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101.
DAPP-1 (dual-adaptor for phosphotyrosine and 3-phosphoinositides-1) is a broadly distributed pleckstrin homology (PH) and Src homology 2 domain containing protein that can bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and can be phosphorylated on tyrosine 139 and internalised in response to activation of type I phosphoinositide 3-kinases (PI3K). Tyrosine phosphorylation of DAPP-1 appears important for appropriate intracellular targeting and creates a potential binding site for Src homology 2 domain-containing proteins. In endothelial cells overexpressing wild-type platelet-derived growth factor beta (PDGFbeta) receptors, which express Bmx and Src as their major Btk (Bruton's tyrosine kinase) family and Src family tyrosine kinases, respectively, PDGF can stimulate PI3K-dependent tyrosine phosphorylation of DAPP-1. Transient overexpression of Src most effectively, compared with Bmx and Syk, augments basal and PDGF-stimulated tyrosine phosphorylation of DAPP-1, whereas overexpression of dominant-negative Src, but not dominant-negative Bmx, inhibits PDGF-stimulated phosphorylation of DAPP-1. Cells expressing mutant PDGFbeta (Y579F/Y581F) receptors (which fail to bind and activate Src-type kinases) fail to tyrosine phosphorylate DAPP-1 in response to PDGF. We show that in DT40 chicken B cell lines, antibody stimulation leads to PI3K-dependent tyrosine phosphorylation of DAPP-1 that is lost in Lyn- or Syk-deficient cell lines but not Btk-deficient cell lines. PI3K-dependent activation of PKB is only lost in Syk-deficient lines. Finally, in vitro we find lipid-modified Src to be the most effective DAPP-1 tyrosine kinase (versus Syk, Lyn, Btk, and Bmx); phosphorylation of DAPP-1 but not Src autophosphorylation is stimulated approximately 10-fold by PtdIns(3,4,5)P(3) (IC(50) = 150 nm) and phosphatidylinositol 3,4-bisphosphate but not by their nonbiological diastereoisomers and depends on PH domain mediated binding of DAPP-1 to PtdIns(3,4,5)P(3)-containing membranes. We conclude that Src family kinases are responsible for tyrosine phosphorylation of DAPP-1 in vivo and that PI3K regulation is at the level of PH domain-mediated translocation of DAPP-1 to PI3K products in the membrane. 相似文献
102.
HU, a nonspecific histone-like DNA binding protein, participates in a number of genomic events as an accessory protein and forms multiple complexes with DNA. The HU-DNA binding interaction was characterized by fluorescence, generated with the guanosine analogue 3-methyl-8-(2-deoxy-beta-D-ribofuranosyl)isoxanthopterin (3-MI) directly incorporated into DNA duplexes. The stoichiometry and equilibrium binding constants of complexes formed between HU and 13 and 34 bp DNA duplexes were determined using fluorescence anisotropy and analytical ultracentrifugation. These measurements reveal that three HU molecules bind to the 34 bp duplexes, while two HU molecules bind to the 13 bp duplex. The data are well described by an independent binding site model, and the association constants for the first binding event for both duplexes are similar (approximately 1 x 10(6) M(-1)), indicating that HU binding affinity is independent of duplex length. Further analysis of the binding curves in terms of a nonspecific binding model is indicative that HU binding to DNA exhibits little to no cooperativity. The fluorescence intensity also increases upon HU binding, consistent with decreased base stacking and increased solvent exposure of the 3-MI fluorescence probe. These results are suggestive of a local bending or unwinding of the DNA. On the basis of these results we propose a model in which bending of DNA accompanies HU binding. Up to five complex bands are observed in gel mobility shift assays of HU binding to the 34 bp duplexes. We suggest that protein-induced bending of the DNA leads to the observation of complexes in the gel, which have the same molecular weight but different relative mobilities. 相似文献
103.
