Src-family kinases mediate an outside-in signal necessary for beta2 integrins to achieve full activation and sustain firm adhesion of polymorphonuclear leucocytes tethered on E-selectin

In cell suspensions subjected to high-shear rotatory motion, human PMN (polymorphonuclear cells) adhered to E-selectin-expressing CHO (Chinese-hamster ovary) cells (CHO-E), and formed homotypic aggregates when challenged by E-selectin-IgG fusion protein, by a mechanism that involved beta2 integrins. Both heterotypic and homotypic PMN adhesion was accompanied by tyrosine phosphorylation of a 110 kDa protein (P110). This event was prevented by blocking anti-(beta2 integrin) antibodies and by inhibitors of Src-family kinases, suggesting that it was part of an 'outside-in' signalling that was initiated by integrin engagement. Interestingly, Src-family kinase inhibitors prevented beta2-integrin-mediated (i) homotypic PMN adhesion triggered by E-selectin-IgG, (ii) heterotypic CHO-E/PMN adhesion in mixed-cell suspensions, and (iii) firm adhesion of PMN to CHO-E monolayers under physiological flow. Similarly to PMN treated with Src-family kinase inhibitors, PMN from hck-/-fgr-/- and hck-/-fgr-/-lyn-/- mice showed significant impairment of beta2-integrin-mediated adhesion to CHO-E. Moreover, the expression of beta2 integrin activation epitopes at the sites of cell-cell contact in CHO-E/PMN conjugates was abolished by Src-family kinase inhibitors. One component of P110 was identified as the FAK (focal adhesion kinase) Pyk2 (proline-rich tyrosine kinase 2), which was phosphorylated in a beta2 integrin- and Src-family-kinase-dependent manner. Thus, Src-family kinases, and perhaps Pyk2, mediate a signal necessary for beta2 integrin function in PMN tethered by E-selectin.     

Csk enhances insulin-stimulated dephosphorylation of focal adhesion proteins.

Insulin has pleiotropic effects on the regulation of cell physiology through binding to its receptor. The wide variety of tyrosine phosphorylation motifs of insulin receptor substrate 1 (IRS-1), a substrate for the activated insulin receptor tyrosine kinase, may account for the multiple functions of insulin. Recent studies have shown that activation of the insulin receptor leads to the regulation of focal adhesion proteins, such as a dephosphorylation of focal adhesion kinase (pp125FAK). We show here that C-terminal Src kinase (Csk), which phosphorylates C-terminal tyrosine residues of Src family protein tyrosine kinases and suppresses their kinase activities, is involved in this insulin-stimulated dephosphorylation of focal adhesion proteins. We demonstrated that the overexpression of Csk enhanced and prolonged the insulin-induced dephosphorylation of pp125FAK. Another focal adhesion protein, paxillin, was also dephosphorylated upon insulin stimulation, and a kinase-negative mutant of Csk was able to inhibit the insulin-induced dephosphorylation of pp125FAK and paxillin. Although we have shown that the Csk Src homology 2 domain can bind to several tyrosine-phosphorylated proteins, including pp125FAK and paxillin, a majority of protein which bound to Csk was IRS-1 when cells were stimulated by insulin. Our data also indicated that tyrosine phosphorylation levels of IRS-1 appear to be paralleled by the dephosphorylation of the focal adhesion proteins. We therefore propose that the kinase activity of Csk, through the insulin-induced complex formation of Csk with IRS-1, is involved in insulin's regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activity of c-Src family kinases.     

Protein tyrosine dephosphorylation and signal transduction

The protein tyrosine phosphatases comprise a family of enzymes that specifically dephosphorylate tyrosyl residues. Determination of the amino acid sequence of a major low molecular mass form isolated from human placenta (PTPase 1B) provided the basis for the first identification of transmembrane proteins that bear intracellular phosphatase domains. The existence of such molecules, bearing the hallmarks of receptors, raises the exciting possibility of a novel mechanism of signal transduction in which the early events involve the ligand-induced dephosphorylation of tyrosyl residues in proteins.     

