Recombinant Squalus acanthias Serine/threonine-protein kinase 3/4 (STK4)

What are the functional roles of STK4 in cellular signaling pathways?

STK4 functions as a stress-activated, pro-apoptotic kinase that plays crucial roles in multiple signaling pathways:

• Hippo Signaling Pathway: STK4 serves as a key component of this pathway, which plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis . The pathway consists of a kinase cascade where STK4/MST1 (and its homolog STK3/MST2), in complex with regulatory protein SAV1, phosphorylates and activates LATS1/2, which then phosphorylates and inactivates YAP1 oncoprotein and WWTR1/TAZ .

• Apoptotic Signaling: Following caspase-cleavage, STK4 enters the nucleus and induces chromatin condensation followed by internucleosomal DNA fragmentation . It phosphorylates 'Ser-14' of histone H2B during apoptosis, a critical step in the apoptotic process .

• Immune Regulation: STK4 regulates T cell immunity by forming a complex with Foxp3 and NF-κB p65, which controls Foxp3 and p65-dependent transcriptional programs in regulatory T cells .

Methodologically, researchers investigating these pathways should employ phosphorylation assays, protein interaction studies, and gene expression analyses to fully characterize STK4's role in specific cellular contexts.

How can researchers effectively measure STK4 kinase activity in experimental settings?

Measuring STK4 kinase activity requires careful experimental design and consideration of several methodological approaches:

In vitro Kinase Assays:

• Substrate-based assays: Utilize known substrates such as histone H2B or LATS1/2. The reaction typically contains:

  • Purified recombinant STK4 (50-100 ng)

  • Substrate protein (1-2 μg)

  • Kinase buffer (commonly containing 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT)

  • ATP (50-100 μM, including trace amounts of [γ-³²P]ATP for radioactive assays)

• ATP consumption measurement: Using non-radioactive methods such as ADP-Glo™ or NADH-coupled assays to measure ATP consumption or ADP production.

Specific Activity Measurement:
Human STK4/MST1 demonstrates a specific activity of approximately 73 pmole/min/μg , which can serve as a reference point for comparative analyses.

Autophosphorylation Assays:
STK4 undergoes robust autoactivation in vitro through intramolecular autophosphorylation on the activation loop of an STK4 dimer . This property can be leveraged to assess kinase functionality.

Important Considerations:

• Include positive and negative controls (kinase-dead mutant such as STK4 K59R)

• Test multiple substrate concentrations to determine Km values

• Consider time-course experiments to establish linear reaction rates

• Ensure proper protein storage conditions to maintain enzymatic activity

When reporting activity, specify the exact assay conditions and quantification methods to enable reproducibility.

What experimental approaches are best suited to study STK4's role in T cell immunity?

Given STK4's critical role in T cell function, several specialized approaches are recommended:

T Cell Isolation and Culture Systems:

• Isolate primary T cells from peripheral blood using negative selection methods

• For regulatory T cells (Tregs), use CD4+CD25+CD127low sorting criteria

• Culture in complete RPMI medium supplemented with IL-2 (100 U/ml) for Tregs

STK4 Knockdown/Knockout Models:

• CRISPR-Cas9 system targeting STK4 in primary T cells or cell lines

• Conditional knockout mice (e.g., Foxp3-Cre × STK4flox/flox) for Treg-specific deletion

• siRNA or shRNA approaches for transient knockdown

STK4-Foxp3-p65 Complex Analysis:

• Co-immunoprecipitation assays following T cell receptor (TCR) stimulation

• Proximity ligation assays to visualize protein interactions in situ

• Chromatin immunoprecipitation (ChIP) to identify target genes

Functional T Cell Assays:

• Suppression assays using STK4-deficient Tregs

• T cell proliferation assays using CFSE dilution

• Flow cytometry to assess activation markers (CD25, CD69)

• Cytokine production measurement using ELISA or intracellular staining

Signaling Studies:

• Western blotting to assess phosphorylation of STK4 targets

• Subcellular fractionation to track STK4 nuclear translocation following TCR stimulation

• Phospho-flow cytometry for single-cell resolution of signaling events

Research has shown that STK4 nuclear translocation in Treg cells can be inhibited by XMU-MP-1, a specific STK4 kinase inhibitor , providing a useful tool for mechanistic studies.

