Recombinant Papio anubis Serine/threonine-protein kinase 4 (STK4)

What is Serine/threonine-protein kinase 4 and what are its key functions?

Serine/threonine-protein kinase 4 (STK4) is a stress-activated, pro-apoptotic kinase that functions as a key component of the Hippo signaling pathway. Following caspase-cleavage, STK4 enters the nucleus and induces chromatin condensation followed by internucleosomal DNA fragmentation. The protein plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis . Within the Hippo pathway, STK4 (also known as MST1) forms a complex with its regulatory protein SAV1, which phosphorylates and activates LATS1/2 in complex with MOB1. This activation cascade ultimately leads to the phosphorylation and inactivation of the YAP1 oncoprotein and WWTR1/TAZ . Understanding these pathways is critical for researchers investigating cellular proliferation, apoptosis, and cancer development mechanisms.

How does Papio anubis STK4 compare structurally to human STK4?

The Papio anubis (Olive baboon) STK4 shows significant homology to human STK4, making it a valuable model for human STK4 research. The recombinant Papio anubis STK4 consists of 487 amino acids and contains the complete functional domains present in the human ortholog . The amino acid sequence includes the kinase domain and regulatory regions that are necessary for its enzymatic activity and interaction with other proteins in the Hippo signaling pathway. This high degree of conservation makes baboon STK4 suitable for investigating mechanisms that may be relevant to human disease states and therapeutic development strategies. When designing experiments, researchers should be aware that while the core functional domains are highly conserved, species-specific variations might impact certain protein-protein interactions or regulatory mechanisms.

What are the subcellular locations of STK4 and why are they important for research?

STK4 can be localized in multiple subcellular compartments, including the cytoplasm, lipid rafts, and nucleus, with each location associated with distinct functions . Research has demonstrated that the subcellular enrichment of STK4 differentially regulates cell growth in vitro and tumor growth in vivo . Cytoplasmic STK4 (CL-STK4), lipid raft-localized STK4 (LR-STK4), and nuclear STK4 (NL-STK4) each affect different sets of gene expression patterns, with NL-STK4 and LR-STK4 showing a much greater number of differentially expressed genes than CL-STK4 . Approximately 90% of differentially expressed genes overlap between NL-STK4 and LR-STK4 cells . These location-specific functions are critical considerations when designing experiments to study STK4's role in various cellular processes and pathological conditions, as targeting STK4 to specific compartments may produce dramatically different outcomes.

What are the optimal storage and handling conditions for recombinant Papio anubis STK4?

For optimal stability and activity of recombinant Papio anubis STK4, storage at -20°C is recommended for regular use, while extended storage should be at -20°C or -80°C . Prior to opening, the vial should be briefly centrifuged to bring contents to the bottom. For reconstitution, the protein should be dissolved in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol (final concentration) for long-term storage . The default final concentration of glycerol is typically 50%. After reconstitution, the protein should be aliquoted to avoid repeated freeze-thaw cycles, which can significantly compromise protein integrity and activity. Working aliquots can be stored at 4°C for up to one week . These handling procedures are essential for maintaining enzymatic activity and structural integrity of the recombinant protein across experimental timeframes.

What reconstitution methods yield optimal STK4 activity for kinase assays?

When preparing recombinant Papio anubis STK4 for kinase assays, reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL is recommended . For optimal enzymatic activity, researchers should consider several methodological factors:

• Buffer composition: The reconstitution buffer should maintain a pH conducive to STK4 stability (typically pH 7.4-8.0)

• Salt concentration: Moderate ionic strength (150-300mM NaCl) helps maintain protein solubility without interfering with kinase activity

• Reducing agents: Addition of a reducing agent such as DTT (1-5mM) can help maintain the protein in its active conformation

• Metal ions: As a kinase, STK4 requires specific metal cofactors (typically Mg²⁺ or Mn²⁺) for optimal activity in phosphorylation reactions

For long-term storage of the reconstituted protein, addition of 5-50% glycerol is recommended to prevent freeze-thaw damage . When designing kinase assays, researchers should optimize the buffer conditions based on their specific experimental requirements, including substrate concentration, incubation time, and detection method.

