Recombinant Pongo abelii Ribosomal protein S6 kinase alpha-5 (RPS6KA5), partial

What is the basic function of RPS6KA5 in Pongo abelii cellular pathways?

RPS6KA5 in Pongo abelii functions as a serine/threonine protein kinase that plays critical roles in multiple cellular processes. It enables ATP binding activity and protein serine/threonine kinase activity, participating in histone-serine phosphorylation, positive regulation of histone modification, and regulation of transcription . Like its human ortholog, the orangutan RPS6KA5 is involved in phosphorylating transcription factors CREB1 and ATF1 in response to mitogenic or stress stimuli such as UV-C irradiation, epidermal growth factor (EGF), and anisomycin . This kinase is also implicated in the regulation of inflammatory genes through its interactions with transcription factors like RELA, STAT3, and ETV1 .

To study its basic function, researchers typically employ phosphorylation assays using purified recombinant protein with ATP and specific substrates, followed by Western blotting with phospho-specific antibodies or mass spectrometry to detect phosphorylation events.

How does Pongo abelii RPS6KA5 compare structurally and functionally to human RPS6KA5?

Pongo abelii (Sumatran orangutan) RPS6KA5 shares high sequence homology with human RPS6KA5, reflecting their evolutionary closeness. Comparative analysis reveals conserved functional domains including:

Functionally, orangutan RPS6KA5 appears to retain the core activities seen in human RPS6KA5, including phosphorylation of transcription factors and involvement in signaling cascades . Paralog analysis from InParanoiDB reveals that within Pongo abelii, RPS6KA5 is part of a family of related proteins including RPS6KA1, RPS6KA2, RPS6KA3, and RPS6KA6, all functioning as ribosomal protein S6 kinases with varying degrees of similarity .

What are the optimal expression systems for producing recombinant Pongo abelii RPS6KA5?

When producing recombinant Pongo abelii RPS6KA5, researchers should consider several expression systems based on experimental goals:

• Yeast expression systems: Commonly used for producing kinases with proper folding and post-translational modifications. The yeast system has been successfully employed for expressing related proteins from Pongo abelii, as evidenced by the production of MAPK6 .

• Bacterial expression systems (E. coli): Suitable for producing larger quantities but may require optimization for solubility and activity. Often used with fusion tags (His, GST, MBP) to improve solubility and facilitate purification.

• Mammalian expression systems: Provide the most authentic post-translational modifications and folding environment for primate proteins.

The optimal expression protocol includes:

• Clone full-length or partial RPS6KA5 cDNA into an appropriate vector with a purification tag

• Transform/transfect host cells and induce protein expression under optimized conditions

• Purify using affinity chromatography followed by size exclusion chromatography

• Validate protein activity through kinase assays with known substrates

Success is highly dependent on construct design, with consideration for including key functional domains while avoiding regions that might impair solubility.

How can quantitative proteomics be optimized to study RPS6KA5 interactions and modifications in Pongo abelii tissues?

Studying RPS6KA5 interactions and modifications requires sophisticated quantitative proteomic approaches. Based on advanced methodological research, a combined PQD-CAD (pulsed-Q dissociation and collisionally activated dissociation) hybrid method in linear ion trap mass spectrometry offers significant advantages for quantifying RPS6KA5 and its interacting partners .

The optimized workflow for RPS6KA5 proteomics includes:

• Sample preparation: Extract proteins from Pongo abelii tissues or cells under conditions that preserve protein-protein interactions and post-translational modifications

• iTRAQ labeling: Apply isobaric tags for relative and absolute quantification to enable multiplexed analysis of different experimental conditions

• Fractionation: Employ multiple dimensional chromatography to enhance proteome coverage

• Mass spectrometry analysis: Utilize the PQD-CAD hybrid method which integrates:

  • PQD for detecting low mass reporter ions from iTRAQ labels

  • CAD for improved peptide fragmentation and identification

• Data analysis: Apply specialized bioinformatics algorithms for comprehensive protein identification and accurate quantification

This approach has demonstrated high accuracy in protein quantification, with previous studies identifying and quantifying over 1,600 proteins in a single proteomic experiment . When applied to RPS6KA5, this method can reveal dynamic changes in interaction partners and phosphorylation states under different cellular conditions.