TGF—β1短时处理降低肝癌细胞与Fn的粘附及FAK的磷酸化

In order to investigate whether TGF-beta 1 could rapidly regulate integrin induced signaling, we treated SMMC-7721 human hepatocellular carcinoma cells with human recombinant TGF-beta 1 for 10 min, and examined cell adhesion, integrin amount and FAK tyrosine phosphorylation. We used cell adhesion assay to estimate the affinity of alpha 5 beta 1 integrin with fibronectin, and analyzed the amount of integrin alpha 5 and beta 1 subunits by performing FACS analysis. Then western blot analysis was carried out to examine tyrosine phosphorylation level of FAK. Our results showed that TGF-beta 1 could rapidly attenuated cell adhesion onto Fn without changing the expression of alpha 5 beta 1 integrin, and at the meantime dephosphorylated FAK. It suggested that TGF-beta 1 rapidly regulated the activation of integrin, and stimulated FAK dephosphorylation, which might induce depolarization in SMMC-7721 hepatocellular carcinoma cells, then facilitates the detachment of tumor cells at early stages of migration.     

Plakophilin3 loss leads to an increase in PRL3 levels promoting K8 dephosphorylation, which is required for transformation and metastasis

The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis.     

Lung-targeted VEGF inactivation leads to an emphysema phenotype in mice.

To test the hypothesis that VEGF is important for the maintenance of alveolar structure and elastic properties in adult mice, lung-targeted ablation of the VEGF gene was accomplished through intratracheal delivery of an adeno-associated cre recombinase virus (AAV/Cre) to VEGFloxP mice, and the effects were followed for 8 wk. Control mice were similarly treated with AAV/Cre. Pulmonary VEGF levels were reduced by 86% at 5 wk postinfection but returned to normal levels by 8 wk. VEGF receptor VEGFR-2 levels were also reduced at 5 wk (by 51%) and returned to control values by 8 wk. However, alveolar septal wall destruction (increased mean linear intercept) and loss of lung elastic recoil (increased compliance) persisted for 8 wk. No decrease in alveolar cell proliferation was detected by Western blot or immunohistochemical analysis of proliferating cell nuclear antigen. Increased alveolar septal cell and bronchial epithelial cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling analysis at 5 wk. Total lung caspase-3 levels and enzyme activity were also increased at 5 wk. No obvious accumulation of inflammatory cells was observed at any time after tracheal instillation of AAV/Cre. Thus a transient decrease in pulmonary VEGF leads to increased alveolar and bronchial cell apoptosis, air space enlargement, and changes in lung elastic recoil (processes that are characteristic of emphysema) that persist for at least 8 wk.     

cGMP-independent inhibition of integrin alphaIIbbeta3-mediated platelet adhesion and outside-in signalling by nitric oxide

We examined the influence of S-nitrosoglutathione (GSNO) on alpha(IIb)beta(3) integrin-mediated platelet adhesion to immobilised fibrinogen. GSNO induced a time- and concentration-dependent inhibition of platelet adhesion. Inhibition was cGMP-independent and associated with both reduced platelet spreading and protein tyrosine phosphorylation. To investigate the cGMP-independent effects of NO we evaluated integrin beta(3) phosphorylation. Adhesion to fibrinogen induced rapid phosphorylation of beta(3) on tyrosines 773 and 785, which was reduced by GSNO in a cGMP independent manner. Similar results were observed in suspended platelets indicating that NO-induced effects were independent of spreading-induced signalling. This is the first demonstration that NO directly regulates integrin beta(3) phosphorylation.     

