Recombinant Bovine Serine/threonine-protein kinase 10 (STK10), partial

What is the basic function of STK10 in cellular processes?

STK10, also known as lymphocyte-oriented kinase (LOK), is a serine/threonine kinase predominantly expressed in lymphoid organs. Its primary function involves phosphorylating the conserved threonine residues at the C-terminal domain of ERM (ezrin, radixin, and moesin) proteins (T567, T564, and T558, respectively), which is essential for their activation . These ERM proteins mediate interactions between the actin cytoskeleton and plasma membrane, playing critical roles in maintaining cytoskeletal structure, cell motility, adhesion, movement, and signal transduction . The phosphorylation activity of STK10 represents a key regulatory mechanism in these cellular processes.

How conserved is STK10 between bovine and human species?

While the search results don't provide specific information about sequence homology between bovine and human STK10, researchers should note that many serine/threonine kinases are highly conserved across mammalian species. When working with recombinant bovine STK10, it's important to consider potential functional similarities and differences compared to human STK10. Sequence alignment analysis using tools like BLAST or Clustal Omega can help determine the degree of conservation, particularly in the catalytic domain and key regulatory regions. This conservation analysis is critical when extrapolating findings from bovine models to human applications.

What expression systems are most effective for producing recombinant bovine STK10?

Based on general recombinant protein production practices, researchers typically use several expression systems for kinases like STK10:

• Bacterial systems (E. coli): Suitable for producing partial domains or non-glycosylated forms

• Insect cell systems: Better for preserving post-translational modifications

• Mammalian cell systems: Optimal for full-length functional kinases with proper folding and modifications

For recombinant bovine STK10, mammalian or insect cell expression systems are generally preferable when conformational integrity and kinase activity are critical for downstream applications. Codon optimization for the expression system of choice may improve yield and quality. When expressing partial STK10 constructs, careful design of the truncation sites is essential to maintain the structural integrity of the included domains.

What is the optimal protocol for generating STK10 knockout cell lines?

The CRISPR-Cas9 gene-editing system has been successfully used to generate STK10 knockout cell lines. Based on published methodologies , researchers should:

• Design guide RNA: Target specific sequences in the STK10 gene. Multiple gRNAs may be designed to target different exons for higher knockout efficiency.

• Clone oligonucleotides: Synthesize and clone the guide-RNA into an appropriate vector (such as pX459) .

• Transfect cells: Introduce the vectors into your cell line of interest using standard transfection methods.

• Selection: Apply appropriate selection (e.g., puromycin) to isolate transfected cells.

• Validation: Confirm indel mutations by DNA sequencing of PCR products from the target DNA region .

• Protein expression verification: Use Western blot with anti-STK10 antibody to confirm the abolishment of STK10 expression .

This approach has been validated in human cervical cancer cell lines (HeLa and Caski) and can be adapted for bovine cell lines with appropriate species-specific modifications to gRNA design.

What methods are most reliable for measuring STK10 kinase activity in vitro?

For measuring STK10 kinase activity, researchers should consider the following methodological approaches:

When working with recombinant bovine STK10, researchers should validate the specificity of substrates and optimize reaction conditions including buffer composition, pH, temperature, and cofactor concentrations to ensure reliable activity measurements.

How does STK10 knockout affect cancer cell migration and invasion?

Studies with STK10 knockout cervical cancer cell lines have demonstrated that target deletion of STK10 promotes the adhesion, migration, and invasion of cancer cells . Importantly, these effects occur without significantly affecting cell proliferation or apoptosis. Researchers investigating bovine STK10 should consider the following experimental approaches:

• Wound healing assay: To measure cell migration capacity

• Transwell assays: With and without Matrigel to assess invasive activity

• Adhesion assays: To quantify cell attachment capabilities

Notably, contrary to expectations, the phosphorylation and expression levels of ezrin and other ERM proteins in STK10 knockout cells were comparable to control cells , suggesting that STK10 may execute various physiological functions beyond ERM protein phosphorylation. RNA-seq analysis of STK10 knockout cells revealed profound alterations in gene expression profiles, which may contribute to the observed phenotypic changes .

What is the role of STK10 in tumor microenvironment regulation?

STK10 deficiency in mice has been shown to promote tumor growth by dysregulating the tumor microenvironment (TME) . Key findings include:

• T cell infiltration: Stk10-deficient mice showed increased percentage of CD3+ cells in tumors, suggesting altered T lymphocyte infiltration patterns .

• CTL activation: Stk10 deletion led to a significant increase in naïve CTLs (CD45+CD3+CD8+CD44−CD62L+), but decreases in activated (CD45+CD3+CD8+CD69+), effector (CD45+CD3+CD8+CD44+CD62L−) and exhausted CTLs (CD45+CD3+CD8+PD-1+LAG3+) .

• Angiogenesis: STK10 knockout can induce angiogenesis in tumor tissue .

These findings suggest STK10 plays a critical role in the activation and/or migration of cytotoxic T lymphocytes, potentially influencing anti-tumor immune responses. Researchers investigating bovine STK10 should consider these immunomodulatory functions when designing experiments in immune-competent models.

How can STK10 targeting be optimized for cancer therapeutic development?

Based on research findings, STK10 presents a potential therapeutic target with complex considerations:

• Context-dependency: In cervical cancer cells, STK10 knockout promotes migration and invasion , while in the tumor microenvironment, Stk10 deficiency promotes tumor growth by dysregulating immune responses . These contrasting roles necessitate careful targeting strategies.