What are the experimental challenges in distinguishing the specific functions of RPS6KA5 from other RPS6K family members in primate cells?

Distinguishing the specific functions of RPS6KA5 from other family members presents significant experimental challenges due to high sequence similarity and functional redundancy. The RPS6K family in Pongo abelii includes multiple members with overlapping functions, as evidenced by InParanoiDB analysis .

To address these challenges, researchers should employ:

• CRISPR/Cas9 gene editing: Generate specific knockout cell lines for RPS6KA5 while keeping other family members intact

• Isoform-specific antibodies: Develop antibodies targeting unique regions of RPS6KA5 for immunoprecipitation and immunoblotting experiments

• Selective inhibitors: Utilize kinase inhibitors with documented selectivity profiles to preferentially inhibit RPS6KA5

• Substrate profiling: Perform comprehensive substrate profiling using peptide arrays or phosphoproteomic approaches to identify unique substrates

• Rescue experiments: Conduct functional rescue experiments with RPS6KA5 constructs in knockout backgrounds to confirm specificity of observed phenotypes

These approaches collectively provide robust methods to distinguish RPS6KA5-specific functions from those shared with other family members.

How does subcellular localization of RPS6KA5 influence its functional activities in Pongo abelii cells?

The subcellular localization of RPS6KA5 is a critical determinant of its functional activities. Based on homology with human RPS6KA5, the protein is localized in both cytoplasm and nucleoplasm , with dynamic shuttling between these compartments in response to cellular stimuli.

Key aspects of RPS6KA5 subcellular localization include:

• Cytoplasmic functions:

  • Interaction with the glucocorticoid receptor NR3C1 in the cytoplasm, contributing to RELA inhibition and repression of inflammatory gene expression

  • Phosphorylation of cytoplasmic substrates in response to mitogenic stimuli

  • Participation in cytoplasmic signaling cascades like ERK signaling pathway

• Nuclear functions:

  • Direct phosphorylation of transcription factors (CREB1, ATF1, RELA, STAT3, ETV1)

  • Contribution to gene activation through histone phosphorylation

  • Regulation of transcription factor activity through nuclear protein phosphorylation

To experimentally investigate the impact of subcellular localization:

• Generate fluorescently-tagged RPS6KA5 constructs with altered nuclear localization signals

• Perform immunofluorescence microscopy with subcellular fractionation validation

• Analyze differential phosphorylation of nuclear vs. cytoplasmic substrates using phospho-specific antibodies

• Employ proximity ligation assays to detect in situ protein-protein interactions in different cellular compartments

• Use kinase activity assays on nuclear and cytoplasmic fractions to measure compartment-specific activity

How has RPS6KA5 evolved in primates, and what functional implications do sequence variations have?

Evolutionary analysis of RPS6KA5 across primates reveals important conservation patterns and species-specific variations with functional implications. The high conservation of RPS6KA5 between Pongo abelii and other primates, including humans, indicates its fundamental importance in cellular signaling .

Comparative analysis shows:

The most conserved regions across primate RPS6KA5 proteins are the N-terminal and C-terminal kinase domains, essential for ATP binding and catalytic activity. The highest variability is observed in linker regions and regulatory domains, suggesting evolution of regulatory mechanisms while preserving core catalytic functions.

Functional implications of these variations include:

• Species-specific phosphorylation patterns

• Differential sensitivity to upstream kinases

• Varied binding affinities for interaction partners

• Modified subcellular localization and trafficking

Researchers should consider these evolutionary aspects when using Pongo abelii RPS6KA5 as a model for human RPS6KA5 function.

What ortholog and paralog relationships should researchers consider when studying RPS6KA5 across species?

