c-Abl and Src-family kinases cross-talk in regulation of myeloid cell migration

Cytoskeleton dynamics are regulated by Src-family tyrosine kinases (SFKs) and c-Abl. We found that the SFK members Hck and c-Fgr regulate tyrosine phosphorylation of c-Abl and c-Abl associates with β1 integrin-bound Hck or c-Fgr in murine macrophages. Studies with selective inhibitors and cells from SFK-deficient mice showed that c-Abl and SFK regulate migration and activation of the small GTPases Cdc42 and Rac in macrophages. Additionally, human neutrophil chemotactic activity was reduced by c-Abl inhibitors, and neutrophils from chronic myeloid leukaemia patients displayed an increased chemotactic ability. Hence, Src-family kinase and c-Abl cross-talk in the regulation of myeloid cell migration.

Structured summary

MINT-7296608: Integrin beta-1 (uniprotkb:P09055) physically interacts (MI:0914) with Hck (uniprotkb:P08103), Abl (uniprotkb:P00520) and Fgr (uniprotkb:P14234) by anti bait coimmunoprecipitation (MI:0006) MINT-7296596: Integrin beta-1 (uniprotkb:P09055) physically interacts (MI:0914) with Fgr (uniprotkb:P14234) and Abl (uniprotkb:P00520) by anti bait coimmunoprecipitation (MI:0006)     

Comparative analysis of the signaling capabilities of the insulin receptor-related receptor

Although insulin receptor (InsR) and type I insulin-like growth factor receptor (IGF-IR) elicit different physiological effects in their target tissues, their signaling capabilities are similar to a large extent. In the present work, we investigated the potential of the third member of the family, insulin receptor-related receptor (IRR), to associate with known interaction partners of the InsR and the IGF-I receptor in a yeast two-hybrid assay. Using the intracellular part of the IRR we found no association with any of the tested signaling molecules. Phosphotyrosine detection revealed a lack in the constitutive activation of the IRR described for analogous constructs of the two other members of the family. Replacement of the kinase domain of the IGF-IR or its C-terminal lobe alone into the IRR caused a complete restoration of the tyrosine phosphorylation of the IRR. The reestablishment of autophosphorylation was paralleled by restoration of interaction with a specific range of signaling molecules.     

Bidirectional regulation of insulin receptor autophosphorylation and kinase activity by peroxynitrite

Accumulating evidence suggests that enhanced peroxynitrite formation occurs during diabetes. This report describes the effect of peroxynitrite on insulin receptor (IR) function. Addition of peroxynitrite to purified IR resulted in concentration-dependent tyrosine nitration and thiol oxidation. Interestingly, the basal and insulin-stimulated IR autophosphorylation and tyrosine kinase activity were upregulated at low peroxynitrite concentrations, but downregulated at high peroxynitrite concentrations. Concomitantly, peroxynitrite dramatically reduced ¹²⁵I-insulin binding capacity and phosphotyrosine phosphatase activity of IR preparations. Moreover, SIN-1 administration decreased blood glucose levels in normal mice via upregulation of IR/IRS-1 tyrosine phosphorylation. In contrast, SIN-1 markedly increased blood glucose levels in diabetic mice concomitant with downregulation of IR/IRS-1 tyrosine phosphorylation. Taken together, these data provide new insights regarding how peroxynitrite influences IR function in vitro and in vivo, suggesting that peroxynitrite plays a dual role in regulation of IR autophosphorylation and tyrosine kinase activity, and SIN-1 has hyperglycemic effect in diabetic mice.     

