PI3KγD946GST Human

What is PI3KγD946GST Human and how does it differ from native PI3Kγ?

PI3KγD946GST Human is a recombinant catalytically inactive mutant of phosphoinositide 3-kinase gamma (PI3Kγ) featuring a D946A mutation in the ATP binding site. This 126.3 kDa protein (without tag) carries an N-terminal GST-Tag that facilitates its application in GST pull-down assays . The key distinction from native PI3Kγ is the D946A mutation that renders it enzymatically inactive while maintaining structural integrity.

Unlike native PI3Kγ, which phosphorylates the 3-position hydroxyl groups of the inositol ring of Phosphatidylinositol (PtdIns) to generate PtdIns(3,4,5)P₃ , PI3KγD946GST cannot catalyze this reaction. This property makes it particularly valuable as a negative control in PI3Kγ kinase activity studies and for investigating protein-protein interactions without the confounding effects of enzymatic activity.

What are the optimal storage and handling conditions for PI3KγD946GST Human?

For optimal preservation of PI3KγD946GST Human's structural integrity and experimental utility, store the protein at 4°C if the entire vial will be used within 2-4 weeks. For longer storage periods, maintain the protein at -20°C in its formulation of 0.4mg/ml solution in 25mM Tris-HCl (pH 8.0), 50mM NaCl, 0.5mM MgCl₂, and 50% glycerol .

To prevent protein degradation during extended storage, addition of a carrier protein (0.1% HSA or BSA) is recommended. Importantly, repeated freeze-thaw cycles should be avoided as they can compromise protein structure and function . When working with the protein, maintain sterile conditions and consider aliquoting the stock solution to minimize freeze-thaw cycles for experiments requiring prolonged timeframes.

How can PI3KγD946GST Human be validated for experimental use?

Validation of PI3KγD946GST Human for experimental use should follow a systematic approach:

• Structural Integrity Assessment: Perform SDS-PAGE analysis to confirm >95% purity and proper molecular weight (approximately 153 kDa including the GST tag) .

• Functional Validation: Verify the absence of kinase activity using standard PI3K activity assays with ATP and PtdIns substrate, comparing results with active PI3Kγ as a positive control.

• Binding Capacity Testing: Confirm the protein's ability to interact with known PI3Kγ binding partners through GST pull-down assays, despite its catalytic inactivity.

• Expression System Verification: Confirm proper production in Sf9 insect cells to ensure appropriate post-translational modifications .

This comprehensive validation ensures that experimental observations using PI3KγD946GST Human can be reliably attributed to its structural properties rather than residual catalytic activity.

How should PI3KγD946GST Human be incorporated as a control in PI3K inhibitor studies?

When designing experiments to evaluate PI3K inhibitors, PI3KγD946GST Human serves as an essential negative control to distinguish between specific inhibitory effects and non-specific binding interactions. The following methodological approach is recommended:

• Parallel Testing Design: Run simultaneous assays with active PI3Kγ and PI3KγD946GST Human under identical conditions with your inhibitor candidates.

• Concentration Gradient Analysis: Test inhibitor binding across a concentration range (typically 0.1-100 μM) similar to that used in studies examining compounds like those described in research on thieno[2,3-d] pyrimidine derivatives .

• Binding Affinity Comparison: Measure direct binding affinities of inhibitors to both active PI3Kγ and PI3KγD946GST using techniques such as isothermal titration calorimetry or surface plasmon resonance.

• Downstream Signaling Assessment: Monitor effects on downstream targets such as Akt phosphorylation in cellular systems expressing either active PI3Kγ or the catalytically inactive mutant.

This approach helps distinguish between compounds that inhibit PI3Kγ through catalytic site interaction versus those that affect enzyme function through allosteric mechanisms or protein-protein interactions.

What cellular models are most appropriate for studying PI3KγD946GST Human effects?

Selection of cellular models for studying PI3KγD946GST Human should be guided by physiological relevance and experimental objectives:

• Leukocyte Models: Since PI3Kγ is highly expressed in leukocytes, myeloid cell lines (THP-1, U937) or primary human/mouse macrophages are particularly suitable . These models are especially relevant for studying inflammatory response modulation.