Understanding ortholog and paralog relationships is essential for comparative studies of RPS6KA5. InParanoiDB analysis reveals important relationships that influence experimental design and data interpretation .

Key ortholog relationships:

Key paralog relationships within Pongo abelii:

When designing experiments:

• Consider generating tools that specifically target unique regions of RPS6KA5

• Account for potential compensatory mechanisms from paralogous proteins

• Use comparative approaches to identify conserved vs. species-specific functions

• Design controls that can distinguish between activities of different family members

These considerations ensure robust experimental design and accurate interpretation of results in cross-species studies.

How does RPS6KA5 participate in stress response signaling networks, and what methodologies best capture this dynamic?

RPS6KA5 plays a crucial role in cellular stress response signaling networks through its ability to phosphorylate key transcription factors in response to various stress stimuli. Understanding these dynamics requires sophisticated experimental approaches.

Key stress-responsive functions of RPS6KA5 include:

• UV-C irradiation response: Phosphorylates CREB1 and ATF1 transcription factors

• Oxidative stress signaling: Required for phosphorylation of RELA at 'Ser-276' during oxidative stress in skeletal myoblasts

• Inflammatory response modulation: Associates with glucocorticoid receptor NR3C1 in the cytoplasm to inhibit RELA and repress inflammatory gene expression

• Growth factor response: Phosphorylates ETV1/ER81 at 'Ser-191' and 'Ser-216' in response to growth factor signaling via the MEK/ERK pathway

To effectively capture these dynamic interactions, researchers should employ:

• Time-course phosphoproteomics: Using the optimized PQD-CAD hybrid method with iTRAQ labeling to quantify phosphorylation changes across multiple time points after stress induction

• Live-cell imaging: With fluorescent biosensors to track RPS6KA5 localization and activity in real-time following stress stimuli

• ChIP-seq combined with RNA-seq: To correlate RPS6KA5-mediated phosphorylation events with transcriptional changes

• Kinase activity assays under stress conditions: Measuring RPS6KA5 activity toward key substrates at different time points following stress exposure

• Proximity-dependent labeling (BioID or APEX): To identify stress-dependent changes in the RPS6KA5 protein interaction network

These methodologies collectively provide a comprehensive view of how RPS6KA5 functions within stress response networks in Pongo abelii cells.

What are the critical considerations when designing inhibitor studies for RPS6KA5 in primate cell models?

Designing inhibitor studies for RPS6KA5 in primate cell models requires careful consideration of several critical factors to ensure specificity, efficacy, and biological relevance.

Key considerations include:

• Inhibitor selectivity profile:

  • Assess cross-reactivity with other RPS6K family members

  • Determine selectivity across the broader kinome

  • Consider species-specific variations in binding pocket structure between human and Pongo abelii RPS6KA5

• Inhibitor mechanism and binding mode:

  Inhibitor Type	Mechanism	Advantages	Limitations
  ATP-competitive	Competes with ATP binding	Well-established design	Lower selectivity
  Allosteric	Binds outside active site	Higher selectivity	May not fully inhibit all functions
  Covalent	Forms chemical bond with target	Long-lasting inhibition	Potential off-target effects
  Substrate-competitive	Blocks substrate binding	High specificity	May be less potent

• Pharmacokinetic considerations:

  • Cell permeability (particularly important for nuclear functions)

  • Stability in cell culture media and intracellular environment

  • Potential metabolism by primate cells

• Validation approaches:

  • Thermal shift assays to confirm direct binding

  • In vitro kinase assays with purified recombinant RPS6KA5

  • Cellular target engagement assays

  • Phosphorylation status of established substrates (CREB1, ATF1, RELA, etc.)

• Appropriate controls:

  • Include structurally related but inactive control compounds

  • Use genetic approaches (siRNA, CRISPR) as orthogonal validation

  • Test multiple chemically distinct inhibitors targeting RPS6KA5

Case study data from gastric cancer research using the kinase inhibitor PHA-665752 demonstrates the importance of temporal analysis in inhibitor studies, as proteomic changes can vary significantly across different time points post-treatment .