蛋白激酶B及其在磷脂酰肌醇3-激酶介导的信号转导中的作用

蛋白激酶Ｂ（ＰＫＢ）是原癌基因ｃ－ａｋｔ的表达产物,它参与由生长因子激活的经磷脂磷肌醇３－激酶（ＰＩ３Ｋ）介导的信号转导过程。与许多蛋白激酶相似,ＰＫＢ分子具有一特殊的ＡＨ／ＰＨ结构域（ＡＨ／ＰＨｄｏｍａｉｎ）,后者能介导信号分子间的相互作用。ＰＫＢ是ＰＩ３Ｋ直接的靶蛋白。ＰＩ３Ｋ产生的脂类第二信使ＰＩ－３,４,Ｐ２和ＰＩ－３,４,５－Ｐ３等均能与ＰＫＢ和磷酸肌醇依赖性蛋白激酶（ＰＤＫ）的ＡＨ／Ｐ     

细胞因子受体的组成,结构功能及信号传导机制

细胞因子受体种类繁多,分属于不同受体超家族。活化受体的功能可分为：ＰＴＫ型受体或其结合蛋白具有ＰＴＫ活性、丝／苏氨酸蛋白激酶型受体以及与Ｇ蛋白耦联的受体等。不同受体与其配体结合后,通过对受体后信号传导成分的可逆的磷酸化反应传递信号,最终通过对其终端成分,如酶活力、基因表达、细胞骨架蛋白的功能、膜通透性等的调节,导致细胞的生物效应。     

Entirely Artificial Signal Transduction with Adrenaline

Multifunctional transmembrane-building blocks with recognition sites for adrenaline on one end and the reaction partners for an S_N2 reaction on the opposite end have been embedded in DPPC-liposomes. These doped vesicles can be quantitatively reduced at their disulfide head groups by externally added reducing agents; their composition and chemical processes taking place within can be monitored by NMR spectroscopy and–with limitations—by UV/Vis spectroscopy. Attempted release of thiopyridine as a second messenger into the interior of the liposome on external adrenaline addition could not be proven unambiguously because the detection system does not fulfill the necessary rigorous specificity and sensitivity requirements.     

Identification of four sites of stimulated tyrosine phosphorylation in the MUC1 cytoplasmic tail

MUC1 is an integral membrane protein expressed on the apical surface of epithelial cells where it acts as a signaling receptor. Its cytoplasmic tail (CT) contains seven, highly conserved tyrosine residues, some of which are constitutively phosphorylated and serve as recognition sites for SH2 domain proteins involved in intracellular signal transduction. However, no studies have determined which MUC1 tyrosines are phosphorylated or which signaling pathways are activated in response to stimulation of its ectodomain. In this report, we used our previously characterized CD8/MUC1 chimeric protein that is tyrosine phosphorylated on the MUC1 CT in response to extracellular treatment with CD8 antibody and performed site-directed mutagenesis of all seven tyrosines, both individually and in multiple combinations, to identify the particular sites of stimulated phosphorylation. We observed four phosphorylation sites, three present in sequence motifs with known signaling potential (Y(20), Y(46), and Y(60)) and one previously uncharacterized (Y(29)). These results are discussed in the context of the role of MUC1 in signal transduction.     

Top-down motif engineering of a cytokine receptor for directing ex vivo expansion of hematopoietic stem cells

The technique to expand hematopoietic stem cells (HSCs) ex vivo is eagerly anticipated to secure an enough amount of HSCs for clinical applications. Previously we developed a scFv-thrombopoietin receptor (c-Mpl) chimera, named S-Mpl, which can transduce a proliferation signal in HSCs in response to a cognate antigen. However, a remaining concern of the S-Mpl chimera may be the magnitude of the cellular expansion level driven by this molecule, which was significantly less than that mediated by endogenous wild-type c-Mpl. In this study, we engineered a tyrosine motif located in the intracellular domain of S-Mpl based on a top-down approach in order to change the signaling properties of the chimera. The truncated mutant (trunc.) and an amino-acid substitution mutant (Q to L) of S-Mpl were constructed to investigate the ability of these mutants to expand HSCs. The result showed that the truncated and Q to L mutants gave higher and considerably lower number of the cells than unmodified S-Mpl, respectively. The proliferation level through the truncated mutant was even higher than that of non-transduced HSCs with the stimulation of a native cytokine, thrombopoietin. Moreover, we analyzed the signaling properties of the S-Mpl mutants in detail using a pro-B cell line Ba/F3. The data indicated that the STAT3 and STAT5 activation levels through the truncated mutant increased, whereas activation of the Q to L mutant was inhibited by a negative regulator of intracellular signaling, SHP-1. This is the first demonstration that a non-natural artificial mutant of a cytokine receptor is effective for ex vivo expansion of hematopoietic cells compared with a native cytokine receptor.     

