Phospho-STK4 (Thr183) Antibody

What is the significance of Thr183 phosphorylation in STK4/MST1 function?

Phosphorylation at Threonine 183 (Thr183) represents a critical activation event in STK4 (also known as MST1) signaling. This post-translational modification occurs within the activation loop of the kinase domain and is essential for catalytic activity. When STK4 is activated by upstream signals, particularly cellular stress or apoptotic stimuli, autophosphorylation at Thr183 triggers a conformational change that enhances kinase activity. This phosphorylation event serves as a molecular switch that propagates downstream signaling, particularly within the Hippo tumor suppressor pathway. The phosphorylation status at this residue is considered a reliable biomarker for STK4 activation in experimental systems and is frequently used to assess pathway activity in research contexts .

How does STK4/MST1 phosphorylation status relate to its role in the Hippo signaling pathway?

STK4/MST1 phosphorylation at Thr183 represents a crucial regulatory step within the Hippo signaling cascade. As a core kinase component of this pathway, phosphorylated STK4 forms complexes with its regulatory protein SAV1, which then phosphorylates and activates downstream LATS1/2 kinases in association with MOB1. This sequential phosphorylation ultimately leads to the phosphorylation and inactivation of transcriptional coactivators YAP1 and WWTR1/TAZ. The phosphorylation of STK4 is therefore positioned as an upstream regulatory event that initiates a phosphorylation cascade culminating in growth suppression and apoptosis promotion. Researchers investigating Hippo pathway dynamics must carefully monitor STK4 phosphorylation as it provides insights into pathway activation status and can serve as an indicator of successful experimental manipulation of this signaling axis .

What is the relationship between STK4 and its paralog STK3 (MST2) in terms of phosphorylation patterns?

STK4 (MST1) and STK3 (MST2) exhibit remarkable conservation in their activation mechanisms, with phosphorylation occurring at analogous residues - Thr183 in STK4 and Thr180 in STK3. This conservation allows for potential compensatory functions between these kinases, though their expression patterns and tissue-specific roles may differ. The close homology between these phosphorylation sites often necessitates careful antibody selection when studying either kinase individually. Many antibodies, including those referenced in the search results, recognize both phosphorylated forms due to sequence similarity surrounding these threonine residues. Studies have demonstrated that STK4 is expressed at approximately 13-fold higher levels than STK3 in regulatory T cells (Treg cells), suggesting differential importance in immune regulation. Additionally, in STK3-deficient contexts, STK4 expression is often upregulated as a compensatory mechanism, highlighting the functional redundancy between these paralogs .

What are the optimal conditions for detecting phospho-STK4 (Thr183) in Western blot applications?

For optimal detection of phospho-STK4 (Thr183) via Western blot, researchers should implement a carefully controlled protocol that preserves phosphorylation status throughout sample processing. Based on validated approaches, samples should be lysed in buffers containing phosphatase inhibitors (sodium fluoride, sodium orthovanadate, and phosphatase inhibitor cocktails) to prevent dephosphorylation during extraction. Cell stimulation with appropriate activators such as staurosporine can enhance phospho-STK4 signal for positive controls. The recommended antibody dilution range for Western blot applications is 1:500-1:2000, with optimal dilution being sample-dependent and requiring titration for each experimental system .

Protein separation should be performed using standard SDS-PAGE (10-12% gels), followed by transfer to PVDF or nitrocellulose membranes. Blocking with 5% BSA in TBST (rather than milk, which contains phosphatases) is crucial for maintaining phospho-epitope integrity. Detection systems should be optimized based on expected expression levels, with chemiluminescence offering good sensitivity for most applications. The expected molecular weight for phospho-STK4 appears at approximately 52-56 kDa bands on Western blots, with potential variations depending on experimental conditions and cell types .

How can researchers verify the specificity of phospho-STK4 (Thr183) antibody reactivity in their experimental systems?

Verifying antibody specificity for phospho-STK4 (Thr183) requires multiple complementary approaches to ensure reliable results. First, researchers should implement appropriate controls including: (1) phosphatase treatment of duplicate samples to demonstrate loss of signal; (2) stimulation of cells with known STK4 activators such as staurosporine to enhance phosphorylation signal; and (3) inclusion of STK4-knockout or knockdown samples as negative controls. For more definitive validation, researchers can utilize phospho-deficient mutants (T183A) expressed in cells lacking endogenous STK4, which should show no reactivity with the phospho-specific antibody .

