Recombinant Danio rerio Casein kinase I isoform delta-A (csnk1da)

What is Recombinant Danio rerio Casein kinase I isoform delta-A?

Recombinant Danio rerio Casein kinase I isoform delta-A (csnk1da) is one of two zebrafish orthologues of the human CK1δ protein. It belongs to the CK1 family of serine/threonine protein kinases that are involved in regulating various signaling pathways including Wnt/β-catenin, Hedgehog, and p53 signaling pathways . The recombinant protein is produced through heterologous expression systems, typically using bacterial or yeast expression platforms, to generate purified protein for biochemical and structural studies .

How does zebrafish CK1δA differ from human CK1δ?

Zebrafish CK1δA shares significant sequence homology and functional characteristics with human CK1δ. Kinetic studies have shown comparable enzymatic parameters between the human and zebrafish orthologues. Inhibition studies revealed that zebrafish CK1δA is inhibited as effectively as human CK1δ by selective inhibitors, with similar IC50 values. For example, the compound G2-2 demonstrates IC50 values of 345 nM for zebrafish CK1δA versus 503 nM for human CK1δ . This high degree of conservation makes zebrafish an excellent model organism for studying CK1δ functions and for screening potential CK1δ inhibitors for therapeutic applications.

What signaling pathways involve CK1δA in zebrafish development?

CK1δA in zebrafish is involved in multiple critical signaling cascades during embryonic development. It participates in regulating the Wnt/β-catenin pathway, which is essential for axis formation and organogenesis, the Hedgehog pathway involved in cell fate determination, and the p53 pathway that controls cell cycle and apoptosis . Inhibition studies have demonstrated that disruption of CK1δA activity in zebrafish embryos results in specific developmental abnormalities including cardiovascular dysfunction, malformation of the tail, necrosis, and early embryonic mortality, indicating its essential role in proper embryonic development .

How can one generate and purify recombinant zebrafish CK1δA for biochemical studies?

The generation of recombinant zebrafish CK1δA typically begins with PCR amplification of the cDNA encoding the protein, followed by insertion into a prokaryotic expression vector such as pET28a(+) via Gibson Assembly or similar cloning techniques . Expression can be induced in E. coli strains such as Rosetta 2(DE3) using IPTG at optimized conditions (e.g., 0.5 μM IPTG, 18°C, 120 rpm for 18 hours) .

For purification of His-tagged recombinant CK1δA, the following protocol is recommended:

• Harvest bacteria by centrifugation and lyse cells using appropriate buffer (e.g., 50 mM sodium phosphate buffer pH 7.0, 350 mM NaCl, 15 mM imidazole, 0.5% NP-40, 10% glycerine, protease inhibitors)

• Clear lysate by centrifugation and incubate with metal affinity resin (such as TALON)

• Wash thoroughly with washing buffer containing 15 mM imidazole

• Elute with buffer containing 300 mM imidazole

• Dialyze against imidazole-free buffer to remove excess imidazole

• Store aliquots at -80°C to maintain activity

For long-term storage, adding 5-50% glycerol (final concentration) to purified protein and aliquoting for storage at -20°C to -80°C is recommended .

What are the optimal conditions for assessing CK1δA kinase activity in vitro?

The optimal conditions for assessing zebrafish CK1δA kinase activity include:

Buffer composition:

• 25 mM Tris-HCl (pH 7.5)

• 10 mM MgCl2

• 0.1 mM EDTA

• 0.1% Triton X-100

• 10 μM ATP (including radioactive ATP for detection)

• 1 mM DTT

Substrate: A peptide substrate containing the consensus sequence pS/T-X-X-S/T is typically used, such as the CK1tide (RRKHAAIGpSAYSITA) or a casein-derived peptide.

Temperature and time: Reactions are typically conducted at 30°C for 15-30 minutes, within the linear range of enzyme activity.

Controls: Include negative controls (no enzyme), positive controls (using commercial CK1), and inhibitor controls (using known CK1 inhibitors like D4476).

Kinetic parameters should be determined using varying substrate concentrations to establish Km and Vmax values. Activity should be measured using either radioactive (³²P-ATP incorporation) or non-radioactive (phospho-specific antibodies or fluorescent/luminescent reporters) detection methods to quantify phosphorylation.

What is the comparative inhibition profile of CK1δA versus CK1δB with known inhibitors?

The comparative inhibition profiles of zebrafish CK1δA and CK1δB with selective inhibitors have been characterized, showing similar inhibition patterns between the two isoforms and their human counterpart:

These data indicate that both zebrafish CK1δ isoforms are inhibited with comparable efficacy to human CK1δ . This inhibition translates to phenotypic effects in vivo, with treated zebrafish embryos developing blood stasis, heart failure, and tail malformations in a dose-dependent manner, confirming the biological relevance of these inhibitors in a whole-organism context .

