Summary Coactivators CBP and p300 play important roles in mediating the transcriptional activity of p53. Until now, however, no detailed structural information has been available on how any of the domains of p300 interact with p53. Here, we report the NMR structure of the complex of the Taz2 (C/H3) domain of p300 and the N-terminal transactivation domain of p53. In the complex, p53 forms a short α-helix and interacts with the Taz2 domain through an extended surface. Mutational analyses demonstrate the importance of hydrophobic residues for complex stabilization. Additionally, they suggest that the increased affinity of Taz2 for p531-39 phosphorylated at Thr18 is due in part to electrostatic interactions of the phosphate with neighboring arginine residues in Taz2. Thermodynamic experiments revealed the importance of hydrophobic interactions in the complex of Taz2 with p53 phosphorylated at Ser15 and Thr18.

Thin film transistors (TFTs) using polycrystalline tin oxides (SnO–SnO2) channels were formed on glass by a conventional sputtering method and subsequent annealing treatments. SnO-channel TFTs showed p-type operation with on/off current ratios of ∼102 and field-effect mobilities of 0.24 cm2 V−1 s−1. Incorporation of excess oxygen to SnO channel layers did not generate holes but did electrons, which in turn led to n-type operation. This result is explained by transformation to a local SnO2-like structure and finally to SnO2. We propose a simple method to fabricate complimentary circuits by simultaneous selective formation of p- and n-channel TFTs.

Tenascin-C (TN-C) is unique for its cell adhesion modulatory function. We have shown that TNIIIA2, a synthetic 22-mer peptide derived from TN-C, stimulated ␤1 integrinmediated cell adhesion of nonadherent and adherent cell types, by inducing activation of ␤1 integrin. The active site of TNIIIA2 appeared cryptic in the TN-C molecule but was exposed by MMP-2 processing of TN-C. The following results suggest that cell surface heparan sulfate (HS) proteoglycan (HSPG), including syndecan-4, participated in TNIIIA2-induced ␤1 integrin activation: 1) TNIIIA2 bound to cell surface HSPG via its HS chains, as examined by photoaffinity labeling; 2) heparitinase I treatment of cells abrogated ␤1 integrin activation induced by TNIIIA2; 3) syndecan-4 was isolated by affinity chromatography using TNIIIA2-immobilized beads; 4) small interfering RNA-based down-regulation of syndecan-4 expression reduced TNIIIA2-induced ␤1 integrin activation, and consequent cell adhesion to fibronectin; 5) overexpression of syndecan-4 core protein enhanced TNIIIA2-induced activation of ␤1 integrin. However, treatments that targeted the cytoplasmic region of syndecan-4, including ectopic expression of its mutant truncated with the cytoplasmic domains and treatment with protein kinase C␣ inhibitor Gö6976, did not influence the TNIIIA2 activity. These results suggest that a TNIIIA2-related matricryptic site of the TN-C molecule, exposed by MMP-2 processing, may have bound to syndecan-4 via its HS chains and then induced conformational change in ␤1 integrin necessary for its functional activation. A lateral interaction of ␤1 integrin with the extracellular region of the syndecan-4 molecule may be involved in this conformation change. Tenascin (TN)-C2 is one of the most intriguing extracellular matrix (ECM) proteins (1-3). TN-C is expressed predominantly during embryogenesis, wound healing, and neoplastic processes, in which alternative mRNA splicing within the fibronectin (FN) type III-like (FN-III) repeats can generate different TN-C isoforms (4). Multifunctional properties have been identified for TN-C, including effects on cell adhesion, migration, proliferation, survival, and differentiation. The effects of TN-C on cell adhesion are particularly complex; the TN-C substrate supports attachment of some cell types, but is nonadhesive or even repulsive for other cell types (5-7). Based on these antipodal effects on cell adhesion, TN-C is multifunctional and is therefore classified as an adhesion modulatory ECM protein, a so-called "matricellular" protein (8). Various TN-C molecule domains, especially FN-III repeats, including the alternative splicing domains, have been implicated in its function as a matricellular protein. However, their contributions to the adhesion modulatory effects of TN-C are not completely understood.Interactions of cells with the ECM are largely mediated by members of the integrin superfamily of adhesive receptors. The most unique feature of integrins is their ability to alter ligand binding and signaling activities. Because ...

A fabrication process of coplanar homojunction thin-film transistors (TFTs) is proposed for amorphous In–Ga–Zn–O (a-IGZO), which employs highly doped contact regions naturally formed by deposition of upper protection layers made of hydrogenated silicon nitride (SiNX:H). The direct deposition of SiNX:H reduced the resistivity of the semiconductive a-IGZO layer down to 6.2×10−3 Ω cm and formed a nearly ideal Ohmic contact with a low parasitic source-to-drain resistance of 34 Ω cm. Simple evaluation of field-effect mobilities (μsat) overestimated their values especially for short-channel TFTs, while the channel resistance method proved that μsat was almost constant at 9.5 cm2 V−1 s−1.

