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-MS3), also provides a roadmap that can be used to rapidly explore the etiologies of additional genetic disorders.
Highlights
Spectrometry (LC-MS3), provides a roadmap that can be used to rapidly explore the etiologies of additional genetic disorders
All phosphatase type 2A (PP2A)-holoenzymes act as serine/threonine protein phosphatases, and most evidence for the involvement of “PP2A” in a particular process has been derived from the use of small-molecule inhibitors including okadaic acid [14], fostriecin [15, 16], and cantharidin [17, 18], which target the catalytic subunit shared among all PP2A-holoenzymes [19, 20], or siRNA, targeting a common core protein
PP2A-phosphatases represent up to 0.2% [52] of total cellular protein, and studies with catalytic inhibitors or siRNA indicate that, as a family, PP2A phosphatases are important for the vast majority of cellular processes [53,54,55,56,57,58,59,60]
Summary
Spectrometry (LC-MS3), provides a roadmap that can be used to rapidly explore the etiologies of additional genetic disorders. Orthogonal validation of the LC-MS/MS data was obtained through western analysis, which revealed similar levels of PPP2R5D in both total cell extracts and IPs generated from all three cell lines (Fig. 1I).
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