Wnt-11 activation of a non-canonical Wnt signalling pathway is required for cardiogenesis

Formation of the vertebrate heart requires a complex interplay of several temporally regulated signalling cascades. In Xenopus laevis, cardiac specification occurs during gastrulation and requires signals from the dorsal lip and underlying endoderm. Among known Xenopus Wnt genes, only Wnt-11 shows a spatiotemporal pattern of expression that correlates with cardiac specification, which indicates that Wnt-11 may be involved in heart development. Here we show, through loss- and gain-of-function experiments, that XWnt-11 is required for heart formation in Xenopus embryos and is sufficient to induce a contractile phenotype in embryonic explants. Treating the mouse embryonic carcinoma stem cell line P19 with murine Wnt-11 conditioned medium triggers cardiogenesis, which indicates that the function of Wnt-11 in heart development has been conserved in higher vertebrates. XWnt-11 mediates this effect by non-canonical Wnt signalling, which is independent of beta-catenin and involves protein kinase C and Jun amino-terminal kinase. Our results indicate that the cardiac developmental program requires non-canonical Wnt signal transduction.     

CDK-dependent phosphorylation of BRCA2 as a regulatory mechanism for recombinational repair

Inherited mutations in BRCA2 are associated with a predisposition to early-onset breast cancers. The underlying basis of tumorigenesis is thought to be linked to defects in DNA double-strand break repair by homologous recombination. Here we show that the carboxy-terminal region of BRCA2, which interacts directly with the essential recombination protein RAD51, contains a site (serine 3291; S3291) that is phosphorylated by cyclin-dependent kinases. Phosphorylation of S3291 is low in S phase when recombination is active, but increases as cells progress towards mitosis. This modification blocks C-terminal interactions between BRCA2 and RAD51. However, DNA damage overcomes cell cycle regulation by decreasing S3291 phosphorylation and stimulating interactions with RAD51. These results indicate that S3291 phosphorylation might provide a molecular switch to regulate RAD51 recombination activity, providing new insight into why BRCA2 C-terminal deletions lead to radiation sensitivity and cancer predisposition.     

Fc receptor phosphorylation during receptor-mediated control of B-cell activation

It is well known that Fc receptors for IgG (FcRII) on macrophages mediate the endocytosis of antibody-antigen complexes and signal the release of inflammatory and cytotoxic agents. FcRII are also expressed at high levels on B cells where they are less involved in endocytosis than in modulating B-cell activation by membrane immunoglobulins. Although crosslinking of membrane immunoglobulins can result in B-cell differentiation and proliferation through stimulation of phospholipase C, mobilization of intracellular Ca2+, and activation of protein kinase C, crosslinking FcR with membrane immunoglobulins confers a dominant inhibitory signal that prevents or aborts activation. This form of regulation may have a role in the induction of tolerance by IgG and in controlling the B-cell repertoire by anti-idiotypes. The different functions of FcR on B cells and macrophages may reflect the fact that these cell types express closely related but distinct FcR isoforms. We have recently found that the main lymphocyte FcR isoform, FcRII-B1, is unable to mediate endocytosis by way of coated pits and coated vesicles owing to an in-frame insertion of 47 amino acids in its cytoplasmic tail. Here we show that this insert, absent from the FcRII-B2 macrophage isoform, also contains serine phosphorylation sites that may have a role in the ability of FcR to regulate B-cell activation through membrane immunoglobulins.     

Ha-Ras augments c-Jun activity and stimulates phosphorylation of its activation domain

Ha-Ras augments c-Jun-mediated transactivation by potentiating the activity of the c-Jun activation domain. Ha-Ras also causes a corresponding increase in phosphorylation of specific sites in that part of the c-Jun protein. A Ha-Ras-induced protein kinase cascade resulting in hyperphosphorylation of the c-Jun activation domain could explain how these oncoproteins cooperate to transform rat embryo fibroblasts.     

Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase

MAP kinase (relative molecular mass, 42,000), a low abundance serine--threonine protein kinase, is transiently activated in many cell types by a variety of mitogens, including insulin, epidermal growth factor, and phorbol esters. In vitro, MAP kinase will phosphorylate and reactivate S6 kinase II previously inactivated by phosphatase treatment. Because many of the stimuli that activate MAP kinase are also stimulators of cell proliferation, and regulation of the cell cycle seems to involve a network of protein kinases, MAP kinase could be important in the transmission of stimuli eventually leading to the progression from G0 to G1 in the cell cycle. Activated MAP kinase contains both phosphotyrosine and phosphothreonine. We report here that MAP kinase can be deactivated completely by treatment with either phosphatase 2A, a protein phosphatase specific for phosphoserine and phosphothreonine, or CD45, a phosphotyrosine-specific protein phosphatase. We demonstrate that MAP kinase is only active when both tyrosyl and threonyl residues are phosphorylated and suggest therefore that the enzyme functions in vivo to integrate signals from two distinct transduction pathways.     

Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction

Signalling by Wnt proteins (Wingless in Drosophila) has diverse roles during embryonic development and in adults, and is implicated in human diseases, including cancer. LDL-receptor-related proteins 5 and 6 (LRP5 and LRP6; Arrow in Drosophila) are key receptors required for transmission of Wnt/beta-catenin signalling in metazoa. Although the role of these receptors in Wnt signalling is well established, their coupling with the cytoplasmic signalling apparatus remains poorly defined. Using a protein modification screen for regulators of LRP6, we describe the identification of Xenopus Casein kinase 1 gamma (CK1gamma), a membrane-bound member of the CK1 family. Gain-of-function and loss-of-function experiments show that CK1gamma is both necessary and sufficient to transduce LRP6 signalling in vertebrates and Drosophila cells. In Xenopus embryos, CK1gamma is required during anterio-posterior patterning to promote posteriorizing Wnt/beta-catenin signalling. CK1gamma is associated with LRP6, which has multiple, modular CK1 phosphorylation sites. Wnt treatment induces the rapid CK1gamma-mediated phosphorylation of these sites within LRP6, which, in turn, promotes the recruitment of the scaffold protein Axin. Our results reveal an evolutionarily conserved mechanism that couples Wnt receptor activation to the cytoplasmic signal transduction apparatus.     

A binding site for the T-cell co-receptor CD8 on the alpha 3 domain of HLA-A2

Adhesion measurements between CD8 and 48 point mutants of HLA-A2.1 show that the CD8 alpha-chain binds to the alpha 3 domain of HLA-A2.1. Three clusters of alpha 3 residues contribute to the binding, with an exposed, negatively charged loop (residues 223-229) playing a dominant role. CD8 binding correlates with cytotoxic T-cell recognition and sensitivity to inhibition by anti-CD8 antibodies. Impaired alloreactive T-cell recognition of an HLA-A2.1 mutant with reduced affinity for CD8 is not restored by functional CD8 binding sites on an antigenically irrelevant class I molecule. Therefore, complexes of CD8 and the T-cell receptor bound to the same class I major histocompatibility complex molecule seem to be necessary for T-cell activation.     

焙烧-活化过程中湿氟石膏的化学相变体特征及改性机理

通过SEM/EDX,XRD和DTA-TGA等表征湿氟石膏化学组成、物相特征、热力学性能;研究外加硅酸盐、含钙铝化合物等改性剂的湿氟石膏在焙烧活化过程中的化学相变体特征及改性机理。研究结果表明:湿氟石膏主要以二水硫酸钙存在;在120~150℃湿氟石膏脱除结晶水生成半水石膏;外加4.2%的改性剂、经200℃下焙烧2 h后得到的改性氟石膏粉完全满足《建筑石膏》(GB 9776—2008)要求,其中,半水硫酸钙质量分数为70.9%;初凝时间为5 min,终凝时间为9 min;遇水2 h后抗折强度为1.9 MPa,抗压强度为4.2 MPa;在焙烧活化过程中,氟石膏粉中游离CaO与硅酸盐通过水化作用形成C-S-H凝胶;含钙、铝化合物与硫酸钙进一步反应生成钙矾石针状骨架结构的硫酸铝钙,增强了半水石膏的水化特性和机械强度。     

Altered tyrosine 527 phosphorylation and mitotic activation of p60c-src.

