Recombinant Mouse Tyrosine-protein phosphatase non-receptor type 7 (Ptpn7)

What is mouse Ptpn7 and what are its primary functions?

Mouse Ptpn7 (also known as hematopoietic protein tyrosine phosphatase or HEPTP) is a member of the protein tyrosine phosphatase (PTP) family. It functions as a signaling molecule that regulates various cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation . Ptpn7 is preferentially expressed in hematopoietic cells and serves as an early response gene in lymphokine-stimulated cells .

The primary function of Ptpn7 is the negative regulation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase signaling in T lymphocytes . This regulatory role makes Ptpn7 important in controlling T cell antigen receptor (TCR) signaling through dephosphorylation of molecules within the MAP kinase pathway . Recent research has also identified Ptpn7 as a negative regulator in platelet functional responses downstream of G protein-coupled receptor (GPCR) agonists, which it accomplishes through inhibition of ERK activation and thromboxane generation .

How do recombinant mouse Ptpn7 proteins differ in structure based on expression systems?

Recombinant mouse Ptpn7 proteins can be produced in various expression systems, including bacterial (E. coli) and mammalian expression systems (such as HEK293 cells) . The structural characteristics of the recombinant protein can vary significantly depending on the expression system used:

For accurate structural and functional studies, researchers should select an expression system that closely mimics the post-translational modifications required for the specific research question. Full-length mouse Ptpn7 consists of 399 amino acids, though some recombinant constructs focus on specific domains such as the catalytic domain (amino acids 79-358) .

What are the typical expression patterns of Ptpn7 in mouse tissues?

Ptpn7 exhibits a distinctive tissue expression pattern, being predominantly expressed in hematopoietic cells . This phosphatase is particularly abundant in:

• T lymphocytes, where it regulates TCR signaling

• Platelets, where it controls ERK1/2 activation and thromboxane generation

• Other hematopoietic lineage cells

Mass spectrometry studies have confirmed the presence of Ptpn7 in mouse platelets . In mouse models, Ptpn7 expression can be observed in bone marrow, thymus, spleen, and lymph nodes. The expression pattern aligns with its functional role in immune cell regulation and hemostasis.

How does mouse Ptpn7 regulate ERK signaling and what are the implications for experimental design?

Mouse Ptpn7 serves as a negative regulator of ERK1/2 signaling, primarily through direct dephosphorylation. When designing experiments to study this regulatory mechanism, researchers should consider the following methodological approaches:

• Phosphorylation analysis: Western blotting using phospho-specific antibodies against ERK1/2 can quantify the impact of Ptpn7 on ERK activation. Comparison between Ptpn7 knockout and wild-type mice has shown that absence of Ptpn7 leads to enhanced ERK phosphorylation, particularly downstream of GPCR agonists in platelets .

• Substrate specificity assays: In vitro phosphatase assays using purified recombinant Ptpn7 and phosphorylated ERK can determine the direct enzymatic activity and kinetics of dephosphorylation.

• Pathway analysis: Since ERK signaling affects numerous downstream targets, researchers should incorporate approaches to measure these targets (transcription factors, cytokine production, etc.) when manipulating Ptpn7 levels.

• Context-dependent regulation: Notably, PTPN7's regulation of ERK is context-dependent. In platelets, GPVI-mediated ERK phosphorylation occurs primarily through thromboxane A2 (TXA2), as demonstrated by experiments showing that stimulation with GPVI agonist collagen-related peptide along with COX inhibitor indomethacin prevented ERK1/2 phosphorylation .

When designing experiments, researchers should account for this pathway complexity and include appropriate controls for both direct and indirect effects of Ptpn7 on ERK signaling.

What phenotypic differences exist between Ptpn7 knockout and wild-type mice that are relevant for research models?

Ptpn7 knockout mice exhibit several phenotypic differences compared to wild-type mice, particularly in platelet function and thrombosis models:

• Platelet aggregation: Ptpn7-null platelets show enhanced aggregation responses to GPCR agonists like thrombin and ADP, but not to GPVI agonists .

• Thrombosis models: In pulmonary thromboembolism models, PTPN7-null mice exhibited significantly faster time to death compared to wild-type mice, indicating enhanced thrombotic potential .

• Hemostasis function: Interestingly, bleeding times did not significantly differ between PTPN7-null and wild-type mice, suggesting compensatory mechanisms may maintain normal hemostasis despite altered platelet reactivity .