How does STK4 deficiency impact interferon signaling and gene expression?

STK4 deficiency significantly alters interferon signaling pathways and gene expression profiles:

Altered Interferon Signaling:

• Defective type I/II and III interferon responses to TLR agonists and pathogens

• Impaired phosphorylation of TBK1 and transcription factor IRF3

• Dysregulated, but not completely abrogated, interferon-regulated gene expression

Transcriptional Dysregulation:
Whole blood and PBMC analyses from STK4-deficient patients reveal:

• Impaired T cell immunity:

  • Downregulation of genes involved in T cell activation and proliferation

  • Increased expression of genes associated with T cell apoptosis

  • Abnormal fractions of T cell subsets

• Dysregulated innate immune signaling:

  • Altered cytokine and chemokine gene expression

  • Impaired regulation of adhesion and leukocyte chemotaxis genes

Methodological Approach for Analysis:

To properly analyze these effects, researchers should employ:

• RNA sequencing of primary cells from STK4-deficient and control subjects

• Comparative analysis of stimulated vs. unstimulated conditions

• Pathway enrichment analysis using tools like ClueGO, Cytoscape, and IPA

• Validation of key findings using RT-qPCR and protein-level assays

A recent study identified two regulatory networks of IFN-α/IFN-β-responsive genes encompassing cytokine, chemokine, and adhesion factor/receptor genes that are indirectly regulated by STK4 . These genes are involved in cytotoxicity and death of immune cells, as well as adhesion and migration of lymphocytes and mononuclear leukocytes.

What methods can researchers use to investigate the Stk4-Foxp3-P65 transcriptional complex formation?

The Stk4-Foxp3-P65 transcriptional complex is critical for regulatory T cell function and can be investigated using the following methodological approaches:

Protein-Protein Interaction Studies:

• Co-immunoprecipitation (Co-IP): Use anti-Stk4, anti-Foxp3, or anti-p65 antibodies to pull down the complex, followed by Western blot analysis to detect interacting partners

• Proximity Ligation Assay (PLA): Visualize protein interactions in situ with single-molecule resolution

• Bimolecular Fluorescence Complementation (BiFC): Tag potential interacting proteins with complementary fragments of a fluorescent protein

Subcellular Localization Analysis:

• Immunofluorescence microscopy: Track TCR signaling-induced translocation of Stk4 into the nuclei of Treg cells and its co-localization with Foxp3

• Subcellular fractionation: Separate nuclear and cytoplasmic fractions to detect complex formation biochemically

• Live-cell imaging: Using fluorescent protein-tagged components to track complex dynamics

Functional Impact Assessment:

• Chromatin Immunoprecipitation (ChIP): Identify genomic binding sites of the complex

• Transcriptional reporter assays: Measure the impact of complex formation on target gene expression

• Mutagenesis studies: Create phosphomimetic mutants (e.g., Foxp3 S418E) to assess the role of specific phosphorylation events

Key Experimental Control:
Use Stk4 K59R mutant that lacks kinase catalytic activity as a negative control . This mutation prevents the TCR-induced nuclear translocation of Stk4.

Important Finding:
The Stk4-Foxp3-p65 complex is stabilized by Stk4-dependent phosphorylation of Foxp3 serine 418 . This phosphorylation event can be monitored using phospho-specific antibodies or mass spectrometry analysis.

What are the methodological approaches to study the impact of STK4 mutations on protein function?

Investigating the impact of STK4 mutations requires a systematic approach combining molecular, cellular, and functional analyses:

Mutation Identification and Characterization:

• In silico analysis: Predict the impact of mutations using tools like CADD scores, which provide evidence of deleteriousness

• Structural modeling: Map mutations onto protein structure to predict functional consequences

• Conservation analysis: Determine if mutations affect evolutionarily conserved residues

Expression Analysis:

• Western blot analysis: Assess protein expression levels using antibodies against specific domains

• RT-qPCR and mRNA-Seq: Measure transcript levels to evaluate effects on mRNA stability

• Pulse-chase experiments: Determine protein half-life and stability

Functional Assays:

• Kinase activity assays: Compare wild-type and mutant protein catalytic activity

