Dissecting the roles of Haspin and VRK1 in histone H3 phosphorylation during mitosis

Cartwright, Tyrell N.; Harris, Rebecca J.; Meyer, Stephanie K.; Mon, Aye M.; Watson, Nikolaus A.; Tan, Cheryl C.; Marcelot, Agathe; Wang, Fangwei; Zinn‐Justin, Sophie; Traktman, Paula; Higgins, Jonathan M.G.

doi:10.1038/s41598-022-15339-8

Cited by 10 publications

(5 citation statements)

References 98 publications

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“…S2B and S2C). Because TP53 is mutated in 28% of glioblastoma ( 22 ), we queried whether VRK1 knockdown correlated with TP53 mutation status. We observed no correlation between TP53 mutation status and VRK1 CRISPR score in 79 CCLE cell lines of the central nervous system (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

VRK1 Is a Synthetic–Lethal Target in VRK2-Deficient Glioblastoma

et al. 2022

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Synthetic lethality is a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone, which can be co-opted for cancer therapeutics. Pairs of paralog genes are among the most straightforward potential synthetic lethal interactions by virtue of their redundant functions. Here we demonstrate a paralog-based synthetic lethality by targeting vaccinia-related kinase 1 (VRK1) in glioblastoma (GBM) deficient of vaccinia-related kinase 2 (VRK2), which is silenced by promoter methylation in approximately two-thirds of GBM. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells resulted in decreased activity of the downstream substrate barrier to autointegration factor (BAF), a regulator of post-mitotic nuclear envelope formation. Reduced BAF activity following VRK1 knockdown caused nuclear lobulation, blebbing, and micronucleation, which subsequently resulted in G2/M arrest and DNA damage. The VRK1-VRK2 synthetic lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic expression of VRK2. In VRK2-methylated GBM cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models, knockdown of VRK1 led to robust tumor growth inhibition. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM.

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Section: Resultsmentioning

confidence: 99%

VRK1 Is a Synthetic–Lethal Target in VRK2-Deficient Glioblastoma

et al. 2022

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“…Previously, we have found that the affinity-purified rabbit polyclonal antibody B8634 selectively binds H3T3ph in vitro and in cells, and that RNAi, inhibition, or CRISPR/Cas9 knockout of the H3T3 kinase Haspin eliminates epitope recognition in cells (Cartwright et al, 2022; Dai et al, 2005; Wang et al, 2012). Using ELISAs on chemically synthesized Histone H3 peptides, we found that the B8634 antibody was indeed specific for H3T3ph, and did not recognize H3S10ph, H3T11ph, or H3T22ph (Figure S1A).…”

Section: Resultsmentioning

confidence: 99%

“…Although there is strong evidence that Haspin is the major H3T3 kinase in mitotic cells (Cartwright et al, 2022; Dai et al, 2005; De Antoni et al, 2012; Markaki et al, 2009; Wang et al, 2012; Zhou et al, 2017), other potential kinases have been proposed (Kang et al, 2007; Karakkat et al, 2019). However, we found that phosphorylation of H3T3 driven by all endogenous kinases active in lysates of mitotic HeLa cells was severely curtailed by H3K4me3 (Figure 3B).…”

Section: Resultsmentioning

confidence: 99%

Release of Histone H3K4-reading transcription factors from chromosomes in mitosis is independent of adjacent H3 phosphorylation

Harris

Heer

Levasseur

et al. 2023

Preprint

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Histone modifications influence the recruitment of reader proteins to chromosomes to regulate events including transcription and cell division. The idea of a histone code, where particular combinations of modifications specify unique downstream functions, is widely accepted and can be demonstratedin vitro. For example, on synthetic peptides, phosphorylation of Histone H3 at threonine-3 (H3T3ph) prevents the binding of reader proteins that recognise trimethylation of the adjacent lysine-4 (H3K4me3), including the TAF3 component of TFIID. To study these combinatorial effects in cells, we analyzed the genome-wide distribution of H3T3ph and H3K4me3 during mitosis. We find that H3K4me3 hinders adjacent H3T3ph deposition in cells, and that the PHD domain of TAF3 can bind H3K4me3 in mitotic chromatin despite the presence of H3T3ph. Unlikein vitro, H3K4 readers are displaced from chromosomes in mitosis in Haspin-depleted cells lacking H3T3ph. H3T3ph is therefore unlikely to be responsible for transcriptional downregulation during cell division.

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“…OPG97 (L3L) is a VACV core component involved in the transcription of early genes. The AlphaFold model of this protein showed significant structural similarity to Ser/Thr proteins kinases, in particular the atypical kinase domain of haspin, an animal chromatin remodeling regulator (26), but the catalytic site residues are partially replaced in the poxvirus protein (Figure 3a). Notably, the aspartate in the active site has not been exchanged for crododilepox virus OPG97 but the lysine at the ATP-binding site is present.…”

Section: Exaptation Of Cellular Proteins For Viral Functionsmentioning

confidence: 99%

Exaptation of inactivated host enzymes for structural roles in orthopoxviruses and novel protein folds revealed by protein structure modeling

Mutz

Resch

Faure

et al. 2022

Preprint

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Viruses with large double-stranded DNA genomes appear to have captured the majority of their genes from the hosts at different stages of evolution. The origin of many virus genes is readily detected through highly significant sequence similarity with cellular homologs. This is the case, in particular, for virus enzymes, such as DNA and RNA polymerases or nucleotide kinases, that retain their catalytic activity after capture by an ancestral virus. However, a large fraction of virus genes have no readily detectable cellular homologs so that their origin remains enigmatic. We sought to explore potential origins of proteins of unknown provenance encoded in the genomes of orthopoxviruses, a thoroughly studied virus genus which includes major human pathogens. To this end, we used AlphaFold2, to predict the structures of all 214 proteins encoded by orthopoxviruses. Among the proteins of unknown provenance, structure prediction yielded a clear indication of origin for 14, along with validating several inferences previously made by sequence analysis. The major trend that emerges from these findings is the exaptation of enzymes from cellular organisms for non-enzymatic, structural roles in virus reproduction which is accompanied by disruption of catalytic sites and overall drastic divergence which precludes detection of homology at the sequence level. Among the 16 orthopoxvirus proteins found to be inactivated enzyme derivatives, are the poxvirus replication processivity factor A20, an inactivated derivative of bacterial NAD-dependent DNA ligase; major core protein A3, an inactivated deubiquitinase; F11, an inactivated prolyl hydroxylase; and more similar cases. However, for nearly one third of the orthopoxvirus virion proteins, no significantly similar structures were identified, suggesting exaptation with subsequent major structural rearrangement, yielding novel protein folds.

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Dissecting the roles of Haspin and VRK1 in histone H3 phosphorylation during mitosis

Cited by 10 publications

References 98 publications

VRK1 Is a Synthetic–Lethal Target in VRK2-Deficient Glioblastoma

VRK1 Is a Synthetic–Lethal Target in VRK2-Deficient Glioblastoma

Release of Histone H3K4-reading transcription factors from chromosomes in mitosis is independent of adjacent H3 phosphorylation

Exaptation of inactivated host enzymes for structural roles in orthopoxviruses and novel protein folds revealed by protein structure modeling

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