How does the purity of recombinant STK4 impact experimental outcomes?

The purity of recombinant Papio anubis STK4 is critical for reliable and reproducible experimental results. Commercial preparations typically ensure >85% purity as confirmed by SDS-PAGE . Lower purity preparations may contain contaminants that could interfere with:

• Enzymatic activity measurements through competing reactions or inhibitory effects

• Protein-protein interaction studies due to non-specific binding

• Structural analyses by introducing heterogeneity in samples

• Cellular assays by triggering unintended signaling events

When designing experiments using recombinant STK4, researchers should verify the purity of their preparation and consider how potential contaminants might impact their specific experimental system. For highly sensitive applications such as structural studies or advanced functional assays, higher purity preparations (>95%) may be necessary, which might require additional purification steps beyond what is provided in commercial preparations.

How does STK4 interact with the Hippo signaling pathway components?

STK4 (also known as MST1) functions as a core kinase in the Hippo signaling pathway, forming complexes with specific regulatory proteins to control downstream signaling events. The pathway typically functions as follows:

• STK4 forms a complex with its regulatory protein SAV1

• This complex phosphorylates and activates LATS1/2 kinases that are in complex with their regulatory protein MOB1

• Activated LATS1/2 then phosphorylates the transcriptional co-activators YAP1 and WWTR1/TAZ

• Phosphorylated YAP1 and WWTR1/TAZ are sequestered in the cytoplasm or degraded, preventing their nuclear translocation and transcriptional activity

This cascade regulates organ size control and tumor suppression by restricting proliferation and promoting apoptosis. When studying this pathway, researchers should be aware that the stoichiometry of these interactions and the presence of other regulatory factors can significantly impact signaling outcomes. Experimental approaches to study these interactions include co-immunoprecipitation, kinase assays with purified components, and cellular models with altered expression of pathway components.

What is the significance of STK4 cleavage in apoptotic signaling?

STK4 undergoes caspase-mediated cleavage during apoptosis, generating a 37kDa N-terminal fragment (STK4-N) and an 18kDa C-terminal fragment . This cleavage event is functionally significant because:

• The N-terminal fragment contains the catalytic kinase domain and becomes constitutively active upon cleavage

• Cleaved STK4 translocates to the nucleus where it phosphorylates histone H2B and other nuclear substrates

• Nuclear STK4 activity promotes chromatin condensation and DNA fragmentation, which are hallmarks of apoptosis

• The cleavage removes auto-inhibitory domains, significantly enhancing kinase activity

For researchers studying apoptotic mechanisms, it's important to note that under standard expression conditions, the cleavage products may not always be detectable by Western blotting . This could be due to the rapid turnover of these fragments or limitations in antibody recognition. Experimental approaches to study STK4 cleavage include using caspase inhibitors, expressing cleavage-resistant STK4 mutants, or directly expressing the cleaved fragments to assess their specific functions.

How do the different subcellular localizations of STK4 affect its signaling outputs?

Research has demonstrated that the subcellular localization of STK4 dramatically influences its signaling activities and downstream gene expression patterns. Studies using engineered prostate cancer cell lines with STK4 targeted to specific cellular compartments revealed:

• Cytoplasmic STK4 (CL-STK4): Shows relatively modest effects on gene expression (using a cutoff of absolute log2 fold change ≥ 1.5; FDR ≤ 0.01)

• Lipid raft-localized STK4 (LR-STK4): Produces extensive changes in gene expression (absolute log2 fold change values ≥ 2, FDR ≤ 0.01)

• Nuclear STK4 (NL-STK4): Similarly produces widespread changes in gene expression patterns (absolute log2 fold change values ≥ 2, FDR ≤ 0.01)

• Approximately 90% of differentially expressed genes overlap between NL-STK4 and LR-STK4 cells, suggesting common downstream pathways despite different localizations

These findings indicate that targeting STK4 to different subcellular compartments can dramatically alter its functional impact on cellular physiology. Researchers studying STK4 signaling should carefully consider compartment-specific effects when interpreting experimental results and designing therapeutic strategies that target this kinase.

