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1.
J Biol Chem ; 299(9): 105154, 2023 09.
Article in English | MEDLINE | ID: mdl-37572851

ABSTRACT

Genetic germline variants of PPP2R5D (encoding: phosphoprotein phosphatase 2 regulatory protein 5D) result in PPP2R5D-related disorder (Jordan's Syndrome), which is characterized by intellectual disability, hypotonia, seizures, macrocephaly, autism spectrum disorder, and delayed motor skill development. The disorder originates from de novo single nucleotide mutations, generating missense variants that act in a dominant manner. Pathogenic mutations altering 13 different amino acids have been identified, with the E198K variant accounting for ∼40% of reported cases. However, the generation of a heterozygous E198K variant cell line to study the molecular effects of the pathogenic mutation has been challenging. Here, we use CRISPR-PRIME genomic editing to introduce a transition (c.592G>A) in a single PPP2R5D allele in HEK293 cells, generating E198K-heterozygous lines to complement existing E420K variant lines. We generate global protein and phosphorylation profiles of WT, E198K, and E420K cell lines and find unique and shared changes between variants and WT cells in kinase- and phosphatase-controlled signaling cascades. We observed ribosomal protein S6 (RPS6) hyperphosphorylation as a shared signaling alteration, indicative of increased ribosomal protein S6-kinase activity. Treatment with rapamycin or an RPS6-kinase inhibitor (LY2584702) suppressed RPS6 phosphorylation in both, suggesting upstream activation of mTORC1/p70S6K. Intriguingly, our data suggests ERK-dependent activation of mTORC1 in both E198K and E420K variant cells, with additional AKT-mediated mTORC1 activation in the E420K variant. Thus, although upstream activation of mTORC1 differs between PPP2R5D-related disorder genotypes, inhibition of mTORC1 or RPS6 kinases warrants further investigation as potential therapeutic strategies for patients.


Subject(s)
Abnormalities, Multiple , Humans , Autism Spectrum Disorder , HEK293 Cells , Mechanistic Target of Rapamycin Complex 1/genetics , Mechanistic Target of Rapamycin Complex 1/metabolism , Phosphorylation , Protein Phosphatase 2/genetics , Protein Phosphatase 2/metabolism , Proteomics , Ribosomal Protein S6/genetics , Ribosomal Protein S6/metabolism , Abnormalities, Multiple/metabolism , Abnormalities, Multiple/pathology
2.
J Biol Chem ; 296: 100313, 2021.
Article in English | MEDLINE | ID: mdl-33482199

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.


Subject(s)
Genetic Diseases, Inborn/genetics , Genetic Predisposition to Disease , Protein Phosphatase 2/genetics , Proteomics , TOR Serine-Threonine Kinases/genetics , Autistic Disorder/genetics , Autistic Disorder/pathology , CRISPR-Cas Systems/genetics , Genetic Diseases, Inborn/pathology , Humans , Intellectual Disability/genetics , Intellectual Disability/pathology , Megalencephaly/genetics , Megalencephaly/pathology , Mutation/genetics , Polymorphism, Single Nucleotide/genetics , Proto-Oncogene Proteins c-akt/genetics
3.
Mol Cancer Ther ; 18(3): 556-566, 2019 03.
Article in English | MEDLINE | ID: mdl-30679389

ABSTRACT

LB-100 is an experimental cancer therapeutic with cytotoxic activity against cancer cells in culture and antitumor activity in animals. The first phase I trial (NCT01837667) evaluating LB-100 recently concluded that safety and efficacy parameters are favorable for further clinical testing. Although LB-100 is widely reported as a specific inhibitor of serine/threonine phosphatase 2A (PP2AC/PPP2CA:PPP2CB), we could find no experimental evidence in the published literature demonstrating the specific engagement of LB-100 with PP2A in vitro, in cultured cells, or in animals. Rather, the premise for LB-100 targeting PP2AC is derived from studies that measure phosphate released from a phosphopeptide (K-R-pT-I-R-R) or inferred from the ability of LB-100 to mimic activity previously reported to result from the inhibition of PP2AC by other means. PP2AC and PPP5C share a common catalytic mechanism. Here, we demonstrate that the phosphopeptide used to ascribe LB-100 specificity for PP2A is also a substrate for PPP5C. Inhibition assays using purified enzymes demonstrate that LB-100 is a catalytic inhibitor of both PP2AC and PPP5C. The structure of PPP5C cocrystallized with LB-100 was solved to a resolution of 1.65Å, revealing that the 7-oxabicyclo[2.2.1]heptane-2,3-dicarbonyl moiety coordinates with the metal ions and key residues that are conserved in both PP2AC and PPP5C. Cell-based studies revealed some known actions of LB-100 are mimicked by the genetic disruption of PPP5C These data demonstrate that LB-100 is a catalytic inhibitor of both PP2AC and PPP5C and suggest that the observed antitumor activity might be due to an additive effect achieved by suppressing both PP2A and PPP5C.


Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/chemistry , Neoplasms/drug therapy , Nuclear Proteins/chemistry , Phosphoprotein Phosphatases/chemistry , Piperazines/chemistry , Protein Phosphatase 2/chemistry , Amino Acid Sequence/genetics , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Catalysis , Catalytic Domain/drug effects , Cell Line, Tumor , Humans , Metals/chemistry , Methylation , Mutagenesis, Site-Directed , Neoplasms/genetics , Neoplasms/pathology , Nuclear Proteins/antagonists & inhibitors , Phosphoprotein Phosphatases/antagonists & inhibitors , Piperazines/pharmacology , Protein Phosphatase 2/antagonists & inhibitors , Protein Phosphatase 2/genetics
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