Recombinant Danio rerio Serine/threonine-protein kinase VRK1 (vrk1)

What is the protein structure of zebrafish VRK1?

Zebrafish VRK1 is a 425 amino acid serine/threonine protein kinase that shares significant homology with human VRK1. The full protein sequence includes a kinase domain and a flexible C-terminal tail that contains an arginine-rich region important for nucleosome binding. This C-terminal region mediates interactions with the nucleosome acidic patch, which is crucial for proper VRK1 localization and function. The protein's structural features enable it to phosphorylate various substrates involved in cell cycle regulation and chromatin dynamics. The recombinant form of zebrafish VRK1 is typically expressed with a His-tag to facilitate purification and experimental applications .

How does zebrafish VRK1 compare to human VRK1?

While zebrafish and human VRK1 proteins share significant functional and structural homology, there are notable species-specific differences in amino acid sequence and expression patterns. Both proteins function as serine/threonine kinases with roles in cell cycle regulation, nuclear envelope dynamics, and chromatin modification. Comparative single-cell RNA-seq analysis between zebrafish and human oocytes has revealed differences in expression patterns that may reflect species-specific developmental requirements . Despite these differences, studies have shown that pathogenic mutations in VRK1 lead to similar phenotypes in both species, including microcephaly and motor dysfunction, making zebrafish an excellent model for studying VRK1-related human diseases .

What are the primary cellular functions of VRK1 in zebrafish?

VRK1 in zebrafish serves several critical cellular functions:

• Cell cycle regulation: VRK1 functions as an early response gene essential for cell cycle progression from G0 to G1 phase, similar to its role in mammalian systems .

• Nuclear envelope dynamics: VRK1 contributes to nuclear envelope assembly and disassembly during mitosis .

• Chromatin modification: Zebrafish VRK1 phosphorylates histone H3 at threonine 3 (H3T3), a modification important for chromosome condensation and segregation during mitosis .

• Neuronal development: VRK1 plays a crucial role in proper brain development, as evidenced by microcephaly in VRK1-deficient zebrafish .

• Dopaminergic system maintenance: VRK1 deficiency leads to reduced brain dopamine content, suggesting a role in dopaminergic neuron development or function .

How can recombinant zebrafish VRK1 be produced for research purposes?

Recombinant zebrafish VRK1 can be produced using several expression systems, with yeast being a commonly used host for the full-length protein (AA 1-425). The protein is typically expressed with a His-tag to facilitate purification through affinity chromatography. The resulting recombinant protein demonstrates >90% purity, making it suitable for various experimental applications . Alternative expression systems include E. coli, mammalian cells, and baculovirus-infected insect cells, each offering different advantages depending on the specific research requirements. When selecting an expression system, researchers should consider factors such as protein folding requirements, post-translational modifications, and the need for enzymatic activity. For kinase activity assays, it is crucial to confirm that the recombinant protein maintains its enzymatic function after purification .

What are the common assays to measure zebrafish VRK1 kinase activity?

Several established methodologies can be used to assess zebrafish VRK1 kinase activity:

• In vitro kinase assays: Using purified recombinant VRK1 with specific substrates such as histone H3 or ATF2. Activity is typically measured through incorporation of radioactive phosphate (³²P-ATP) or using phospho-specific antibodies .

• Phosphorylation-specific detection: Western blotting with antibodies against phosphorylated substrates (e.g., phospho-H3T3) can be used to detect VRK1 activity both in vitro and in vivo .

• Transcriptional activation assays: Since VRK1 phosphorylates transcription factors like ATF2, reporter gene assays measuring the activation of target promoters (such as collagenase gene promoter) can be used as functional readouts of VRK1 activity .

• Cellular phenotype assays: Measuring cell cycle progression, nuclear envelope dynamics, or chromatin modification in cells expressing wild-type versus mutant VRK1 can provide functional assessment of kinase activity .

When performing these assays, it is important to include appropriate controls, such as kinase-dead VRK1 mutants (e.g., K179E) and specific inhibitors to validate that observed effects are specifically due to VRK1 kinase activity .

