Summary The histone H2A variant H2AX is rapidly phosphorylated in response to DNA double-stranded breaks to produce γ-H2AX. γ-H2AX stabilizes cell cycle checkpoint proteins and DNA repair factors at the break site. We previously found that the protein phosphatase PP2A is required to resolve γ-H2AX foci and complete DNA repair after exogenous DNA damage. Here we describe a three-protein PP4 phosphatase complex in mammalian cells, containing PP4C, PP4R2 and PP4R3β, that specifically dephosphorylates ATR-mediated γ-H2AX generated during DNA replication. PP4 efficiently dephosphorylates γ-H2AX within mononucleosomes in vitro. The effect of PP4 on γ-H2AX is independent of ATR and checkpoint kinase activity. When the PP4 complex is silenced, repair of DNA replication mediated breaks is inefficient, and cells are hypersensitive to DNA replication inhibitors, but not radiomimetic drugs. Therefore γ-H2AX elimination at DNA damage foci is required for DNA damage repair, but accomplishing this task involves distinct phosphatases with potentially overlapping roles.
In schizophrenia, genetic predisposition has been linked to chromosome 22q11 and myelin-specific genes are misexpressed in schizophrenia. Nogo-66 receptor 1 (NGR or RTN4R) has been considered to be a 22q11 candidate gene for schizophrenia susceptibility because it encodes an axonal protein that mediates myelin inhibition of axonal sprouting. Confirming previous studies, we found that variation at the NGR locus is associated with schizophrenia in a Caucasian case-control analysis, and this association is not attributed to population stratification. Within a limited set of schizophrenia-derived DNA samples, we identified several rare NGR nonconservative coding sequence variants. Neuronal cultures demonstrate that four different schizophrenia-derived NgR1 variants fail to transduce myelin signals into axon inhibition, and function as dominant negatives to disrupt endogenous NgR1. This provides the first evidence that certain disease-derived human NgR1 variants are dysfunctional proteins in vitro. Mice lacking NgR1 protein exhibit reduced working memory function, consistent with a potential endophenotype of schizophrenia. For a restricted subset of individuals diagnosed with schizophrenia, the expression of dysfunctional NGR variants may contribute to increased disease risk.
Nogo, MAG, and OMgp are myelin-associated proteins that bind to a neuronal Nogo-66 receptor (NgR/NgR1) to limit axonal regeneration after central nervous system injury. Within Nogo-A, two separate domains are known interact with NgR1. NgR1 is the founding member of the three-member NgR family, whereas Nogo-A (RTN4A) belongs to a fourmember reticulon family. Here, we systematically mapped the interactions between these superfamilies, demonstrating novel nanomolar interactions of RTN2 and RTN3 with NgR1. Because RTN3 is expressed in spinal cord white matter, it may have a role in myelin inhibition of axonal growth. Further analysis of the Nogo-A and NgR1 interactions revealed a novel third interaction site between the proteins, suggesting a trivalent Nogo-A interaction with NgR1. We also confirmed here that MAG binds to NgR2, but not to NgR3. Unexpectedly, we found that OMgp interacts with MAG with a higher affinity compared with NgR1. To better define how these multiple structurally distinct ligands bind to NgR1, we examined a series of Ala-substituted NgR1 mutants for ligand binding activity. We found that the core of the binding domain is centered in the middle of the concave surface of the NgR1 leucine-rich repeat domain and surrounded by differentially utilized residues. This detailed knowledge of the molecular interactions between NgR1 and its ligands is imperative when assessing options for development of NgR1-based therapeutics for central nervous system injuries.
CLASPIN is an essential mediator in the DNA replication checkpoint, responsible for ATR (ataxia telangiectasia and Rad3-related protein)-dependent activation of CHK1 (checkpoint kinase 1). Here we found a dynamic signaling pathway that regulates CLASPIN turn over. Under unperturbed conditions, the E3 ubiquitin ligase HERC2 regulates the stability of the deubiquitinating enzyme USP20 by promoting ubiquitination-mediated proteasomal degradation. Under replication stress, ATR-mediated phosphorylation of USP20 results in the disassociation of HERC2 from USP20. USP20 in turn deubiquitinates K48-linked-polyubiquitinated CLASPIN, stabilizing CLASPIN and ultimately promoting CHK1 phosphorylation and CHK1-directed checkpoint activation. Inhibition of USP20 expression promotes chromosome instability and xenograft tumor growth. Taken together, our findings demonstrated a novel function of HERC2/USP20 in coordinating CHK1 activation by modulating CLASPIN stability, which ultimately promotes genome stability and suppresses tumor growth.
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