Recombinant Pongo abelii Serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B beta isoform (PPP2R2B)

What is the basic structure of PPP2R2B and how does it function within the PP2A complex?

PPP2R2B is one of the regulatory B subunits of the heterotrimeric protein phosphatase 2A (PP2A) complex, which catalyzes approximately half of all Ser/Thr dephosphorylations in eukaryotic cells. The protein adopts a 7-bladed β-propeller structure as revealed by AlphaFold2 structural modeling . Within the PP2A holoenzyme, PPP2R2B interacts with a scaffolding A subunit and catalytic C subunit to form a functional trimeric complex. The B regulatory subunit modulates substrate selectivity and catalytic activity, and also directs the localization of the catalytic enzyme to specific subcellular compartments . The PP2A complex incorporating PPP2R2B is particularly important in neurons, where it regulates various phosphorylation-dependent processes essential for neuronal development and function .

How are the different isoforms of PPP2R2B generated and what are their functional differences?

PPP2R2B is diversified through alternative splicing and promoter use at its N-terminus. In humans, at least five N-terminal coding variants have been annotated with lengths ranging from 442 to 509 residues . The two major isoforms are:

• Bβ1: Primarily localizes to the cytosol

• Bβ2: Contains a mitochondrial targeting sequence that directs the PP2A holoenzyme to the outer mitochondrial membrane

The Bβ2 isoform has been particularly well-characterized and plays a critical role in mitochondrial dynamics by dephosphorylating and activating dynamin-related protein 1 (Drp1), which is involved in mitochondrial fission . This isoform-specific localization is regulated by phosphorylation of Ser20-22 in the targeting sequence, where phosphorylation antagonizes mitochondrial localization and auto-dephosphorylation restores it .

What methods can be used to assess PPP2R2B interaction with other PP2A subunits?

Several complementary approaches have been demonstrated for studying PPP2R2B interactions within the PP2A complex:

• NanoBiT split luciferase complementation assay: This novel approach allows investigation of PPP2R2B association with A and C subunits in intact cells. The optimized configuration involves C-terminally LgBiT-tagged Bβ2 combined with N-terminally SmBiT-tagged Cα, co-transfected with untagged Aα for stoichiometric expression .

• Co-immunoprecipitation: FLAG-tagged Bβ2 can be used to assess binding to endogenous A and C subunits. After transfection, complexes are immunoprecipitated with anti-FLAG antibodies and then analyzed by Western blotting .

• Pulse-chase experiments: HaloTag fusion proteins can measure PPP2R2B turnover, providing indirect evidence of complex formation since monomeric PPP2R2 family regulatory subunits are rapidly degraded by the ubiquitin-proteasome pathway .

How can we effectively measure PPP2R2B-associated phosphatase activity?

A robust approach to measuring PPP2R2B-associated phosphatase activity involves:

• Generation of cell lines with inducible expression of wild-type or mutant PPP2R2B

• Isolation of PPP2R2B complexes using immunoprecipitation (e.g., with GFP nanobody-based agarose resin for GFP-tagged constructs)

• Incubation of bead-bound complexes with phospho-peptide substrates (e.g., phospho-threonine peptide RRA(pT)VA)

• Measurement of phosphatase activity using a molybdate/malachite green-based colorimetric assay

• Quantification of inorganic phosphate released over time (0-45 min) by measuring absorbance at 630 nm

This method allows direct comparison of wild-type and mutant PPP2R2B phosphatase activity, correlating with their ability to incorporate into functional PP2A complexes.

How do CAG repeat expansions in PPP2R2B lead to spinocerebellar ataxia type 12 (SCA12)?

Spinocerebellar ataxia type 12 (SCA12) is caused by expansion of a CAG repeat in the PPP2R2B gene . Research has revealed several mechanisms by which this expansion may cause disease:

• Altered gene expression: The CAG repeat functions as a cis element to up-regulate PPP2R2B expression. Studies using deletion/site-directed mutagenesis, in silico searches, and cDNA overexpression revealed that transcription factors CREB1 and SP1 bind to conserved sequences upstream of the CAG repeats to up-regulate PPP2R2B expression .