Current understanding of the role of tyrosine kinase 2 signaling in immune responses

Immune system is a complex network that clears pathogens,toxic substrates,and cancer cells.Distinguishing self-antigens from non-self-antigens is critical for the immune cell-mediated response against foreign antigens.The innate immune system elicits an early-phase response to various stimuli,whereas the adaptive immune response is tailored to previously encountered antigens.During immune responses,B cells differentiate into antibody-secreting cells,while na?ve T cells differentiate into functionally specific effector cells[T helper 1(Th1),Th2,Th17,and regulatory T cells].However,enhanced or prolonged immune responses can result in autoimmune disorders,which are characterized by lymphocytemediated immune responses against self-antigens.Signal transduction of cytokines,which regulate the inflammatory cascades,is dependent on the members of the Janus family of protein kinases.Tyrosine kinase 2(Tyk2)is associated with receptor subunits of immune-related cytokines,such as type I interferon,interleukin(IL)-6,IL-10,IL-12,and IL-23.Clinical studies on the therapeutic effects and the underlying mechanisms of Tyk2 inhibitors in autoimmune or chronic inflammatory diseases are currently ongoing.This review summarizes the findings of studies examining the role of Tyk2 in immune and/or inflammatory responses using Tyk2-deficient cells and mice.     

Structural analysis of receptor tyrosine kinases

Receptor tyrosine kinases (RTKs) are single-pass transmembrane receptors that possess intrinsic cytoplasmic enzymatic activity, catalyzing the transfer of the γ-phosphate of ATP to tyrosine residues in protein substrates. RTKs are essential components of signal transduction pathways that affect cell proliferation, differentiation, migration and metabolism. Included in this large protein family are the insulin receptor and the receptors for growth factors such as epidermal growth factor, fibroblast growth factor and vascular endothelial growth factor. Receptor activation occurs through ligand binding, which facilitates receptor dimerization and autophosphorylation of specific tyrosine residues in the cytoplasmic portion. The phosphotyrosine residues either enhance receptor catalytic activity or provide docking sites for downstream signaling proteins. Over the past several years, structural studies employing X-ray crystallography have advanced our understanding of the molecular mechanisms by which RTKs recognize their ligands and are activated by dimerization and tyrosine autophosphorylation. This review will highlight the key results that have emerged from these structural studies.     

Cross-talk between the autocrine (mitogenic) pheromone loop of the ciliate Euplotes raikovi and the intracellular cyclic AMP concentration

Cell type-specific protein signals, called pheromones, are constitutively secreted by Euplotes raikovi and bound back in autocrine fashion, with a positive effect on the vegetative (mitotic) cell growth. In cells growing suspended with their secreted pheromone, it was found that any interruption of this autocrine signaling loop was immediately followed by an effective enhancement of the basal intracellular cyclic AMP (cAMP) level. To establish a cause-effect relationship between these pheromone-induced variations in the cytoplasmic cAMP level and cell growth, cells ready to pass from a resting stage to a new growth cycle were conditioned either to incorporate a cAMP analog resistant to phosphodiesterase degradation, or to utilize cAMP released (following cell irradiation) from incorporated "caged" cAMP. Cells responded at every induced increase in their basal cAMP level by markedly decreasing their commitment to start a new growth cycle. It was deduced that the autocrine signaling of E. raikovi pheromones involves cAMP as inhibitor of its mitogenic activity.     