• Autophosphorylation analysis: Evaluate the capacity for autoactivation

• Substrate phosphorylation: Test ability to phosphorylate known substrates (e.g., LATS1/2)

Cellular Localization and Interaction Studies:

• Nuclear translocation assays: Assess ability to translocate to the nucleus following stimulation

• Co-immunoprecipitation: Evaluate interactions with partner proteins

• Immunofluorescence microscopy: Visualize subcellular localization

Functional Complementation:

• Rescue experiments: Reintroduce wild-type or mutant STK4 into deficient cells to assess restoration of function

• Cellular phenotype analysis: Measure apoptosis, proliferation, and other STK4-dependent processes

Case Study:
A homozygous nonsense STK4 mutation (NM_006282.2:c.871C > T, p.Arg291*) identified in a pediatric patient resulted in partial loss of STK4 expression and complete loss of STK4 function . This mutation was associated with recurrent viral and bacterial infections, notably persistent Epstein-Barr virus viremia and pulmonary tuberculosis.

How can researchers effectively isolate and purify recombinant STK4 for structural and functional studies?

Efficient isolation and purification of recombinant STK4 requires careful consideration of expression systems, purification tags, and chromatographic techniques:

1. Expression System Selection:
For optimal results with STK4, consider:

• Sf9 insect cells: Yield properly folded protein with enzymatic activity (≥50% purity)

• E. coli: Higher yield but may require refolding for functional studies

• Mammalian cells: Better for complex interaction studies but lower yield

2. Fusion Tag Selection:
Common tags for STK4 purification include:

• GST-tag: Enhances solubility and enables single-step affinity purification

• His-tag: Allows for metal affinity chromatography under native or denaturing conditions

• Avi-tag: Enables in vivo biotinylation for streptavidin-based purification and detection

3. Purification Protocol:
A typical workflow for GST-tagged human STK4:

4. Stabilization Considerations:
To maintain STK4 stability and activity:

• Include 3-5 mM DTT in all buffers

• Add 10-15% glycerol to storage buffers

• Flash-freeze in small aliquots and store at -80°C

• Avoid repeated freeze/thaw cycles

5. Activity Validation:
Prior to experimental use, validate purified STK4:

• Measure specific activity (expect ~73 pmole/min/μg for human STK4)

• Confirm autophosphorylation capacity

• Verify substrate phosphorylation

What experimental design considerations are important when investigating STK4's role in immune disorders?

When investigating STK4's role in immune disorders, researchers should consider a comprehensive experimental approach:

Patient-Derived Sample Analysis:

• Genetic screening: Identify STK4 mutations using whole genome/exome sequencing

• Protein expression: Quantify STK4 levels in different immune cell populations

• Functional assays: Compare immune cell responses between patients and controls

Immune Cell Phenotyping:

• Flow cytometry analysis: Assess distributions of T cell subsets, plasmacytoid dendritic cells, and NK cells

• Activation marker analysis: Evaluate surface markers like CCR7 and CD62L on T cells

• Cell death analysis: Measure apoptosis rates in different immune cell populations

Infection and Inflammation Models:

• Pathogen challenge experiments: Test responses to viral and bacterial pathogens

• Ex vivo stimulation: Use purified PRR agonists (LPS, poly(I:C)) to assess innate immune responses

• Cytokine production: Measure IFN-α/IFN-β responses following stimulation

Transcriptomic and Proteomic Approaches:

• RNA-Seq analysis: Compare gene expression profiles between STK4-deficient and control cells

• Pathway enrichment analysis: Identify dysregulated signaling networks

• Phosphoproteomics: Map global changes in phosphorylation patterns

Therapeutic Intervention Models:

• Gene complementation: Restore STK4 expression in deficient cells

• Small molecule inhibitors: Use STK4 inhibitors like XMU-MP-1 in control cells

• Adoptive transfer experiments: Test if STK4-deficient cells can be functionally restored

Key Clinical Correlations:
STK4 deficiency is associated with:

• Combined immunodeficiency with severe T cell lymphopenia

• Recurrent bacterial and viral infections

• Persistent EBV viremia often leading to B cell lymphoma development

• Mucocutaneous candidiasis

• Congenital heart disease in some patients

Understanding these relationships requires careful consideration of both direct STK4 functions and downstream effects on multiple immune pathways.