Recombinant Mouse Inositol-trisphosphate 3-kinase C (Itpkc)

What is the primary function of Itpkc in mouse models?

Itpkc (Inositol-trisphosphate 3-kinase C) primarily functions as a kinase that phosphorylates inositol 1,4,5-trisphosphate (IP3) to produce inositol 1,3,4,5-tetrakisphosphate (IP4). This conversion plays a critical role in calcium signaling pathways, particularly in immune cells. Similar to its family member ITPKB, Itpkc is involved in the regulation of calcium mobilization, which subsequently affects downstream signaling cascades including the nuclear factor of activated T-cells (NFAT) pathway . In experimental models, Itpkc activity has been shown to modulate calcium-dependent cellular responses, making it a significant target for understanding immune cell function and inflammatory processes.

How does Itpkc differ from other inositol-trisphosphate 3-kinase family members?

While Itpkc shares the core catalytic function of phosphorylating IP3 to IP4 with other family members (Itpka and Itpkb), it exhibits distinct tissue distribution, regulatory mechanisms, and physiological roles. Unlike ITPKB, which has been more extensively studied in dendritic cells and immune regulation , Itpkc has unique expression patterns and may be regulated differently at both transcriptional and post-translational levels.

The three mammalian inositol trisphosphate 3-kinases (Itpka, Itpkb, and Itpkc) likely evolved from a common ancestral gene but have diverged to serve specialized functions in different tissues and signaling contexts. Recent evolutionary studies have traced the diversification of these enzymes across plants and animals, suggesting functional specialization throughout vertebrate evolution . Each isoform contains conserved catalytic domains but differs in regulatory regions, accounting for their distinctive roles in calcium signaling networks.

What are the recommended protocols for expressing and purifying recombinant mouse Itpkc?

For successful expression and purification of recombinant mouse Itpkc, the following methodological approach is recommended:

• Vector Selection and Cloning:

  • Clone the full-length mouse Itpkc cDNA into an expression vector with an appropriate tag (His, GST, or FLAG)

  • Verify the sequence integrity through DNA sequencing to confirm the absence of mutations

• Expression System:

  • For mammalian expression: Use HEK293 or CHO cells transfected with the expression construct

  • For bacterial expression: Use E. coli BL21(DE3) strain with optimization of induction conditions (0.1-0.5 mM IPTG at 16-18°C overnight)

• Purification Strategy:

  • Lyse cells in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, protease inhibitors

  • For His-tagged protein: Use Ni-NTA affinity chromatography

  • Include a size exclusion chromatography step for higher purity

  • Verify purity by SDS-PAGE and confirm activity through enzyme assays

• Storage Conditions:

  • Store in buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM DTT, 10% glycerol

  • Flash-freeze aliquots and store at -80°C to maintain enzyme activity

The enzyme activity should be assessed using IP3 kinase assays measuring the conversion of IP3 to IP4, similar to methods used for assessing ITPKB activity in previous studies .

In Vitro Assays:

• Radiometric Assay:

  • Incubate purified Itpkc with [³H]-labeled IP3 substrate

  • Separate reaction products by anion exchange chromatography or HPLC

  • Quantify [³H]-IP4 formation as a measure of enzyme activity

• Fluorescence-Based Assays:

  • Use fluorescently labeled IP3 analogs

  • Monitor reaction progress in real-time by detecting changes in fluorescence properties

• Coupled Enzyme Assays:

  • Link ATP consumption during IP3 phosphorylation to NADH oxidation

  • Monitor decrease in NADH fluorescence (340/460 nm) spectrophotometrically

In Vivo/Cellular Assays:

• IP4 Quantification:

  • Extract cellular inositol phosphates using perchloric acid

  • Separate and quantify IP4 levels by HPLC or mass spectrometry

  • Compare IP4/IP3 ratios between experimental conditions

• Calcium Flux Measurements:

  • Load cells with calcium-sensitive dyes (Fura-2 or Fluo-4)

  • Stimulate with appropriate agonists to induce calcium signals

  • Compare calcium dynamics between wild-type and Itpkc-manipulated cells

• NFAT Translocation Assays:

  • Monitor NFAT nuclear translocation by immunofluorescence or with NFAT-GFP constructs

  • Quantify nuclear:cytoplasmic NFAT ratio as readout of calcium-dependent signaling downstream of Itpkc

For both types of assays, appropriate controls including enzyme inhibitors, catalytically inactive Itpkc mutants, and genetic knockdown/knockout models are essential for confirming specificity.

