Recombinant Pig cAMP-regulated phosphoprotein 19 (ARPP19)

What is the structural nature of ARPP19 and how does it relate to protein function?

ARPP19 is an intrinsically disordered protein (IDP) that lacks a well-defined three-dimensional structure. Biophysical characterization using NMR spectroscopy reveals highly overlapped cross peaks in 2D 15N-HSQC spectra due to degeneracy of proton spectra dispersion, confirming its disordered nature . SAXS (Small Angle X-ray Scattering) analysis further supports this, with Kratky plots exhibiting rising curves with increasing angles - a characteristic scattering pattern of IDPs .

Despite being intrinsically disordered, ARPP19 has a propensity to form three transient α-helices, as determined by Secondary Structure Propensity (SSP) calculations using 1Hα, 13Cα, and 13Cβ chemical shifts . Computational analysis shows that ARPP19 adopts various conformations organized into six ensembles composed of 8-20 models, but with high average CαCα RMSD (approximately 20.0 Å), representing substantial conformational flexibility .

This structural flexibility is functionally significant as it enables ARPP19 to:

• Interact with multiple binding partners, particularly PP2A

• Respond dynamically to phosphorylation by different kinases (PKA and Greatwall)

• Adopt different conformational ensembles (compact and extended)

• Function as a signaling hub integrating multiple regulatory inputs

For recombinant pig ARPP19 research, this structural plasticity must be considered when designing expression constructs, as truncations might disrupt these transient structural elements essential for function.

What are the key phosphorylation sites in ARPP19 and their functional significance?

ARPP19 contains multiple phosphorylation sites that are crucial for its regulatory functions. Based on extensive studies in Xenopus and mammalian systems, two primary phosphorylation sites have been identified:

The interplay between these phosphorylation sites creates a sophisticated regulatory system:

• In prophase-arrested oocytes, ARPP19 is phosphorylated at S109 by PKA

• Progesterone stimulation leads to partial dephosphorylation of S109 (approximately 49% reduction)

• This dephosphorylation permits formation of a threshold level of active Cdk1

• Active Cdk1 initiates the MPF (Maturation Promoting Factor) autoamplification loop

• The autoamplification process activates Greatwall kinase, which phosphorylates ARPP19 at S67

• S67-phosphorylated ARPP19 inhibits PP2A-B55, maintaining Cdk1 activity

Interestingly, once the MPF autoamplification loop is initiated, S109 phosphorylation loses its inhibitory effect, suggesting a complex multi-step regulatory mechanism . For recombinant pig ARPP19, characterizing these phosphorylation sites would be essential for functional studies.

What expression systems are most suitable for producing recombinant pig ARPP19?

Based on successful approaches with human ARPP19, Escherichia coli expression systems represent the most reliable starting point for recombinant pig ARPP19 production. The following methodological considerations apply:

• Expression vector selection:

  • pET28a vector has been successfully used for human ARPP19 expression

  • This vector provides a 6×His tag for affinity purification

  • T7 promoter-driven expression enables high protein yields

• Protein characteristics to consider:

  • Full-length expression (1-112 amino acids for human ARPP19)

  • Appropriate tag selection (His or GST) based on downstream applications

  • Potential inclusion of protease cleavage sites if tag removal is required

• Expression conditions optimization:

  • Temperature adjustment (often lowered to 16-25°C) to enhance proper folding

  • IPTG concentration titration to balance yield and solubility

  • Growth media selection (rich media like TB or minimal media depending on application)

For applications requiring phosphorylated ARPP19, in vitro phosphorylation using recombinant kinases (PKA for S107/S109 or Greatwall for S62/S67) can be performed post-purification. Alternatively, co-expression with appropriate kinases might be considered, though this approach has not been explicitly described in the literature for ARPP19.

What purification strategies yield the highest purity and activity for recombinant ARPP19?

