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1.  Aurora B-dependent phosphorylation of Ataxin-10 promotes the interaction between Ataxin-10 and Plk1 in cytokinesis 
Scientific Reports  2015;5:8360.
Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurologic disorder caused by ATTCT expansion in the ATXN10 gene. Previous investigations have identified that depletion of Ataxin-10, the gene product, leads to cellular apoptosis and cytokinesis failure. Herein we identify the mitotic kinase Aurora B as an Ataxin-10 interacting partner. Aurora B interacts with and phosphorylates Ataxin-10 at S12, as evidenced by in vitro kinase and mass spectrometry analysis. Both endogenous and S12-phosphorylated Ataxin-10 localizes to the midbody during cytokinesis, and cytokinetic defects induced by inhibition of ATXN10 expression is not rescued by the S12A mutant. Inhibition of Aurora B or expression of the S12A mutant renders reduced interaction between Ataxin-10 and polo-like kinase 1 (Plk1), a kinase previously identified to regulate Ataxin-10 in cytokinesis. Taken together, we propose a model that Aurora B phosphorylates Ataxin-10 at S12 to promote the interaction between Ataxin-10 and Plk1 in cytokinesis. These findings identify an Aurora B-dependent mechanism that implicates Ataxin-10 in cytokinesis.
PMCID: PMC4322367  PMID: 25666058
2.  HERC2/USP20 coordinates CHK1 activation by modulating CLASPIN stability 
Nucleic Acids Research  2014;42(21):13074-13081.
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.
PMCID: PMC4245974  PMID: 25326330
3.  The Pou5f1 distal enhancer is sufficient to drive Pou5f1 promoter-EGFP expression in embryonic stem cells 
The POU5F1 transcription factor is the gatekeeper of the pluripotent state in mammals. It is essential for epigenetic reprogramming events and also for germ cell viability. The Pou5f1 gene’s expression is tightly controlled during embryogenesis, but its regulatory regions are not fully deciphered. The GOF18ΔPE-EGFP transgene, harboring the enhanced green fluorescence protein reporter gene inserted into an 17- kilobase long mouse Pou5f1 genomic sequence, has been widely used to visualize pluripotent embryonic cells and primordial germ cells in the mouse and other mammalian species. This construct includes the Pou5f1 promoter under the control of the distal enhancer and also includes the Pou5f1 gene body and flanking sequences. In search of the essential regulatory regions of Pou5f1 we generated four shorter forms of this construct. We found that the shortest form, containing the Pou5f1 promoter and distal enhancer but lacking the gene body and upstream flanking sequences, correctly expressed EGFP in transiently transformed undifferentiated ES cells, correctly switched it off upon ES cell differentiation, and correctly kept it silenced in differentiated Hep3B cells. Similarly to the original GOF18ΔPE-EGFP, this shortest form was expressed in the fetal mouse gonad. Our data suggest that the Pou5f1 distal enhancer and proximal promoter may be sufficient to specify transgene expression in pluripotent cells.
PMCID: PMC4017653  PMID: 24395560
Pou5f1; Oct4; EGFP transgene; pluripotency
4.  Protein phosphatase PP6 is required for homology-directed repair of DNA double-strand breaks 
Cell Cycle  2011;10(9):1411-1419.
DNA double-strand breaks (DSBs) are among the most lethal lesions associated with genome stability, which, when destabilized, predisposes organs to cancers. DSBs are primarily fixed either with little fidelity by non-homologous end joining (NHEJ) repair or with high fidelity by homology-directed repair (HDR). The phosphorylated form of H2AX on serine 139 (γ-H2AX) is a marker of DSBs. In this study, we explored if the protein phosphatase PP6 is involved in DSB repair by depletion of its expression in human cancer cell lines, and determined PP6 expression in human breast cancer tissues by immunohistochemistry staining. We found that bacterially produced PP6c (the catalytic subunit of PP6)-containing heterotrimeric combinations exhibit phosphatase activity against γ-H2AX in the in vitro phosphatase assays. Depletion of PP6c or PP6R2 led to persistent high levels of γ-H2AX after DNA damage and a defective HDR. Chromatin immunoprecipitation assays demonstrated that PP6c was recruited to the region adjacent to the DSB sites. Expression of PP6c, PP6R2 and PP6R3 in human breast tumors was significantly lower than those in benign breast diseases. Taken together, our results suggest that γ-H2AX is a physiological substrate of PP6 and PP6 is required for HDR and its expression may harbor a protective role during the development of breast cancer.
PMCID: PMC3117043  PMID: 21451261
protein phosphatase; PP6; γ-H2AX; DNA double-strand break; homology-directed repair
5.  Structural mechanism of the phosphorylation-dependent dimerization of the MDC1 forkhead-associated domain 
Nucleic Acids Research  2012;40(9):3898-3912.
MDC1 is a key mediator of the DNA-damage response in mammals with several phosphorylation-dependent protein interaction domains. The function of its N-terminal forkhead-associated (FHA) domain remains elusive. Here, we show with structural, biochemical and cellular data that the FHA domain mediates phosphorylation-dependent dimerization of MDC1 in response to DNA damage. Crystal structures of the FHA domain reveal a face-to-face dimer with pseudo-dyad symmetry. We found that the FHA domain recognizes phosphothreonine 4 (pT4) at the N-terminus of MDC1 and determined its crystal structure in complex with a pT4 peptide. Biochemical analysis further revealed that in the dimer, the FHA domain binds in trans to pT4 from the other subunit, which greatly stabilizes the otherwise unstable dimer. We show that T4 is phosphorylated primarily by ATM upon DNA damage. MDC1 mutants with the FHA domain deleted or impaired in its ability to dimerize formed fewer foci at DNA-damage sites, but the localization defect was largely rescued by an artificial dimerization module, suggesting that dimerization is the primary function of the MDC1 FHA domain. Our results suggest a novel mechanism for the regulation of MDC1 function through T4 phosphorylation and FHA-mediated dimerization.
