Depletion of the protein kinase VRK1 disrupts nuclear envelope morphology and leads to BAF retention on mitotic chromosomes

Molitor, Tyler P.; Traktman, Paula

doi:10.1091/mbc.e13-10-0603

Cited by 93 publications

(110 citation statements)

References 59 publications

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“…Like endogenous BAF (14,29,(38)(39)(40), WT BAF is expressed in the nucleus, nuclear envelope, and cytoplasm ( Fig. 2B), and such localization is also consistent with the published results on GFP-tagged and 3ϫ FLAG-tagged BAF (27,28,30,41). With regard to the mutant BAF, we found that MAAAQ is found almost exclusively in the nucleus (Fig.…”

Section: Stable Expression Of Baf Phosphorylation Mutants In Cv1 Cellssupporting

confidence: 91%

“…Based on both direct in vitro studies (28) and indirect evidence in live cells (30)(31)(32)(33), phosphorylation is a potent inhibitor of BAF interaction with DNA. Here, we have examined BAF-DNA interaction of our mutant proteins by utilizing a ChIP protocol.…”

Section: Discussionmentioning

confidence: 99%

“…For example, BAF is a substrate of the cellular vaccinia-related kinases (VRKs) (28,(30)(31)(32)(33) and protein phosphatases PP2A and PP4 (32,34). These enzymes are critical regulators of BAF's mitotic function, controlling its interaction with DNA and other proteins (28,31,32,35).…”

mentioning

confidence: 99%

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Cell- and Virus-Mediated Regulation of the Barrier-to-Autointegration Factor's Phosphorylation State Controls Its DNA Binding, Dimerization, Subcellular Localization, and Antipoxviral Activity

Jamin

Wicklund

Wiebe

2014

J Virol

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Barrier-to-autointegration factor (BAF) is a DNA binding protein with multiple cellular functions, including the ability to act as a potent defense against vaccinia virus infection. This antiviral function involves BAF's ability to condense double-stranded DNA and subsequently prevent viral DNA replication. In recent years, it has become increasingly evident that dynamic phosphorylation involving the vaccinia virus B1 kinase and cellular enzymes is likely a key regulator of multiple BAF functions; however, the precise mechanisms are poorly understood. Here we analyzed how phosphorylation impacts BAF's DNA binding, subcellular localization, dimerization, and antipoxviral activity through the characterization of BAF phosphomimetic and unphosphorylatable mutants. Our studies demonstrate that increased phosphorylation enhances BAF's mobilization from the nucleus to the cytosol, while dephosphorylation restricts BAF to the nucleus. Phosphorylation also impairs both BAF's dimerization and its DNA binding activity. Furthermore, our studies of BAF's antiviral activity revealed that hyperphosphorylated BAF is unable to suppress viral DNA replication or virus production. Interestingly, the unphosphorylatable BAF mutant, which is capable of binding DNA but localizes predominantly to the nucleus, was also incapable of suppressing viral replication. Thus, both DNA binding and localization are important determinants of BAF's antiviral function. Finally, our examination of how phosphatases are involved in regulating BAF revealed that PP2A dephosphorylates BAF during vaccinia infection, thus counterbalancing the activity of the B1 kinase. Altogether, these data demonstrate that phosphoregulation of BAF by viral and cellular enzymes modulates this protein at multiple molecular levels, thus determining its effectiveness as an antiviral factor and likely other functions as well. IMPORTANCEThe barrier-to-autointegration factor (BAF) contributes to cellular genomic integrity in multiple ways, the best characterized of which are as a host defense against cytoplasmic DNA and as a regulator of mitotic nuclear reassembly. Although dynamic phosphorylation involving both viral and cellular enzymes is likely a key regulator of multiple BAF functions, the precise mechanisms involved are poorly understood. Here we demonstrate that phosphorylation coordinately regulates BAF's DNA binding, subcellular localization, dimerization, and antipoxviral activity. Overall, our findings provide new insights into how phosphoregulation of BAF modulates this protein at multiple levels and governs its effectiveness as an antiviral factor against foreign DNA.

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Section: Stable Expression Of Baf Phosphorylation Mutants In Cv1 Cellssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

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Cell- and Virus-Mediated Regulation of the Barrier-to-Autointegration Factor's Phosphorylation State Controls Its DNA Binding, Dimerization, Subcellular Localization, and Antipoxviral Activity

Jamin

Wicklund

Wiebe

2014

J Virol

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“…These NE breaks occur at regions that are remote from centrosomes in a microtubule-dependent manner. The detachment of chromatin from the NE is likely regulated by phosphorylation events, including the phosphorylation of BAF by VRK1, which reduces the affinity between BAF and chromatin (Nichols et al 2006;Molitor and Traktman 2014). Components of the nuclear lamina are phosphorylated by Cdk1 and possibly other kinases (Heald and McKeon 1990;Peter et al 1990), leading to their dissociation from the NE.…”

