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B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability

Gunnar Houge et al. J Clin Invest. .

Abstract

Here we report inherited dysregulation of protein phosphatase activity as a cause of intellectual disability (ID). De novo missense mutations in 2 subunits of serine/threonine (Ser/Thr) protein phosphatase 2A (PP2A) were identified in 16 individuals with mild to severe ID, long-lasting hypotonia, epileptic susceptibility, frontal bossing, mild hypertelorism, and downslanting palpebral fissures. PP2A comprises catalytic (C), scaffolding (A), and regulatory (B) subunits that determine subcellular anchoring, substrate specificity, and physiological function. Ten patients had mutations within a highly conserved acidic loop of the PPP2R5D-encoded B56δ regulatory subunit, with the same E198K mutation present in 6 individuals. Five patients had mutations in the PPP2R1A-encoded scaffolding Aα subunit, with the same R182W mutation in 3 individuals. Some Aα cases presented with large ventricles, causing macrocephaly and hydrocephalus suspicion, and all cases exhibited partial or complete corpus callosum agenesis. Functional evaluation revealed that mutant A and B subunits were stable and uncoupled from phosphatase activity. Mutant B56δ was A and C binding-deficient, while mutant Aα subunits bound B56δ well but were unable to bind C or bound a catalytically impaired C, suggesting a dominant-negative effect where mutant subunits hinder dephosphorylation of B56δ-anchored substrates. Moreover, mutant subunit overexpression resulted in hyperphosphorylation of GSK3β, a B56δ-regulated substrate. This effect was in line with clinical observations, supporting a correlation between the ID degree and biochemical disturbance.