What cell models are most appropriate for studying STK4 functions?

Selecting appropriate cell models is crucial for studying STK4 functions effectively. Based on published research, several considerations should guide model selection:

• Endogenous STK4 expression levels: C4-2 cells (a castration-resistant prostate cancer cell line) express significantly lower levels of STK4 than their parental LNCaP cells, making them suitable for overexpression studies

• Inducible expression systems: Tetracycline-inducible systems allow controlled expression of STK4, as demonstrated with doxycycline-inducible C4-2 cell models expressing compartment-specific STK4 variants

• Subcellular targeting: Engineering cells with compartment-specific STK4 variants (cytoplasmic, lipid raft, or nuclear) provides insights into location-dependent functions

• Physiological relevance: Models should reflect the disease or developmental context being studied, as STK4 functions may vary by tissue type and pathological state

For optimal experimental design, researchers should verify endogenous STK4 expression in their chosen model, ensure appropriate controls (such as vector-only transduced cells), and validate subcellular localization of engineered STK4 variants using techniques such as immunofluorescence imaging and subcellular fractionation followed by Western blotting .

What methods are most effective for analyzing STK4 kinase activity?

Analyzing STK4 kinase activity requires specialized techniques that assess enzymatic function rather than just protein levels. Several methodological approaches are recommended:

• In vitro kinase assays: Purified recombinant STK4 can be incubated with appropriate substrates (such as histone H2B or LATS1/2) in the presence of ATP, followed by detection of phosphorylated products using:

  • Phospho-specific antibodies in Western blotting

  • Radioactive [γ-³²P]ATP incorporation

  • Mass spectrometry to identify phosphorylation sites

• Cellular phosphorylation assays: Monitoring phosphorylation of known STK4 substrates in cells with manipulated STK4 expression using phospho-specific antibodies

• Kinase activity sensors: Genetically encoded FRET-based sensors can monitor STK4 activity in living cells with spatiotemporal resolution

• Chemical genetics approaches: Using ATP analog-sensitive STK4 mutants that can utilize bulky ATP analogs, allowing specific labeling or inhibition of the engineered kinase

When analyzing STK4 activity, researchers should consider factors such as cellular context, the presence of regulatory partners (like SAV1), and potential post-translational modifications that might affect kinase function. Controls should include kinase-dead mutants and specific inhibitors to confirm that observed phosphorylation events are STK4-dependent.

How can RNAseq data be effectively analyzed to understand STK4-mediated gene expression changes?

RNAseq data analysis for STK4-mediated gene expression changes should follow rigorous bioinformatic workflows, as demonstrated in studies of compartment-specific STK4 signaling . Key methodological considerations include:

• Quality control: Ensure high correlation between replicates to minimize technical variability (as shown in the referenced study where normalized gene counts between replicates were highly correlated)

• Differential expression analysis: Tools like DESeq2 can identify differentially expressed (DE) genes by comparing STK4-expressing cells to vector controls

• Statistical cutoffs: Define appropriate threshold criteria such as:

  • For stringent analysis: absolute log2 fold change values ≥ 2, False Discovery Rate (FDR) ≤ 0.01

  • For more inclusive analysis: absolute log2 fold change ≥ 1.5; FDR ≤ 0.01

• Visualization techniques:

  • Volcano plots to display significance versus fold change

  • Heatmaps to visualize expression patterns across conditions

  • Venn diagrams to compare overlapping DE genes between different conditions

• Functional annotation clustering: Group DE genes into molecular pathways to identify biological processes affected by STK4 signaling, such as tumor suppression, oncogenesis, and cellular metabolism

When analyzing location-specific effects, researchers should be prepared for substantial differences in the number and identity of DE genes based on STK4 subcellular localization, as studies have shown that nuclear and lipid raft-localized STK4 affect many more genes than cytoplasmic STK4 .