Cross-reactivity assessment is particularly important due to the high homology between STK4 (Thr183) and STK3 (Thr180) phosphorylation sites. Researchers should note that many commercial antibodies, including those referenced in the search results, detect both phosphorylated forms. When specific detection of only phospho-STK4 is required, additional experimental approaches such as immunoprecipitation with STK4-specific antibodies prior to phospho-detection may be necessary. Additionally, peptide competition assays using phosphorylated and non-phosphorylated peptides spanning the Thr183 region can further confirm specificity. Each lot of antibody should be validated in the specific cellular contexts being studied, as reactivity can vary between experimental systems .

What methodological approaches can be used to study STK4 phosphorylation in relation to Treg cell function?

Investigating STK4 phosphorylation in regulatory T (Treg) cells requires specialized methodologies that accommodate the unique biology of these immune cells. Based on published research, several approaches have proven effective: (1) Flow cytometry with phospho-specific antibodies allows single-cell analysis of Treg populations using Foxp3 co-staining; (2) Western blot analysis of sorted Treg cells following T cell receptor (TCR) stimulation can reveal phosphorylation dynamics; and (3) Immunoprecipitation followed by immunoblotting enables detection of specific complexes between phospho-STK4, Foxp3, and NF-κB p65 .

More advanced approaches involve functional assessment of how STK4 phosphorylation impacts Treg cell activity. Researchers can employ adoptive transfer models where wild-type and phosphorylation-deficient STK4 mutants are expressed in STK4-deficient Treg cells prior to transfer into recipient mice. Subsequent assessment of suppressive function, stability, and transcriptional programs can reveal the biological significance of STK4 phosphorylation. Chromatin immunoprecipitation (ChIP) assays have also been valuable in demonstrating how phospho-STK4 contributes to transcriptional regulation in Treg cells, specifically through formation of a trimolecular complex with Foxp3 and p65. When studying human Treg cells, techniques must be adapted for limited cell numbers, with phospho-flow cytometry offering advantages for clinical samples .

How does TCR signaling regulate STK4 phosphorylation and nuclear translocation in Treg cells?

T cell receptor (TCR) signaling initiates a complex cascade that dynamically regulates STK4 phosphorylation and subcellular localization in regulatory T cells. Upon TCR engagement, STK4 undergoes autophosphorylation at Thr183, which enables its nuclear translocation. This activation process is critically important for Treg cell function and involves several sequential steps. First, TCR stimulation activates upstream kinases that may facilitate STK4 autophosphorylation. Once phosphorylated, STK4 forms a trimolecular complex with NF-κB p65 and Foxp3, which is stabilized by STK4-mediated phosphorylation of Foxp3 at serine 418 .

Research has demonstrated that deficiency of STK4 in Treg cells severely impairs this signaling axis, resulting in decreased p65 expression, reduced nuclear translocation, and defective formation of the p65/Foxp3 complex. These defects ultimately compromise Treg cell activation and immune regulatory function. Experimental approaches to study this process include subcellular fractionation followed by immunoblotting to track STK4 movement between cytoplasm and nucleus, co-immunoprecipitation to detect complex formation, and confocal microscopy to visualize the translocation process in real time. Understanding this regulatory mechanism has significant implications for therapeutic approaches targeting Treg cell function in autoimmunity and cancer .

What are the molecular mechanisms by which phosphorylated STK4 regulates Foxp3 activity in Treg cells?

Phosphorylated STK4 regulates Foxp3 activity through multiple interconnected molecular mechanisms that ultimately control Treg cell stability and function. The primary mechanism involves direct phosphorylation of Foxp3 at serine 418 (S418) by activated STK4. This post-translational modification was identified using phospho-specific antibodies and confirmed through reconstitution experiments in Jurkat T cells with either wild-type or kinase-inactive (K59R mutant) STK4. The phosphorylation of S418 on Foxp3 critically stabilizes the formation of a trimolecular complex consisting of STK4, Foxp3, and NF-κB p65 .

This complex serves as a transcriptional regulatory unit that controls Treg-specific gene expression patterns. Experimental evidence using STK3/4-deficient Treg cells revealed that absence of these kinases severely impairs Foxp3 phosphorylation as detected by pan anti-phospho-serine and phospho-threonine antibodies following TCR stimulation. Importantly, expression of phosphomimetic Foxp3^S418E^ in STK3/4-deficient Treg cells partially rescued their immune regulatory defects in adoptive transfer models, demonstrating the functional significance of this phosphorylation event. This mechanistic understanding provides critical insights into how STK4 serves as a molecular sensor coupling TCR activation to transcriptional programming in Treg cells, with significant implications for manipulation of Treg function in therapeutic contexts .

How do STK4/MST1 phosphorylation patterns differ between its roles in immune regulation versus the canonical Hippo pathway?