Hartl MG Hutchinson S Hawkins LE 《Journal of experimental marine biology and ecology》2001,256(2):267-278
Chronic (5 weeks) exposure of freshwater-adapted European flounder, Platichthys flesus (L.), to environmental concentrations of sediment-associated tri-n-butyltin chloride (TBTCl) and triphenyltin chloride (TPhTCl) caused significant changes to hydromineral fluxes and membrane permeability, mechanisms that maintain osmotic homeostasis. The half-time of exchange of tritiated water (THO) in TBTCl- and TPhTCl-exposed fish was significantly increased during the first 2 weeks of the experiment and then decreased steadily, eventually reaching the level that the control group had constantly maintained throughout the experiment. This change in apparent water permeability was accompanied by a significant decrease in diffusional water flux across the membranes. Passive Na(+)-efflux across the gills was increased significantly but effluxes in the control group were near constant over the same time span. Drinking rates in the organotin groups increased significantly while the rate of urine production did not change. This lead to an increased net water balance in the organotin groups and consequently to a significant reduction of the blood osmolality of both organotin groups when compared to a control. There would appear to be a metabolic cost attached to the changes produced by exposure to environmental levels of organotin compounds which are manifested as a minimal increase in body length compared to the controls. 相似文献
104.
Structure-activity relationships in flexible protein domains: regulation of rho GTPases by RhoGDI and D4 GDI 总被引:1,自引:0,他引:1
Golovanov AP Chuang TH DerMardirossian C Barsukov I Hawkins D Badii R Bokoch GM Lian LY Roberts GC 《Journal of molecular biology》2001,305(1):121-135
The guanine dissociation inhibitors RhoGDI and D4GDI inhibit guanosine 5'-diphosphate dissociation from Rho GTPases, keeping these small GTPases in an inactive state. The GDIs are made up of two domains: a flexible N-terminal domain of about 70 amino acid residues and a folded 134-residue C-terminal domain. Here, we characterize the conformation of the N-terminal regions of both RhoGDI and D4GDI using a series of NMR experiments which include (15)N relaxation and amide solvent accessibility measurements. In each protein, two regions with tendencies to form helices are identified: residues 36 to 58 and 9 to 20 in RhoGDI, and residues 36 to 57 and 20 to 25 in D4GDI. To examine the functional roles of the N-terminal domain of RhoGDI, in vitro and in vivo functional assays have been carried out with N-terminally truncated proteins. These studies show that the first 30 amino acid residues are not required for inhibition of GDP dissociation but appear to be important for GTP hydrolysis, whilst removal of the first 41 residues completely abolish the ability of RhoGDI to inhibit GDP dissociation. The combination of structural and functional studies allows us to explain why RhoGDI and D4GDI are able to interact in similar ways with the guanosine 5'-diphosphate-bound GTPase, but differ in their ability to regulate GTP-bound forms; these functional differences are attributed to the conformational differences of the N-terminal domains of the guanosine 5'-diphosphate dissociation inhibitors. Therefore, the two transient helices, appear to be associated with different biological effects of RhoGDI, providing a clear example of structure-activity relationships in a flexible protein domain. 相似文献
105.
Sargent DJ Geibel M Hawkins JA Wilkinson MJ Battey NH Simpson DW 《Annals of botany》2004,94(6):787-796
BACKGROUND AND AIMS: The aims of this investigation were to highlight the qualitative and quantitative diversity apparent between nine diploid Fragaria species and produce interspecific populations segregating for a large number of morphological characters suitable for quantitative trait loci analysis. METHODS: A qualitative comparison of eight described diploid Fragaria species was performed and measurements were taken of 23 morphological traits from 19 accessions including eight described species and one previously undescribed species. A principal components analysis was performed on 14 mathematically unrelated traits from these accessions, which partitioned the species accessions into distinct morphological groups. Interspecific crosses were performed with accessions of species that displayed significant quantitative divergence and, from these, populations that should segregate for a range of quantitative traits were raised. KEY RESULTS: Significant differences between species were observed for all 23 morphological traits quantified and three distinct groups of species accessions were observed after the principal components analysis. Interspecific crosses were performed between these groups, and F2 and backcross populations were raised that should segregate for a range of morphological characters. In addition, the study highlighted a number of distinctive morphological characters in many of the species studied. CONCLUSIONS: Diploid Fragaria species are morphologically diverse, yet remain highly interfertile, making the group an ideal model for the study of the genetic basis of phenotypic differences between species through map-based investigation using quantitative trait loci. The segregating interspecific populations raised will be ideal for such investigations and could also provide insights into the nature and extent of genome evolution within this group. 相似文献
106.