What is the role of STK4 in cancer development and progression?

STK4 functions primarily as a tumor suppressor through its central role in the Hippo signaling pathway, with its dysregulation being implicated in various cancers. Research demonstrates that:

• STK4 restricts proliferation and promotes apoptosis, serving as a barrier to uncontrolled cell growth

• In prostate cancer models, STK4 enriched in different subcellular compartments differentially regulated cell growth in vitro and tumor growth in vivo

• The tumor-suppressive function operates through multiple mechanisms:

  • Direct phosphorylation and activation of downstream kinases LATS1/2

  • Inhibition of oncogenic transcription factors YAP1 and WWTR1/TAZ

  • Promotion of apoptosis through nuclear translocation and histone phosphorylation

• Gene expression profiling revealed that STK4 regulates genes associated with both tumor suppressor and oncogenesis pathways

When investigating STK4 in cancer contexts, researchers should consider both loss-of-function and gain-of-function approaches, as well as the impact of subcellular localization on STK4's tumor-suppressive activities. The cell-type specific effects and interactions with other cancer-related pathways should also be taken into account when designing experiments and interpreting results.

How can STK4 be targeted for therapeutic development?

Therapeutic targeting of STK4 presents unique challenges and opportunities that researchers should consider:

• Activation strategies: Since STK4 primarily functions as a tumor suppressor, therapeutic approaches in cancer might focus on activating or restoring STK4 function through:

  • Small molecule activators that mimic caspase cleavage activation

  • Targeting negative regulators of STK4 such as Raf-1 or certain phosphatases

  • Promoting nuclear translocation of STK4 to enhance its pro-apoptotic function

• Subcellular targeting: Given the differential effects of STK4 based on its localization , strategies that direct STK4 to specific cellular compartments might yield more precise therapeutic outcomes

• Combination approaches: STK4 activation might be combined with other therapies to enhance effects:

  • DNA damaging agents that trigger apoptotic pathways

  • Inhibitors of YAP/TAZ activity to complement upstream STK4 activation

  • Immunotherapies, as STK4 has been implicated in immune cell function

• Disease-specific considerations: The therapeutic strategy may need to be tailored to specific cancer types based on the molecular context and status of the Hippo pathway

When developing STK4-targeted therapeutics, researchers should carefully evaluate potential off-target effects, given STK4's broad involvement in fundamental cellular processes and its expression across multiple tissues. In vitro kinase assays with recombinant Papio anubis STK4 could serve as a preliminary screening platform for identifying potential therapeutic compounds.

What methodological approaches are used to investigate STK4 interactions with other signaling networks?

Investigating STK4's cross-talk with other signaling networks requires specialized methodological approaches:

• Proteomics-based interaction mapping:

  • Immunoprecipitation coupled with mass spectrometry to identify STK4 binding partners

  • Proximity labeling techniques (BioID, APEX) to identify compartment-specific interaction networks

  • Phosphoproteomics to map STK4-dependent phosphorylation events across the proteome

• Transcriptomic approaches:

  • RNAseq analysis as demonstrated in studies comparing gene expression changes induced by differently localized STK4

  • Chromatin immunoprecipitation sequencing (ChIP-seq) to identify genomic regions affected by STK4-regulated transcription factors

  • Single-cell transcriptomics to capture cell-to-cell variability in STK4 signaling responses

• Genetic interaction screens:

  • CRISPR-based screens to identify synthetic lethal interactions with STK4 modulation

  • Combinatorial perturbation of STK4 and other pathway components to map genetic interactions

• Computational approaches:

  • Pathway enrichment analysis of differential expression data

  • Network modeling to predict STK4's role in integrated signaling networks

  • Machine learning algorithms to identify patterns in large-scale datasets

These approaches should be selected based on the specific research question, with consideration for the technical limitations and appropriate controls for each method. Integration of multiple methodologies often provides the most comprehensive understanding of STK4's role within complex signaling networks.