The p53 tumor suppressor protein is regulated by multiple post-translational modifications, including lysine methylation. We previously found that monomethylation of p53 at lysine 382 (p53K382me1) by the protein lysine methyltransferase (PKMT) SET8/PR-Set7 represses p53 transactivation of target genes. However, the molecular mechanism linking p53K382 monomethylation to repression is not known. Here we show in biochemical and crystallographic studies the preferential recognition of p53K382me1 by the triple malignant brain tumor (MBT) repeats of the chromatin compaction factor L3MBTL1. We demonstrate that SET8-mediated methylation of p53 at Lys-382 promotes the interaction between L3MBTL1 and p53 in cells, and the chromatin occupancy of L3MBTL1 at p53 target promoters. In the absence of DNA damage, L3MBTL1 interacts with p53K382me1 and p53-target genes are repressed, whereas depletion of L3MBTL1 results in a p53-dependent increase in p21 and PUMA transcript levels. Activation of p53 by DNA damage is coupled to a decrease in p53K382me1 levels, abrogation of the L3MBTL1-p53 interaction, and disassociation of L3MBTL1 from p53-target promoters. Together, we identify L3MBTL1 as the second known methyl-p53 effector protein, and provide a molecular explanation for the mechanism by which p53K382me1 is transduced to regulate p53 activity.The reversible and dynamic methylation of proteins on the nitrogen side-chain of lysine residues can greatly increase the signaling potential of the modified factor (1, 2). Lysine residues can accept up to three methyl groups, forming mono-, di-, and trimethylated derivatives, with a unique activity frequently being coupled to the specific methylation state. The chemical addition of methyl moieties to histones is not believed to intrinsically affect chromatin structure. Rather, the principal mechanism by which histone lysine methylation is thought to manifest functionally occurs through regulation of modular protein-protein interactions (3-5). In this regard, the protein(s) that recognize a methylated lysine within a specific sequence context can define the functional outcome associated with that specific lysine methylation event. Thus, mechanistic insight into how lysine methylation influences a biological program requires knowledge of the proteins and domains that recognize and transduce this modification.In addition to histones, several other proteins such as the tumor suppressor p53 undergo lysine methylation, arguing that this modification may be a common mechanism for modulating protein-protein interactions and key cellular signaling pathways (6 -8). p53 plays a pivotal role in the regulation of cellular responses to various forms of genotoxic stresses, and its activity is coordinated by a complex network of post-translational modifications (9, 10). We previously demonstrated that the protein lysine methyltransferase (PKMT) 5 PR-Set7/SET8 monomethylates p53 exclusively at lysine 382 (p53K382me1), and that the placement of this modification negatively regulates p53 activity (11). Spec...

The functions of androgen receptor (AR) in stromal cells are still debated in spite of the demonstrated importance of these cells in organ development and diseases. Here, we show that physiological androgen concentration (10 nM R1881 or DHT) fails to induce DNA synthesis, while it consistently stimulates cell migration in mesenchymal and transformed mesenchymal cells. Ten nanomolar R1881 triggers p27 Ser10 phosphorylation and its stabilization in NIH3T3 fibroblasts. Activation of Rac and its downstream effector DYRK 1B is responsible for p27 Ser10 phosphorylation and cell quiescence. Ten nanomolar androgen also inhibits transformation induced by oncogenic Ras in NIH3T3 fibroblasts. Overexpression of an AR mutant unable to interact with filamin A, use of a small peptide displacing AR/filamin A interaction, and filamin A knockdown indicate that the androgen-triggered AR/filamin A complex regulates the pathway leading to p27 Ser10 phosphorylation and cell cycle arrest. As the AR/filamin A complex is also responsible for migration stimulated by 10 nM androgen, our report shows that the androgen-triggered AR/filamin A complex controls, through Rac 1, the decision of cells to halt cell cycle and migration. This study reveals a new and unexpected role of androgen/AR signalling in coordinating stromal cell functions.

High-performance and excellent-uniformity thin-film transistors (TFTs) having bottom-gate structures are fabricated using an amorphous indium-gallium-zinc-oxide (IGZO) film and an amorphous-silicon dioxide film as the channel layer and the gate insulator layer, respectively. All of the 94 TFTs fabricated with an area 1 cm 2 show almost identical transfer characteristics: the average saturation mobility is 14.6 cm 2 /(V-sec) with a small standard deviation of 0.11 cm 2 /(V-sec). A five-stage ring-oscillator composed of these TFTs operates at 410 kHz at an input voltage of 18 V. Pixel-driving circuits based on these TFTs are also fabricated with organic light-emitting diodes (OLED) which are monolithically integrated on the same substrate. It is demonstrated that light emission from the OLED cells can be switched and modulated by a 120-Hz ac signal input. Amorphous-IGZO-based TFTs are prominent candidates for building blocks of large-area OLED-display electronics.

The tumor suppressor p53 functions as a transcriptional activator for many genes, including several key genes involved in cell cycle arrest and apoptosis. Following DNA damage-induced stress, p53 undergoes extensive post-translational modification, resulting in increased stability and activity. Two critical cofactors for p53-mediated transactivation are the histone acetyltransferase paralogs CREB-binding protein (CBP) and p300. The N-terminal transactivation domain of p53 interacts with several domains of CBP/p300, including the Taz2 domain. Here, we report the effects of specific p53 phosphorylations on its interaction with the Taz2 domain of p300. Using a competitive fluorescence anisotropy assay, we determined that monophosphorylation of p53 at Ser15 or Thr18 increased the affinity of p53(1-39) for Taz2, and diphosphorylations at Ser15 and Ser37 or Thr18 and Ser20 further increased the affinity. In addition, we identified a second binding site for Taz2 within p53 residues 35-59. This second site bound Taz2 with a similar affinity as the first site, but the binding was unaffected by phosphorylation. Thus, p53 post-translational modification modulates only one of the two binding sites for p300 Taz2. Further investigation of Taz2 binding to p53(1-39) or p53(35-59) by isothermal titration calorimetry indicated that upon complex formation the change in heat capacity at constant pressure, ΔCp, was negative for both sites, suggesting the importance of hydrophobic interactions. However, the more negative value of ΔCp for Taz2 binding to the first (−330 cal/mol K) compared to the second site (−234 cal/mol K) suggests that the importance of nonpolar and polar interactions differs between the two sites.