Recombinant Mouse Serine/threonine-protein kinase PAK 7 (Pak7)

What is PAK7 and what are its main functions in cellular signaling?

PAK7, also known as PAK5, is a member of the p21-activated kinase family of serine/threonine protein kinases that act as effectors of the small GTPases Rac and CDC42. The human PAK7 gene is located on chromosome 20p12, spans approximately 300 kb, contains 12 exons, and encodes an 80 kDa protein . Mouse PAK7 shares significant homology with its human counterpart.

PAK7 exhibits several critical cellular functions:

• Promotion of neurite outgrowth and microtubule stabilization

• Activation of cell survival signaling pathways

• Regulation of cytoskeleton organization

• Modulation of cell cycle progression

• Inhibition of apoptosis

• Facilitation of cell migration and invasion

As a member of group B PAKs, PAK7's activation mechanism can be partially independent of Cdc42/Rac binding, distinguishing it from group A PAKs .

How does mouse PAK7 differ structurally and functionally from other PAK family members?

While the search results don't provide comprehensive comparative information, PAK7 belongs to the group B PAK family (which includes PAK4, PAK6, and PAK7), which is functionally distinct from group A PAKs (PAK1-3). Group B PAKs typically have:

• Different regulatory mechanisms - their activation may be less dependent on Cdc42/Rac binding

• Unique substrate preferences

• Distinct expression patterns with PAK7 being particularly enriched in neuronal tissues

• Different cellular localization patterns

For comparison, mouse PAK3 (another PAK family member) plays critical roles in dendrite spine morphogenesis and synapse formation, acting as a downstream effector of CDC42 and RAC1 .

What expression systems are recommended for producing recombinant mouse PAK7?

While specific information about PAK7 expression systems isn't detailed in the search results, successful strategies for related proteins suggest:

• Baculovirus-infected Sf9 insect cells have been effectively used for producing recombinant mouse PAK3 with >75% purity

• Mammalian expression systems are advantageous for proteins requiring proper folding and post-translational modifications

Key considerations when selecting an expression system include:

• Need for proper protein folding and post-translational modifications

• Required yield and purity

• Downstream application requirements

• Presence of binding partners or activators

What methods are recommended for studying PAK7 interactions with signaling pathways?

For investigating PAK7's interactions with signaling pathways such as Wnt/β-catenin, researchers have employed multiple complementary approaches:

• Western blotting: To detect changes in β-catenin levels and GSK3β phosphorylation status

• Reporter assays: TOP/FOP flash assays to measure Wnt/β-catenin signaling activity

• Co-immunoprecipitation (co-IP): To detect physical interactions between PAK7, GSK3β, and β-catenin

• Co-localization assays: To visualize cellular distribution of these proteins

Research has revealed that PAK7 can directly bind to GSK3β and β-catenin, regulating β-catenin degradation through GSK3β phosphorylation .

How does PAK7 contribute to cancer progression and metastasis in experimental models?

PAK7 has been implicated in multiple cancer types with several consistent mechanisms:

A study of colon cancer found that PAK7 knockdown significantly suppressed the progression of epithelial-to-mesenchymal transition (EMT), migration, and invasion of cancer cells in vitro and inhibited tumor growth in vivo .

What role does PAK7 play in DNA damage response and radiotherapy sensitivity?

PAK7 has been shown to influence radiotherapy sensitivity in hepatocellular carcinoma (HCC):

• PAK7 downregulation enhanced radiosensitivity by:

  • Inhibiting cell viability

  • Promoting apoptosis

  • Arresting cells in G2/M phase

  • Inducing DNA damage (measured by increased γ-H2AX expression)

Mechanistically, PAK7 appears to:

• Reduce DNA double-strand breaks (DSB) in HCC cells

• Accelerate the repair of DNA damage

• Weaken cellular sensitivity to radiotherapy when highly expressed

These findings suggest targeting PAK7 could be a potential strategy to enhance the efficacy of radiotherapy in HCC patients .

How do PAK7 mutations correlate with tumor immunogenicity and response to immunotherapy?

A study on non-small cell lung cancer (NSCLC) found significant correlations between PAK7 mutations and immunotherapy response:

PAK7 mutations were also associated with:

• Lower copy number variation

• Higher mutation rates in DNA damage response (DDR)-related pathways

• Positive correlation with immune-related gene expression

These findings suggest PAK7 mutations might serve as a potential biomarker for predicting immunotherapy efficacy in NSCLC patients .