STK4/MST1 exhibits context-specific phosphorylation patterns and signaling outcomes depending on whether it functions in immune regulation or within the canonical Hippo tumor suppressor pathway. In the immune context, particularly in Treg cells, TCR stimulation induces STK4 Thr183 phosphorylation, leading to formation of a regulatory complex with Foxp3 and NF-κB p65. This complex primarily influences transcriptional programs governing Treg cell activation and stability. In contrast, within the canonical Hippo pathway, STK4 phosphorylation at Thr183 initiates a distinct signaling cascade involving SAV1, LATS1/2, and MOB1, ultimately regulating YAP1/WWTR1 transcriptional activity to control cell proliferation and apoptosis .

These divergent pathways likely involve different upstream activators and regulatory mechanisms. In Treg cells, the predominant trigger appears to be TCR engagement, while in the Hippo context, cell density, mechanical forces, and various stress signals are primary activators. The downstream effects also differ substantially: in Treg cells, STK4 phosphorylation promotes immune tolerance, whereas in the Hippo pathway, it typically restricts cell growth and induces apoptosis. These distinct roles are further supported by the phenotypic differences observed in tissue-specific knockout models. For instance, Treg-specific deletion of STK4 leads to autoimmune lymphoproliferative disease, while liver-specific deletion results in hepatocyte proliferation and potential tumor formation .

What are common challenges in detecting phospho-STK4 (Thr183) and how can they be addressed?

Researchers frequently encounter several challenges when detecting phospho-STK4 (Thr183) in experimental systems. The most prevalent issues include: (1) rapid dephosphorylation during sample preparation, (2) antibody cross-reactivity with phospho-STK3 (Thr180), (3) low signal-to-noise ratio in certain cell types, and (4) context-dependent phosphorylation dynamics. To address these challenges, several technical modifications should be implemented .

For sample preparation, immediate lysis in buffers containing robust phosphatase inhibitor cocktails is essential. These should include sodium fluoride (50mM), sodium orthovanadate (1mM), and commercial phosphatase inhibitor mixtures. Processing samples at 4°C throughout all steps helps preserve phosphorylation status. When dealing with antibody cross-reactivity between phospho-STK4 and phospho-STK3, researchers should consider immunoprecipitation with isoform-specific antibodies prior to phospho-detection or carefully validate their findings using genetic approaches (knockout/knockdown models). For improving signal detection, stimulus optimization (e.g., staurosporine treatment for positive controls) and signal amplification methods such as enhanced chemiluminescence systems may be beneficial. Finally, researchers should consider the biological context, as STK4 phosphorylation may be transient or stimulus-dependent, requiring careful time-course experiments to capture phosphorylation events .

How can phospho-STK4 (Thr183) detection be optimized in different experimental systems?

Optimizing phospho-STK4 (Thr183) detection requires tailored approaches for different experimental systems, each presenting unique challenges and opportunities. For Western blot applications, sample-dependent titration of antibody concentrations (ranging from 1:500 to 1:2000) is recommended to determine optimal signal-to-noise ratios. Blocking with 5% BSA rather than milk proteins preserves phospho-epitopes and enhances detection sensitivity .

In cell-based imaging techniques such as immunofluorescence, fixation methods significantly impact phospho-epitope preservation. Brief fixation with 4% paraformaldehyde followed by methanol permeabilization typically yields superior results compared to longer fixation protocols. For flow cytometry applications, single-cell phospho-protein analysis benefits from rapid fixation with formaldehyde followed by methanol permeabilization to access intracellular epitopes while maintaining phosphorylation status .

When working with tissue samples, phospho-STK4 detection presents additional challenges. Rapid tissue processing and fixation are critical, with optimal results achieved using phospho-friendly fixatives and antigen retrieval methods specifically optimized for phospho-epitopes. For all systems, researchers should implement appropriate positive controls (such as staurosporine-treated cells) and negative controls (dephosphorylated samples and/or genetic knockouts) to validate detection methods. The experimental context also matters significantly - in Treg cells, for example, TCR stimulation is necessary to observe robust phosphorylation signals, while in other cell types, different stimuli may be required to induce detectable STK4 phosphorylation .

What methodological approaches can reconcile contradictory data in STK4 phosphorylation studies?

Contradictory findings in STK4 phosphorylation studies often stem from methodological differences, biological context variations, and technical limitations. To reconcile such discrepancies, researchers should implement a systematic approach incorporating multiple complementary methods and careful experimental controls. First, standardization of detection methods is essential - researchers should clearly document antibody specificity validation, stimulation protocols, and detection techniques to enable meaningful cross-study comparisons .

When contradictory data arise regarding STK4 phosphorylation patterns or functional outcomes, several approaches can help resolve discrepancies: (1) Conducting parallel experiments using multiple antibodies targeting the same phosphorylation site but from different sources or with different epitope recognition properties; (2) Employing both antibody-dependent and antibody-independent detection methods, such as mass spectrometry-based phospho-proteomics; (3) Utilizing genetic approaches with phospho-mimetic (T183E) and phospho-deficient (T183A) mutants to validate functional findings; and (4) Carefully controlling for cell type specificity and activation state, as STK4 function differs markedly between contexts .