• Knockout Complementation Systems: PI3Kγ-deficient cells (either naturally occurring or CRISPR-generated) reconstituted with either wild-type PI3Kγ or PI3KγD946GST provide an excellent system to attribute phenotypic differences specifically to catalytic activity.

• G-Protein Coupled Receptor (GPCR) Signaling Models: Cell types with robust GPCR signaling are valuable as PI3Kγ facilitates signaling downstream of GPCRs, particularly in chemokine receptor responses .

• Cancer Cell Lines: Selected cancer cell lines with PI3K pathway alterations, such as those used in the National Cancer Institute's NCI-60 panel, can provide insights into the role of PI3Kγ catalytic activity in malignant transformation .

For optimal results, expression levels of endogenous PI3Kγ should be characterized in the selected model prior to experimental manipulation to ensure physiological relevance and proper interpretation of results.

How can PI3KγD946GST Human be used to differentiate between kinase-dependent and scaffolding functions of PI3Kγ?

PI3KγD946GST Human provides a powerful tool to dissect kinase-dependent versus scaffolding functions of PI3Kγ through the following methodological approaches:

• Complementation Analysis: Express PI3KγD946GST in PI3Kγ-deficient cells or organisms (such as the human patient described with bi-allelic loss-of-function mutations in PIK3CG ) and assess which phenotypes are rescued versus which remain impaired.

• Protein Complex Analysis:

  • Perform immunoprecipitation studies to compare protein interaction partners between wild-type PI3Kγ and PI3KγD946GST

  • Utilize proximity labeling approaches (BioID or APEX) with both proteins to identify their proximal interactomes in living cells

• Subcellular Localization Studies: Compare the dynamic localization patterns of fluorescently tagged wild-type PI3Kγ versus PI3KγD946GST during cellular activation by chemokines or other stimuli.

• Functional Reconstitution Assays: Assess cellular functions known to depend on PI3Kγ, such as:

  • Chemotaxis responses to GPCR ligands

  • Inflammatory cytokine production (IL-12, IL-23)

  • Regulatory T cell development and function

This systematic comparative approach helps establish which cellular functions depend specifically on PI3Kγ's catalytic activity versus its physical presence in signaling complexes.

What experimental approaches can resolve contradictory findings about PI3Kγ's role in inflammation?

The literature reports both pro-inflammatory and anti-inflammatory roles for PI3Kγ . To resolve these apparent contradictions using PI3KγD946GST Human, consider the following methodological framework:

• Context-Specific Analysis:

  • Compare wild-type PI3Kγ and PI3KγD946GST effects across different cell types (macrophages, neutrophils, T cells)

  • Evaluate responses under varying inflammatory stimuli (TLR agonists, cytokines, microbial components)

  • Test acute versus chronic inflammatory conditions

• Pathway-Focused Approach:

  • Examine GSK3α/β activation status, as PI3Kγ-deficient macrophages show elevated IL-12 and IL-23 production in a GSK3α/β-dependent manner

  • Monitor NF-κB and MAPK pathway activation in parallel

  • Assess transcriptional responses using RNA-seq or targeted gene expression analysis

• Microbiota-Dependent Effects:

  • Consider microbiome influence, as PI3Kγ-deficient mice recapitulate human disease features only after exposure to natural microbiota

  • Design experiments that control for or deliberately manipulate microbiome composition

• Temporal Analysis:

  • Implement time-course studies to distinguish between early and late effects of PI3Kγ activity on inflammatory responses

  • Use inducible expression systems to introduce PI3KγD946GST at different stages of the inflammatory response

This comprehensive approach can help reconcile seemingly contradictory findings by identifying specific contexts where PI3Kγ promotes versus restrains inflammation.

How does PI3KγD946GST Human compare with other PI3K isoform mutants in research applications?

When selecting between different PI3K isoform mutants for research applications, researchers should consider the following comparative aspects:

When designing experiments, select the appropriate mutant based on tissue expression patterns, signaling pathway specificity, and the biological process under investigation. For studies focused on immune modulation, particularly in myeloid cells and T lymphocyte regulation, PI3KγD946GST Human offers distinct advantages over other isoform mutants .

What are the key methodological differences when using PI3KγD946GST Human versus small molecule PI3Kγ inhibitors?