How can phosphoproteomic approaches be optimized to identify novel RPS6KA5 substrates in Pongo abelii cells?

Identifying novel RPS6KA5 substrates requires sophisticated phosphoproteomic approaches tailored to capture transient kinase-substrate relationships. Based on advances in proteomic methodologies, researchers can implement the following optimized workflow:

• Experimental design for substrate identification:

  • Generate cell models with inducible RPS6KA5 expression or activity

  • Implement analog-sensitive kinase technology by engineering RPS6KA5 to accept bulky ATP analogs

  • Create paired experimental conditions with active vs. inhibited RPS6KA5

• Enhanced sample preparation:

  • Employ phosphopeptide enrichment strategies (TiO2, IMAC, or combined approaches)

  • Implement rapid lysis conditions to preserve transient phosphorylation events

  • Consider subcellular fractionation to identify compartment-specific substrates

• Advanced MS methodology:

  • Utilize the optimized PQD-CAD hybrid method in linear ion trap MS for iTRAQ quantification

  • Apply multiple dimensional chromatography to enhance phosphoproteome coverage

  • Implement parallel reaction monitoring for targeted analysis of predicted substrates

• Bioinformatic substrate validation:

  • Analyze phosphorylation motifs for consensus RPS6KA5 target sequences

  • Cross-reference with kinase prediction algorithms

  • Integrate with protein-protein interaction data

  • Compare with known RPS6KA5 substrates across species

• Functional validation of novel substrates:

  • Perform in vitro kinase assays with recombinant substrates

  • Generate phospho-deficient and phospho-mimetic mutants for functional studies

  • Analyze phenotypic consequences of disrupting specific phosphorylation events

This integrated approach has proven successful in related studies and can be particularly valuable for identifying novel RPS6KA5 substrates involved in primate-specific cellular processes.

What insights can Pongo abelii RPS6KA5 studies provide for understanding human RPS6KA5-associated diseases?

Studies of Pongo abelii RPS6KA5 provide valuable insights for understanding human diseases associated with RPS6KA5 dysfunction due to the high conservation between species. These translational insights include:

• Coffin-Lowry Syndrome: Although primarily associated with mutations in RPS6KA3, studying the functional relationship between RPS6KA5 and RPS6KA3 in Pongo abelii can reveal compensatory mechanisms relevant to this neurodevelopmental disorder .

• Bladder Cancer: RPS6KA5 has been implicated in bladder cancer . Comparative studies of signaling pathway integration in primate cells can illuminate how alterations in RPS6KA5 activity contribute to oncogenesis.

• Inflammatory Disorders: Given RPS6KA5's role in regulating inflammatory genes through interaction with glucocorticoid receptor NR3C1 and inhibition of RELA , Pongo abelii models can provide insights into inflammatory disease mechanisms.

• Stress Response Disorders: The role of RPS6KA5 in phosphorylating CREB1 and ATF1 in response to stress stimuli makes it relevant to stress-related disorders. Primate models provide evolutionarily relevant contexts for studying these pathways.

Methodological approaches linking Pongo abelii studies to human disease include:

• Comparative phosphoproteomics to identify conserved vs. species-specific substrates

• Analysis of disease-associated variants in conserved functional domains

• Evaluation of compensatory mechanisms involving paralogous proteins

• Testing potential therapeutic approaches in primate cell models before human studies

These translational studies bridge the evolutionary gap while leveraging the high degree of conservation in RPS6KA5 structure and function.

How can recombinant Pongo abelii RPS6KA5 be used to develop screening assays for potential therapeutic compounds?

Recombinant Pongo abelii RPS6KA5 provides an excellent platform for developing screening assays to identify therapeutic compounds with potential applications in human diseases. The high conservation between orangutan and human RPS6KA5 makes this approach particularly valuable.