The timing of analysis is particularly important, as phosphorylation events may be transient and follow different kinetics in various cell types or under different stimulation conditions. Time-course experiments with multiple sampling points can help resolve apparent contradictions that result from temporal differences in phosphorylation dynamics. Additionally, researchers should consider potential crosstalk with other signaling pathways that might influence STK4 phosphorylation in a context-dependent manner, particularly given its involvement in both immune regulation and the Hippo tumor suppressor pathway .

How does STK4 phosphorylation status correlate with autoimmune disease progression?

The phosphorylation status of STK4 demonstrates significant correlation with autoimmune disease progression, particularly in contexts involving regulatory T cell dysfunction. Research using mouse models has revealed that Treg-specific deletion of STK4 precipitates a fatal autoimmune lymphoproliferative disease characterized by decreased Treg cell frequencies, expanded T effector memory cell populations, dysregulated IFNγ expression, and hyper-immunoglobulinemia. The severity and progression rate of this autoimmune phenotype appears directly linked to STK4 phosphorylation-dependent functions, as evidenced by the more severe disease presentation in STK3/4 double-knockout mice compared to STK4 single-knockout animals .

Mechanistically, impaired STK4 phosphorylation in Treg cells compromises their activation and immune regulatory capacity through defective formation of the STK4-Foxp3-p65 complex. This molecular defect translates to reduced expression of Treg activation markers, including CD25 and CD73, alongside increased Treg cell turnover with heightened apoptosis rates. Importantly, studies in female heterozygous mice (which serve as models with reduced inflammatory background) confirmed the intrinsic requirement for STK4 in Treg cell fitness even under non-inflammatory conditions. These findings suggest that monitoring STK4 phosphorylation status in Treg cells could potentially serve as a biomarker for autoimmune disease progression and treatment response. Furthermore, the observation that patients with STK4 deficiency manifest similar Treg cell defects to those observed in mouse models underscores the translational relevance of these findings for human autoimmune conditions .

What are the emerging technologies for studying spatial and temporal dynamics of STK4 phosphorylation?

Cutting-edge technologies are revolutionizing our ability to investigate the spatial and temporal dynamics of STK4 phosphorylation with unprecedented resolution. Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) represent one of the most promising approaches for visualizing STK4 phosphorylation in living cells. These biosensors typically incorporate the STK4 phosphorylation motif between fluorescent protein pairs, allowing real-time monitoring of phosphorylation events in response to various stimuli. This technology enables researchers to track the kinetics of STK4 activation with subcellular resolution, revealing previously unappreciated spatial regulation of its phosphorylation .

Mass spectrometry-based phosphoproteomics has also evolved to provide multiplexed analysis of STK4 phosphorylation alongside hundreds of other phosphorylation events. Techniques such as TMT (tandem mass tag) labeling allow quantitative comparison across multiple conditions and timepoints, generating comprehensive phosphorylation profiles. When combined with subcellular fractionation, these approaches can map compartment-specific phosphorylation dynamics of STK4 .

Advanced microscopy techniques, including super-resolution methods such as STORM (Stochastic Optical Reconstruction Microscopy) and lattice light-sheet microscopy, now enable visualization of phospho-STK4 localization with nanometer precision and minimal phototoxicity. These approaches reveal how phosphorylation influences STK4 molecular interactions and subcellular organization in previously unattainable detail. For in vivo applications, intravital microscopy combined with phospho-specific antibodies allows tracking of STK4 activation in tissues of living organisms. Together, these technological advances are providing unprecedented insights into how STK4 phosphorylation is dynamically regulated in diverse biological contexts, from immune cell signaling to Hippo pathway activation .

How might targeting STK4 phosphorylation be leveraged for therapeutic applications in autoimmunity and cancer?

Therapeutic targeting of STK4 phosphorylation represents a promising frontier for intervention in both autoimmune disorders and cancer, though with opposing strategies required for each disease context. In autoimmune conditions, enhancing STK4 phosphorylation and activity in Treg cells could potentially restore immune tolerance. This approach is supported by evidence that phospho-STK4 promotes Foxp3 stability and function through direct phosphorylation at serine 418. Potential therapeutic strategies include development of small molecule STK4 activators or targeted delivery of constitutively active STK4 to Treg cells. Alternatively, gene therapy approaches using phosphomimetic Foxp3^S418E^ could bypass the requirement for STK4 activity, as demonstrated in experimental models where expression of this construct ameliorated immune regulatory defects in STK3/4-deficient Treg cells .