How can zebrafish be used as a model system for CK1δA inhibitor screening?

Zebrafish provides an excellent in vivo platform for CK1δA inhibitor screening due to its rapid development, optical transparency, and genetic amenability. A comprehensive screening protocol would include:

This approach allows for rapid in vivo assessment of inhibitor efficacy, specificity, and toxicity in a vertebrate system before proceeding to mammalian models.

What phenotypic markers indicate successful CK1δA inhibition in zebrafish embryos?

Successful CK1δA inhibition in zebrafish embryos manifests through several characteristic phenotypic markers that can be monitored and quantified:

These phenotypic markers correlate with the developmental impairments observed following CK1δA downregulation using morpholinos, confirming the specificity of the inhibitor effects . The severity of these phenotypes typically develops in a dose-dependent manner, with higher inhibitor concentrations producing more pronounced effects.

How do mutations in CK1δA affect signaling pathway regulation in zebrafish?

Mutations in CK1δA disrupt multiple signaling pathways in zebrafish, with the following effects:

• Wnt/β-catenin pathway: CK1δA mutations can dysregulate β-catenin phosphorylation and degradation, potentially leading to constitutive pathway activation. This disruption manifests as axis duplication, posterior truncation, or head defects depending on the specific mutation and timing of effect.

• Hedgehog pathway: CK1δA normally phosphorylates Gli transcription factors and Smoothened. Mutations can lead to abnormal Hedgehog signaling, resulting in defects in floor plate specification, motor neuron development, and somite patterning.

• p53 pathway: CK1δA mutations can impact p53 regulation, potentially affecting cell cycle control and apoptotic responses during development. This may be observed as increased sensitivity to DNA damage or altered patterns of programmed cell death.

• Circadian rhythm: CK1δA plays a crucial role in phosphorylating clock proteins. Mutations can disrupt circadian gene expression patterns and behavioral rhythms in developing and adult zebrafish.

The specific effects depend on the nature of the mutation (loss-of-function vs. gain-of-function) and can be analyzed through phosphorylation status of downstream targets, reporter gene expression, and transcriptomic analysis of pathway components.

What quantitative methods can be used to assess CK1δA inhibition efficacy in zebrafish?

Several quantitative methods can be employed to rigorously assess CK1δA inhibition efficacy in zebrafish:

• Biochemical validation:

  • Western blotting with phospho-specific antibodies to quantify reduced phosphorylation of known CK1δA substrates

  • In vitro kinase assays using tissue lysates to measure residual CK1δA activity

  • Mass spectrometry-based phosphoproteomics to identify global changes in phosphorylation patterns

• Phenotypic quantification:

  • Heart rate measurements (beats per minute)

  • Blood flow velocity using transgenic lines with fluorescent blood cells

  • Quantitative morphometrics of tail length and curvature

  • Survival rates and developmental timing

• Molecular readouts:

  • qRT-PCR analysis of target gene expression in relevant pathways

  • Luciferase reporter assays for pathway activity

  • RNA-seq for global transcriptional changes

  • Whole-mount in situ hybridization with densitometric analysis

• Functional assays:

  • Behavioral analyses for circadian rhythm disruption

  • Cell proliferation and apoptosis quantification using TUNEL or BrdU incorporation

  • Calcium imaging for heart function assessment

Data from these assays can be compiled to create dose-response curves and determine EC50 values for phenotypic endpoints, which can then be compared with biochemical IC50 values to establish pharmacokinetic/pharmacodynamic relationships for CK1δA inhibitors.

What are common challenges in expressing and purifying active recombinant zebrafish CK1δA?

Researchers face several technical challenges when working with recombinant zebrafish CK1δA:

• Solubility issues: CK1δA can form inclusion bodies during bacterial expression, reducing yield of soluble protein.

  • Solution: Optimize expression conditions by lowering temperature (15-18°C), reducing IPTG concentration (0.1-0.5 mM), and using specialized E. coli strains like Rosetta 2(DE3) that supply rare codons .

• Protein stability: Purified CK1δA may show reduced activity over time during storage.

  • Solution: Add stabilizing agents such as glycerol (5-50%), avoid repeated freeze-thaw cycles, and store working aliquots at 4°C for up to one week .

• Post-translational modifications: Bacterial expression systems lack mammalian post-translational modifications that may be important for regulation.

  • Solution: Consider eukaryotic expression systems (yeast, insect, or mammalian cells) for studies requiring native-like modifications.

• Autophosphorylation: CK1δA undergoes autophosphorylation, which can lead to heterogeneous protein preparations.

  • Solution: Include phosphatase treatment steps during purification or use kinase-dead mutants for structural studies.

• Contaminating kinases: E. coli-derived kinases may co-purify with the target protein.

  • Solution: Use multiple purification steps, including ion exchange chromatography after initial affinity purification.

