B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability
Gunnar Houge, … , the Deciphering Developmental Disorders (DDD) study, Veerle Janssens
Gunnar Houge, … , the Deciphering Developmental Disorders (DDD) study, Veerle Janssens
Published July 13, 2015
Citation Information: J Clin Invest. 2015;125(8):3051-3062. https://doi.org/10.1172/JCI79860.
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Research Article Neuroscience

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

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.

Authors

Gunnar Houge, Dorien Haesen, Lisenka E.L.M. Vissers, Sarju Mehta, Michael J. Parker, Michael Wright, Julie Vogt, Shane McKee, John L. Tolmie, Nuno Cordeiro, Tjitske Kleefstra, Marjolein H. Willemsen, Margot R.F. Reijnders, Siren Berland, Eli Hayman, Eli Lahat, Eva H. Brilstra, Koen L.I. van Gassen, Evelien Zonneveld-Huijssoon, Charlotte I. de Bie, Alexander Hoischen, Evan E. Eichler, Rita Holdhus, Vidar M. Steen, Stein Ove Døskeland, Matthew E. Hurles, David R. FitzPatrick, the Deciphering Developmental Disorders (DDD) study, Veerle Janssens

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Figure 4

Analysis of Aα mutant complexes.

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Analysis of Aα mutant complexes. (A) Endogenous B subunit binding assays...
(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).