• Bidirectional transcription: The repeat region in the PPP2R2B gene locus is bidirectionally transcribed, producing both sense (CAG-containing) and antisense (CUG-containing, named PPP2R2B-AS1) transcripts. This has been demonstrated in SCA12 human induced pluripotent stem cells (iPSCs), iPSC-derived neurons, and SCA12 knock-in mouse brains .

• RNA toxicity: The expanded PPP2R2B-AS1 (expPPP2R2B-AS1) transcripts form CUG RNA foci in cells, a marker of toxic processes involving mutant RNAs .

• RAN translation: The expPPP2R2B-AS1 transcript is subject to repeat-associated non-ATG (RAN) translation in the Alanine ORF, potentially producing toxic peptides .

• Neurodegeneration: Neuropathological investigation of an autopsied SCA12 brain revealed enlarged ventricles, marked cerebral cortical atrophy, Purkinje cell loss, less-prominent cerebellar and pontine atrophy, and neuronal intranuclear ubiquitin-positive inclusions .

What is the mechanism by which de novo missense variants in PPP2R2B cause intellectual disability?

Recent research has established that monoallelic missense variants in PPP2R2B cause intellectual disability with developmental delay. Five variants (R149P, T246K, N310K, E37K, I427T) have been clinically characterized, with four confirmed as de novo . The pathogenic mechanisms include:

• Impaired PP2A holoenzyme assembly: Cell-based assays demonstrate that these variants significantly impair the ability of PPP2R2B to incorporate into the PP2A holoenzyme. The R149P variant showed the most severe defect, reducing binding to background levels in both NanoBiT complementation and co-immunoprecipitation assays .

• Accelerated protein turnover: Pulse-chase experiments showed that variants, particularly R149P, significantly accelerated PPP2R2B turnover (wild-type t₁/₂ = 9.5h vs. R149P t₁/₂ = 4.8h), indicating instability and preferential degradation .

• Reduced mitochondrial localization: The R149P variant significantly reduced Bβ2 targeting to mitochondria, impairing its function in mitochondrial dynamics .

• Decreased phosphatase activity: Purified complexes containing variant PPP2R2B showed reduced ability to dephosphorylate substrates, including the mitochondrial fission enzyme Drp1 .

These mechanisms result in a neurodevelopmental syndrome characterized by moderate to severe intellectual disability, developmental delay, seizures, microcephaly, aggression, hypotonia, and broad-based or stiff gait .

How does bidirectional transcription at the PPP2R2B locus contribute to disease pathogenesis?

Bidirectional transcription at the PPP2R2B locus produces both sense and antisense transcripts that may contribute to disease through complementary mechanisms:

• Expression pattern: The PPP2R2B antisense transcript (PPP2R2B-AS1) contains a CUG repeat (complementary to the CAG repeat on the sense strand) and has been detected in SCA12 iPSCs, iPSC-derived NGN2 neurons, and SCA12 knock-in mouse brains using strand-specific RT-PCR .

• RNA toxicity: Expanded PPP2R2B-AS1 transcripts form CUG RNA foci in cells, similar to those observed in other repeat expansion disorders like myotonic dystrophy. These foci may sequester RNA-binding proteins, disrupting normal RNA processing .

• Apoptotic effects: Transfected expanded PPP2R2B-AS1 transcripts induce apoptosis in neuroblastoma cells, an effect that appears to be mediated, at least in part, by RNA secondary structure .

• RAN translation: The expanded PPP2R2B-AS1 transcript undergoes repeat-associated non-ATG (RAN) translation in the Alanine ORF. This process is diminished by single nucleotide interruptions within the CUG repeat and by MBNL1 overexpression, suggesting potential therapeutic approaches .

The finding that both sense and antisense transcripts contribute to pathogenesis suggests that targeting both transcripts may be necessary for effective therapy in SCA12.