• ERK activation and thromboxane generation: Platelets from Ptpn7 knockout mice show increased ERK1/2 phosphorylation and enhanced thromboxane generation in response to GPCR stimulation .

These phenotypic differences make Ptpn7 knockout mice valuable models for studying:

• The role of ERK signaling in platelet function

• Mechanisms of thromboxane regulation

• Potential therapeutic targets for thrombotic disorders

• The specificity of phosphatase regulation in different receptor-mediated pathways

When designing studies using these mouse models, researchers should consider the specific pathway being investigated and the potential for compensatory mechanisms in genetic knockout models.

How can researchers effectively use recombinant mouse Ptpn7 to study immune cell regulation?

Recombinant mouse Ptpn7 serves as a valuable tool for studying immune cell regulation, particularly in T lymphocytes. The following methodological approaches are recommended:

• In vitro phosphatase assays: Purified recombinant Ptpn7 can be used to assess direct dephosphorylation of potential substrates in immune signaling pathways, particularly those in the MAP kinase cascade.

• Structure-function relationship studies: Various recombinant constructs of Ptpn7 (full-length vs. catalytic domain only) can help determine which domains are critical for specific interactions with immune cell signaling components.

• Competitive binding assays: Recombinant Ptpn7 can be used to compete with endogenous Ptpn7 for binding to partners, allowing researchers to map interaction sites and disrupt specific signaling pathways.

• T cell activation models: When studying T cell function, researchers should note that the non-catalytic N-terminus of Ptpn7 can interact with MAP kinases and suppress MAP kinase activities, affecting T cell antigen receptor (TCR) signaling .

• Analysis of interaction partners: Techniques such as pull-down assays using GST-tagged recombinant Ptpn7 (available commercially) can identify novel binding partners in immune cell lysates.

For meaningful results in immune regulation studies, it's crucial to maintain the physiological relevance of the experimental conditions, as Ptpn7 functions can be context-dependent.

What is the role of Ptpn7 in cancer biology and how can it be studied as a potential biomarker?

Recent research has identified Ptpn7 as a potential biomarker in cancer biology, particularly in breast cancer and other malignancies. The following approaches are valuable for investigating its role:

• Expression analysis: Transcriptomic data from The Cancer Genome Atlas (TCGA) has shown that PTPN7 expression is upregulated in tumor tissues compared to paraneoplastic tissues in breast cancer and multiple other cancer types, including STAD, CHOL, HNSC, ESCA, KIPR, KIRC, LUAD, LIHC, CESE, and GMB .

• Correlation with immune infiltration: PTPN7 expression shows positive correlation with immune cell infiltration and negative correlation with tumor purity across multiple cancer types . Researchers can employ techniques like:

  • TIMER (Tumor Immune Estimation Resource) database analysis

  • Immunohistochemistry for spatial distribution

  • Flow cytometry for quantitative assessment of immune cell populations

• Immunotherapy response prediction: PTPN7 expression levels may predict response to immunotherapy. In GSE35640 and other datasets, PTPN7 was upregulated in patients with better immunotherapeutic responses . Study designs should incorporate:

  • Pre- and post-treatment biopsies

  • Correlation with established markers like PD-L1

  • Survival analysis based on PTPN7 expression

• Functional mechanism investigation: PTPN7 appears to be associated with "immuno-hot" tumors, potentially through regulation of T cell function. Experimental approaches should include:

  • Co-culture systems with tumor and immune cells

  • Analysis of checkpoint molecule expression in relation to PTPN7 levels

  • Investigation of PTPN7's effect on tumor mutation burden (TMB)

The data indicates that PTPN7 correlates positively with multiple immune checkpoints including PD-L1 and CTLA-4 expression across almost all cancer types, making it a promising biomarker for immunotherapy response .

What experimental approaches are most effective for studying the role of Ptpn7 in platelet function?

To investigate Ptpn7's role in platelet function, researchers should consider the following methodological approaches:

• Platelet aggregation assays: Comparative studies between Ptpn7 knockout and wild-type platelets have shown enhanced aggregation responses to GPCR agonists (thrombin, ADP) but not to GPVI agonists in Ptpn7-null platelets . Standard light transmission aggregometry can quantify these differences.