107.
Increased attention to spatial context increases both place field stability and spatial memory 总被引:2,自引:0,他引:2
The hippocampal formation is critical for the acquisition and consolidation of memories. When recorded in freely moving animals, hippocampal pyramidal neurons fire in a location-specific manner: they are "place" cells, comprising a hippocampal representation of the animal's environment. To explore the relationship between place cells and spatial memory, we recorded from mice in several behavioral contexts. We found that long-term stability of place cell firing fields correlates with the degree of attentional demands and that successful spatial task performance was associated with stable place fields. Furthermore, conditions that maximize place field stability greatly increase orientation to novel cues. This suggests that storage and retrieval of place cells is modulated by a top-down cognitive process resembling attention and that place cells are neural correlates of spatial memory. We propose a model whereby attention provides the requisite neuromodulatation to switch short-term homosynaptic plasticity to long-term heterosynaptic plasticity, and we implicate dopamine in this process. 相似文献
108.
Jabbour AM Ho PK Puryer MA Ashley DM Ekert PG Hawkins CJ 《Cell death and differentiation》2004,11(12):1309-1316
A genetically defined pathway orchestrates the removal of 131 of the 1090 somatic cells generated during the development of the hermaphrodite nematode Caenorhabditis elegans. Regulation of apoptosis is highly evolutionarily conserved and the nematode cell death pathway is a valuable model for studying mammalian apoptotic pathways, the dysregulation of which can contribute to numerous diseases. The nematode caspase CED-3 is ultimately responsible for the destruction of worm cells in response to apoptotic signals, but it must first be activated by CED-4. CED-9 inhibits programmed cell death and considerable data have demonstrated that CED-9 can directly bind and inhibit CED-4. However, it has been suggested that CED-9 may also directly inhibit CED-3. In this study, we used a yeast-based system and biochemical approaches to explore this second potential mechanism of action. While we confirmed the ability of CED-9 to inhibit CED-4, our data argue that CED-9 can not directly inhibit CED-3. 相似文献
109.
110.
LL5beta is a phosphatidylinositol (3,4,5)-trisphosphate sensor that can bind the cytoskeletal adaptor,gamma-filamin 总被引:1,自引:0,他引:1
Paranavitane V Coadwell WJ Eguinoa A Hawkins PT Stephens L 《The Journal of biological chemistry》2003,278(2):1328-1335
We identified a potential phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) binding pleckstrin homology domain in the data bases and have cloned and expressed its full coding sequence (LL5beta). The protein bound PtdIns(3,4,5)P(3) selectively in vitro. Strikingly, a substantial proportion of LL5beta became associated with an unidentified intracellular vesicle population in the context of low PtdIns(3,4,5)P(3) levels produced by the addition of wortmannin or LY294002. In addition, expression of platelet-derived growth factor-receptor mutants unable to activate type 1A phosphoinositide 3-kinase (PI3K) or serum starvation in porcine aortic endothelial cells lead to redistribution of LL5beta to this vesicle population. Importantly, pleckstrin homology domain mutants of LL5beta that could not bind PtdIns(3,4,5)P(3) were constitutively localized to this vesicle population. At increased PtdIns(3,4,5)P(3) levels, LL5beta was redirected to a predominantly cytoplasmic distribution, presumably through a PI3K-dependent block on its targeting to the vesicular compartment. Furthermore, at high, hormone-stimulated PtdIns(3,4,5)P(3) levels, it became significantly plasma-membrane localized. The distribution of LL5beta is thus dramatically and uniquely sensitive to low levels of PtdIns(3,4,5)P(3) indicating it can act as a sensor of both low and hormone-stimulated levels of PtdIns(3,4,5)P(3). In addition, LL5beta bound to the cytoskeletal adaptor, gamma-filamin, tightly and in a PI3K-independent fashion, both in vitro and in vivo. This interaction could co-localize heterologously expressed gamma-filamin with GFP-LL5beta in the unidentified vesicles. 相似文献