Purification of recombinant pig ARPP19 should follow established protocols that have achieved >90% purity for human ARPP19 . The recommended multi-step purification approach includes:

• Initial capture by affinity chromatography:

  • For His-tagged ARPP19: Nickel or cobalt affinity resin with imidazole elution (300mM imidazole)

  • For GST-tagged ARPP19: Glutathione-agarose with glutathione elution

• Buffer optimization:

  • PBS-based buffers (58mM Na₂HPO₄, 17mM NaH₂PO₄, 68mM NaCl, pH 7.4) have proven suitable

  • Addition of reducing agents (DTT or β-mercaptoethanol) at 1-5mM to prevent oxidation

  • Potential addition of protease inhibitors during initial extraction steps

• Secondary purification (if higher purity is required):

  • Size exclusion chromatography to remove aggregates and improve homogeneity

  • Ion exchange chromatography as a polishing step

• Quality control assessment:

  • SDS-PAGE with Coomassie staining to verify purity (target >90%)

  • Mass spectrometry to confirm protein identity

  • Functional assays to verify activity (e.g., PP2A binding or inhibition)

• Storage optimization:

  • Lyophilization from PBS with 5% trehalose and 5% mannitol as protectants

  • Storage of lyophilized protein at -20°C to -80°C (stable for up to 12 months)

  • For reconstituted protein, short-term storage at 2-8°C (1-2 weeks) or long-term storage at -20°C to -80°C with added glycerol (up to 3 months)

  • Avoiding repeated freeze-thaw cycles

This methodological approach ensures both high purity and maintained activity of recombinant ARPP19 for subsequent experimental applications.

What are the established methods for measuring ARPP19 phosphorylation states?

Accurate quantification of ARPP19 phosphorylation is critical for understanding its regulatory mechanisms. The following methodological approaches have been validated:

• Western blotting with phospho-specific antibodies:

  • Development of phospho-specific antibodies against key sites (e.g., phospho-S109/S107 and phospho-S67/S62)

  • Antibody generation typically involves immunizing rabbits with phosphopeptides (e.g., CQDLPQRKPpSLVASK for phospho-S109)

  • Affinity purification of antibodies to enhance specificity

  • Validation through phosphatase treatment and phosphomimetic/non-phosphorylatable mutants

• Quantitative phosphorylation analysis:

  • Normalization of phospho-specific signal to total ARPP19 levels

  • Implementation of ImageJ software-based densitometry

  • Standardization against controls (e.g., prophase state or GVBD timepoint)

  • Calculation of percent dephosphorylation relative to baseline

• Radiometric phosphorylation assays:

  • In vitro phosphorylation using purified kinases and [γ-³²P]ATP

  • Detection of phosphorylation by autoradiography

  • Quantification of phosphorylation dynamics through time-course experiments

  • Measurement of dephosphorylation rates in kinase-inactivated extracts

• Mass spectrometry-based approaches:

  • Identification of phosphorylation sites via LC-MS/MS

  • Label-free quantification of phosphorylation stoichiometry

  • Phospho-enrichment techniques (TiO₂, IMAC) to enhance detection sensitivity

  • Multiple reaction monitoring (MRM) for targeted quantification

For pig ARPP19, these methodologies would require adaptation with species-specific considerations, particularly for antibody development and validation.

How can the interaction between ARPP19 and PP2A be quantitatively assessed?

The interaction between ARPP19 and PP2A is critical for understanding cell cycle regulation mechanisms. Several complementary methods can be employed for rigorous quantitative assessment:

• Co-immunoprecipitation (Co-IP) with quantitative readout:

  • Immunoprecipitation of ARPP19 followed by immunoblotting for PP2A subunits

  • Quantification of binding efficiency under different phosphorylation states

  • Comparison of wild-type vs. mutant ARPP19 binding capacities

  • Controls including phosphatase treatment and competing peptides

• In vitro binding assays:

  • GST-pulldown assays using recombinant ARPP19 and purified PP2A

  • Surface plasmon resonance (SPR) to determine binding kinetics and affinity constants

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Fluorescence-based interaction assays (FRET, FP) for real-time monitoring

• Functional PP2A inhibition assays:

  • Measurement of PP2A activity using artificial substrates (e.g., pNPP, DiFMUP)

  • Quantification of PP2A-specific substrate dephosphorylation

  • Dose-response curves with varying concentrations of ARPP19

  • Comparison of differentially phosphorylated ARPP19 forms

• Cellular assays:

  • Transfection of wild-type or mutant ARPP19 into cells

  • Assessment of downstream PP2A substrate phosphorylation

  • Measurement of cell cycle progression markers (Cdk1 activation, Y15 dephosphorylation)

  • Correlation between ARPP19-PP2A binding and functional outcomes

These methodological approaches provide complementary data on both the physical interaction and functional consequences of ARPP19-PP2A binding, essential for understanding the molecular mechanisms involved in cell cycle regulation.