• Signaling pathway analysis: Western blotting for phosphorylated ERK1/2 in platelets is essential, as Ptpn7 regulates this pathway. Experiments should include:

  • Time course of ERK1/2 phosphorylation after agonist stimulation

  • Comparison between multiple agonists (GPCR vs. GPVI)

  • Inclusion of pathway inhibitors (e.g., indomethacin for COX inhibition)

• Thromboxane generation measurement: Since Ptpn7 affects thromboxane production, researchers should quantify TXB2 (the stable metabolite of TXA2) using enzyme immunoassays after platelet stimulation .

• In vivo thrombosis models: The pulmonary thromboembolism model has proven valuable in demonstrating enhanced thrombotic potential in Ptpn7 knockout mice . Other models to consider include:

  • Ferric chloride-induced arterial thrombosis

  • Laser-induced microvascular injury

  • Tail bleeding time assays

• Receptor-specific studies: To distinguish between different activation pathways, researchers should design experiments that selectively activate:

  • GPCR pathways (using thrombin, ADP, thromboxane analogs)

  • GPVI pathway (using collagen-related peptide)

  • Integrin signaling (using direct activators of GPIIb/IIIa)

This comprehensive approach will help elucidate the specific mechanisms by which Ptpn7 regulates platelet function, particularly focusing on its role as a negative regulator downstream of GPCR signaling.

What are the optimal storage and handling conditions for recombinant mouse Ptpn7?

For maintaining optimal activity of recombinant mouse Ptpn7, researchers should follow these technical guidelines:

• Storage temperature: Store aliquoted protein at -80°C for long-term storage. Avoid repeated freeze-thaw cycles which can lead to protein denaturation and loss of enzymatic activity.

• Buffer conditions: Phosphatase activity is optimal in buffers containing:

  • 50 mM HEPES or Tris, pH 7.0-7.5

  • 1-5 mM DTT to maintain reduced state of catalytic cysteine

  • 100-150 mM NaCl for physiological ionic strength

  • 0.01% non-ionic detergent (e.g., Triton X-100) to prevent aggregation

• Avoid phosphatase inhibitors: Common phosphatase inhibitors like vanadate, molybdate, and fluoride should be excluded from buffers when enzymatic activity is being measured.

• Protein concentration: Working concentrations typically range from 10-100 nM for enzymatic assays, though this may vary depending on substrate and assay conditions.

• Reducing agents: Always include fresh reducing agents in buffers as the catalytic cysteine must be reduced for activity.

These conditions are particularly important when working with GST-tagged or His-tagged recombinant mouse Ptpn7, which are commonly used in research settings .

How can researchers validate the specificity and activity of recombinant mouse Ptpn7?

Validating both the specificity and activity of recombinant mouse Ptpn7 is crucial for meaningful experimental outcomes. Recommended approaches include:

• Phosphatase activity assay: Use the artificial substrate para-nitrophenyl phosphate (pNPP) to assess basic enzymatic activity. Active Ptpn7 will cleave pNPP, producing a colored product measurable at 405 nm.

• Substrate specificity assay: Test dephosphorylation of physiologically relevant substrates:

  • Phosphorylated ERK1/2 peptides

  • Purified phospho-MAP kinases

  • Immunoprecipitated phosphorylated signaling proteins

• Inhibitor sensitivity: Validate that the recombinant protein responds appropriately to known phosphatase inhibitors, which serves as a fingerprint for authenticity.

• Western blot verification: Confirm protein identity and purity using antibodies specific to mouse Ptpn7 and any epitope tags (GST, His, etc.).

• Negative controls: Include catalytically inactive Ptpn7 mutants (typically Cys to Ser mutation in the catalytic site) as negative controls in enzymatic assays.

• Functional rescue experiments: In Ptpn7-deficient cells or tissues, demonstrate that the addition of recombinant Ptpn7 can restore normal signaling patterns, particularly in ERK1/2 phosphorylation.

These validation steps ensure that experimental outcomes can be confidently attributed to Ptpn7 activity rather than contaminating phosphatases or non-specific effects.

How can recombinant mouse Ptpn7 be utilized in immunotherapy research?

Recombinant mouse Ptpn7 has emerging applications in immunotherapy research, particularly given its correlation with immunotherapy response markers. Researchers can utilize it in the following ways:

• Biomarker development: PTPN7 expression correlates with response to immunotherapy in multiple cancer types . Researchers can develop assays using recombinant PTPN7 as standards for quantification of endogenous PTPN7 levels.