How do post-translational modifications affect STK4 function and how can they be studied?

Post-translational modifications (PTMs) significantly impact STK4 function, activity, and localization. Understanding and studying these modifications requires specialized approaches:

• Key STK4 modifications and their functional impacts:

  • Phosphorylation at Thr183 in the activation loop is critical for kinase activity

  • Caspase-mediated cleavage generates active N-terminal fragments that translocate to the nucleus

  • Oxidation of conserved cysteine residues can regulate STK4 activity in response to oxidative stress

  • Acetylation may affect protein stability and interaction capabilities

• Methodological approaches for studying STK4 PTMs:

  • Site-specific phospho-antibodies for detecting activation status (e.g., Phospho-STK4 (Thr183) Antibody)

  • Mass spectrometry-based proteomics to comprehensively map modification sites

  • Mutagenesis studies replacing modifiable residues with non-modifiable variants

  • Inhibitor studies using specific enzyme inhibitors that prevent particular modifications

• Considerations for experimental design:

  • Cell stimulation conditions that promote specific modifications

  • Subcellular fractionation to track modification-dependent localization changes

  • Time-course analyses to determine modification dynamics

  • Use of phosphatase inhibitors during protein extraction to preserve phosphorylation states

When studying PTMs of recombinant Papio anubis STK4, researchers should consider whether the expression system used for production (yeast, E. coli, baculovirus, or mammalian cells) accurately reflects the physiological modification patterns observed in vivo.

What are the challenges in comparing STK4 function across different species models?

Cross-species comparison of STK4 function presents several methodological challenges that researchers should address:

• Sequence and structural variations:

  • While the core functional domains are conserved, species-specific variations exist in regulatory regions

  • The full sequence of Papio anubis STK4 (487 amino acids) may differ in key regulatory sites compared to human or mouse orthologs

  • These variations can affect protein-protein interactions, substrate specificity, and regulation by PTMs

• Experimental considerations:

  • Antibody cross-reactivity must be verified when using antibodies developed against one species for detecting STK4 in another

  • Expression systems should be selected based on the need for species-appropriate post-translational modifications

  • Protein tags and fusion constructs may affect function differently depending on the species variant

• Functional conservation assessment:

  • Complementation studies where the STK4 ortholog from one species is expressed in cells from another species

  • Comparative biochemical assays with purified proteins from different species

  • Structural analyses to identify conserved and divergent regions that might affect function

What quality control metrics should be applied when working with recombinant STK4 preparations?

Ensuring consistent quality of recombinant STK4 preparations is essential for reproducible research. The following quality control metrics should be applied:

• Purity assessment:

  • SDS-PAGE analysis (>85% purity is typically considered acceptable for most applications)

  • Size exclusion chromatography to detect aggregates or breakdown products

  • Mass spectrometry to verify protein identity and detect modifications

• Functional validation:

  • In vitro kinase activity assays using known substrates

  • Thermal shift assays to assess protein stability

  • Circular dichroism to confirm proper protein folding

• Storage stability monitoring:

  • Regular testing of activity after storage at recommended conditions (-20°C/-80°C)

  • Assessment of freeze-thaw stability using activity assays

  • Monitoring for precipitation or aggregation

• Batch-to-batch consistency:

  • Standardized production and purification protocols

  • Reference standards for comparative analysis

  • Detailed documentation of source, expression system, and purification method

The table below summarizes key quality parameters for recombinant Papio anubis STK4:

Implementing these quality control measures ensures that experimental outcomes reflect true biological phenomena rather than artifacts of variable protein quality.