• Selective inhibition approaches:

  • ATP-competitive inhibitors targeting the catalytic domain

  • Allosteric modulators affecting protein-protein interactions

  • Degraders (PROTACs) for targeted protein degradation

• Combination strategies: Considering STK10's role in immune cell regulation, combining STK10 modulators with immune checkpoint inhibitors might provide synergistic effects.

• Biomarker development: Expression analysis of STK10 in various cancer types shows correlations with immune cell infiltration , suggesting potential use as a predictive biomarker for immunotherapy response.

When developing STK10-targeting strategies using bovine models or recombinant bovine STK10, researchers should carefully consider these context-dependent effects and validate findings across multiple experimental systems.

What are the challenges in extrapolating bovine STK10 research to human applications?

Researchers face several key challenges when translating findings from bovine STK10 studies to human applications:

• Species-specific differences: While core kinase domains are often conserved, regulatory regions and post-translational modification sites may differ, affecting drug binding and functional outcomes.

• Tissue expression variances: Expression patterns of STK10 across tissues can vary between bovine and human subjects. According to studies, STK10 is expressed in approximately 17 cancer types in humans , but bovine expression patterns may differ.

• Interaction partners: Differences in the complement of interaction partners and downstream effectors between species may lead to divergent functional outcomes.

• Immune system differences: Given STK10's role in immune regulation , differences in bovine and human immune systems must be considered when studying immunomodulatory functions.

To address these challenges, researchers should conduct comparative studies using both bovine and human STK10, focusing on structure-function relationships, interaction networks, and downstream signaling pathways.

What controls are essential when studying recombinant bovine STK10 in experimental systems?

Robust experimental design requires the following controls:

• Kinase-dead mutant: A catalytically inactive STK10 mutant (typically with a mutation in the ATP-binding site) serves as a negative control for kinase activity-dependent effects.

• Wild-type comparison: Always include wild-type STK10 when studying partial or truncated constructs to differentiate domain-specific functions.

• Species controls: When possible, compare bovine STK10 with human STK10 to identify species-specific effects.

• Substrate specificity controls: Include non-phosphorylatable substrate mutants (e.g., ERM proteins with T→A mutations) to confirm kinase specificity.

• System validation: For knockout studies, rescue experiments with wild-type STK10 reintroduction can confirm phenotype specificity. Studies have successfully used this approach to validate STK10 knockout systems .

These controls help ensure experimental observations are specifically attributed to bovine STK10 activity rather than artifacts or indirect effects.

How should researchers address contradictory findings in STK10 research across different cancer types?

Research has revealed seemingly contradictory roles for STK10 across different experimental systems, particularly in cancer research. To address these contradictions:

• Cell type specificity: STK10 functions may be cell type-dependent. For example, knockout studies in cervical cancer cell lines showed enhanced migration and invasion , while STK10 deletion in mice promoted tumor growth through immune system dysregulation .

• Detailed phenotypic profiling: Conduct comprehensive analysis of multiple cellular processes (proliferation, apoptosis, migration, invasion, immune interactions) to develop a complete functional profile.

• Pathway analysis: Investigate downstream signaling networks through techniques like phosphoproteomics, RNA-seq, and protein-protein interaction studies to identify context-specific regulatory mechanisms .

• Microenvironment considerations: Assess STK10 function both in isolated cancer cells and in the context of the tumor microenvironment, as its effects may differ substantially between these settings .

• Multi-omics integration: Combine genomic, transcriptomic, and proteomic data to build comprehensive models of STK10 function across different cellular contexts.

By systematically addressing these considerations, researchers can reconcile apparently contradictory findings and develop a more nuanced understanding of STK10 biology.

What are promising approaches for identifying novel substrates of bovine STK10 beyond ERM proteins?

While ERM proteins are established STK10 substrates, research suggests additional functional roles . To identify novel substrates:

• Phosphoproteomic screening: Compare the phosphoproteome of wild-type and STK10-knockout cells to identify differentially phosphorylated proteins.

• Kinase substrate prediction: Use computational tools to predict potential substrates based on consensus phosphorylation motifs.

• Proximity labeling: Employ BioID or TurboID fused to STK10 to identify proteins in close proximity, potentially including substrates.

• Synthetic peptide arrays: Screen peptide libraries for STK10 phosphorylation to identify preferred substrate motifs.

• Yeast two-hybrid or mammalian two-hybrid systems: Identify proteins that directly interact with STK10, which may include substrates.

For researchers working with bovine STK10, comparing identified substrates with those of human STK10 could reveal conserved and species-specific targets, providing insights into the transferability of findings across species.

How might STK10 function in immune cell migration and activation be therapeutically exploited?

STK10's role in immune cell function presents opportunities for therapeutic applications:

• T cell-based immunotherapies: Studies show that Stk10 deletion affects CTL activation and differentiation . Modulating STK10 activity could potentially enhance CAR-T or TCR-T cell persistence and efficacy.

• Tumor microenvironment modulation: Given STK10's impact on immune cell infiltration in tumors , targeted inhibition might alter the immune composition of the tumor microenvironment to favor anti-tumor responses.

• Combination with checkpoint inhibitors: Since Stk10 deficiency affects exhausted CTL populations (PD-1+LAG3+) , combining STK10 modulators with checkpoint inhibitors might have synergistic effects.

• Autoimmune disease applications: Beyond cancer, STK10's role in lymphocyte function suggests potential applications in autoimmune disease treatment.

Research using recombinant bovine STK10 in relevant model systems could help identify and validate these potential therapeutic approaches before translation to human applications.