Mercury leads to abnormal red blood cell adhesion to laminin mediated by membrane sulfatides

Exposure to mercury is associated with numerous health problems, affecting different parts of the human body, including the nervous and cardiovascular systems in adults and children; however, the underlying mechanisms are yet to be fully elucidated. We investigated the role of membrane sulfatide on mercuric ion (Hg²⁺) mediated red blood cell (RBC) adhesion to a sub-endothelial matrix protein, laminin, using a microfluidic system that mimics microphysiological flow conditions. We exposed whole blood to mercury (HgCl₂), at a range of concentrations to mimic acute (high dose) and chronic (low dose) exposure, and examined RBC adhesion to immobilized laminin in microchannels at physiological flow conditions. Exposure of RBCs to both acute and chronic levels of Hg²⁺ resulted in elevated adhesive interactions between RBCs and laminin depending on the concentration of HgCl₂ and exposure duration. BCAM-Lu chimer significantly inhibited the adhesion of RBCs that had been treated with 50 μM of HgCl₂ solution for 1 h at 37 °C, while it did not prevent the adhesion of 3 h and 24 h Hg²⁺-treated RBCs. Sulfatide significantly inhibited the adhesion of RBC that had been treated with 50 μM of HgCl₂ solution for 1 h at 37 °C and 0.5 μM of HgCl₂ solution for 24 h at room temperature (RT). We demonstrated that RBC BCAM-Lu and RBC sulfatides bind to immobilized laminin, following exposure of RBCs to mercuric ions. The results of this study are significant considering the potential associations between sulfatides, red blood cells, mercury exposure, and cardiovascular diseases.     

Urokinase receptor (CD87) clustering in detergent-insoluble adhesion patches leads to cell adhesion independently of integrins

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidyl inositol-anchored protein that mediates cell adhesion to the extracellular matrix protein vitronectin (VN). We demonstrate here that this cell adhesion process is accompanied by the formation of an adhesion patch characterized by an accumulation of uPAR into areas of direct contact between the cell and the matrix. The adhesion patch requires the glycolipid anchor and develops only on a VN-coated substrate, but not on fibronectin. It consists of detergent-insoluble microdomains that accumulate F-actin and tyrosine-phosphorylated proteins, but not β₁ integrins. Lack of inhibition of adhesion in the presence of integrin-blocking reagents and adhesion on a VN fragment without the RGD sequence indicated that the adhesion of uPAR-bearing cells on VN could occur independently of integrins. Hence, uPAR-mediated cell adhesion on VN relies on the formation of a unique cellular structure that we have termed “detergent-insoluble adhesion patch” (DIAP).     

The regulation of cadherin-mediated adhesion by tyrosine phosphorylation/dephosphorylation of beta-catenin

The formation of stable cell-cell adhesions by type I cadherins depends on the association of their cytoplasmic domain with beta-catenin, and of beta-catenin with alpha-catenin. The binding of beta-catenin to these partners is regulated by phosphorylation of at least three critical tyrosine residues. Each of these residues is targeted by one or more specific kinases: Y142 by Fyn, Fer and cMet; Y489 by Abl; and Y654 by Src and the epidermal growth factor receptor. Developmental and physiological signals have been identified that initiate the specific phosphorylation and dephosphorylation of these residues, regulating cadherin function during neurite outgrowth, permeability of airway epithelium and synapse remodeling, and possibly initiating epithelial cell migration during development and metastasis.     

Histone H1 dephosphorylation is mediated through a radiation-induced signal transduction pathway dependent on ATM.

Ionizing radiation is known to activate multiple signal transduction pathways, but the targets of these pathways are poorly understood. Phosphorylation of histone H1 is thought to have a role in chromatin condensation/decondensation, and we asked whether ionizing radiation (IR) would alter H1 phosphorylation. Our data demonstrate that low doses of IR result in a dramatic, but transient, dephosphorylation of H1 isoforms. The in vivo IR-induced dephosphorylation of H1 is completely blocked by wortmannin and is abrogated in ataxia telangiectasia cells. Furthermore, we measured radiation-induced inhibition of cyclin dependent kinase activity and activation of histone H1 phosphatase activity. Both activities were affected by radiation-induced signals in an ATM-dependent manner. Thus, the rapid IR-induced dephosphorylation of H1 involves a pathway including ATM and a wortmannin-sensitive step leading to both inhibition of cyclin-dependent kinase activities as well as activation of H1 phosphatase(s).     