How does Itpkc modulate calcium signaling in immune cell function?

Itpkc functions as a critical regulator of calcium signaling in immune cells through its enzymatic activity converting IP3 to IP4. This modulation follows several mechanistic pathways:

• IP3 Receptor Regulation:

  • By converting IP3 to IP4, Itpkc reduces the available IP3 pool that would otherwise activate IP3 receptors (IP3Rs)

  • IP4 produced by Itpkc may directly interact with IP3Rs to modulate channel activity

  • In dendritic cells, IP4 produced by the related enzyme ITPKB has been shown to stimulate the IP3 receptor subtype 3 (IP3R3), which colocalizes with CD14 on the plasma membrane

• Calcium-NFAT Axis Regulation:

  • Calcium influx triggered by IP4 leads to calcineurin activation

  • Activated calcineurin dephosphorylates NFAT, allowing its nuclear translocation

  • Nuclear NFAT drives transcription of proinflammatory genes

  • Studies with ITPKB have demonstrated that LPS-induced nuclear translocation of NFAT in dendritic cells depends on calcium influx triggered by IP4

• Inflammatory Response Modulation:

  • Itpkc activity influences the magnitude and duration of calcium signals

  • This in turn affects immune cell functions including cytokine production, differentiation, and migration

  • Pharmacological inhibition of the related enzyme ITPKB in mice reduced LPS-induced tissue swelling and inflammatory arthritis severity, suggesting similar potential roles for Itpkc

These mechanisms highlight the potential of Itpkc as a therapeutic target for modulating inflammation in various disease contexts.

What are the main differences in signaling outcomes between Itpkb and Itpkc in experimental models?

While both Itpkb and Itpkc catalyze the same biochemical reaction (conversion of IP3 to IP4), they exhibit distinct signaling outcomes due to differences in:

• Expression Patterns:

  • Itpkb is expressed more broadly across immune cell types

  • Itpkc shows more restricted tissue expression patterns

  • These differential expression profiles lead to cell type-specific signaling outcomes

• Subcellular Localization:

  • Itpkb has been observed to colocalize with IP3R3 and CD14 on the plasma membrane of dendritic cells

  • The subcellular localization of Itpkc may differ, affecting its access to substrate pools and proximity to downstream effectors

• Signaling Outcomes:

  • In dendritic cells, ITPKB-mediated IP4 production is necessary for calcium mobilization and NFAT activation in response to LPS

  • The specialized role of Itpkc in signaling pathways is still being fully characterized, but evidence suggests it may regulate distinct aspects of calcium signaling

  • Studies in knockout models suggest that the enzymes are not fully redundant, indicating specialized functions

• Disease Associations:

  • ITPKC polymorphisms (e.g., rs7251246) have been associated with increased risk of coronary artery aneurysms in Kawasaki disease

  • These polymorphisms affect ITPKC mRNA expression levels, with the CC genotype showing lower ITPKC mRNA levels in children with Kawasaki disease

  • The disease associations of Itpkb appear to be different, suggesting distinct physiological roles

Understanding these differences is critical for developing targeted therapeutic approaches that modulate specific aspects of calcium signaling pathways.

How should researchers interpret contradictory data regarding Itpkc function in different mouse tissues?

When facing contradictory data about Itpkc function across different mouse tissues, researchers should consider the following analytical approach:

• Experimental Context Analysis:

  • Evaluate differences in experimental systems (in vitro vs. in vivo, primary cells vs. cell lines)

  • Assess genetic background variations between mouse strains which may contain modifier genes

  • Consider developmental stage differences, as Itpkc function may vary during development

• Methodological Considerations:

  • Examine detection methods (antibody specificity, PCR primer design)

  • Evaluate knockdown/knockout verification strategies

  • Compare acute vs. chronic manipulation approaches (pharmacological inhibition vs. genetic deletion)

• Signaling Network Compensation:

  • Analyze potential compensatory upregulation of related kinases (Itpka, Itpkb)

  • Assess adaptive changes in IP3R expression or localization

  • Consider alternative calcium signaling pathways that may become dominant in specific tissues

• Multifactorial Data Integration:

  • Create a systematic comparison table of conflicting results

  • Weight evidence based on methodological rigor

  • Identify patterns that may explain tissue-specific differences

• Validation Strategies:

  • Design experiments that directly address contradictions

  • Utilize multiple complementary approaches (genetic, pharmacological, biochemical)

  • Consider tissue-specific conditional knockout models to eliminate developmental compensation

By systematically addressing these factors, researchers can develop unifying hypotheses that accommodate seemingly contradictory observations about Itpkc function.

