Biomedical subjects

Xiaoyang Wu

Publications and source records attributed to Xiaoyang Wu.

11 recordsLinked to original sources

PubMed2007

A novel role of the actin-nucleating Arp2/3 complex in the regulation of RNA polymerase II-dependent transcription.

It has been well documented that actin is present in the nucleus and involved in numerous nuclear functions including regulation of transcription. The actin-nucleating Arp2/3 complex is an essential, evolutionarily conserved seven-subunit protein complex that promotes actin cytoskeleton assembly in the cytoplasm upon stimulation by WASP family proteins. Our recent study indicates that the nuclear localized neural Wiskott-Aldrich syndrome protein (N-WASP) can induce de novo actin polymerization in the nucleus, and this function is important for the role of N-WASP in the regulation of RNA polymerase II-dependent transcription. Here, we have presented evidence to show that the Arp2/3 complex is also localized in the nucleus and plays an essential role in mediating nuclear actin polymerization induced by N-WASP. We have also demonstrated that the Arp2/3 complex physically associates with RNA polymerase II and is involved in the RNA polymerase II-dependent transcriptional regulation both in vivo and in vitro. Together, these data provide strong support for the hypothesis that N-WASP and the Arp2/3 complex regulate transcription, at least in part, through the regulation of nuclear actin polymerization in a manner similar to their function in the cytoplasm.

Actin-Related Protein 2-3 Complex↗

PubMed2006

Regulation of RNA-polymerase-II-dependent transcription by N-WASP and its nuclear-binding partners.

The presence of actin in the nucleus has been well established, and several studies have implicated nuclear actin in transcriptional regulation. Neuronal Wiskott-Aldrich syndrome protein (N-WASP) is a member of the WASP family of proteins; these proteins function in the cytoplasm as key regulators of cortical actin filament. Interestingly, N-WASP has also been observed in the nucleus. However, a potential nuclear function for N-WASP has not been established. Here, we report the identification of nuclear N-WASP within a large nuclear-protein complex containing PSF-NonO (polypyrimidine-tract-binding-protein-associated splicing factor-non-Pou-domain octamer-binding protein/p54(nrb)), nuclear actin and RNA polymerase II. The PSF-NonO complex is involved in the regulation of many cellular processes, such as transcription, RNA processing, DNA unwinding and repair. We demonstrate that the interaction of N-WASP with the PSF-NonO complex can couple N-WASP with RNA polymerase II to regulate transcription. We also provide evidence that the potential function of N-WASP in promoting polymerization of nuclear actins has an important role in this process. Based on these results, we propose a nuclear function for N-WASP in transcriptional regulation.

Actin Cytoskeleton↗

PubMed2006

Interaction of N-WASP with hnRNPK and its role in filopodia formation and cell spreading.

N-WASP is a member of the WASP family of proteins, which play essential roles in actin dynamics during cell adhesion and migration. hnRNPK is a member of the heterogeneous nuclear ribonucleoprotein complex, which has also been implicated in the regulation of cell spreading. Here, we identify a direct interaction between N-WASP and hnRNPK. We show that this interaction is mediated by the N-terminal WH1 domain of N-WASP and the segment of hnRNPK containing its K interaction (KI) domain. Furthermore, these two proteins are co-localized at the cell periphery in the spreading initiation center during the early stage of cell spreading. We found that co-expression of hnRNPK with N-WASP reverses the stimulation of cell spreading by N-WASP, and this effect is correlated with hnRNPK binding to N-WASP. Expression of hnRNPK does not affect subcellular localization of N-WASP protein. However, co-expression of hnRNPK with N-WASP reduced filopodia formation stimulated by N-WASP in spreading cells. Together, these results identify hnRNPK as a new negative regulator of N-WASP and suggest that hnRNPK may regulate the initial stage of cell spreading by direct association with N-WASP in the spreading initiation center.

Animals↗

PubMed2005

Mechanism of cell cycle regulation by FIP200 in human breast cancer cells.