How can I design a zebrafish VRK1 knockdown/knockout model?

Designing effective zebrafish VRK1 knockdown or knockout models requires careful consideration of several methodological approaches:

• CRISPR/Cas9 gene editing: This is currently the most efficient method for generating vrk1-/- zebrafish. Design guide RNAs targeting conserved regions of the vrk1 gene, particularly within the kinase domain. Verify knockouts through sequencing and assess protein absence via Western blotting .

• Morpholino oligonucleotides: For transient knockdowns, antisense morpholinos targeting vrk1 mRNA splicing or translation can be microinjected into one-cell stage embryos. This approach allows for rapid assessment of phenotypes but may have off-target effects .

• Transgenic rescue experiments: To confirm phenotype specificity, design rescue experiments by co-expressing wild-type vrk1 mRNA in knockout/knockdown models. This is essential for validating that observed phenotypes are specifically due to VRK1 deficiency .

• Phenotypic assessment: Evaluate model effectiveness by examining established VRK1-deficiency phenotypes, including:

  • Microcephaly (brain size measurements)

  • Motor function impairments (swimming behavior tests)

  • Cell proliferation defects (using BrdU or EdU incorporation)

  • Nuclear envelope integrity (immunostaining for nuclear envelope proteins)

  • Dopamine content in the brain (HPLC or immunohistochemistry)

When establishing these models, careful consideration of developmental timing is crucial, as complete vrk1 knockout may affect early embryonic development, potentially complicating analysis of later developmental stages .

How does VRK1 contribute to cell cycle progression in zebrafish?

VRK1 plays a critical role in cell cycle regulation in zebrafish, particularly in the G0 to G1 transition and progression through the cell cycle. Similar to mammalian systems, zebrafish VRK1 functions as an early response gene in cell cycle progression. It is expressed simultaneously with early response genes like myc and fos, and its expression precedes that of cyclin D1 . VRK1 is necessary for cells to exit the G0 phase and enter the G1 phase of the cell cycle. Experimental evidence from studies shows that downregulation of VRK1 results in a proliferation block where cells cannot exit G1 phase .

The molecular mechanism involves VRK1-mediated phosphorylation of transcription factors like CREB and ATF2, which regulate cyclin D1 expression. From late G1 to early S phase, VRK1 phosphorylates CREB, enhancing its binding to the cAMP response element (CRE) of the cyclin D1 promoter and activating transcription. Additionally, VRK1 modulates the association of ATF2 with the CRE of cyclin D1 . VRK1 expression itself is stimulated by Myc binding to its promoter, creating a regulatory network that controls cell cycle progression .

What is the role of VRK1 in nuclear envelope dynamics?

VRK1 plays a crucial role in nuclear envelope (NE) dynamics during cell division in zebrafish, similar to its function in other vertebrates. The nuclear envelope separates the nucleus from the cytoplasm and consists of an outer and inner nuclear membrane. VRK1 is involved in both the disassembly of the nuclear envelope during mitotic entry and its reassembly during mitotic exit .

In vrk1-deficient zebrafish, defects in nuclear envelope formation have been observed, highlighting the protein's importance in this process . Mechanistically, VRK1 phosphorylates components of the nuclear envelope, including nuclear lamins and other nuclear envelope proteins, regulating their interactions and organization during cell division. This phosphorylation is essential for the proper breakdown of the nuclear envelope at the onset of mitosis and its subsequent reformation after chromosome segregation .

The disruption of nuclear envelope dynamics in VRK1-deficient cells contributes to genomic instability and may be one of the underlying mechanisms of neurodevelopmental abnormalities seen in vrk1-/- zebrafish models . Furthermore, the interaction of VRK1 with chromatin through both linker DNA and the nucleosome acidic patch is important for its proper localization during mitosis, which in turn is crucial for its function in nuclear envelope dynamics .

How does VRK1 interact with chromatin in zebrafish cells?