Funneled landscape leads to robustness of cellular networks: MAPK signal transduction

We uncover the underlying potential energy landscape for a cellular network. We find that the potential energy landscape of the mitogen-activated protein-kinase signal transduction network is funneled toward the global minimum. The funneled landscape is quite robust against random perturbations. This naturally explains robustness from a physical point of view. The ratio of slope versus roughness of the landscape becomes a quantitative measure of robustness of the network. Funneled landscape is a realization of the Darwinian principle of natural selection at the cellular network level. It provides an optimal criterion for network connections and design. Our approach is general and can be applied to other cellular networks.     

粟酒裂殖酵母核糖体蛋白RPL21表达不足引发细胞粘附

【目的】随机选择裂殖酵母核糖体蛋白RPL21作为研究对象,分析其表达不足对细胞的影响。【方法】通过同源臂交换的方法,敲除裂殖酵母基因组中RPL21蛋白的编码基因rpl21-1和rpl21-2,观察突变菌株rpl21-1Δ和rpl21-2Δ细胞内的核糖体合成情况以及细胞表型变化。【结果】突变菌株rpl21-1Δ和rpl21-2Δ细胞内总的rpl21(rpl21-1+rpl21-2)表达水平与野生型菌株相比分别减少了66.5%和58.7%,合成的核糖体总量较野生型菌株分别下降了62.8%和50.4%。突变菌株在YEPD液体培养基中培养时发生细胞粘附现象,而基因回补的重组菌株rpl21-1Δ/RPL21-1和rpl21-2Δ/RPL21-2突变株细胞中粘附现象消失。【结论】核糖体蛋白损伤造成核糖体合成受阻,进而引发细胞生长过程中的粘附在粟酒裂殖酵母中是普遍存在的现象。     

Blocking dephosphorylation at Serine 120 residue in t-SNARE SNAP-23 leads to massive inhibition in exocytosis from mast cells

Mast cells (MCs) respond to allergen challenge by release of pre-stored inflammatory mediators from their secretory granules, on cross-linking of Fcε receptor I (FcεRI) receptors. The target-SNARE (t-SNARE) SNAP-23 has been shown to play an important role in MC exocytosis and undergoes transient phosphorylation at Serine 95 (S95) and Serine 120 (S120), concomitant with mediator release. During current study we explored the importance of transient nature of phosphorylation at S120 in MC exocytosis. A phosphomimetic SNAP-23-S120D mutant of rodent SNAP-23 was cloned into EGFP vector and its effect on the exocytosis and the mechanisms involved was studied in RBL-2H3 MC line. Secretion reporter assay with SNAP-23-S120D transfected MCs revealed a very significant inhibition of exocytosis, and reduced ruffling in response to FcεRI cross-linking. Further, the effect of this mutation on localization of SNAP-23 in MCs was studied. Immunofluorescence microscopy studies and membrane-cytosol fractionation of green fluorescent protein-tagged SNAP-23-S120D (GFP-SNAP-23-S120D) transfected MCs showed that a large proportion of GFP-SNAP-23-S120D was residing in cytosol unlike wild-type SNAP-23, in resting and activated MCs and even the membrane associated portion was on internal lysosomal membranes than plasma membrane. These studies imply that dephosphorylation of S120 is important for SNAP-23 membrane association dynamics and subsequently MC degranulation.     

Activation of the prolyl-hydroxylase oxygen-sensing signal cascade leads to AMPK activation in cardiomyocytes