FIP200 is a novel protein inhibitor for focal adhesion kinase (FAK), which binds to FAK directly and inhibits its kinase activity and associated cellular functions, such as cell adhesion, spreading, and motility in fibroblasts. Here we show that FIP200 inhibits G1-S phase progression, proliferation, and clonogenic survival in human breast cancer cells. Consistent with the G1 arrest induced by FIP200, we found that FIP200 increased p21 and decreased cyclin D1 protein levels in breast cancer cells. In addition, FIP200 significantly induced p21 promoter activity in MCF-7 cells and this response was abolished upon deletion of p53 binding sites within p21 promoter. Furthermore, we found that FIP200 could interact with exogenous and endogenous p53 protein and significantly increase its half-life compared with the control cells. We also found that the NH2-terminal 154 residues of FIP200 were sufficient to mediate p53 interaction and G1 arrest in cells. The increase in p53 half-life correlated with the increased phosphorylation at Ser15 and decreased proteasomal degradation via ubiquitin and Hdm2-independent mechanism. Stabilization of p53 by FIP200 could be partially reversed by NQO1 inhibitor, dicoumarol. In contrast to p53, FIP200 decreased cyclin D1 protein half-life by promoting proteasome-dependent degradation of cyclin D1. In summary, our results suggest that FIP200 increases p21 protein levels via stabilization of its upstream regulator p53 and decreases cyclin D1 protein by promoting its degradation. Both effects are critical for FIP200-induced G1 arrest and may contribute to the putative antitumor activities of FIP200 in breast cancer.

Autophagy-Related Proteins↗

PubMed2005

Identification of FIP200 interaction with the TSC1-TSC2 complex and its role in regulation of cell size control.

FIP200 (focal adhesion kinase [FAK] family interacting protein of 200 kD) is a newly identified protein that binds to the kinase domain of FAK and inhibits its kinase activity and associated cellular functions. Here, we identify an interaction between FIP200 and the TSC1-TSC2 complex through FIP200 binding to TSC1. We found that association of FIP200 with the TSC1-TSC2 complex correlated with its ability to increase cell size and up-regulate S6 kinase phosphorylation but was not involved in the regulation of cell cycle progression. Conversely, knockdown of endogenous FIP200 by RNA interference reduced S6 kinase phosphorylation and cell size, which required TSC1 but was independent of FAK. Furthermore, overexpression of FIP200 reduced TSC1-TSC2 complex formation, although knockdown of endogenous FIP200 by RNA interference did not affect TSC1-TSC2 complex formation. Lastly, we showed that FIP200 is important in nutrient stimulation-induced, but not energy- or serum-induced, S6 kinase activation. Together, these results suggest a cellular function of FIP200 in the regulation of cell size by interaction with the TSC1-TSC2 complex.

Animals↗

PubMed2005

FAK-mediated src phosphorylation of endophilin A2 inhibits endocytosis of MT1-MMP and promotes ECM degradation.

Focal adhesion kinase (FAK) is an important mediator of integrin signaling in the regulation of cell proliferation, survival, migration, and invasion. To understand how FAK contributes to cell invasion, we explored the regulation of matrix metalloproteinases (MMPs) by FAK. We found that v-Src-transformed cells activate a FAK-dependent mechanism that attenuates endocytosis of MT1-MMP. This in turn increases cell-surface expression of MT1-MMP and cellular degradation of extracellular matrix. Further, we identified an interaction between FAK's second Pro-rich motif and endophilin A2's SH3 domain. This interaction served as an autophosphorylation-dependent scaffold to allow Src phosphorylation of endophilin A2 at Tyr315. Tyr315 phosphorylation inhibited endophilin/dynamin interactions, and blockade of Tyr315 phosphorylation promoted endocytosis of MT1-MMP. Together, these results suggest a regulatory mechanism of cell invasion whereby FAK promotes cell-surface presentation of MT1-MMP by inhibiting endophilin A2-dependent endocytosis.

Animals↗

PubMed2005

Wound-healing assay.

The wound-healing assay is simple, inexpensive, and one of the earliest developed methods to study directional cell migration in vitro. This method mimics cell migration during wound healing in vivo. The basic steps involve creating a "wound" in a cell monolayer, capturing the images at the beginning and at regular intervals during cell migration to close the wound, and comparing the images to quantify the migration rate of the cells. It is particularly suitable for studies on the effects of cell-matrix and cell-cell interactions on cell migration. A variation of this method that tracks the migration of individual cells in the leading edge of the wound is also described in this chapter.

3T3 Cells↗

PubMed2004

Identification of a novel family of ankyrin repeats containing cofactors for p160 nuclear receptor coactivators.

Members of the p160 nuclear receptor coactivators interact with liganded nuclear receptors to enhance transcription of target genes. Here we identify a novel family of ankyrin repeats containing cofactors (ANCOs) that interact with the p160 coactivators. ANCO-1 binds to the conserved Per-Arnt-Sim (PAS) region of the p160 coactivators. It encodes a large nuclear protein with five ankyrin repeats, and parts of its sequences have been reported as nasopharyngeal carcinoma susceptibility protein and medulloblastoma antigen. Immunofluorescence staining reveals discrete nuclear foci of ANCO-1 that are distinct from known nuclear structures. Intriguingly, ANCO-1 also colocalizes and interacts with histone deacetylases. Transient reporter gene assay shows that ANCO-1 expression inhibits ligand-dependent transactivation by both steroid and nonsteroid nuclear receptors. Taken together, we have identified a novel family of ankyrin repeats containing cofactors that may recruit histone deacetylases to the p160 coactivators/nuclear receptor complex to inhibit ligand-dependent transactivation.