Understanding the methodological differences between genetic tools like PI3KγD946GST and pharmacological approaches using small molecule inhibitors is crucial for experimental design:

• Specificity Considerations:

  • PI3KγD946GST provides absolute isoform specificity, while small molecule inhibitors often show cross-reactivity with other PI3K isoforms. For example, thieno[2,3-d] pyrimidine derivatives showed varying levels of activity against different PI3K isoforms (compound VIb exhibited 72% inhibition of PI3Kβ and 84% inhibition of PI3Kγ) .

  • Use PI3KγD946GST when strict isoform specificity is required and small molecule inhibitors when broader PI3K pathway inhibition is acceptable.

• Temporal Control:

  • Small molecule inhibitors offer rapid and reversible inhibition, suitable for acute intervention studies.

  • PI3KγD946GST requires expression system establishment, making it better suited for long-term studies of PI3Kγ function.

• Functional Implications:

  • PI3KγD946GST maintains scaffolding functions while eliminating catalytic activity.

  • Small molecule inhibitors may disrupt protein-protein interactions in addition to inhibiting catalytic activity, potentially affecting scaffolding functions.

• Experimental Readouts:

  • When using PI3KγD946GST, compare phenotypes to wild-type PI3Kγ expression and PI3Kγ knockout.

  • For small molecule inhibitors, implement dose-response studies and include structurally related inactive analogs as controls.

These methodological distinctions highlight the complementary nature of these approaches, where combining both genetic tools and pharmacological inhibitors often provides the most comprehensive understanding of PI3Kγ biology.

What are common pitfalls when interpreting data from experiments using PI3KγD946GST Human?

When interpreting experimental results involving PI3KγD946GST Human, researchers should be vigilant for these common pitfalls:

• Expression Level Disparities:

  • Problem: Unequal expression levels between PI3KγD946GST and wild-type PI3Kγ can lead to misinterpretation of phenotypic differences.

  • Solution: Quantify protein expression by Western blot and normalize functional readouts to expression levels, or use inducible expression systems to achieve comparable levels.

• Dominant Negative Effects:

  • Problem: PI3KγD946GST may sequester binding partners of endogenous PI3Kγ, creating effects beyond simple loss of catalytic activity.

  • Solution: Include appropriate controls with PI3Kγ knockdown/knockout and overexpression of wild-type PI3Kγ to distinguish between absence of activity versus competitive inhibition effects.

• Cell Type-Specific Compensatory Mechanisms:

  • Problem: Different cell types may compensate differently for loss of PI3Kγ catalytic activity, as seen in the variable effects of PI3Kγ deficiency across immune cell subsets .

  • Solution: Validate findings across multiple cell types and consider examining adaptive responses through time-course experiments.

• Microbiome Confounding Factors:

  • Problem: Microbiota composition can significantly influence phenotypes in PI3Kγ-deficient systems .

  • Solution: Control for microbiome effects by standardizing housing conditions, using littermate controls, or explicitly manipulating microbiota through cohousing or gnotobiotic approaches.

Awareness of these potential pitfalls allows for experimental design modifications that strengthen data interpretation and enhance reproducibility.

How can researchers address inconsistent results between in vitro and in vivo studies using PI3KγD946GST?

When faced with discrepancies between in vitro and in vivo findings with PI3KγD946GST, consider this systematic resolution framework:

• Physiological Context Assessment:

  • Analyze differences in signaling pathway components between in vitro and in vivo systems

  • Consider compensatory mechanisms that may operate in intact organisms but not in isolated cell systems

  • Examine potential intercellular communications present in vivo but absent in vitro

• Technical Reconciliation Approach:

  • Adopt intermediate models such as organoids or ex vivo tissue cultures that bridge the complexity gap

  • Implement tissue-specific or inducible expression systems for in vivo studies to better match temporal aspects of in vitro work

  • Validate protein expression and localization patterns across both systems

• Microenvironmental Factors Analysis:

  • Evaluate the impact of microbiota, as PI3Kγ-deficient phenotypes in mouse models were shown to be microbiota-dependent

  • Consider the influence of tissue-specific factors, cytokines, and metabolites present in vivo but absent in vitro

  • Examine the role of mechanical forces and three-dimensional organization absent in most in vitro systems

• Cross-Validation with Complementary Approaches:

  • Compare results with both pharmacological inhibitors of PI3Kγ and genetic knockout models

  • Assess phenotypes at multiple levels (molecular, cellular, tissue, organismal)

  • Implement rescue experiments with varying levels of PI3Kγ activity to establish dose-response relationships

This structured approach helps identify the specific factors responsible for experimental discrepancies and can guide refinement of both in vitro and in vivo models to improve translational relevance.