What are the key considerations when designing experiments to study PAK7's role in cell cycle regulation?

When investigating PAK7's role in cell cycle regulation, researchers should consider:

• Cell line selection: Different cell lines exhibit varying levels of PAK7 expression (PAK7^low, PAK7^moderate, PAK7^high), which may influence experimental outcomes

• Cell cycle analysis methods:

  • Flow cytometry with propidium iodide (PI) staining

  • Western blot analysis of cell cycle regulators

• Molecular markers to monitor:

  • CDK2, CDC25A, and cyclin D1 (downregulated after PAK7 knockdown in gastric cancer)

  • γ-H2AX (indicator of DNA damage)

  • Cell cycle checkpoint proteins

• Context-dependent effects: PAK7 knockdown has been associated with G0/G1 arrest in gastric cancer but G2/M arrest in hepatocellular carcinoma , suggesting tissue-specific mechanisms

• Potential interactions: Consider the role of Cdc42, as its inhibition has been shown to affect cell cycle arrest in G2/M phase

How can researchers use recombinant PAK7 to investigate the competitive binding of inhibitors?

While the search results don't directly address this question, standard approaches for kinase inhibitor studies would include:

• In vitro kinase assays: Using purified recombinant PAK7 with:

  • Known substrates

  • Varying concentrations of potential inhibitors

  • Appropriate controls (including known inhibitors if available)

• Binding affinity measurements:

  • Surface plasmon resonance (SPR)

  • Isothermal titration calorimetry (ITC)

  • Thermal shift assays

• Structural studies:

  • Co-crystallization of PAK7 with inhibitors

  • Structure-activity relationship analyses

• Cellular validation:

  • Confirmation that in vitro findings translate to cellular effects

  • Assessment of specificity against other PAK family members

• Species differences: Consider potential differences between mouse and human PAK7 when developing inhibitors for eventual clinical applications

What are common challenges in maintaining PAK7 activity during recombinant protein production?

While specific challenges for PAK7 aren't detailed in the search results, general considerations for kinase production include:

• Phosphorylation status: Autophosphorylation or phosphorylation by other kinases in the expression system may affect activity

• Protein folding: Ensuring proper folding, especially of the kinase domain

• Buffer optimization: Testing various buffer conditions to maintain stability and activity:

  • pH ranges

  • Salt concentrations

  • Reducing agents

  • Stabilizing additives

• Storage conditions: Determining optimal temperature and additives for long-term stability

• Activation state: Consider co-expression with activators or inhibitors depending on experimental needs

How can researchers differentiate between the functions of PAK7 and other PAK family members?

To distinguish PAK7 functions from other PAK family members:

• Specific genetic manipulation:

  • Use targeted shRNA or CRISPR approaches specific to PAK7

  • Verify specificity by confirming no change in expression of other PAK family members

• Assess compensatory mechanisms:

  • Determine whether other PAK family members show altered expression when PAK7 is inhibited

  • Look for changes in downstream signaling that might indicate compensation

• Identify unique interactions:

  • Study PAK7-specific binding partners

  • Investigate PAK7-specific substrates

  • Examine unique activation mechanisms

• Consider context-dependency:

  • Tissue-specific roles

  • Cancer-type specific functions

  • Developmental stage-specific activities

PAK7 activation may be partially independent of Cdc42/Rac binding, which distinguishes it from some other PAK family members and expands its cellular biological roles in tumor development .

What emerging applications exist for recombinant PAK7 in therapeutic development?

Based on the research findings, several therapeutic directions show promise:

• Cancer therapy:

  • PAK7 inhibition could enhance radiosensitivity in hepatocellular carcinoma

  • PAK7 targeting might reduce metastasis in colon and breast cancers

  • PAK7 mutation status could serve as a biomarker for immunotherapy response

• Combination approaches:

  • Combining PAK7 inhibition with DNA damage-inducing therapies

  • Pairing PAK7 targeting with Wnt/β-catenin pathway modulators

• Selective inhibitor development:

  • Creating inhibitors specific to PAK7 versus other PAK family members

  • Designing inhibitors that target cancer-specific functions while preserving normal cellular roles

• Diagnostic applications:

  • Using PAK7 mutation status as a predictive biomarker for immunotherapy response

  • Developing PAK7 expression assays to guide treatment decisions

The significant correlation between PAK7 mutations and improved immunotherapy response suggests particularly promising applications in precision oncology and biomarker development .