ABSTRACT
DNA damage triggers chromatin remodeling by mechanisms that are poorly understood. The oncogene and chromatin remodeler ALC1/CHD1L massively decompacts chromatin in vivo yet is inactive prior to DNA-damage-mediated PARP1 induction. We show that the interaction of the ALC1 macrodomain with the ATPase module mediates auto-inhibition. PARP1 activation suppresses this inhibitory interaction. Crucially, release from auto-inhibition requires a poly-ADP-ribose (PAR) binding macrodomain. We identify tri-ADP-ribose as a potent PAR-mimic and synthetic allosteric effector that abrogates ATPase-macrodomain interactions, promotes an ungated conformation, and activates the remodeler's ATPase. ALC1 fragments lacking the regulatory macrodomain relax chromatin in vivo without requiring PARP1 activation. Further, the ATPase restricts the macrodomain's interaction with PARP1 under non-DNA damage conditions. Somatic cancer mutants disrupt ALC1's auto-inhibition and activate chromatin remodeling. Our data show that the NAD+-metabolite and nucleic acid PAR triggers ALC1 to drive chromatin relaxation. Modular allostery in this oncogene tightly controls its robust, DNA-damage-dependent activation.
Subject(s)
Chromatin Assembly and Disassembly , DNA Damage , DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , Neoplasms/enzymology , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Allosteric Regulation , Binding Sites , Cell Line, Tumor , DNA Helicases/chemistry , DNA Helicases/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Enzyme Activation , Humans , Mutation , Neoplasms/genetics , Neoplasms/pathology , Nucleic Acid Conformation , Poly (ADP-Ribose) Polymerase-1/chemistry , Poly (ADP-Ribose) Polymerase-1/genetics , Poly ADP Ribosylation , Poly Adenosine Diphosphate Ribose/chemistry , Protein Binding , Structure-Activity Relationship , Time FactorsABSTRACT
DNA damage induces chemical and structural changes in our chromatin-embedded genome. In a recent issue of Nature Communications, Grundy et al. (2016) identify a role for PARP3 in the repair of single-strand breaks and reveal that PARP3 mono-ADP-ribosylates nucleosomal histone H2B.
Subject(s)
Cell Cycle Proteins/genetics , Chromatin/chemistry , DNA Repair , DNA/genetics , Histones/genetics , Poly(ADP-ribose) Polymerases/genetics , Animals , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Chickens , Chromatin/metabolism , DNA/metabolism , DNA Breaks, Single-Stranded , Gene Expression Regulation , Histones/metabolism , Humans , Poly(ADP-ribose) Polymerases/metabolism , Xenopus laevisABSTRACT
Phosphatases and kinases contribute to the regulation of protein phosphorylation homeostasis in the cell. Phosphorylation is a key post-translational modification underlying the regulation of many cellular processes. Thus, a comprehensive picture of phosphatase function and the identification of their target substrates would aid a systematic approach to a mechanistic description of cell signalling. Here we present a website designed to facilitate the retrieval of information about human protein phosphatases. To this end we developed a search engine to recover and integrate information annotated in several publicly available web resources. In addition we present a text-mining-assisted annotation effort aimed at extracting phosphatase related data reported in the scientific literature. The HuPho (human phosphatases) website can be accessed at http://hupho.uniroma2.it.
Subject(s)
Computational Biology/methods , Internet , Phosphoric Monoester Hydrolases/metabolism , Databases, Protein , Humans , Information Storage and Retrieval/methods , Phosphoric Monoester Hydrolases/chemistry , Phosphoric Monoester Hydrolases/classification , Phosphorylation , Protein Binding , Proteomics , Substrate SpecificityABSTRACT
Activating mutations in PTPN11 cause Noonan syndrome, the most common nonchromosomal disorder affecting development and growth. PTPN11 encodes SHP2, an Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase that positively modulates RAS function. Here, we characterized functionally all possible amino acid substitutions arising from single-base changes affecting codons 62 and 63 to explore the molecular mechanisms lying behind the largely invariant occurrence of the Y62D and Y63C substitutions recurring in Noonan syndrome. We provide structural and biochemical data indicating that the autoinhibitory interaction between the N-SH2 and protein-tyrosine phosphatase (PTP) domains is perturbed in both mutants as a result of an extensive structural rearrangement of the N-SH2 domain. Most mutations affecting Tyr(63) exerted an unpredicted disrupting effect on the structure of the N-SH2 phosphopeptide-binding cleft mediating the interaction of SHP2 with signaling partners. Among all the amino acid changes affecting that codon, the disease-causing mutation was the only substitution that perturbed the stability of the inactive conformation of SHP2 without severely impairing proper phosphopeptide binding of N-SH2. On the other hand, the disruptive effect of the Y62D change on the autoinhibited conformation of the protein was balanced, in part, by less efficient binding properties of the mutant. Overall, our data demonstrate that the selection-by-function mechanism acting as driving force for PTPN11 mutations affecting codons 62 and 63 implies balancing of counteracting effects operating on the allosteric control of the function of SHP2.
Subject(s)
Noonan Syndrome/enzymology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , src Homology Domains , Amino Acid Sequence , Humans , Models, Molecular , Molecular Dynamics Simulation , Molecular Sequence Data , Mutation , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 11/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 11/geneticsABSTRACT
Density-enhanced phosphatase-1 (DEP-1) is a trans-membrane receptor protein-tyrosine phosphatase that plays a recognized prominent role as a tumor suppressor. However, the mechanistic details underlying its function are poorly understood because its primary physiological substrate(s) have not been firmly established. To shed light on the mechanisms underlying the anti-proliferative role of this phosphatase, we set out to identify new DEP-1 substrates by a novel approach based on screening of high density peptide arrays. The results of the array experiment were combined with a bioinformatics filter to identify eight potential DEP-1 targets among the proteins annotated in the MAPK pathway. In this study we show that one of these potential targets, the ERK1/2, is indeed a direct DEP-1 substrate in vivo. Pulldown and in vitro dephosphorylation assays confirmed our prediction and demonstrated an overall specificity of DEP-1 in targeting the phosphorylated tyrosine 204 of ERK1/2. After epidermal growth factor stimulation, the phosphorylation of the activation loop of ERK1/2 can be modulated by changing the concentration of DEP-1, without affecting the activity of the upstream kinase MEK. In addition, we show that DEP-1 contains a KIM-like motif to recruit ERK1/2 proteins by a docking mechanism mediated by the common docking domain in ERK1/2. ERK proteins that are mutated in the conserved docking domain become insensitive to DEP-1 de-phosphorylation. Overall this study provides novel insights into the anti-proliferative role of this phosphatase and proposes a new mechanism that may also be relevant for the regulation of density-dependent growth inhibition.