How might PI3KγD946GST Human be utilized in developing targeted therapies for glioblastoma?

Given the interest in PI3K inhibition for brain tumors, particularly glioblastoma multiforme (GBM) , PI3KγD946GST Human offers several methodological approaches for therapeutic development:

• Mechanism Dissection Strategy:

  • Compare the effects of wild-type PI3Kγ versus PI3KγD946GST expression in GBM cell lines and patient-derived xenografts

  • Identify which GBM-promoting phenotypes depend specifically on PI3Kγ catalytic activity versus scaffolding functions

  • Determine whether PI3Kγ catalytic activity is required for blood-brain barrier penetration and brain tumor invasion

• Drug Target Validation Approach:

  • Use PI3KγD946GST as a control in high-throughput screening to identify compounds that specifically target PI3Kγ catalytic activity

  • Employ PI3KγD946GST-expressing cells to distinguish desired on-target effects from off-target cytotoxicity of PI3K inhibitor candidates

  • Evaluate differential responses between wild-type PI3Kγ and PI3KγD946GST-expressing GBM cells to establish predictive biomarkers for inhibitor sensitivity

• Combination Therapy Optimization:

  • Assess synergistic potential between PI3Kγ inhibition and standard-of-care treatments by comparing treatment responses in wild-type versus PI3KγD946GST-expressing GBM models

  • Investigate compensatory pathways activated specifically in response to loss of PI3Kγ catalytic activity to identify rational combination approaches

• Blood-Brain Barrier Considerations:

  • Leverage insights from BBB-penetrating PI3K inhibitors like GDC-0084 to develop optimal delivery strategies for targeting PI3Kγ in brain tumors

  • Examine whether PI3Kγ scaffolding functions independent of catalytic activity influence BBB integrity and permeability

This comprehensive approach could advance the development of brain-penetrant PI3Kγ inhibitors while minimizing off-target effects that have limited current therapeutic options.

What role might PI3KγD946GST Human play in understanding immune dysregulation in PI3Kγ deficiency?

The identification of human patients with bi-allelic loss-of-function mutations in PI3Kγ (PIK3CG) opens new research directions where PI3KγD946GST Human can help dissect mechanisms of immune dysregulation:

• Functional Complementation Analysis:

  • Express either wild-type PI3Kγ or PI3KγD946GST in patient-derived cells to determine which immune phenotypes can be rescued by the catalytically inactive mutant

  • Specific focus on:

    • Memory B cell development

    • Regulatory T cell maintenance

    • CXCR3+ T cell trafficking to tissues

    • Inflammatory cytokine production by myeloid cells

• GSK3α/β Pathway Investigation:

  • Examine whether the heightened IL-12 and IL-23 production by PI3Kγ-deficient macrophages in a GSK3α/β-dependent manner requires catalytic activity or protein scaffolding

  • Use PI3KγD946GST to determine the molecular mechanism by which PI3Kγ normally restrains GSK3α/β activity

• Microbiota-Immune Interaction Studies:

  • Investigate how PI3Kγ catalytic activity influences host responses to microbiota, given that PI3Kγ-deficient mice recapitulate human disease features after exposure to natural microbiota

  • Compare microbiome composition and immune responses between wild-type, PI3Kγ-knockout, and PI3KγD946GST-expressing systems

• Therapeutic Implication Assessment:

  • Evaluate whether transient versus sustained inhibition of PI3Kγ catalytic activity differentially affects immune homeostasis

  • Compare the immunological effects of small molecule PI3Kγ inhibitors to the genetic model of catalytic inactivation using PI3KγD946GST

This research direction could lead to improved understanding of "Inactivated PI3K-gamma Syndrome" (IPGS) and inform the development of safer PI3Kγ-targeted therapies with reduced immunological side effects.