• Immune checkpoint correlation studies: Recombinant Ptpn7 can be used to investigate its direct interaction with immune checkpoint molecules like PD-L1 and CTLA-4, as PTPN7 shows strong positive correlation with these checkpoints across cancer types .

• T cell engineering: Understanding how Ptpn7 regulates T cell function can inform CAR-T cell engineering approaches. Modulating Ptpn7 levels or activity in engineered T cells might enhance their anti-tumor functions.

• Therapeutic target exploration: As a regulator of ERK signaling, Ptpn7 could be targeted to enhance or suppress specific immune cell functions. Recombinant protein can be used to screen for compounds that modulate its activity.

• Tumor microenvironment studies: Since PTPN7 correlates with "immuno-hot" tumors and immune cell infiltration , recombinant protein can be used in assays to understand mechanisms driving this correlation.

The relationship between PTPN7 expression and immunotherapy response suggests significant potential in this research area, particularly in breast cancer and lung cancer settings where clinical data shows promising correlations .

What are the most reliable methods for measuring Ptpn7 activity in complex biological samples?

Measuring Ptpn7 activity in complex biological samples presents challenges due to the presence of multiple phosphatases. The following methodological approaches enhance specificity and reliability:

• Immunoprecipitation followed by activity assay:

  • Isolate Ptpn7 from complex samples using specific antibodies

  • Measure phosphatase activity against known substrates like phospho-ERK peptides

  • Include controls with phosphatase inhibitors to confirm specificity

• In-gel phosphatase assays:

  • Separate proteins by non-denaturing PAGE

  • Overlay gel with radiolabeled phosphopeptide substrates

  • Visualize zones of dephosphorylation corresponding to Ptpn7's molecular weight

• FRET-based assays:

  • Use fluorescently labeled phosphopeptide substrates designed for Ptpn7 specificity

  • Measure fluorescence changes upon dephosphorylation

  • Include competitive inhibitors to confirm signal specificity

• Targeted mass spectrometry:

  • Identify Ptpn7-specific peptides for quantification

  • Monitor changes in phosphorylation status of known Ptpn7 substrates

  • Provides both activity inference and abundance measurement

• Genetic validation approaches:

  • Compare activity in wild-type versus Ptpn7 knockout samples

  • Reconstitute activity by adding recombinant Ptpn7 to knockout samples

  • Use siRNA knockdown to confirm specificity of activity measurements

When measuring Ptpn7 activity specifically in platelets or immune cells, researchers should account for the high expression of other phosphatases that might have overlapping substrate specificity. Including multiple controls and validation steps is essential for reliable activity measurement.

How does mouse Ptpn7 compare to human PTPN7 for translational research applications?

Understanding the similarities and differences between mouse Ptpn7 and human PTPN7 is critical for translational research. Key comparisons include:

For translational applications, researchers should consider:

The high degree of conservation between mouse and human PTPN7 supports the translational value of mouse models for studying this phosphatase, particularly in immune and cancer contexts.

What are the emerging research directions for mouse Ptpn7 studies?

Based on current literature, several promising research directions for mouse Ptpn7 are emerging:

• Cancer immunotherapy biomarker development: PTPN7's strong correlation with immune checkpoint expression and immunotherapy response positions it as a potentially valuable predictive biomarker. Future studies should focus on validating its predictive power across diverse cancer types and immunotherapy approaches.

• Thrombosis and hemostasis regulation: The role of Ptpn7 in regulating platelet activation through ERK1/2 and thromboxane generation suggests potential applications in thrombotic disorder research. Further investigation of this pathway could identify novel therapeutic targets.

• T cell engineering and immunomodulation: Understanding how Ptpn7 regulates T cell receptor signaling could inform strategies for enhancing or suppressing T cell responses in various disease contexts, from autoimmunity to cancer.

• Structure-based drug design: Detailed structural studies of mouse Ptpn7 could facilitate the development of specific inhibitors or activators with potential therapeutic applications.

• Systems biology of phosphatase networks: Placing Ptpn7 within larger signaling networks could reveal how this phosphatase coordinates with other regulatory mechanisms to control cellular responses.

The intersection of Ptpn7's functions in immune regulation, cancer biology, and hemostasis presents particularly rich opportunities for integrative research approaches that could eventually translate to clinical applications.