A comprehensive screening platform includes:

• Primary screening assays:

  • In vitro kinase activity assays: Using purified recombinant RPS6KA5 with known substrates (e.g., peptides derived from CREB1, ATF1, or RELA)

  • ATP consumption assays: Measuring remaining ATP after kinase reaction using luminescence-based detection

  • Phospho-specific antibody-based detection: ELISA or AlphaScreen formats measuring substrate phosphorylation

  • Thermal shift assays: Identifying compounds that bind to and stabilize RPS6KA5

• Secondary cellular assays:

  • Reporter gene assays: Measuring effects on CREB, ATF1, or NF-κB transcriptional activity

  • Phosphorylation of endogenous substrates: Western blotting with phospho-specific antibodies

  • Cellular localization: Effects on nuclear-cytoplasmic shuttling of RPS6KA5

• Assay optimization parameters:

  Parameter	Optimization Approach	Important Considerations
  Buffer composition	Systematic testing of pH, salt, reducing agents	May affect compound binding
  Substrate selection	Testing multiple known substrates	Different substrates may reveal different inhibitor profiles
  ATP concentration	Kinetic analysis to determine appropriate [ATP]	Critical for ATP-competitive inhibitors
  Incubation conditions	Time course and temperature optimization	Ensure linear reaction conditions
  Detection method	Compare direct vs. coupled detection systems	Balance sensitivity with artifact potential

• Data analysis and hit validation:

  • Implement dose-response testing to determine IC50 values

  • Assess selectivity using panel of related kinases

  • Conduct detailed mechanism of action studies for promising compounds

  • Evaluate effects in cell-based disease models

Implementing MS-based methods like the PQD-CAD hybrid approach enables comprehensive evaluation of compound effects on the phosphoproteome, providing deeper insights into compound specificity and off-target effects.

What emerging technologies will advance our understanding of RPS6KA5 function in primate models?

Several cutting-edge technologies are poised to revolutionize our understanding of RPS6KA5 function in primate models, opening new avenues for research:

• CRISPR-based technologies:

  • Base editing for introducing specific point mutations in RPS6KA5

  • CRISPRi/CRISPRa for temporal control of expression

  • CRISPR screens to identify synthetic interactions with RPS6KA5

• Single-cell multi-omics:

  • Integrating transcriptomics, proteomics, and phosphoproteomics at single-cell resolution

  • Revealing cell type-specific functions of RPS6KA5

  • Identifying rare cell populations with unique RPS6KA5 activity profiles

• Advanced structural biology approaches:

  • Cryo-EM studies of RPS6KA5 complexes with interaction partners

  • Hydrogen-deuterium exchange mass spectrometry for dynamic conformational analysis

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

• Spatially resolved proteomics:

  • Imaging mass spectrometry to map RPS6KA5 activity in tissues

  • Spatial transcriptomics to correlate RPS6KA5 activity with gene expression patterns

  • Multiplexed ion beam imaging for simultaneous detection of multiple phosphorylation events

• Advanced mathematical modeling:

  • Integration of multi-omics data into comprehensive signaling network models

  • Prediction of species-specific differences in RPS6KA5 pathway dynamics

  • In silico prediction of therapeutic targets within RPS6KA5 networks

These technologies will collectively provide unprecedented insights into the dynamic function of RPS6KA5 in primate biology, potentially revealing novel therapeutic opportunities for human diseases involving this important kinase.

What are the most significant unresolved questions about RPS6KA5 biology that researchers should prioritize?

Despite advances in understanding RPS6KA5, several critical questions remain unresolved. Prioritizing these questions will drive significant progress in the field:

• Substrate specificity mechanisms:

  • How does RPS6KA5 achieve specificity for its diverse substrates (CREB1, ATF1, RELA, STAT3, ETV1) ?

  • What structural features determine differential phosphorylation of targets under various conditions?

  • How conserved is substrate selection across primate species?

• Regulatory mechanisms:

  • What is the precise mechanism of RPS6KA5 activation in response to different upstream signals?