What emerging technologies could advance our understanding of STK4 biology?

Several cutting-edge technologies hold promise for deepening our understanding of STK4 biology:

• Single-cell multi-omics:

  • Single-cell RNA-seq combined with protein measurements to correlate STK4 expression with transcriptional outputs

  • Spatial transcriptomics to map STK4 activity in tissue contexts

  • Single-cell phosphoproteomics to detect cell-to-cell variation in STK4 signaling

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize STK4 localization with nanometer precision

  • Live-cell FRET biosensors to monitor STK4 activity in real-time

  • Lattice light-sheet microscopy for long-term imaging of STK4 dynamics

• Structural biology approaches:

  • Cryo-EM studies of STK4 in complex with regulatory partners

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes

  • AlphaFold2 and other AI-based structure prediction to model species-specific variations

• Genome editing technologies:

  • CRISPR base editing for precise modification of regulatory sites

  • CRISPR activation/inhibition systems for temporally controlled modulation of STK4 expression

  • CRISPR screens to systematically identify genetic interactors of STK4

These technologies could help resolve outstanding questions about compartment-specific STK4 functions, the temporal dynamics of STK4 activation, and the context-dependent roles of STK4 in normal physiology and disease states.

How might STK4 research inform personalized medicine approaches?

STK4 research has several potential applications in personalized medicine:

• Biomarker development:

  • STK4 expression or activation status as prognostic indicators in cancer

  • Phosphorylation of STK4 substrates as pharmacodynamic markers for drug response

  • Genetic variants affecting STK4 pathway components as predictors of disease susceptibility

• Therapeutic stratification:

  • Identification of patient subgroups likely to respond to therapies targeting the Hippo pathway

  • Combination therapy approaches based on STK4 pathway status

  • Resistance mechanisms related to STK4 pathway alterations

• Precision therapeutic approaches:

  • Cell compartment-specific targeting based on findings that different localizations of STK4 produce distinct effects

  • Development of STK4 activators for cancers with impaired Hippo signaling

  • Genetic circuit designs incorporating STK4 signaling for cell-based therapies

• Model systems for drug testing:

  • Patient-derived organoids with engineered STK4 variants

  • Humanized mouse models with patient-specific STK4 pathway alterations

  • In silico modeling of patient-specific STK4 network responses to therapeutic interventions

Research using recombinant Papio anubis STK4 and other model systems provides fundamental knowledge that can be translated into these personalized medicine applications through careful validation in human samples and clinical trials.

What unresolved questions remain in our understanding of STK4 regulation and function?

Despite significant advances, several important questions about STK4 biology remain unresolved:

• Compartment-specific regulation:

  • How is STK4 differentially regulated in distinct subcellular compartments?

  • What factors determine the localization of STK4 to cytoplasm, lipid rafts, or nucleus?

  • Why do lipid raft-localized and nuclear-localized STK4 show greater and overlapping effects on gene expression compared to cytoplasmic STK4?

• Context-dependent functions:

  • How does STK4 function differ across tissue types and developmental stages?

  • What determines whether STK4 promotes apoptosis versus other cellular responses?

  • How does STK4 interact with tissue-specific transcription factors and signaling networks?

• Evolutionary considerations:

  • What functional differences exist between Papio anubis STK4 and human STK4?

  • How has the Hippo pathway evolved across species, and what can this tell us about fundamental versus specialized functions?

• Therapeutic implications:

  • Can STK4 be effectively targeted for therapeutic benefit?

  • What biomarkers can predict response to STK4-targeted therapies?

  • How can compartment-specific STK4 functions be selectively modulated?

Addressing these questions will require integrated approaches combining biochemical, cellular, and in vivo studies, potentially using recombinant Papio anubis STK4 as a model system alongside human samples and diverse experimental platforms.