What are the best approaches for studying interactions between Itpkc and IP3 receptors in native cellular contexts?

To effectively study Itpkc-IP3R interactions in native cellular contexts, researchers should consider these methodological approaches:

• Proximity-Based Interaction Assays:

  • Proximity Ligation Assay (PLA): Detects proteins within 40 nm proximity in fixed cells

  • FRET/BRET: For measuring dynamic interactions in living cells

  • BiFC (Bimolecular Fluorescence Complementation): To visualize direct protein interactions

• Co-Immunoprecipitation Strategies:

  • Use membrane-compatible detergents to preserve interaction integrity

  • Consider crosslinking approaches for transient interactions

  • Implement IP3R subtype-specific antibodies to determine specificity

  • Compare results under resting and stimulated conditions to detect dynamic interactions

• Advanced Microscopy Techniques:

  • STED Microscopy: For super-resolution imaging of protein co-localization, similar to techniques used to demonstrate IP3R3 colocalization with ITPKB and CD14 on the plasma membrane

  • Live-Cell Imaging: With fluorescently tagged proteins to track dynamic interactions

  • Calcium Microdomain Imaging: To correlate interactions with local calcium signals

• Functional Interaction Assessment:

  • Domain Mapping: Generate deletion/mutation constructs to identify interaction interfaces

  • Competitive Peptide Interference: Design peptides mimicking interaction domains

  • Manipulation of IP4 Levels: Use IP4 analogs or metabolically stable derivatives to assess functional consequences

• Mathematical Modeling:

  • Develop computational models incorporating Itpkc-IP3R interactions

  • Simulate calcium dynamics under different interaction scenarios

  • Validate model predictions experimentally

These approaches, used in combination, can provide robust evidence of functional interactions between Itpkc and IP3 receptors in physiologically relevant contexts.

How might targeting Itpkc activity be applied in inflammatory disease treatment strategies?

Based on emerging research, targeting Itpkc activity presents several promising therapeutic strategies for inflammatory diseases:

• Mechanistic Rationale:

  • Itpkc modulates calcium signaling through IP4 production

  • Calcium signaling drives NFAT activation and proinflammatory gene expression

  • Inhibition of the related enzyme ITPKB reduced LPS-induced tissue swelling and inflammatory arthritis severity in mice

  • ITPKC polymorphisms affecting expression levels are associated with inflammatory conditions like Kawasaki disease

• Potential Therapeutic Approaches:

  • Small Molecule Inhibitors: Develop selective Itpkc catalytic domain inhibitors

  • Allosteric Modulators: Target regulatory domains to fine-tune activity rather than completely block function

  • Gene Therapy: Correct disease-associated variants in appropriate contexts

  • Cell-Type Specific Delivery: Use nanoparticle-based approaches similar to those used to deliver NFAT-inhibiting peptides specifically to phagocytic cells

• Disease Applications:

  • Autoimmune Disorders: Modulate aberrant immune cell activation

  • Inflammatory Vascular Diseases: Target vasculitis pathways, particularly in Kawasaki disease where ITPKC downregulation is observed

  • Inflammatory Arthritis: Build on findings that ITPKB inhibition reduced inflammatory arthritis severity

• Combination Strategies:

  • Combine Itpkc modulation with existing anti-inflammatory approaches

  • Target multiple nodes in calcium-dependent inflammatory cascades

  • Use temporal modulation to limit side effects

These approaches would need careful development to ensure specificity and limit off-target effects on physiological calcium signaling pathways.

What genetic models are most suitable for investigating Itpkc function in complex disease phenotypes?

To effectively investigate Itpkc function in complex disease phenotypes, researchers should consider these genetic model systems:

• Mouse Genetic Models:

  • Conventional Knockout: Complete deletion of Itpkc gene

  • Conditional Knockout: Tissue-specific and/or temporally controlled deletion using Cre-loxP technology

  • Knockin Models: Introduction of disease-associated mutations (e.g., equivalent to human polymorphisms like rs7251246 )

  • Reporter Models: Knockin of reporter genes to track Itpkc expression patterns

• Selection Criteria for Disease Models:

  Disease Context	Recommended Model	Key Advantage
  Inflammatory disorders	Myeloid-specific conditional KO	Targets innate immune cells while preserving function elsewhere
  Vascular pathologies	Endothelial-specific inducible KO	Allows temporal control to separate developmental from homeostatic roles
  Neurological disorders	Brain region-specific KO	Addresses specific neural circuits while avoiding developmental compensation
  Metabolic dysfunction	Global hypomorphic alleles	Partially reduces function to mimic human polymorphisms

• Advanced Genetic Approaches:

  • CRISPR-based screens: For identifying genetic modifiers of Itpkc function

  • Humanized mouse models: Replacement of mouse Itpkc with human ITPKC gene

  • Allelic series: Creation of multiple strains with varying levels of Itpkc activity

• Physiological Assessment:

  • Comprehensive phenotyping across multiple physiological systems

  • Challenge models that reveal phenotypes not apparent under homeostatic conditions

  • Multi-omics approaches to capture system-wide effects of Itpkc manipulation

• Translational Considerations:

  • Correlate findings with human genetic studies

  • Validate in human cellular systems when possible

  • Consider pharmacological validation alongside genetic approaches

Selection of the appropriate model should be guided by the specific disease context and research question, with consideration of potential compensatory mechanisms that may mask phenotypes in conventional knockout models.

What are the major challenges in developing specific antibodies for mouse Itpkc and how can they be overcome?

Developing specific antibodies for mouse Itpkc presents several technical challenges due to its structural similarity with other inositol trisphosphate 3-kinase family members. Researchers can address these challenges through the following approaches:

• Challenges in Antibody Development:

  • High sequence homology between Itpka, Itpkb, and Itpkc in conserved catalytic domains

  • Limited immunogenicity of unique regions

  • Potential post-translational modifications affecting epitope accessibility

  • Low native expression levels in many tissues

• Antigen Selection Strategies:

  • Unique Peptide Selection: Target Itpkc-specific sequences outside the catalytic domain

  • Recombinant Protein Domains: Use unique regulatory domains as immunogens

  • Differential Screening: Develop antibodies against multiple isoforms and screen for specificity

  • Post-translational Modification-specific: Generate antibodies against Itpkc-specific phosphorylation sites

• Validation Methodology:

  • Multi-platform Validation: Test antibodies by Western blot, IP, IHC, and flow cytometry

  • Knockout Controls: Use Itpkc knockout tissues/cells as negative controls

  • Isoform Cross-reactivity Testing: Test against recombinant Itpka and Itpkb

  • Epitope Mapping: Confirm binding to the intended region

• Alternative Approaches:

  • Genetic Tagging: Generate knock-in mice with epitope-tagged Itpkc

  • Nanobody Development: Use camelid antibody fragments for improved specificity

  • Aptamer Selection: Develop DNA/RNA aptamers as alternative binding reagents

By implementing these strategic approaches, researchers can overcome the challenges in developing specific antibodies for mouse Itpkc, enabling more reliable detection and functional studies of this important signaling enzyme.

What considerations are important when designing experimental controls for Itpkc activity assays?

Designing robust experimental controls is critical for accurate interpretation of Itpkc activity assays. Researchers should implement the following control strategies:

• Enzyme Source Controls:

  • Positive Controls: Purified recombinant Itpkc with confirmed activity

  • Negative Controls: Heat-inactivated enzyme or catalytically inactive mutants

  • Specificity Controls: Related kinases (Itpka, Itpkb) to assess assay selectivity

• Substrate and Reaction Controls:

  • Substrate Purity: Verify IP3 substrate purity by analytical methods

  • Product Standards: Include synthetic IP4 standards for calibration

  • Non-specific Hydrolysis: Monitor IP3 stability in assay conditions without enzyme

  • ATP Dependence: Confirm ATP requirement by omitting ATP from reaction

• Inhibitor Controls:

  • Known Inhibitors: Include established inhibitors at varying concentrations

  • Structurally Related Inactive Compounds: Test specificity of inhibition

  • Solvent Controls: Include vehicle controls for compounds dissolved in DMSO or ethanol

• Cellular Assay Controls:

  • Genetic Controls: Use Itpkc knockout/knockdown cells alongside wild-type

  • Overexpression Controls: Compare endogenous to overexpressed enzyme activity

  • Stimulus Controls: Include unstimulated cells and maximal stimulus controls

  • Time Course: Establish appropriate kinetics for cellular responses

• Analytical Controls:

  Control Type	Purpose	Implementation
  Internal Standards	Normalize between experiments	Add known quantities of labeled standards
  Recovery Controls	Assess extraction efficiency	Spike samples with known analyte amounts
  Matrix Effects	Control for sample composition	Prepare standards in matched sample matrix
  Technical Replicates	Assess assay precision	Perform multiple measurements of same sample
  Biological Replicates	Account for biological variability	Use independent biological samples

Implementing these comprehensive control strategies will enhance the reliability and reproducibility of Itpkc activity measurements across different experimental systems.