  • How is nuclear-cytoplasmic shuttling of RPS6KA5 regulated in different cell types?

  • What post-translational modifications beyond phosphorylation regulate RPS6KA5 activity?

• Evolutionary significance:

  • What evolutionary pressures have shaped RPS6KA5 function in primates?

  • Do primate-specific features of RPS6KA5 contribute to unique aspects of primate biology?

  • How has the relationship between RPS6KA5 and its paralogs evolved in primates?

• Disease relevance:

  • What is the precise contribution of RPS6KA5 dysfunction to diseases like bladder cancer ?

  • Can RPS6KA5 compensation rescue defects caused by mutations in related kinases (e.g., RPS6KA3 in Coffin-Lowry Syndrome) ?

  • Are there primate-specific aspects of RPS6KA5 function relevant to human disease?

• Technological challenges:

  • How can we develop truly selective tools (inhibitors, activators, biosensors) for RPS6KA5?

  • What approaches can overcome the challenge of distinguishing RPS6KA5 functions from those of closely related paralogs?

  • How can we capture the dynamic, context-dependent functions of RPS6KA5 in complex tissues?

Addressing these questions requires integrative approaches combining structural biology, biochemistry, cell biology, and systems biology in relevant primate models.

What are the recommended protocols for purification and activity assessment of recombinant Pongo abelii RPS6KA5?

The following detailed protocols provide a comprehensive workflow for purification and activity assessment of recombinant Pongo abelii RPS6KA5:

Purification Protocol:

• Expression system selection:

  • Yeast expression systems have shown success with Pongo abelii proteins

  • Consider S. cerevisiae or P. pastoris for proper folding and post-translational modifications

• Construct design:

  • Full-length construct (for complete functional studies)

  • N-terminal kinase domain (residues 1-400, for specific activity studies)

  • C-terminal kinase domain (residues 401-802, for regulatory studies)

  • Add affinity tag (His6, GST, or FLAG) with a precision protease cleavage site

• Expression conditions:

  • Optimize temperature (typically 16-30°C)

  • Induction time (12-72 hours depending on system)

  • Media composition (consider supplementation with ATP precursors)

• Purification steps:

  Step	Method	Buffer Composition	Critical Parameters
  Cell lysis	Sonication or mechanical disruption	50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol, 1 mM DTT, protease inhibitors	Complete lysis while maintaining protein integrity
  Affinity chromatography	Ni-NTA, Glutathione, or anti-FLAG	Lysis buffer + 10-30 mM imidazole (for His tag)	Slow flow rate, thorough washing
  Tag cleavage	PreScission or TEV protease	50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT	Complete cleavage without degradation
  Ion exchange	Q or S Sepharose	20 mM Tris-HCl pH 7.5, 50-500 mM NaCl gradient, 1 mM DTT	pH selection based on isoelectric point
  Size exclusion	Superdex 200	20 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM DTT	Final polishing and buffer exchange

• Quality control:

  • SDS-PAGE for purity assessment (>95% purity)

  • Western blot with anti-RPS6KA5 antibodies

  • Mass spectrometry to confirm identity and integrity

  • Dynamic light scattering for aggregation assessment

Activity Assessment Protocols:

• In vitro kinase assay:

  • Reaction components: 5-50 ng purified RPS6KA5, 1-5 μg substrate (CREB1-derived peptide), 100 μM ATP, 10 mM MgCl2, 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT

  • Detection methods:

    • [γ-32P]ATP incorporation for sensitive detection

    • Anti-phospho-substrate antibodies for specific phosphorylation sites

    • Mass spectrometry for site identification and quantification

• Thermal stability assay:

  • Differential scanning fluorimetry using SYPRO Orange

  • Test stabilization by ATP, substrate peptides, and potential inhibitors

  • Compare with human RPS6KA5 to identify species-specific differences

• Substrate specificity profiling:

  • Peptide array screening with consensus motifs

  • Quantitative comparison of phosphorylation efficiency across substrates

  • Validation with full-length protein substrates