The tyrosine kinasee activity of p60c-src, the protein product of the c-src gene, increases during mitosis; this may be important in initiating at least some of the cellular changes that occur during this phase of the cell cycle. Although there is evidence that p60c-src is phosphorylated at several sites during mitosis, phosphorylation in vitro does not increase its kinase activity. We now report that the kinase activity of a p60c-src mutant with residue tyrosine 527 changed to phenylanine does not change during the cell cycle, suggesting that changes in the phosphorylation state of this residue may be responsible for the activation of p60c-src at mitosis. Although changes in phosphorylation at Tyr 527 cannot be detected with the wild-type protein we find that phosphorylation at Tyr 527 of a mutant with reduced kinase activity decreases threefold during mitosis. On the basis of these results we suggest that activation of p60c-src at mitosis results from decreased phosphorylation on Tyr 527, and that p60c-src may be or may activate the kinase that phosphorylates Tyr 527.     

Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation

Many bacterial pathogens secrete proteins that activate host trypsinogen-like enzyme precursors, most notably the proenzymes of the blood coagulation and fibrinolysis systems. Staphylococcus aureus, an important human pathogen implicated in sepsis and endocarditis, secretes the cofactor staphylocoagulase, which activates prothrombin, without the usual proteolytic cleavages, to directly initiate blood clotting. Here we present the 2.2 A crystal structures of human alpha-thrombin and prethrombin-2 bound to a fully active staphylocoagulase variant. The cofactor consists of two domains, each with three-helix bundles; this is a novel fold that is distinct from known serine proteinase activators, particularly the streptococcal plasminogen activator streptokinase. The staphylocoagulase fold is conserved in other bacterial plasma-protein-binding factors and extracellular-matrix-binding factors. Kinetic studies confirm the importance of isoleucine 1 and valine 2 at the amino terminus of staphylocoagulase for zymogen activation. In addition to making contacts with the 148 loop and (pro)exosite I of prethrombin-2, staphylocoagulase inserts its N-terminal peptide into the activation pocket of bound prethrombin-2, allosterically inducing functional catalytic machinery. These investigations demonstrate unambiguously the validity of the zymogen-activation mechanism known as 'molecular sexuality'.     

A role for Wnt signalling in self-renewal of haematopoietic stem cells

Haematopoietic stem cells (HSCs) have the ability to renew themselves and to give rise to all lineages of the blood; however, the signals that regulate HSC self-renewal remain unclear. Here we show that the Wnt signalling pathway has an important role in this process. Overexpression of activated beta-catenin expands the pool of HSCs in long-term cultures by both phenotype and function. Furthermore, HSCs in their normal microenvironment activate a LEF-1/TCF reporter, which indicates that HCSs respond to Wnt signalling in vivo. To demonstrate the physiological significance of this pathway for HSC proliferation we show that the ectopic expression of axin or a frizzled ligand-binding domain, inhibitors of the Wnt signalling pathway, leads to inhibition of HSC growth in vitro and reduced reconstitution in vivo. Furthermore, activation of Wnt signalling in HSCs induces increased expression of HoxB4 and Notch1, genes previously implicated in self-renewal of HSCs. We conclude that the Wnt signalling pathway is critical for normal HSC homeostasis in vitro and in vivo, and provide insight into a potential molecular hierarchy of regulation of HSC development.     