The proline hydroxylase domain-containing enzymes (PHD) act as cellular oxygen sensors and initiate a hypoxic signal cascade to induce a range of cellular responses to hypoxia especially in the aspect of energy and metabolic homeostasis regulation. AMP-activated protein kinase (AMPK) is recognized as a major energetic sensor and regulator of cardiac metabolism. However, the effect of PHD signal on AMPK has never been studied before. A PHD inhibitor (PHI), dimethyloxalylglycine and PHD2-specific RNA interference (RNAi) have been used to activate PHD signalling in neonatal rat cardiomyocytes. Both PHI and PHD2-RNAi activated AMPK pathway in cardiomyocytes effectively. In addition, the increased glucose uptake during normoxia and enhanced myocyte viability during hypoxia induced by PHI pretreatment were abrogated substantially upon AMPK inhibition with an adenoviral vector expressing a dominant negative mutant of AMPK-α1. Furthermore, chelation of intracellular Ca2+ by BAPTA, inhibition of calmodulin-dependent kinase kinase (CaMKK) with STO-609, or RNAi-mediated down-regulation of CaMKK α inhibited PHI-induced AMPK activation significantly. In contrast, down-regulation of LKB1 with adenoviruses expressing the dominant negative form did not affect PHI-induced AMPK activation. We establish for the first time that activation of PHD signal cascade can activate AMPK pathway mainly through a Ca(2+)/CaMKK-dependent mechanism in cardiomyocytes. Furthermore, activation of AMPK plays an essential role in hypoxic protective responses induced by PHI.     

Stimulation of human red blood cells leads to Ca2+-mediated intercellular adhesion

Red blood cells (RBCs) are a major component of blood clots, which form physiologically as a response to injury or pathologically in thrombosis. The active participation of RBCs in thrombus solidification has been previously proposed but not yet experimentally proven. Holographic optical tweezers and single-cell force spectroscopy were used to study potential cell-cell adhesion between RBCs. Irreversible intercellular adhesion of RBCs could be induced by stimulation with lysophosphatidic acid (LPA), a compound known to be released by activated platelets. We identified Ca²⁺ as an essential player in the signaling cascade by directly inducing Ca²⁺ influx using A23187. Elevation of the internal Ca²⁺ concentration leads to an intercellular adhesion of RBCs similar to that induced by LPA stimulation. Using single-cell force spectroscopy, the adhesion of the RBCs was identified to be approximately 100 pN, a value large enough to be of significance inside a blood clot or in pathological situations like the vasco-occlusive crisis in sickle cell disease patients.     

Regulated binding of PTP1B-like phosphatase to N-cadherin: control of cadherin-mediated adhesion by dephosphorylation of beta-catenin

Cadherins are a family of cell-cell adhesion molecules which play a central role in controlling morphogenetic movements during development. Cadherin function is regulated by its association with the actin containing cytoskeleton, an association mediated by a complex of cytoplasmic proteins, the catenins: alpha, beta, and gamma. Phosphorylated tyrosine residues on beta-catenin are correlated with loss of cadherin function. Consistent with this, we find that only nontyrosine phosphorylated beta-catenin is associated with N-cadherin in E10 chick retina tissue. Moreover, we demonstrate that a PTP1B-like tyrosine phosphatase associates with N-cadherin and may function as a regulatory switch controlling cadherin function by dephosphorylating beta-catenin, thereby maintaining cells in an adhesion-competent state. The PTP1B-like phosphatase is itself tyrosine phosphorylated. Moreover, both direct binding experiments performed with phosphorylated and dephosphorylated molecules, and treatment of cells with tyrosine kinase inhibitors indicate that the interaction of the PTP1B-like phosphatase with N-cadherin depends on its tyrosine phosphorylation. Concomitant with the tyrosine kinase inhibitor-induced loss of the PTP1B-like phosphatase from its association with N-cadherin, phosphorylated tyrosine residues are retained on beta-catenin, the association of N- cadherin with the actin containing cytoskeleton is lost and N-cadherin- mediated cell adhesion is prevented. Tyrosine phosphatase inhibitors also result in the accumulation of phosphorylated tyrosine residues on beta-catenin, loss of the association of N-cadherin with the actin- containing cytoskeleton, and prevent N-cadherin mediated adhesion, presumably by directly blocking the function of the PTP1B-like phosphatase. We previously showed that the binding of two ligands to the cell surface N-acetylgalactosaminylphosphotransferase (GalNAcPTase), the monoclonal antibody 1B11 and a proteoglycan with a 250-kD core protein, results in the accumulation of phosphorylated tyrosine residues on beta-catenin, uncoupling of N-cadherin from its association with the actin containing cytoskeleton, and loss of N- cadherin function. We now report that binding of these ligands to the GalNAcPTase results in the absence of the PTP1B-like phosphatase from its association with N-cadherin as well as the loss of the tyrosine kinase and tyrosine phosphatase activities that otherwise co- precipitate with N-cadherin. Control antibodies and proteoglycans have no such effect. This effect is similar to that observed with tyrosine kinase inhibitors, suggesting that the GalNAcPTase/proteoglycan interaction inhibits a tyrosine kinase, thereby preventing the phosphorylation of the PTP1B-like phosphatase, and its association with N-cadherin. Taken together these data indicate that a PTP1B-like tyrosine phosphatase can regulate N-cadherin function through its ability to dephosphorylate beta-catenin and that the association of the phosphatase with N-cadherin is regulated via the interaction of the GalNAcPTase with its proteoglycan ligand. In this manner the GalNAcPTase-proteoglycan interaction may play a major role in morphogenetic cell and tissue interactions during development.     