What are the implications of PPP2R2B variants for mitochondrial dynamics and neuronal function?

PPP2R2B, particularly the Bβ2 isoform, plays a critical role in neuronal mitochondrial dynamics:

• Mitochondrial fission regulation: Bβ2 localizes the PP2A complex to the outer mitochondrial membrane where it dephosphorylates and activates Drp1, a key mediator of mitochondrial fission .

• Isoform-specific effects: Mice with selective knockout of the Bβ2 isoform of PPP2R2B display an allele dose-dependent increase in the length of neuronal mitochondria, confirming its role in mitochondrial fission .

• Pathogenic variants: De novo missense variants in PPP2R2B impair its ability to:

  • Incorporate into functional PP2A holoenzyme complexes

  • Localize to mitochondria

  • Induce fission of neuronal mitochondria

  • Dephosphorylate Drp1

• Neurodevelopmental consequences: The disruption of mitochondrial dynamics likely contributes to the intellectual disability phenotype, as proper mitochondrial function is essential for neuronal development, synaptic plasticity, and energy production in the brain .

These findings suggest that PPP2R2B-related disorders may involve a spectrum of mechanisms from gain-of-function (in SCA12) to loss-of-function (in intellectual disability), both affecting neuronal health but manifesting as different clinical entities.

How can we assess the pathogenicity of novel PPP2R2B variants?

Assessment of novel PPP2R2B variants can be approached through a combination of computational, biochemical, and cellular methods:

• Computational prediction: AlphaMissense, a deep learning algorithm built on AlphaFold2 structure prediction, can effectively predict pathogenicity of PPP2R2B variants. The five clinically characterized variants received pathogenicity scores above 0.5, and an additional seven unreported variants were identified as potentially pathogenic .

• Evolutionary conservation: Analysis of conservation across species reveals that pathogenic variants typically affect highly conserved residues. Alignment of human PPP2R2A/B/C/D with orthologues from Drosophila and C. elegans shows that residues affected by clinically characterized missense mutations are either phylogenetically invariant (I427), highly conserved (E37, T246, N310), or moderately conserved (R149) .

• Structural impact: Mapping variants onto protein structure models helps predict their effects. For example, proline substitutions (as in R149P) tend to be pathogenic because they disrupt secondary structures .

• Functional assays: A comprehensive assessment should include:

  • PP2A holoenzyme incorporation (NanoBiT assay)

  • A/C subunit binding (co-immunoprecipitation)

  • Protein stability (pulse-chase)

  • Subcellular localization (mitochondrial targeting)

  • Phosphatase activity

The correlation between computational predictions and experimental results suggests that bioinformatic approaches can provide valuable initial screening, guiding subsequent functional validation.

What considerations should be taken when analyzing PPP2R2B expression data across different experimental models?

When analyzing PPP2R2B expression data, researchers should consider several key factors:

• Isoform specificity: PPP2R2B has multiple isoforms with distinct functions and subcellular localizations. Studies should clearly specify which isoform(s) are being examined, as results may not generalize across all isoforms .

• Cell type dependency: The CAG repeat region in PPP2R2B functions as a promoter, but its activity increases with longer repeat length and is dependent on cell type, repeat sequence, and sequences flanking the repeat. Reporter assays have demonstrated that promoter activity varies significantly between cell types .

• Bidirectional transcription: The PPP2R2B locus undergoes bidirectional transcription, producing both sense and antisense transcripts. Analysis should consider both transcripts and their potential interactions .

• Regulatory elements: Transcription factors CREB1 and SP1 bind to conserved sequences upstream of the CAG repeats to up-regulate PPP2R2B expression, while TFAP4 binds to conserved sequences downstream to down-regulate expression. These regulatory interactions may vary across models and conditions .

• Methodology considerations: For detecting antisense transcripts, strand-specific RT-PCR is essential. Standard RT-PCR may not distinguish between sense and antisense products .