ABSTRACT
Protein phosphatase 1 regulatory subunit 3F (PPP1R3F) is a member of the glycogen targeting subunits (GTSs), which belong to the large group of regulatory subunits of protein phosphatase 1 (PP1), a major eukaryotic serine/threonine protein phosphatase that regulates diverse cellular processes. Here, we describe the identification of hemizygous variants in PPP1R3F associated with a novel X-linked recessive neurodevelopmental disorder in 13 unrelated individuals. This disorder is characterized by developmental delay, mild intellectual disability, neurobehavioral issues such as autism spectrum disorder, seizures and other neurological findings including tone, gait and cerebellar abnormalities. PPP1R3F variants segregated with disease in affected hemizygous males that inherited the variants from their heterozygous carrier mothers. We show that PPP1R3F is predominantly expressed in brain astrocytes and localizes to the endoplasmic reticulum in cells. Glycogen content in PPP1R3F knockout astrocytoma cells appears to be more sensitive to fluxes in extracellular glucose levels than in wild-type cells, suggesting that PPP1R3F functions in maintaining steady brain glycogen levels under changing glucose conditions. We performed functional studies on nine of the identified variants and observed defects in PP1 binding, protein stability, subcellular localization and regulation of glycogen metabolism in most of them. Collectively, the genetic and molecular data indicate that deleterious variants in PPP1R3F are associated with a new X-linked disorder of glycogen metabolism, highlighting the critical role of GTSs in neurological development. This research expands our understanding of neurodevelopmental disorders and the role of PP1 in brain development and proper function.
Subject(s)
Autism Spectrum Disorder , Autistic Disorder , Intellectual Disability , Neurodevelopmental Disorders , Male , Humans , Intellectual Disability/genetics , Intellectual Disability/complications , Protein Phosphatase 1/genetics , Autism Spectrum Disorder/genetics , Autistic Disorder/genetics , Glucose , Glycogen , Neurodevelopmental Disorders/genetics , Neurodevelopmental Disorders/complicationsABSTRACT
PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tightly regulated dual-specificity phosphatase and key regulator of the PI3K/AKT/mTOR signaling pathway. PTEN phosphorylation at its carboxy-terminal tail (CTT) serine/threonine cluster negatively regulates its tumor suppressor function by inducing a stable, closed, and inactive conformation. Germline PTEN mutations predispose individuals to PTEN hamartoma tumor syndrome (PHTS), a rare inherited cancer syndrome and, intriguingly, one of the most common causes of autism spectrum disorder (ASD). However, the mechanistic details that govern phosphorylated CTT catalytic conformational dynamics in the context of PHTS-associated mutations are unknown. Here, we utilized a comparative protein structure network (PSN)-based approach to investigate PTEN CTT phosphorylation-induced conformational dynamics specific to PTEN-ASD compared to PTEN-cancer phenotypes. Results from our study show differences in structural flexibility, inter-residue contacts, and allosteric communication patterns mediated by CTT phosphorylation, differentiating PTEN-ASD and PTEN-cancer phenotypes. Further, we identified perturbations among global metapaths and community network connections within the active site and inter-domain regions, indicating the significance of these regions in transmitting information across the PSN. Together, our studies provide a mechanistic underpinning of allosteric regulation through the coupled interplay of CTT phosphorylation conformational dynamics in PTEN-ASD and PTEN-cancer mutations. Importantly, the detailed atomistic interactions and structural consequences of PTEN variants reveal potential allosteric druggable target sites as a viable and currently unexplored treatment approach for individuals with different PHTS-associated mutations.
Subject(s)
Autism Spectrum Disorder , Autistic Disorder , Neoplasms , Humans , Autism Spectrum Disorder/genetics , Autistic Disorder/genetics , Mutation , Neoplasms/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/chemistry , PTEN Phosphohydrolase/metabolismABSTRACT
The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) tumor suppressor gene encodes a tightly regulated dual-specificity phosphatase that serves as the master regulator of PI3K/AKT/mTOR signaling. The carboxy-terminal tail (CTT) is key to regulation and harbors multiple phosphorylation sites (Ser/Thr residues 380-385). CTT phosphorylation suppresses the phosphatase activity by inducing a stable, closed conformation. However, little is known about the mechanisms of phosphorylation-induced CTT-deactivation dynamics. Using explicit solvent microsecond molecular dynamics simulations, we show that CTT phosphorylation leads to a partially collapsed conformation, which alters the secondary structure of PTEN and induces long-range conformational rearrangements that encompass the active site. The active site rearrangements prevent localization of PTEN to the membrane, precluding lipid phosphatase activity. Notably, we have identified phosphorylation-induced allosteric coupling between the interdomain region and a hydrophobic site neighboring the active site in the phosphatase domain. Collectively, the results provide a mechanistic understanding of CTT phosphorylation dynamics and reveal potential druggable allosteric sites in a previously believed clinically undruggable protein.
Subject(s)
PTEN Phosphohydrolase , Phosphatidylinositol 3-Kinases , Molecular Dynamics Simulation , PTEN Phosphohydrolase/chemistry , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Protein Structure, Secondary , Signal TransductionABSTRACT
The phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene encodes a tumor suppressor phosphatase that has recently been found to be frequently mutated in patients with endometriosis, endometrial cancer and ovarian cancer. Here, we present the first computational analysis of 13 somatic missense PTEN mutations associated with these phenotypes. We found that a majority of the mutations are associated in conserved positions within the active site and are clustered within the signature motif, which contain residues that play a crucial role in loop conformation and are essential for catalysis. In silico analyses were utilized to identify the putative effects of these mutations. In addition, coarse-grained models of both wild-type (WT) PTEN and mutants were constructed using elastic network models to explore the interplay of the structural and global dynamic effects that the mutations have on the relationship between genotype and phenotype. The effects of the mutations reveal that the local structure and interactions affect polarity, protein structure stability, electrostatic surface potential, and global dynamics of the protein. Our results offer new insight into the role in which PTEN missense mutations contribute to the molecular mechanism and genotypic-phenotypic correlation of endometriosis, endometrial cancer, and ovarian cancer. Proteins 2016; 84:1625-1643. © 2016 Wiley Periodicals, Inc.