VRK1 interacts with chromatin through a dual-binding mechanism that involves both linker DNA and the nucleosome acidic patch. This interaction is crucial for VRK1's function in histone modification, particularly the phosphorylation of histone H3 at threonine 3 (H3T3) . The mechanism of chromatin engagement by VRK1 has been elucidated through cryo-electron microscopy studies combined with biochemical and cellular assays .

The interaction with the nucleosome acidic patch is mediated by an arginine-rich flexible C-terminal tail of VRK1. This interaction is functionally significant, as mutations in this acidic patch recognition motif have been identified in patients with distal spinal muscular atrophy . These mutations interfere with nucleosome acidic patch binding, leading to mislocalization of VRK1 during mitosis, which provides a potential molecular mechanism for pathogenesis .

In zebrafish cells, as in other vertebrate cells, this chromatin interaction is essential for VRK1's role in regulating chromatin structure and function, particularly during mitosis. VRK1-deficient zebrafish exhibit defects in heterochromatin formation in the brain, indicating the importance of this kinase in chromatin organization . The proper interaction of VRK1 with chromatin is also necessary for its role in cell proliferation, as evidenced by decreased cell proliferation in the brains of vrk1-/- zebrafish .

How does VRK1 deficiency affect zebrafish brain development?

VRK1 deficiency has significant impacts on zebrafish brain development, with vrk1-/- zebrafish exhibiting mild microcephaly (reduced brain size) . This phenotype mirrors the microcephaly observed in human patients with VRK1 mutations and in Vrk1 knockdown mice, suggesting evolutionary conservation of VRK1's role in brain development across vertebrates .

The molecular and cellular mechanisms underlying VRK1-dependent brain development include:

• Reduced neural cell proliferation: VRK1-deficient zebrafish show decreased cell proliferation in the brain, consistent with VRK1's known role in cell cycle progression .

• Nuclear envelope defects: Abnormalities in nuclear envelope formation observed in vrk1-/- zebrafish brain cells likely contribute to disrupted neural progenitor proliferation and differentiation .

• Heterochromatin formation defects: VRK1 deficiency leads to impaired heterochromatin formation in the brain, which can alter gene expression patterns critical for proper brain development .

These findings from zebrafish models provide valuable insights into the pathophysiological mechanisms of VRK1-related microcephaly in humans, highlighting the utility of zebrafish as a model system for studying neurodevelopmental disorders associated with VRK1 mutations .

What is known about VRK1's role in motor function and dopaminergic signaling?

VRK1 plays a significant role in motor function and dopaminergic signaling in zebrafish. Studies with vrk1-deficient (vrk1-/-) zebrafish have demonstrated that these animals exhibit impaired motor function accompanied by low brain dopamine content . This finding establishes a previously unknown link between VRK1 and dopaminergic neuron development or function.

The relationship between VRK1 and motor dysfunction is particularly relevant in the context of human disease, as VRK1 mutations have been associated with motor neuron disorders, including spinal muscular atrophy . The zebrafish model has provided valuable insights into the potential mechanisms underlying these disorders.

Key observations regarding VRK1's role in motor function and dopaminergic signaling include:

• Reduced dopamine levels: VRK1-deficient zebrafish show significantly lower brain dopamine content compared to wild-type controls, suggesting a role for VRK1 in dopaminergic neuron development or function .

• Motor behavior deficits: vrk1-/- zebrafish display impaired swimming behavior and reduced motor activity, consistent with motor dysfunction .

• Potential mechanistic links: The connection between VRK1, dopaminergic signaling, and motor function may involve:

  • VRK1-dependent regulation of genes involved in dopamine synthesis or signaling

  • Effects on dopaminergic neuron development through VRK1's role in cell proliferation

  • Altered nuclear envelope or chromatin organization in dopaminergic neurons

These findings from zebrafish models contribute significantly to understanding the pathophysiological mechanisms underlying VRK1-mediated neurodegenerative diseases associated with motor dysfunction in humans .

How do VRK1 mutations contribute to neurodegenerative diseases?