Phosphorylation of focal adhesion kinase at Tyrosine 407 negatively regulates Ras transformation of fibroblasts

Focal adhesion kinase (FAK) mediates signal transduction in response to multiple extracellular inputs, via tyrosine phosphorylation at specific residues. We recently reported that FAK Tyr-407 phosphorylation negatively regulates the enzymatic and biological activities of FAK, unlike phosphorylation of other tyrosine residues. In this study, we further investigated the effect of FAK Tyr-407 phosphorylation on cell transformation. We found that FAK Tyr-407 phosphorylation was lower in H-Ras transformed NIH3T3 and K-Ras transformed rat-2 fibroblasts than in the respective untransformed control cells. Consistently, FAK Tyr-407 phosphorylation was decreased in parallel with cell transformation in H-Ras-inducible NIH3T3 cells and increased during trichostatin A-induced detransformation of both K-Ras transformed rat-2 fibroblasts and H-Ras transformed NIH3T3 cells. In addition, overexpression of a phosphorylation-mimicking FAK Tyr-407 mutant inhibited morphological transformation of H-Ras-inducible NIH3T3 cells and inhibited invasion activity and anchorage-independent growth of H-Ras-transformed NIH3T3 cells. Taken together, these data strongly suggest that FAK Tyr-407 phosphorylation negatively regulates transformation of fibroblasts.     

脱落酸信号转导研究进展

脱落酸（ABA）对植物生长发育和适应环境胁迫等多方面有重要的调节作用,其信号转导机制非常引人注目,近年来这方面研究进展很快,本文利用现有文献,对脱落酶不敏感和超敏感性突变体,脱落酸的结合位点与受体,ABA信号转涉及的细胞第二信使（Ca^2 ,磷酸肌醇,cADPR,阴离子通道与H^ ), 蛋白质可逆磷酸化以及ABA诱导基因表达所必需的顺序作用元件（cis-acting elenment)和反式作用因子（trans-acting factor)等几方面的最新研究进展作了介绍。     

Oleoylethanolamide,a natural ligand for PPAR-alpha,inhibits insulin receptor signalling in HTC rat hepatoma cells

Oleoylethanolamide (OEA) is a lipid mediator belonging to the fatty acid ethanolamides family. It is produced by intestine and adipose tissue. It inhibits food intake and body weight gain, and has hypolipemiant action in vivo, as well as a lipolytic effect in vitro. OEA is a PPAR-alpha agonist, and recently it has been found that OEA is an endogenous ligand of an orphan receptor. Previously, we have shown that OEA inhibits insulin-stimulated glucose uptake in isolated adipocytes, and produces glucose intolerance in rats. In the present work, we have studied another insulin target cell, the hepatocyte using a rat hepatoma cell line (HTC), and we have studied the cross-talk of OEA signalling with metabolic and mitotic signal transduction of insulin receptor. OEA dose-dependently activates JNK and p38 MAPK, and inhibits insulin receptor phosphorylation. OEA inhibits insulin receptor activation, blunting insulin signalling in the downstream PI3K pathway, decreasing phosphorylation of PKB and its target GSK-3. OEA also inhibits insulin-dependent MAPK pathway, as assessed by immunoblot of phosphorylated MEK and MAPK. These effects were reversed by blocking JNK or p38 MAPK using pharmacological inhibitors (SP 600125, and SB 203580). Since OEA is an endogenous PPAR-alpha agonist, we investigated whether a pharmacologic agonist (WY 14643) may mimic the OEA effect on insulin receptor signalling. Activation of PPAR-alpha by the pharmacological agonist WY14643 in HTC hepatoma cells is sufficient to inhibit insulin signalling and this effect is also dependent on p38 MAPK but not JNK kinase. In summary, OEA inhibits insulin metabolic and mitogenic signalling by activation of JNK and p38 MAPK via PPAR-alpha.     