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Figures

Figure 6
Figure 6. Mechanistic model.
Top panels model the physiological situation in which the B-type subunit dictates subcellular targeting, substrate specificity, and substrate dephosphorylation by the C subunit. Conceivably, certain B subunits (like B56δ) or A-B dimers could dock to substrates independent of holoenzymes (alternatives labeled with question marks). Bottom panel displays the pathological situation in which (i) a B subunit mutation hindering interaction of the A and C subunit, (ii) an A subunit mutation hindering interaction of the C subunit but not the B subunit, or (iii) an A subunit mutation resulting in the incorporation of a catalytically impaired C subunit into the trimeric complex result in protection of B subunit–directed substrate dephosphorylation by a competition-based, dominant-negative mechanism. The common feature in all ID cases described here is hindered access of the PP2A activity to B56δ-specific PP2A substrates. S, substrate; P, phosphate.
Figure 5
Figure 5. Increased phosphorylation of GSK-3β Ser9 upon expression of B56δ-E198K or Aα-R182W.
GFP-tagged WT B56δ, E198K mutant B56δ, or GFP alone (–) (left); or HA-tagged WT Aα, R182W mutant Aα, or HA alone (–) (right) were expressed in HEK293 cells, and the effect on GSK-3β phosphorylation determined by immunoblotting (IB) with the indicated antibodies. Total GSK-3β and phospho-Ser9 GSK-3β signals were determined on different blots, which were both developed for vinculin to correct for loading differences. In cells expressing the ID-associated mutants, higher phospho/total GSK-3β ratios were found, relative to cells transfected with empty expression vector (in which case, this ratio was set to 100%) or cells expressing WT subunits. Mean values and one representative image of 3 independent experiments are shown (1-way multiple-comparisons ANOVA; *P < 0.05, **P < 0.01).
Figure 4
Figure 4. Analysis of Aα mutant complexes.
(A) Endogenous B subunit binding assays: HA-tagged WT Aα, Aα mutants (P179L, R182W, and R258H), or an empty HA-vector (–) were transfected into HEK293 cells. Following anti-HA immunoprecipitation, presence of endogenous B56δ and B55α subunits in the immunoprecipitates was examined by immunoblotting (IB). (B) Formation of B56δ-(mutant Aα)-C complexes: HEK293 cells stably expressing EGFP-TEV-B56δ were transfected with HA-Aα, HA-Aα mutants, or empty HA-vector (–). Following EGFP-trapping and cleavage of the trapped complexes with TEV protease, the eluates were subjected to HA immunoprecipitation and the immunoprecipitates analyzed by IB with anti-HA, anti-C, and anti-B56δ antibodies. After quantification of the band intensities (ImageJ), the ratios between C and HA signals were determined and calculated relative to WT Aα control (set to 100% in each IP-on-IP experiment). Mean values and a representative image of 4 independent experiments are shown (1-way multiple-comparisons ANOVA; *P < 0.05). (C) PP2A activity measurements in B56δ-(mutant Aα)-C complexes. The pmol number of released phosphate from K-R-pT-I-R-R phosphopeptide (350 μM) was determined by Malachite Green for each B56δ-(mutant Aα)-C complex (retrieved as in B). To obtain specific C activities, this number was divided by the amount of C in the respective samples, as determined by IB and following quantification by ImageJ software. All specific activities were eventually recalculated relative to WT Aα control (set to 100%). Mean values and one representative image of 3 independent experiments are shown (1-way multiple-comparisons ANOVA; **P < 0.01).
Figure 3
Figure 3. Binding of mutant and WT Aα to C and B subunits.
(A) PP2A-C subunit binding assays: HA-tagged WT Aα, 3 ID-associated Aα mutants (P179L, R182W, and R258H), or an empty HA-vector (–) were transfected into HEK293 cells. Following anti-HA immunoprecipitation, the presence of endogenous C subunit in the immunoprecipitates was examined by immunoblotting (IB). After quantification of the band intensities with ImageJ software, the ratios between HA and C signals were determined and calculated relative to WT Aα control. Mean values and a representative image of 3 independent experiments are shown (1-way multiple-comparisons ANOVA, **P < 0.01). (B) PP2A B subunit binding assays: Several GST-tagged B subunits, belonging to 3 different families (B55 or B, B56 or B’, and B’’) or GST alone (–) were coexpressed in HEK293 cells with HA-tagged WT Aα, or ID-associated Aα-P179L, R182W, and R258H mutants. The presence of HA-Aα (WT or mutant) in the complete lysates and the isolated GST pulldown complexes was determined by IB. After quantification of the band intensities with ImageJ software, the ratios between GST and HA signals were determined and calculated relative to WT Aα control (which were set to 100% for each B-type subunit pulldown). Mean values and a representative image of 3 independent experiments are shown (1-way multiple-comparisons ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001).
Figure 2
Figure 2. Binding of mutant and WT B56δ to the A or C subunits.
(A) Model of the highly conserved B56 acidic loop, harboring 4 ID-associated B56δ missense mutations, based on crystallographic data from PP2A-B56γ (PDB code: 2IAE)(5). The corresponding residues in B56γ (highlighted in white) are displayed with amino acid numbering according to B56δ. Note that the E198 residue directly contacts the C subunit. Color code: C subunit, blue; A subunit, yellow; B56γ, red. The structure was analyzed and visualized with Molsoft MolBrowser 3.7. (B) Cellular binding assays of ID-associated B56δ mutants and endogenous A and C subunits. EGFP-tagged WT B56δ, 5 ID-associated B56δ mutants (P53S, E198K, E200K, P201R, and W207R), or EGFP alone (–) were ectopically expressed in HEK293 cells. Following EGFP trapping, the presence of endogenous A and C subunits in the trapped complexes was examined by immunoblotting (IB). After quantification of the band intensities with ImageJ software, the ratios between EGFP and C signals — and between EGFP and A signals — were determined and calculated relative to B56δ WT control. Mean values and a representative image of 4 independent experiments are shown (1-way multiple-comparisons ANOVA; *P < 0.05, **P < 0.01).
Figure 1
Figure 1. Facial photographs.
The display shows the facial features of the PPP2R5D cases that consented to facial photographs being shown and the PPP2R1A cases.
See this image and copyright information in PMC

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References

    1. Janssens V, Goris J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J. 2001;353(pt 3):417–439. - PMC - PubMed
    1. Yang J, Phiel C. Functions of B56-containing PP2As in major developmental and cancer signaling pathways. Life Sci. 2010;87(23):659–666. - PMC - PubMed
    1. Deciphering Developmental Disorders Study Large-scale discovery of novel genetic causes of developmental disorders. Nature. 2015;519(7542):223–228. - PMC - PubMed
    1. Xu Y, et al. Structure of the protein phosphatase 2A holoenzyme. Cell. 2006;127(6):1239–1251. doi: 10.1016/j.cell.2006.11.033. - DOI - PubMed
    1. Cho US, Xu W. Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme. Nature. 2007;445(7123):53–57. doi: 10.1038/nature05351. - DOI - PubMed

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