How does ARPP19 integrate into signaling networks to regulate the cell cycle?

ARPP19 functions as a critical integration point within signaling networks that regulate cell cycle progression. Its position at the intersection of multiple regulatory pathways enables precise control of mitotic and meiotic events:

• Integration of PKA and Greatwall kinase signaling pathways:

  • ARPP19 receives inputs from both PKA (S109/S107 phosphorylation) and Greatwall (S67/S62 phosphorylation)

  • In Xenopus oocytes, PKA activity maintains prophase arrest through ARPP19 phosphorylation at S109

  • Progesterone stimulation reduces PKA activity, leading to partial S109 dephosphorylation (49% reduction)

  • This dephosphorylation permits formation of threshold active Cdk1 levels

  • Cdk1 activation triggers the MPF autoamplification loop including Greatwall activation

  • Greatwall phosphorylates ARPP19 at S67, converting it to a potent PP2A-B55 inhibitor

• Regulation of the kinase-phosphatase balance:

  • ARPP19 maintains the critical balance between Cdk1 kinase and PP2A phosphatase activities

  • When phosphorylated at S67/S62, ARPP19 inhibits PP2A-B55, allowing Cdk1 substrates to remain phosphorylated

  • This inhibition is essential for M-phase entry and maintenance

  • The temporal pattern of protein dephosphorylation during mitotic progression depends on ARPP19-mediated PP2A-B55 inhibition

• Differential roles compared to ENSA paralog:

  • Despite biochemical similarities, ARPP19 and ENSA paralogs display distinct functions in vivo

  • ARPP19, but not ENSA, is essential for mouse embryogenesis and early development

  • ARPP19 knockout is embryonic lethal, with 100% of ARPP19 Δ/Δ embryos showing severe abnormalities by E8.5

  • ARPP19 ablation dramatically decreases MEF viability

  • ENSA cannot compensate for ARPP19 loss in mitotic division, despite their shared biochemical mechanism

  • ARPP19 knockout doesn't perturb S-phase, unlike ENSA ablation

This complex integration enables ARPP19 to function as a molecular switch that coordinates cell cycle progression through precise temporal regulation of PP2A activity in response to upstream signals.

What are the molecular mechanisms governing the dual roles of ARPP19 in meiosis versus mitosis?

ARPP19 exhibits sophisticated molecular switching mechanisms that enable its distinct but related functions in meiotic and mitotic processes:

Meiotic Regulation (based on Xenopus oocyte model)

• Prophase arrest maintenance:

  • ARPP19 phosphorylation at S109 by PKA is essential for maintaining prophase arrest

  • Phosphorylated ARPP19 serves as a molecular lock preventing meiotic progression

  • High PKA activity in prophase-arrested oocytes maintains this phosphorylation

• Meiotic resumption mechanism:

  • Progesterone stimulation decreases PKA activity, leading to partial (~49%) dephosphorylation of ARPP19 at S109

  • The extent of dephosphorylation correlates with progesterone concentration

  • S109 dephosphorylation permits formation of threshold active Cdk1 levels

  • Active Cdk1 initiates the MPF autoamplification loop independently of protein synthesis and PKA activity

• Transition to Greatwall-dependent regulation:

  • Once the MPF autoamplification loop is initiated, ARPP19 is phosphorylated by Greatwall at S67

  • S67-phosphorylated ARPP19 becomes a potent PP2A-B55 inhibitor

  • Intriguingly, ARPP19 is rephosphorylated at S109 during GVBD, but this no longer inhibits meiotic progression

  • This suggests a one-way molecular switch where S67 phosphorylation overrides S109 phosphorylation effects

Mitotic Regulation

• Essential role in embryonic development:

  • ARPP19 knockout in mice is embryonic lethal

  • 100% of ARPP19 Δ/Δ embryos show severe abnormalities by E8.5

  • Most knockout embryos fail to undergo gastrulation

• Mitotic progression control:

  • ARPP19 depletion in MEFs significantly reduces cell viability

  • Knockout embryos show increased mitotic cells in the epidermal basal layer, suggesting mitotic arrest or delay

  • ARPP19 ablation perturbs the temporal pattern of protein dephosphorylation during mitotic progression

  • These effects cannot be compensated by ENSA, despite its shared biochemical mechanism

The dual functionality of ARPP19 in meiosis and mitosis demonstrates how a single regulatory protein can be repurposed for different cell cycle contexts through subtle changes in its regulation and interaction partners.