VRK1 mutations have been implicated in several neurodegenerative conditions, with the zebrafish model providing significant insights into the underlying pathophysiological mechanisms. Multiple pathways connect VRK1 dysfunction to neurodegeneration:

• Disrupted nucleosome binding: Certain VRK1 mutations interfere with nucleosome acidic patch binding through the protein's arginine-rich C-terminal tail . This disruption leads to mislocalization of VRK1 during mitosis, potentially triggering cellular dysfunction in neural tissues .

• Nuclear envelope integrity: VRK1 mutations can compromise nuclear envelope formation and dynamics, critical for neuronal development and function . The nuclear envelope defects observed in vrk1-/- zebrafish brains suggest that nuclear membrane abnormalities contribute to neurodegeneration .

• Heterochromatin formation: VRK1-deficient zebrafish exhibit defects in heterochromatin formation in the brain, which may alter gene expression patterns and disrupt neuronal homeostasis . This chromatin disorganization likely contributes to progressive neuronal dysfunction.

• Cell cycle regulation impairment: Mutations affecting VRK1's role in cell cycle regulation may compromise neural progenitor proliferation, leading to developmental abnormalities and potentially triggering premature neuronal death .

• Dopaminergic signaling: The reduced dopamine content observed in vrk1-/- zebrafish brains suggests that VRK1 mutations may specifically impact dopaminergic neurons, explaining some of the motor manifestations of VRK1-related disorders .

The zebrafish vrk1-/- model has been particularly valuable in elucidating these mechanisms, as it recapitulates key features of human VRK1-related neurodegenerative diseases, including microcephaly and motor dysfunction . These insights contribute significantly to understanding the pathogenesis of conditions such as spinal muscular atrophy associated with VRK1 mutations .

How can phosphoproteomic approaches be used to identify VRK1 substrates in zebrafish?

Phosphoproteomic approaches offer powerful strategies for comprehensively identifying VRK1 substrates in zebrafish. A methodological framework for such investigations would include:

• Comparative phosphoproteomics: Compare phosphoproteome profiles between wild-type and vrk1-/- zebrafish using mass spectrometry-based approaches. This differential analysis can identify phosphorylation sites that are significantly reduced in VRK1-deficient samples, suggesting direct or indirect VRK1 substrates .

• Substrate consensus motif analysis: Based on known VRK1 phosphorylation sites, such as H3T3 and ATF2 Thr-73, develop a consensus motif for VRK1 phosphorylation . Use this motif to predict potential VRK1 substrates in the zebrafish proteome, followed by validation experiments.

• Kinase assays with candidate substrates: Express and purify recombinant zebrafish VRK1 and potential substrate proteins for in vitro kinase assays . Confirm phosphorylation sites using mass spectrometry or phospho-specific antibodies.

• Proximity-based labeling: Employ BioID or TurboID approaches with VRK1 as the bait protein to identify proteins in close proximity to VRK1 in zebrafish cells, which may include potential substrates. Combine this approach with phosphoproteomics to identify which proximal proteins are also phosphorylated in a VRK1-dependent manner .

• Temporal analysis during development: Conduct phosphoproteomic analysis at different developmental stages in wild-type and vrk1-/- zebrafish to identify stage-specific VRK1 substrates, particularly in the context of neurodevelopment .

This integrated approach allows for the systematic identification and validation of VRK1 substrates in zebrafish, providing insights into the molecular mechanisms underlying VRK1's diverse cellular functions and pathological roles .

What approaches can be used to study VRK1's role in transcriptional regulation?

VRK1 influences transcriptional regulation through the phosphorylation of transcription factors and chromatin modification. Several sophisticated approaches can be utilized to investigate this function in zebrafish:

• ChIP-seq of VRK1-regulated transcription factors: Chromatin immunoprecipitation followed by sequencing (ChIP-seq) can be performed for transcription factors known to be regulated by VRK1, such as ATF2 and CREB, in wild-type versus vrk1-/- zebrafish . This approach identifies genome-wide binding sites that are dependent on VRK1-mediated phosphorylation.