Proper quality control through activity assays, mass spectrometry, and SDS-PAGE analysis should be performed to ensure protein homogeneity and activity before experimental use.

How can researchers differentiate between the effects of CK1δA and CK1δB inhibition in zebrafish?

Differentiating between CK1δA and CK1δB inhibition effects presents a significant challenge due to their sequence similarity and potential functional redundancy. Researchers can employ the following strategies:

• Isoform-specific genetic approaches:

  • Generate isoform-specific knockout lines using CRISPR/Cas9

  • Use splice-blocking morpholinos targeting unique exon junctions

  • Create isoform-specific dominant-negative constructs

• Rescue experiments:

  • Perform rescue experiments in morphants/mutants with isoform-specific mRNA

  • Test if human CK1δ can rescue either or both isoform deficiencies

• Expression and localization analysis:

  • Generate isoform-specific antibodies for immunolocalization

  • Create transgenic reporter lines with isoform promoters driving fluorescent proteins

  • Perform in situ hybridization with isoform-specific probes to determine tissue-specific expression patterns

• Biochemical approaches:

  • Develop isoform-selective inhibitors through structure-based design

  • Characterize substrate preferences of each isoform through in vitro kinase assays

  • Identify isoform-specific interaction partners through IP-MS approaches

• Temporal analysis:

  • Use heat-shock inducible or chemically-inducible systems for temporal control of isoform inhibition

  • Apply inhibitors at different developmental stages to identify time-sensitive processes

By combining these approaches, researchers can build a comprehensive understanding of the specific roles of CK1δA versus CK1δB in zebrafish development and physiology.

How might zebrafish CK1δA studies translate to human disease models?

Zebrafish CK1δA research has significant translational potential for human disease models due to the high conservation of CK1 signaling pathways across vertebrates. Several promising translational applications include:

• Neurodegenerative diseases:

  • CK1δA is implicated in tau phosphorylation and α-synuclein processing, relevant to Alzheimer's and Parkinson's diseases

  • Zebrafish models expressing human disease proteins can be used to test CK1δ inhibitors for reducing pathological phosphorylation

  • High-throughput behavioral assays in zebrafish can assess cognitive and motor improvements

• Cancer models:

  • Dysregulation of CK1δ is associated with various cancers through effects on Wnt signaling and p53 regulation

  • Zebrafish xenograft models allow for testing CK1δ inhibitors against human cancer cells in vivo

  • Transgenic zebrafish expressing oncogenes can reveal how CK1δA modulates cancer progression

• Circadian rhythm disorders:

  • CK1δA regulates period proteins critical for circadian rhythms

  • Zebrafish circadian mutants can model human sleep disorders

  • Small molecule screens can identify compounds that modulate CK1δA activity to restore normal rhythms

• Inflammatory conditions:

  • CK1δA influences inflammatory signaling pathways

  • Zebrafish inflammation models provide visual readouts of neutrophil recruitment and resolution

  • Anti-inflammatory effects of CK1δA modulation can be rapidly assessed

The combination of genetic conservation, rapid development, and optical transparency makes zebrafish an ideal bridge between in vitro studies and mammalian disease models, accelerating the translation of basic CK1δA research to human therapeutic applications.

What emerging technologies might enhance zebrafish CK1δA research?

Several cutting-edge technologies are poised to significantly advance zebrafish CK1δA research:

• CRISPR-based technologies:

  • Base editing and prime editing for precise modification of CK1δA without double-strand breaks

  • CRISPRi/CRISPRa for temporal and tissue-specific regulation of CK1δA expression

  • CRISPR screening approaches to identify genetic interactors of CK1δA

• Advanced imaging techniques:

  • Light sheet microscopy for long-term, high-resolution imaging of CK1δA activity reporters

  • Super-resolution microscopy to visualize CK1δA subcellular localization

  • Correlative light and electron microscopy (CLEM) to connect CK1δA function with ultrastructural changes

• Single-cell technologies:

  • Single-cell RNA-seq to map CK1δA expression patterns across development

  • Single-cell proteomics to profile CK1δA-dependent phosphorylation events

  • Spatial transcriptomics to correlate CK1δA activity with gene expression in intact tissues

• Optogenetics and chemogenetics:

  • Optogenetic control of CK1δA activity for precise spatiotemporal manipulation

  • Chemically-induced proximity systems for rapid activation/inhibition of CK1δA

• Microfluidic approaches:

  • Automated embryo handling and drug delivery for high-throughput screening

  • Organ-on-chip models incorporating zebrafish cells for specialized assays

• Computational approaches:

  • Machine learning algorithms for automated phenotype classification

  • Molecular dynamics simulations to predict isoform-specific inhibitor binding

  • Systems biology modeling of CK1δA in signaling networks

These technologies will enable more precise manipulation of CK1δA function, higher-resolution analysis of its activities, and more comprehensive understanding of its roles in development and disease.