How do phosphorylation patterns determine ARPP19's multiple regulatory states?

ARPP19 functions as a sophisticated molecular switch through a multi-state phosphorylation code that determines its regulatory activities:

Phosphorylation-Dependent Regulatory States

Molecular Mechanisms of State Transitions

• Inhibitory to permissive transition:

  • Progesterone stimulation reduces PKA activity, leading to partial (~49%) S109 dephosphorylation

  • This dephosphorylation is concentration-dependent (higher progesterone causes greater dephosphorylation)

  • S109D phosphomimetic completely blocks meiotic resumption, confirming this site's inhibitory role

  • Dephosphorylation permits formation of threshold active Cdk1 levels

• Permissive to active transition:

  • Active Cdk1 initiates the MPF autoamplification loop

  • This activates Greatwall kinase, which phosphorylates ARPP19 at S67

  • S67A non-phosphorylatable mutant blocks meiotic resumption

  • The S67 phosphorylation converts ARPP19 into a potent PP2A-B55 inhibitor

• Override mechanism:

  • Once S67 is phosphorylated, the inhibitory effect of S109 phosphorylation is overridden

  • ARPP19 is actually rephosphorylated at S109 during GVBD, reaching or exceeding the prophase level

  • This rephosphorylation occurs despite PKA inhibition, suggesting another kinase may be involved

  • S109 phosphorylation does not prevent S67 phosphorylation by Greatwall

• Mutant analysis evidence:

  • Double mutants (S109A/S67A or S109D/S67A) completely block meiotic resumption

  • Single mutant S67A blocks meiotic resumption regardless of S109 phosphorylation status

  • Once the MPF autoamplification loop is activated, S109 phosphorylation status becomes irrelevant

This multi-state regulatory system allows ARPP19 to function as a sophisticated molecular switch integrating multiple inputs and controlling critical cell cycle transitions through precise temporal regulation of PP2A activity.

What are the key experimental design considerations for studying phospho-regulation of recombinant pig ARPP19?

Studying the phospho-regulation of recombinant pig ARPP19 requires careful experimental design that addresses several critical considerations:

• Phosphorylation site identification and conservation:

  • Sequence alignment of pig ARPP19 with well-characterized orthologs (human, Xenopus)

  • Mass spectrometry analysis to confirm conservation of key phosphorylation sites (S62/S67 and S107/S109)

  • Generation of phospho-specific antibodies against pig ARPP19 phosphorylation sites

  • Development of phosphomimetic (S→D) and non-phosphorylatable (S→A) mutants

• Kinase-specific phosphorylation assays:

  • In vitro phosphorylation with purified kinases (PKA for S107, Greatwall for S62)

  • Optimization of buffer conditions (pH, salt, divalent cations) for each kinase

  • Time-course analysis to determine phosphorylation kinetics

  • Quantification using radiometric ([γ-³²P]ATP) or phospho-specific antibody detection

• Dephosphorylation dynamics assessment:

  • Measurement of site-specific dephosphorylation rates in cell extracts

  • Identification of phosphatases responsible for each site

  • Analysis of dephosphorylation in response to physiological signals

  • Comparison with dephosphorylation patterns in other species

• Functional consequence analysis:

  • PP2A-B55 binding assays with different phosphorylation states

  • PP2A inhibition assays using purified components

  • Cell-based assays with phospho-mutants to assess cell cycle effects

  • Correlation between phosphorylation status and functional outcomes

• Physiological context consideration:

  • Cell type-specific phosphorylation patterns in pig tissues

  • Hormonal regulation of ARPP19 phosphorylation in reproductive tissues

  • Species-specific differences in phosphorylation dynamics

  • Relevance to pig-specific physiological processes

Experimental Model System Selection Table:

These methodological considerations provide a framework for designing rigorous experiments to characterize the phospho-regulation of recombinant pig ARPP19 in various experimental contexts.