PMCID: PMC3351156  PMID: 22234877
6.  A PP4-phosphatase complex dephosphorylates γ-H2AX generated during DNA replication 
Molecular cell  2008;31(1):33-46.
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.
PMCID: PMC3242369  PMID: 18614045
7.  Human RIF1 encodes an anti-apoptotic factor required for DNA repair 
Carcinogenesis  2009;30(8):1314-1319.
Human Rap1-interacting protein 1 (RIF1) contributes to the ataxia telangiectasia, mutated-mediated DNA damage response against the dexterous effect of DNA lesions and plays a critical role in the S-phase checkpoint. However, the molecular mechanisms by which human RIF1 conquers DNA aberrations remain largely unknown. We here showed that inhibition of RIF1 expression by small interfering RNA led to defective homologous recombination-mediated DNA double-strand break repair and sensitized cancer cells to camptothecin or staurosporine treatment. RIF1 underwent caspase-dependent cleavage upon apoptosis. We further found that RIF1 was highly expressed in human breast tumors, and its expression status was positively correlated with differentiation degrees of invasive ductal carcinoma of the breast. Our results suggest that RIF1 encodes an anti-apoptotic factor required for DNA repair and is a potential target for cancer treatment.
PMCID: PMC2718077  PMID: 19483192
8.  Genetic variants of Nogo-66 Receptor with possible association to schizophrenia block myelin inhibition of axon growth 
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 as a 22q11 candidate gene for schizophrenia susceptibility since 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 non-conservative coding sequence variants. Neuronal cultures demonstrate that 4 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.
PMCID: PMC2892845  PMID: 19052207
Schizophrenia; Myelin; Genetic linkage; Axonal growth; Nogo; Plasticity
9.  CDK5RAP2 is required for spindle checkpoint function 
Cell Cycle  2009;8(8):1206-1216.
The combination of paclitaxel and doxorubicin is among the most successful chemotherapy regimens in cancer treatment. CDK5RAP2, when mutated, causes primary microcephaly. We show here that inhibition of CDK5RAP2 expression causes chromosome mis-segregation, fails to maintain the spindle checkpoint, and is associated with reduced expression of the spindle checkpoint proteins BUBR1 and MAD2 and an increase in chromatin-associated CDC20. CDK5RAP2 resides on the BUBR1 and MAD2 promoters and regulates their transcription. Furthermore, CDK5RAP2-knockdown cells have increased resistance to paclitaxel and doxorubicin, and this resistance is partially rescued upon restoration of CDK5RAP2 expression. Cancer cells cultured in the presence of paclitaxel or doxorubicin exhibit dramatically decreased CDK5RAP2 levels. These results suggest that CDK5RAP2 is required for spindle checkpoint function and is a common target in paclitaxel and doxorubicin resistance.
PMCID: PMC3820842  PMID: 19282672
CDK5RAP2; primary microcephaly; spindle checkpoint; BUBR1; MAD2; drug resistance; paclitaxel; doxorubicin
10.  Nogo-A Interacts with the Nogo-66 Receptor through Multiple Sites to Create an Isoform-Selective Subnanomolar Agonist 
Nogo is a myelin-derived protein that limits axonal regeneration after CNS injury. A short hydrophilic Nogo-66 loop between two hydrophobic domains of Nogo binds to a Nogo-66 receptor (NgR) to inhibit axonal outgrowth. Inhibition of axon outgrowth and cell spreading by a second Nogo domain, termed Amino-Nogo-A, is thought to be mediated by a distinct receptor complex. Here, we define a novel Nogo-A-specific domain in Amino-Nogo that binds to NgR with nanomolar affinity. This second domain of 24 amino acids does not alter cell spreading or axonal outgrowth. Fusion of the two NgR-binding Nogo-A domains creates a ligand with substantially enhanced affinity for NgR and converts a NgR antagonist peptide to an agonist. Thus, NgR activation by Nogo-A involves multiple sites of interaction between Nogo-A and NgR.
PMCID: PMC2855126  PMID: 15930377
myelin; axon; regeneration; spinal cord injury; outgrowth inhibitor; cell spreading
The Journal of biological chemistry  2006;282(8):5715-5725.
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 (CNS) injury. Within Nogo-A two separate domains are known interact with NgR1. NgR1 is the founding member of three-member NgR family, whereas Nogo-A (RTN4-A) belongs to a four-member reticulon family. Here, we systematically map the interactions between these superfamilies, demonstrating novel nanomolar interactions of RTN2 and RTN3 with NgR1. Since RTN3 is expressed in the 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 confirm here that MAG binds to NgR2, but not to NgR3. Unexpectedly, we found that OMgp interacts with MAG with a higher affinity than its affinity to 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 NgR1 leucine-rich repeat (LRR) domain and surrounded by differentially utilized residues. This detailed knowledge of the molecular interactions between NgR1 and its ligands is imperative when assessing the options to develop NgR1-based therapeutics for CNS injuries.
PMCID: PMC2852886  PMID: 17189258

Results 1-11 (11)