How might systems biology approaches advance our understanding of Itpkc in calcium signaling networks?

Systems biology approaches offer powerful frameworks for understanding Itpkc's role within complex calcium signaling networks:

• Network Modeling Approaches:

  • Ordinary Differential Equation (ODE) Models: Capture the dynamics of Itpkc-mediated IP3 to IP4 conversion and downstream calcium oscillations

  • Agent-Based Models: Simulate subcellular localization and microdomains of calcium signaling components

  • Bayesian Network Analysis: Infer causal relationships between Itpkc activity and downstream signaling events

  • Constraint-Based Modeling: Identify fundamental constraints in Itpkc regulation

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, metabolomics (focusing on inositol phosphates), and phosphoproteomics data

  • Map Itpkc-dependent alterations across multiple molecular levels

  • Identify emergent properties and feedback loops not apparent from single-omics approaches

• Spatiotemporal Signaling Analysis:

  • Real-time Biosensor Development: Create specific biosensors for IP4 similar to those developed for other signaling molecules

  • Multiplexed Imaging: Simultaneously track multiple components of the signaling pathway

  • 4D Analysis: Incorporate temporal dynamics with spatial distribution of signaling components

• Cross-species Network Comparison:

  • Leverage evolutionary insights into inositol trisphosphate 3-kinase diversification across species

  • Identify conserved network motifs vs. species-specific adaptations

  • Correlate network architecture with physiological demands

• Therapeutic Network Perturbation Analysis:

  • Predict system-wide effects of pharmacological Itpkc inhibition

  • Identify optimal multi-target intervention strategies

  • Quantify robustness and fragility in calcium signaling networks

These systems approaches will help transition from reductionist understandings of Itpkc to comprehensive network-level insights, potentially revealing new therapeutic strategies and fundamental principles of calcium signal transduction.

What emerging technologies might enhance our ability to study Itpkc function at the single-cell level?

Emerging technologies are revolutionizing the study of signaling enzymes like Itpkc at single-cell resolution, offering unprecedented insights into functional heterogeneity:

• Advanced Single-Cell Genomics:

  • Single-cell RNA-seq: Profile transcriptional consequences of Itpkc activity variations

  • Single-cell ATAC-seq: Link chromatin accessibility to Itpkc-dependent NFAT translocation

  • Spatial Transcriptomics: Map Itpkc expression and activity signatures with tissue context preservation

  • Multi-modal Single-cell Analysis: Simultaneously profile RNA, protein, and metabolites

• High-Resolution Imaging Technologies:

  • Lattice Light Sheet Microscopy: Capture 3D dynamics of Itpkc localization with minimal phototoxicity

  • STORM/PALM Super-resolution: Resolve nanoscale interactions between Itpkc and IP3 receptors

  • Expansion Microscopy: Physically expand specimens to resolve subcellular details

  • Correlative Light-Electron Microscopy: Combine functional imaging with ultrastructural context

• Live-Cell Biochemical Probes:

  • IP4-specific Biosensors: Develop FRET-based sensors to detect IP4 production in real-time

  • Optogenetic Itpkc Modulators: Control Itpkc activity with light to precisely manipulate signaling dynamics

  • Split Fluorescent Protein Systems: Monitor protein-protein interactions in native contexts

  • Calcium Microdomain Sensors: Target calcium indicators to specific subcellular compartments

• Microfluidic and Nanoscale Technologies:

  • Droplet Microfluidics: Isolate single cells for biochemical assays of Itpkc activity

  • Organ-on-a-chip: Study Itpkc in physiologically relevant tissue microenvironments

  • Nanoparticle-based Delivery: Target Itpkc inhibitors to specific cell populations

• Computational Analysis Tools:

  • Deep Learning Image Analysis: Extract subtle patterns from complex imaging data

  • Trajectory Inference Algorithms: Map cellular states during Itpkc-mediated signaling responses

  • Causal Network Inference: Deduce regulatory relationships from single-cell perturbation data

These emerging technologies, especially when used in combination, will provide unprecedented insights into how Itpkc functions in heterogeneous cell populations and complex tissues, potentially revealing new principles of calcium signal encoding and decoding.