How can researchers address species-specific variations when extrapolating ARPP19 research findings?

When extrapolating research findings between species, researchers must implement systematic approaches to address potential species-specific variations in ARPP19 function:

• Comparative sequence analysis:

  • Conduct comprehensive sequence alignments of ARPP19 across species (human, mouse, Xenopus, pig)

  • Identify conserved domains, motifs, and phosphorylation sites

  • Quantify sequence identity/similarity percentages at key functional regions

  • Map species-specific insertions, deletions, or substitutions onto structural models

• Functional domain conservation assessment:

  • Generate chimeric ARPP19 proteins with domains from different species

  • Test phosphorylation efficiency of conserved sites by relevant kinases

  • Measure PP2A binding and inhibition capacities across orthologs

  • Compare protein stability and conformational properties using biophysical methods

• Cellular context evaluation:

  • Perform cross-species complementation experiments (e.g., pig ARPP19 in mouse knockout cells)

  • Analyze subcellular localization patterns in different species

  • Assess interaction networks using co-immunoprecipitation and proteomics

  • Compare cell cycle phenotypes upon manipulation of ARPP19 orthologs

• Regulatory network comparison:

  • Characterize expression profiles of ARPP19 interaction partners across species

  • Measure relative activities of relevant kinases (PKA, Greatwall) and phosphatases

  • Determine phosphorylation dynamics in response to common stimuli

  • Identify species-specific regulatory mechanisms

• Methodological validation across species:

  • Validate antibody cross-reactivity with multiple species' ARPP19

  • Optimize assay conditions for each species context

  • Develop species-specific tools when cross-reactivity is limited

  • Implement parallel experimental approaches in multiple species

Decision Framework for Cross-Species Extrapolation:

By systematically addressing these considerations, researchers can make informed decisions about the validity of cross-species extrapolations and identify aspects of ARPP19 biology that may be species-specific versus broadly conserved.

What are the emerging research directions for ARPP19 beyond cell cycle regulation?

While ARPP19's role in cell cycle regulation is well-established, several emerging research directions point to broader functions that merit investigation, particularly in species-specific contexts like pigs:

• Neuroscience applications:

  • ARPP19 belongs to a family of cAMP-regulated phosphoproteins important in neuronal signaling

  • Related proteins like DARPP-32 play crucial roles in dopamine signaling in the striatum

  • Investigation of ARPP19's potential roles in pig neuronal function and development

  • Exploration of species-specific neuronal expression patterns and signaling pathways

• Metabolic regulation:

  • Studies in yeast suggest ARPP19 phosphorylation by PKA and Greatwall are involved in protein synthesis regulation when cells exit quiescence

  • This indicates potential broader metabolic regulatory functions

  • Investigation of ARPP19's role in metabolic pathways in porcine tissues

  • Analysis of ARPP19 regulation in response to metabolic stress or nutritional status

• Reproductive biology:

  • ARPP19's critical role in meiosis in Xenopus oocytes suggests potential importance in mammalian reproduction

  • Investigation of ARPP19 expression and function in pig reproductive tissues

  • Analysis of potential roles in oocyte maturation, fertilization, and early embryonic development

  • Correlation with reproductive performance in agricultural contexts

• Cancer research applications:

  • The cell cycle regulatory function of ARPP19 suggests potential roles in cancer progression

  • Expression analysis in normal versus neoplastic porcine tissues

  • Investigation as a potential biomarker or therapeutic target

  • Comparative oncology applications between human and porcine cancer models

• Agricultural biotechnology:

  • ARPP19's essential role in early embryonic development suggests applications in reproductive biotechnologies

  • Potential applications in improving artificial reproductive technologies in pigs

  • Investigation as a target for modifying cell cycle regulation in biotechnological applications

  • Development of molecular tools for agricultural research based on ARPP19 biology

Methodological Approaches for Novel ARPP19 Research Directions:

These emerging directions highlight the potential for ARPP19 research to expand beyond traditional cell cycle biology into diverse fields with significant basic science and applied implications.