A disorder-related variant (E420K) of a PP2A-regulatory subunit (PPP2R5D) causes constitutively active AKT-mTOR signaling and uncoordinated cell growth

Abstract

Functional genomic approaches have facilitated the discovery of rare genetic disorders and improved efforts to decipher their underlying etiology. PPP2R5D-related disorder is an early childhood onset condition characterized by intellectual disability, hypotonia, autism-spectrum disorder, macrocephaly, and dysmorphic features. The disorder is caused by de novo single nucleotide changes in PPP2R5D , which generate heterozygous dominant missense variants. PPP2R5D is known to encode a B’-type (B’56δ) regulatory subunit of a PP2A-serine/threonine phosphatase. To help elucidate the molecular mechanisms altered in PPP2R5D-related disorder, we used a CRISPR-single-base editor to generate HEK-293 cells in which a single transition (c.1258G>A) was introduced into one allele, precisely recapitulating a clinically relevant E420K variant. Unbiased quantitative proteomic and phosphoproteomic analyses of endogenously expressed proteins revealed heterozygous-dominant changes in kinase/phosphatase signaling. These data combined with orthogonal validation studies revealed a previously unrecognized interaction of PPP2R5D with AKT in human cells, leading to constitutively active AKT-mTOR signaling, increased cell size, and uncoordinated cellular growth in E420K-variant cells. Rapamycin reduced cell size and dose-dependently reduced RPS6 phosphorylation in E420K-variant cells, suggesting that inhibition of mTOR1 can suppress both the observed RPS6 hyperphosphorylation and increased cell size. Together, our findings provide a deeper understanding of PPP2R5D and insight into how the E420K-variant alters signaling networks influenced by PPP2R5D. Our comprehensive approach, which combines precise genome editing, isobaric tandem mass tag labeling of peptides generated from endogenously expressed proteins, and concurrent liquid chromatography–mass spectrometry (LC-MS 3 ), also provides a roadmap that can be used to rapidly explore the etiologies of additional genetic disorders.

Neuron-specific protein network mapping of autism risk genes identifies shared biological mechanisms and disease relevant pathologies

“…4l and Supplementary Table 8 and 9), suggesting altered localization. Previous studies have described an overactive AKT pathway caused by the PPP2R5D E420K variant 79 . However, measurement of phospho-AKT levels in HEK293 cells expressing the variants revealed no difference (Extended Fig.…”

Protein phosphatase 2A – structure, function and role in neurodevelopmental disorders

“…Further functional studies have suggested that most PPP2R5D mutations cause Jordan's syndrome by dysregulating PI3K-AKT-mTOR signaling, which regulates cellular proliferation and growth (Loveday et al, 2015;Yeung et al, 2017). Furthermore, a recent study has shown that holoenzyme containing E420K PP2A-B′δ forms a complex with AKT family proteins and suppresses the dephosphorylation of AKT (Papke et al, 2021). Interestingly, mice with global KO of both PPP2R5D alleles are viable and fertile, and display no behavioral or cognitive abnormalities (Louis et al, 2011).…”

Protein Phosphatase 2A (PP2A) mutations in brain function, development, and neurologic disease

“…Of the three other reported B56δ variants, one (p.E250K) affects a conserved region immediately downstream of the SLiM-binding domain in the primary amino acid sequence that forms the intra-helical loop of HR3 and is in close contact with the acidic loop and the C subunit (Figure 3); one (p.E420K) affects the second helix of HR7, a conserved region in all B56 isoforms, of which the precise function is unclear (Figure 3); and another (p.P53S) is uniquely present in the specific N-terminal domain of the B56δ isoform (and is hence not present in any of the known B56 crystal structures). While the p.P53S variant showed normal A/C binding [84], p.E420K exhibited a mild A/C subunit binding defect and decreased phosphatase activity measured on DiFMUP (6,8-Difluoro-4-Methylumbelliferyl Phosphate) as a substrate [99]. How the p.E250K variant might be biochemically impaired remains to be determined, although an A/C-binding defect seems likely.…”

“…The first functional or downstream consequences of PPP2R5D mutation were recently described for the p.E420K variant in HEK293 cells [99]. Unbiased quantitative proteomic and phospho-proteomic analyses of heterozygous and homozygous E420K knockin (KI) cells revealed heterozygous-dominant changes in kinase/phosphatase signalling.…”

“…Particularly, AKT-mTOR signalling was constitutively activated in p.E420K-expressing cells, correlating with increased cell size and an increased representation of phospho-peptides containing the AGC kinase consensus motif (R-x-R-x-x-S/T-B, where x represents any amino acid and B represent amino acids with a hydrophobic R-group) [99]. Interestingly, addition of rapamycin, a pharmacologic inhibitor of mTOR1, reduced cell size and phospho-S6 levels, but not phospho-AKT or phospho-GSK-3β, suggesting the p.E420K variant would affect signalling upstream of mTOR1 [99]. In this respect, a clear association between AKT1 and PP2A-B56δ or the p.E420K variant was found, suggesting AKT as the major PP2A-B56δ substrate.…”