气相中Zr活化CH_4分子C—H键的理论研究

采用密度泛函理论和高级电子相关耦合簇方法,在CCSD/6-311++G(3df,3pd)//B3LYP/6-311++G(3df,3pd)理论水平下,研究了两个自旋态下的Zr原子活化CH4分子中C—H键逐个夺取H原子的微观反应机理.通过计算,讨论了势能面交叉和可能的自旋翻转过程.用Harvey等的方法优化出最低能量交叉点(MECP),并计算了MECP处相应的自旋-轨道耦合常数(SOC),进一步讨论了Zr与CH4的反应中不同势能面之间的"系间窜越"(ISC)的可能性.     

A conserved mechanism of DEAD-box ATPase activation by nucleoporins and InsP6 in mRNA export

Superfamily 1 and superfamily 2 RNA helicases are ubiquitous messenger-RNA-protein complex (mRNP) remodelling enzymes that have critical roles in all aspects of RNA metabolism. The superfamily 2 DEAD-box ATPase Dbp5 (human DDX19) functions in mRNA export and is thought to remodel mRNPs at the nuclear pore complex (NPC). Dbp5 is localized to the NPC via an interaction with Nup159 (NUP214 in vertebrates) and is locally activated there by Gle1 together with the small-molecule inositol hexakisphosphate (InsP(6)). Local activation of Dbp5 at the NPC by Gle1 is essential for mRNA export in vivo; however, the mechanistic role of Dbp5 in mRNP export is poorly understood and it is not known how Gle1(InsP6) and Nup159 regulate the activity of Dbp5. Here we report, from yeast, structures of Dbp5 in complex with Gle1(InsP6), Nup159/Gle1(InsP6) and RNA. These structures reveal that InsP(6) functions as a small-molecule tether for the Gle1-Dbp5 interaction. Surprisingly, the Gle1(InsP6)-Dbp5 complex is structurally similar to another DEAD-box ATPase complex essential for translation initiation, eIF4G-eIF4A, and we demonstrate that Gle1(InsP6) and eIF4G both activate their DEAD-box partner by stimulating RNA release. Furthermore, Gle1(InsP6) relieves Dbp5 autoregulation and cooperates with Nup159 in stabilizing an open Dbp5 intermediate that precludes RNA binding. These findings explain how Gle1(InsP6), Nup159 and Dbp5 collaborate in mRNA export and provide a general mechanism for DEAD-box ATPase regulation by Gle1/eIF4G-like activators.     

粉红丝带引发的思考——Wnt信号通路在乳腺发育与乳腺癌变中的角色

 Wnt信号转导通路是参与乳腺发育和肿瘤形成的重要机制。本文综述了Wnt信号转导通路调控乳腺发育和干细胞稳态研究的进展,讨论了Wnt信号转导中成员分子在乳腺癌形成中的角色。     

Midzone activation of aurora B in anaphase produces an intracellular phosphorylation gradient

Proper partitioning of the contents of a cell between two daughters requires integration of spatial and temporal cues. The anaphase array of microtubules that self-organize at the spindle midzone contributes to positioning the cell-division plane midway between the segregating chromosomes. How this signalling occurs over length scales of micrometres, from the midzone to the cell cortex, is not known. Here we examine the anaphase dynamics of protein phosphorylation by aurora B kinase, a key mitotic regulator, using fluorescence resonance energy transfer (FRET)-based sensors in living HeLa cells and immunofluorescence of native aurora B substrates. Quantitative analysis of phosphorylation dynamics, using chromosome- and centromere-targeted sensors, reveals that changes are due primarily to position along the division axis rather than time. These dynamics result in the formation of a spatial phosphorylation gradient early in anaphase that is centred at the spindle midzone. This gradient depends on aurora B targeting to a subpopulation of microtubules that activate it. Aurora kinase activity organizes the targeted microtubules to generate a structure-based feedback loop. We propose that feedback between aurora B kinase activation and midzone microtubules generates a gradient of post-translational marks that provides spatial information for events in anaphase and cytokinesis.