• RNA-seq comparative analysis: Transcriptome profiling of wild-type and vrk1-deficient zebrafish tissues, particularly brain tissue, can reveal genes and pathways whose expression is regulated by VRK1 . Time-course RNA-seq during development can further elucidate stage-specific transcriptional programs controlled by VRK1.

• Integrated histone modification analysis: Since VRK1 phosphorylates histone H3T3, ChIP-seq for this modification along with other histone marks can reveal how VRK1-mediated chromatin modification influences gene expression patterns . Combining this with RNA-seq data provides a comprehensive view of VRK1's impact on the epigenetic landscape.

• Promoter-reporter assays: Using luciferase or GFP reporter constructs driven by promoters of interest (e.g., cyclin D1, collagenase promoters), the effect of VRK1 on specific transcriptional elements can be quantified in zebrafish cell lines or in vivo . This approach is particularly useful for validating direct transcriptional targets.

• Single-cell transcriptomics: Applying single-cell RNA-seq to specific tissues from wild-type and vrk1-/- zebrafish can reveal cell type-specific transcriptional programs regulated by VRK1, particularly in heterogeneous tissues like the brain . This approach can identify subtle cell population-specific effects that might be missed in bulk RNA-seq.

• Transcription factor activity assays: Phosphorylation-dependent dimerization assays for transcription factors like ATF2 can directly measure how VRK1-mediated phosphorylation affects transcription factor activity . These functional assays provide mechanistic insights into VRK1's role in transcriptional regulation.

By integrating these approaches, researchers can build a comprehensive understanding of how VRK1 influences gene expression programs in zebrafish, contributing to its roles in development, cell cycle regulation, and disease pathogenesis .

How can advanced imaging techniques enhance understanding of VRK1 localization and dynamics?

Advanced imaging techniques provide powerful tools for investigating VRK1 localization, dynamics, and interactions in zebrafish models. These approaches can reveal spatiotemporal aspects of VRK1 function that are not accessible through biochemical methods alone:

• Live cell imaging with fluorescent protein fusions: Generating transgenic zebrafish expressing VRK1-GFP fusion proteins allows for real-time visualization of VRK1 localization during development and in response to various stimuli . This approach is particularly valuable for studying VRK1's dynamic localization during cell cycle progression and mitosis.

• Super-resolution microscopy: Techniques such as structured illumination microscopy (SIM), stimulated emission depletion (STED), or photoactivated localization microscopy (PALM) can resolve VRK1's subnuclear localization with nanometer precision . This is crucial for understanding VRK1's interactions with chromatin, the nuclear envelope, and other nuclear structures.

• Fluorescence resonance energy transfer (FRET): FRET-based biosensors can detect VRK1-substrate interactions or conformational changes in VRK1 in living zebrafish cells . This approach provides direct evidence of VRK1 activity and substrate engagement in physiologically relevant contexts.

• Fluorescence recovery after photobleaching (FRAP): FRAP experiments with fluorescently tagged VRK1 can assess the protein's mobility and binding dynamics within different nuclear compartments . This technique can reveal how VRK1 mutations affect its interaction with chromatin or other nuclear components.

• Proximity ligation assay (PLA): PLA can detect endogenous protein-protein interactions between VRK1 and its binding partners or substrates with high specificity and sensitivity in fixed zebrafish tissues . This is particularly useful for validating interactions identified through biochemical approaches.

• Correlative light and electron microscopy (CLEM): CLEM combines fluorescence microscopy with electron microscopy to correlate VRK1 localization with ultrastructural features, such as nuclear envelope integrity or chromatin organization . This approach is valuable for understanding structural defects in vrk1-deficient zebrafish.

• Light-sheet microscopy: For whole-embryo imaging, light-sheet microscopy allows for high-speed, low-phototoxicity imaging of VRK1 dynamics throughout zebrafish development . This technique is ideal for studying VRK1's role in developmental processes, particularly in the context of neurogenesis.

By integrating these advanced imaging approaches with genetic and biochemical methods, researchers can gain unprecedented insights into VRK1's spatiotemporal dynamics, interactions, and functions in zebrafish models of normal development and disease .