Section: Ne Breakdownmentioning

confidence: 99%

Cell Biology of theCaenorhabditis elegansNucleus

Cohen-Fix

Askjaer

2017

Genetics

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Studies on the Caenorhabditis elegans nucleus have provided fascinating insight to the organization and activities of eukaryotic cells. Being the organelle that holds the genetic blueprint of the cell, the nucleus is critical for basically every aspect of cell biology. The stereotypical development of C. elegans from a one cell-stage embryo to a fertile hermaphrodite with 959 somatic nuclei has allowed the identification of mutants with specific alterations in gene expression programs, nuclear morphology, or nuclear positioning. Moreover, the early C. elegans embryo is an excellent model to dissect the mitotic processes of nuclear disassembly and reformation with high spatiotemporal resolution. We review here several features of the C. elegans nucleus, including its composition, structure, and dynamics. We also discuss the spatial organization of chromatin and regulation of gene expression and how this depends on tight control of nucleocytoplasmic transport. Finally, the extensive connections of the nucleus with the cytoskeleton and their implications during development are described. Most processes of the C. elegans nucleus are evolutionarily conserved, highlighting the relevance of this powerful and versatile model organism to human biology.KEYWORDS Caenorhabditis elegans; chromatin; gene expression; lamin; LEM-domain proteins; LINC; P granule; nuclear pore complex; nuclear envelope; nucleocytoplasmic transport; nucleolus; WormBook TABLE OF CONTENTSAbstract 25 C. elegans as a model system to study cell biology of the nucleus 26 Structural components of the nucleus 27

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“…For example, VRK1 is located predominantly in the nucleus, while the major isoform of VRK2 is associated with the endoplasmic reticulum (ER) and nuclear envelope (11,14). Interestingly, both VRK1 and VRK2 possess strong catalytic activity found to modulate cellular processes, including mitosis and apoptosis, via multiple substrates (15)(16)(17)(18). It has also been demonstrated that VRK2 limits cell death during Epstein-Barr virus (EBV) infection (19) and delays myxoma virus replication in a breast cancer cell line (20), thus providing other examples that VRKs may regulate pathways important during viral infection.…”

mentioning

confidence: 99%

Deletion of the Vaccinia Virus B1 Kinase Reveals Essential Functions of This Enzyme Complemented Partly by the Homologous Cellular Kinase VRK2

Olson

Rico

Wang

et al. 2017

J Virol

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The vaccinia virus B1 kinase is highly conserved among poxviruses and is essential for the viral life cycle. B1 exhibits a remarkable degree of similarity to vaccinia virus-related kinases (VRKs), a family of cellular kinases, suggesting that the viral enzyme has evolved to mimic VRK activity. Indeed, B1 and VRKs have been demonstrated to target a shared substrate, the DNA binding protein BAF, elucidating a signaling pathway important for both mitosis and the antiviral response. In this study, we further characterize the role of B1 during vaccinia infection to gain novel insights into its regulation and integration with cellular signaling pathways. We begin by describing the construction and characterization of the first B1 deletion virus (vvΔB1) produced using a complementing cell line expressing the viral kinase. Examination of vvΔB1 revealed that B1 is critical for the production of infectious virions in various cell types and is sufficient for BAF phosphorylation. Interestingly, the severity of the defect in DNA replication following the loss of B1 varied between cell types, leading us to posit that cellular VRKs partly complement for the absence of B1 in some cell lines. Using cell lines devoid of either VRK1 or VRK2, we tested this hypothesis and discovered that VRK2 expression facilitates DNA replication and allows later stages of the viral life cycle to proceed in the absence of B1. Finally, we present evidence that the impact of VRK2 on vaccinia virus is largely independent of BAF phosphorylation. These data support a model in which B1 and VRK2 share additional substrates important for the replication of cytoplasmic poxviruses.IMPORTANCE Viral mimicry of cellular signaling modulators provides clear evidence that the pathogen targets an important host pathway during infection. Poxviruses employ numerous viral homologs of cellular proteins, the study of which have yielded insights into signaling pathways used by both virus and cells alike. The vaccinia virus B1 protein is a homolog of cellular vaccinia virus-related kinases (VRKs) and is needed for viral DNA replication and likely other stages of the viral life cycle. However, much remains to be learned about how B1 and VRKs overlap functionally. This study utilizes new tools, including a B1 deletion virus and VRK knockout cells, to further characterize the functional links between the viral and cellular enzymes. As a result, we have discovered that B1 and VRK2 target a common set of substrates vital to productive infection of this large cytoplasmic DNA virus.

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Depletion of the protein kinase VRK1 disrupts nuclear envelope morphology and leads to BAF retention on mitotic chromosomes

Cited by 93 publications

References 59 publications

Cell- and Virus-Mediated Regulation of the Barrier-to-Autointegration Factor's Phosphorylation State Controls Its DNA Binding, Dimerization, Subcellular Localization, and Antipoxviral Activity

Cell- and Virus-Mediated Regulation of the Barrier-to-Autointegration Factor's Phosphorylation State Controls Its DNA Binding, Dimerization, Subcellular Localization, and Antipoxviral Activity

Cell Biology of theCaenorhabditis elegansNucleus

Deletion of the Vaccinia Virus B1 Kinase Reveals Essential Functions of This Enzyme Complemented Partly by the Homologous Cellular Kinase VRK2

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