs through the dissociation of BAF. More importantly, the ATP-induced disruption was not observed with the PICs from VRK1 knockdown cells. Our in vitro results therefore suggest the presence of cellular kinases including VRKs that can inactivate the retroviral integration complex via BAF phosphorylation.     

Spinal Muscular Atrophy with Pontocerebellar Hypoplasia Is Caused by a Mutation in the VRK1 Gene

The spinal muscular atrophies (SMAs) are a genetically and clinically heterogeneous group of disorders characterized by degeneration and loss of anterior horn cells in the spinal cord, leading to muscle weakness and atrophy. Spinal muscular atrophy with pontocerebellar hypoplasia (SMA-PCH, also known as pontocerebellar hypoplasia type 1 [PCH1]) is one of the rare infantile SMA variants that include additional clinical manifestations, and its genetic basis is unknown. We used a homozygosity mapping and positional cloning approach in a consanguineous family of Ashkenazi Jewish origin and identified a nonsense mutation in the vaccinia-related kinase 1 gene (VRK1) as a cause of SMA-PCH. VRK1, one of three members of the mammalian VRK family, is a serine/threonine kinase that phosphorylates p53 and CREB and is essential for nuclear envelope formation. Its identification as a gene involved in SMA-PCH implies new roles for the VRK proteins in neuronal development and maintenance and suggests the VRK genes as candidates for related phenotypes.