Amino Acid Sequence↗

PubMed2003

Focal adhesion kinase regulation of N-WASP subcellular localization and function.

N-WASP is a member of the WASP family of proteins that regulate actin cytoskeleton remodeling. FAK is a cytoplasmic tyrosine kinase implicated in integrin signaling during cell migration. Here we identify a direct interaction between N-WASP and FAK and show that N-WASP is phosphorylated by FAK at a conserved tyrosine residue, Tyr(256). We found that phosphorylation of Tyr(256) affected N-WASP nuclear localization, suggesting that phosphorylation of N-WASP by FAK may regulate its activity in vivo by altering its subcellular localization. We also showed that the nuclear localization of N-WASP is dependent on its being in the open conformation either after its activation by Cdc42 or the truncation of the C-terminal VCA domain. Phosphorylation of Tyr(256) of N-WASP could reduce its interaction with nuclear importin NPI-1, which might be responsible for its decreased nuclear localization. Lastly, we show that phosphorylation of Tyr(256) plays an important role in promoting cell migration. Together, these results suggest a novel regulatory mechanism of N-WASP by tyrosine phosphorylation and subcellular localization and its potential role in the regulation of cell migration.

Animals↗

PubMed2003

Chemical reactivity of Ro-26-9228, 1alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol in aqueous solution.

The degradation of Ro-26-9228, 1alpha-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol, 2, was studied in aqueous solution in the pH range of 1.17-10.56 and in alcohol solutions, at 25, 40, and 50 degrees C. The degradation of Ro-26-9228 was found to be acid catalyzed and to be independent of potassium acetate buffer concentration. Above pH 4, the reaction rate is independent of pH, with a T90 of 14.3 h at 25 degrees C in pH 7.75 buffer. 19F nuclear magnetic resonance was used to study the ratio of the vitamin (6-s-trans) to previtamin form in acetonitrile at 40 degrees C. The equilibrium percentage of previtamin and the rate of approach to equilibrium were 13.8% and 0.2 h(-1), respectively. Nuclear magnetic resonance was used to elucidate the structure of the degradation products. Novel products were formed from the elimination of the fluorine and addition of solvent to C9, with formation occurring through the previtamin form. Additional degradation products result from reaction of the side chain 25-hydroxyl and addition of solvent to C1.

2-Propanol↗

PubMed2002

Influence of ferrous sulfate on the solubility, partition coefficient, and stability of mycophenolic acid and the ester mycophenolate mofetil.

Studies were performed to (1) evaluate whether the presence of iron affected the physicochemical properties of mycophenolate mofetil (MMF) and mycophenolic acid (MPA), and (2) determine whether alteration of these properties was indicative of formation of an MMF-iron complex. The solubility, stability (chemical reactivity), and partitioning properties of MMF and MPA were evaluated over a pH range of 2-7 in the presence and absence of ferrous sulfate. In addition, the solubility and partitioning properties of MMF were assessed after the MMF drug product, CellCept capsules, was combined with an iron tablet (Fero-Gradumet, ferrous sulfate, tablets). The results of studies showed that: The solubility of MMF in the presence of ferrous sulfate was generally unaffected over a pH range of 2-7; a small increase in solubility was observed in pH 5.2 buffer solution. The solubility of MPA decreased in pH 5.2 and 7.0 buffer solutions. Both MMF and MPA were more stable in the presence of ferrous sulfate at pH 2.0; ferrous sulfate had no effect on the stability of MMF and MPA at pH 7.0. Overall, the partitioning of MMF and MPA was unaffected by the addition of ferrous sulfate. The solubility and partitioning of MMF from CellCept capsules combined with Fero-Gradumet (ferrous sulfate) tablets showed a twofold increase in aqueous solubility of MMF as well as increased concentration of MMF in both the n-octanol and aqueous phases, leading to a decrease in the octanol/water partition coefficient due to a reduction in pH of the aqueous phase. Based on these results, it was concluded that the physicochemical properties of MMF and MPA were generally not affected by the presence of ferrous sulfate. Further, the presence of ferrous sulfate did not suggest the formation of an MMF-iron complex.

Chemistry, Pharmaceutical↗