Hormonal regulation of glycogen synthase: Insulin decreases protein kinase sensitivity to cyclic AMP

Rat hemidiaphragms incubated with epinephrine exhibited increases in cyclic AMP content and protein kinase activity which were proportional to the logarithm of the hormone concentration from 0.1–2 μM. The fraction of glycogen synthase made independent of glucose-6-P for activity (%I) decreased concomitantly, but correlated only with epinephrine concentrations up to 0.2 μM. Insulin (0–100 mU/ml) increased glycogen synthase %I in a dose-dependent manner with no change in cyclic AMP concentration. Protein kinase activity increased slightly at the lowest insulin concentration, then decreased slightly as glycogen synthase %I increased. Insulin was without effect when administered with a supramaximal dose of epinephrine. In the presence of submaximal epinephrine, insulin produced a dose-dependent increase in glycogen synthase %I which correlated with a decrease in protein kinase activity, without changing cyclic AMP. Insulin had no effect on the increases in cyclic AMP produced by varying levels of epinephrine. However, the activation of protein kinase activity by endogenous cyclic AMP was inhibited in the presence of insulin. The glycogen synthase %I response to epinephrine also was less sensitive in the presence of insulin. Insulin antagonizes the activation of cyclic AMP-dependent protein kinase by epinephrine without altering cyclic AMP levels.     

Mutational analysis of the interaction between insulin receptor and IGF-I receptor with c-Crk and Crk-L in a yeast two-hybrid system

The SH2/SH3 adapter proteins of the Crk family are potent signal transducers after receptor tyrosine kinase stimulation with insulin or IGF-1. We have employed a yeast two-hybrid approach and mutational analysis to dissect the capabilities of the insulin receptor and the IGF-I receptor to directly associate with Crk isoforms. Insulin receptor stably recruits full length Crk by association with its SH2 domain in an auto-phosphorylation dependent manner. In contrast, interaction of the IGF-I receptor with the Crk-IISH2 domain was only detectable when Crk-II was truncated in its C-terminal part, indicating the transient nature of this interaction. From these data it can be concluded that members of the insulin receptor family activate Crk proteins in a differential manner.     

Altered signaling through IL-12 receptor in children with very high serum IgE levels

An alteration of Th1/Th2 homeostasis may lead to diseases in humans. In this study, we investigated whether an impaired IL-12R signaling occurred in children with elevated serum IgE levels divided on the basis of the IgE levels (group A: >2000 kU/l; group B: <2000 kU/l). We evaluated the integrity of the IL-12R signaling through the analysis of phosphorylation/activation of STAT4, and mRNA expression and membrane assembly of the receptor chains. At a functional level, a proliferative defect of lymphocytes from group A patients was observed. In these patients, an abnormal IL-12R signaling was documented, and this finding was associated with abnormal expression of the IL-12Rβ2 chain. Our data indicate that in patients with very high IgE levels the generation of Th1 response is impaired, and that this abnormality associates with abnormal IL-12R signaling.     

Reviews in Molecular Biology and Biotechnology: Transmembrane Signaling by G Protein-Coupled Receptors

As the most diverse type of cell surface receptor, the importance heptahelical G protein-coupled receptors (GPCRs) to clinical medicine cannot be overestimated. Visual, olfactory and gustatory sensation, intermediary metabolism, cell growth and differentiation are all influenced by GPCR signals. The basic receptor-G protein-effector mechanism of GPCR signaling is tuned by a complex interplay of positive and negative regulatory events that amplify the effect of a hormone binding the receptor or that dampen cellular responsiveness. The association of heptahelical receptors with a variety of intracellular partners other than G proteins has led to the discovery of potential mechanisms of GPCR signaling that extend beyond the classical paradigms. While the physiologic relevance of many of these novel mechanisms of GPCR signaling remains to be established, their existence suggests that the mechanisms of GPCR signaling are even more diverse than previously imagined.