Interleukin-4 proliferative signal transduction involves the activation of a tyrosine-specific phosphatase and the dephosphorylation of an 80-kDa protein

Interleukin-4 (IL-4) is a cytokine that expresses its biological effects by binding to specific membrane receptors. Although the diverse biological properties of this molecule have been characterized extensively the biochemical mechanisms by which extracellular binding events lead to biological responses remain unclear. IL-4 can stimulate the proliferation of several hemopoietic cell types, and we have taken advantage of its ability to induce the growth of leukemic cell lines to investigate the role that protein phosphorylation events might play in IL-4 mitogenic signal transduction. We show that the addition of IL-4 to several leukemic cell lines of different origin causes the rapid dephosphorylation of an 80-kDa phosphoprotein (p80) from tyrosine residues. This event occurs in a dose-responsive manner closely correlating to that of biological activity, and both are blocked by an anti-IL-4-specific antiserum. The ability of sodium orthovanadate to prevent IL-4-induced dephosphorylation of p80 suggests that this event is mediated by a protein-tyrosine-phosphatase (EC 3.1.3.48). The importance of the role that tyrosine-specific dephosphorylation plays in mediating IL-4 mitogenic signal transduction is substantiated by the ability of sodium orthovanadate in cell culture to block effectively IL-4-induced proliferation at doses that enhance the proliferation stimulated by either granulocyte-macrophage colony-stimulating factor or interleukin-3.     

Inhibition of the catalytic activity of cell adhesion kinase beta by protein-tyrosine phosphatase-PEST-mediated dephosphorylation

Protein-tyrosine phosphatase (PTP)-PEST is a cytoplasmic tyrosine phosphatase that can bind and dephosphorylate the focal adhesion-associated proteins p130(CAS) and paxillin. Focal adhesion kinase (FAK) and cell adhesion kinase beta (CAKbeta)/PYK2/CADTK/RAFTK are protein-tyrosine kinases that can colocalize with, bind to, and induce tyrosine phosphorylation of p130(CAS) and paxillin. Thus, we considered the possibility that these kinases might be substrates for PTP-PEST. Using a combination of substrate-trapping assays and overexpression of PTP-PEST in mammalian cells, CAKbeta was found to be a substrate for PTP-PEST. Both the major autophosphorylation site of CAKbeta (Tyr(402)) and activation loop tyrosine residues, Tyr(579) and Tyr(580), were targeted for dephosphorylation by PTP-PEST. Dephosphorylation of CAKbeta by PTP-PEST dramatically inhibited CAKbeta kinase activity. In contrast, FAK was a poor substrate for PTP-PEST, and treatment with PTP-PEST had no effect on FAK kinase activity. Tyrosine phosphorylation of paxillin, which is greatly enhanced by CAKbeta overexpression, was dramatically reduced upon coexpression of PTP-PEST. Finally, endogenous PTP-PEST and endogenous CAKbeta were found to localize to similar cellular compartments in epithelial and smooth muscle cells. These results suggest that CAKbeta is a substrate of PTP-PEST and that FAK is a poor PTP-PEST substrate. Further, PTP-PEST can negatively regulate CAKbeta signaling by inhibiting the catalytic activity of the kinase.