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

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

  • J Biol Chem. 2021 Jan-Jun;296:100313. doi: 10.1016/j.jbc.2021.100313.
Cinta M Papke 1 Kali A Smolen 2 Mark R Swingle 1 Lauren Cressey 2 Richard A Heng 1 Mourad Toporsian 1 Liyong Deng 3 Jacob Hagen 3 Yufeng Shen 4 Wendy K Chung 5 Arminja N Kettenbach 6 Richard E Honkanen 7
Affiliations

Affiliations

  • 1 Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, Alabama, USA.
  • 2 Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA; Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire, USA.
  • 3 Department of Pediatrics, Columbia University Irving Medical Center, New York, New York, USA.
  • 4 Department of Systems Biology, Columbia University Irving Medical Center, New York, New York, USA.
  • 5 Department of Pediatrics, Columbia University Irving Medical Center, New York, New York, USA; Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, New York, USA; Department of Medicine, Columbia University Medical Center, New York, New York, USA.
  • 6 Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA; Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, New Hampshire, USA. Electronic address: Arminja.N.Kettenbach@dartmouth.edu.
  • 7 Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, Alabama, USA. Electronic address: rhonkanen@southalabama.edu.
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.

Keywords

AKT-mTOR; Jordan’s syndrome; PP2A; PPP2R5D; PPP2R5D-intellectual disability; PPP2R5D-related neurodevelopmental disorder; phosphatase.

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