Integrin Signaling and Lipid Rafts

Integrin-mediated adhesion regulates the recruitment of the small GTPase Rac to the plasma membrane and subsequent activation of downstream signaling. We recently reported that Rac binds preferentially to cholesterol-rich membranes (“lipid rafts”), and integrins regulate Rac function by preventing the internalization of its binding sites within these domains. Regulation of lipid rafts by integrins may be important for the spatial control of cell migration and signaling pathways involved in anchorage-dependent cell growth.     

Genetic Analyses of Integrin Signaling

The development of multicellular organisms, as well as maintenance of organ architecture and function, requires robust regulation of cell fates. This is in part achieved by conserved signaling pathways through which cells process extracellular information and translate this information into changes in proliferation, differentiation, migration, and cell shape. Gene deletion studies in higher eukaryotes have assigned critical roles for components of the extracellular matrix (ECM) and their cellular receptors in a vast number of developmental processes, indicating that a large proportion of this signaling is regulated by cell-ECM interactions. In addition, genetic alterations in components of this signaling axis play causative roles in several human diseases. This review will discuss what genetic analyses in mice and lower organisms have taught us about adhesion signaling in development and disease.Almost all cells in multicellular organisms are surrounded by a three-dimensional organized meshwork of macromolecules that constitute the extracellular matrix (ECM). The ECM is a dynamic structure that is generated and constantly remodeled by cells that secrete and manipulate its components into a precise configuration. It functions as a structural framework that provides cells with positional and environmental information, but also forms specialized structures such as cartilage, tendons, basement membranes (BM), bone, and teeth. In addition to its structural properties, the ECM acts as a signaling platform that regulates a large number of cellular functions. It is capable of binding growth factors, chemokines, and cytokines thereby modulating their bioavailability and activity. On the other hand, the ECM is recognized by multiple cell surface receptors that transmit information from the extracellular environment by propagating intracellular signals (for a review, see Hynes 2009).The major cell surface receptors that recognize and assemble the ECM are integrins. Integrins are heterodimeric transmembrane proteins composed of α and β subunits. Eighteen α subunits and eight β subunits can assemble in 24 different combinations with overlapping substrate specificity and cell-type-specific expression patterns (Hynes 2002; Humphries et al. 2006). This, together with the ability of different heterodimers to assemble specific intracellular signaling complexes, provides multiple layers of signaling specificity to these receptors. Conversely, the integrin expression profile of a given cell type determines which ECM components it can bind. Signals arising from integrins regulate virtually all aspects of cell behavior, including cell migration, survival, cell cycle progression, and differentiation.Genetics has proven to be a powerful tool to dissect the functions of ECM–cell interactions in complex organisms. To date, all of the integrin subunits and their major ligands have been deleted in mice. Given the large variety of cellular processes regulated by adhesion signaling, it is not surprising that a significant subset of these proteins has proven to be essential for embryonic development and/or tissue maintenance. However, in addition to underlining the importance of cell-ECM interactions in development, genetic studies also revealed critical roles for tissue- and cell-type-specific modes of adhesion signaling and provided important insights into human disease.     

Integrin and Cadherin Synergy Regulates Contact Inhibition of Migration and Motile Activity

Integrin receptors play a central role in cell migration through their roles as adhesive receptors for both other cells and extracellular matrix components. In this study, we demonstrate that integrin and cadherin receptors coordinately regulate contact-mediated inhibition of cell migration. In addition to promoting proliferation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A. Horwitz. 1996. J. Cell Biol. 133:169–184), ectopic expression of the α5 integrin in cultures of primary quail myoblasts promotes a striking contact-mediated inhibition of cell migration. Myoblasts ectopically expressing α5 integrin (α5 myoblasts) move normally when not in contact, but upon contact, they show inhibition of migration and motile activity (i.e., extension and retraction of membrane protrusions). As a consequence, these cells tend to grow in aggregates and do not migrate to close a wound. This phenotype is also seen with ectopic expression of β1 integrin, paxillin, or activated FAK (CD2 FAK) and therefore appears to result from enhanced integrin-mediated signaling. The contact inhibition observed in the α5 myoblasts is mediated by N-cadherin, whose expression is upregulated more than fivefold. Perturbation studies using low calcium conditions, antibody inhibition, and ectopic expression of wild-type and mutant N-cadherins all implicate N-cadherin in the contact inhibition of migration. Ectopic expression of N-cadherin also produces cells that show inhibited migration upon contact; however, they do not show suppressed motile activity, suggesting that integrins and cadherins coordinately regulate motile activity. These observations have potential importance to normal and pathologic processes during embryonic development and tumor metastasis.     

An RGD Motif Present in Cadherin 17 Induces Integrin Activation and Tumor Growth

Little is known about the mechanism of integrin activation by cadherin 17 (CDH17). Here we observed the presence of a tri-peptide motif, RGD, in domain 6 of the human CDH17 sequence and other cadherins such as cadherin 5 and cadherin 6. The use of CDH17 RAD mutants demonstrated a considerable decrease of proliferation and adhesion in RKO and KM12SM colon cancer cells. Furthermore, RGD peptides inhibited the adhesion of both cell lines to recombinant CDH17 domain 6. The RGD motif added exogenously to the cells provoked a change in β1 integrin to an active, high-affinity conformation and an increase in focal adhesion kinase and ERK1/2 activation. In vivo experiments with Swiss nude mice demonstrated that cancer cells expressing the CDH17 RAD mutant showed a considerable delay in tumor growth and liver homing. CDH17 RGD effects were also active in pancreatic cancer cells. Our results suggest that α2β1 integrin interacts with two different ligands, collagen IV and CDH17, using two different binding sites. In summary, the RGD binding motif constitutes a switch for integrin pathway activation and shows a novel capacity of CDH17 as an integrin ligand. This motif could be targeted to avoid metastatic dissemination in tumors overexpressing CDH17 and other RGD-containing cadherins.     

Cross Talk Between Substance P and Melittin-Activated Cellular Signaling Pathways in Rat Lactotroph-Enriched Cell Cultures

Abstract: We have investigated the possible interaction (cross talk) between the phospholipase A₂ (PLA₂) and inositol 1,4,5-trisphosphate/protein kinase C (PKC) signaling pathways in rat lactotroph-enriched cell cultures. Melittin, a bee venom peptide, stimulated release of [³H]-arachidonic acid ([³H]AA) from [³H]AA-labeled enriched lactotrophs in a dose-dependent manner. Moreover, melittin and exogenous AA induced a redistribution of PKC catalytic activity and PKCα and β immunoreactivity from the soluble to the particulate fraction in resting and substance P (SP)-stimulated cells. Mellitin had no effect on phospholipase C (PLC) activity. Pretreatment of cell cultures with the PLA₂ inhibitors quinacrine and aristolochic acid resulted in a dose-dependent inhibition of melittin-stimulated PKC isozyme translocation as did the inhibitor of lipoxygenase, nordihydroguaiaretic acid, whereas the cyclooxygenase inhibitor indomethacin had no effect. SP and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) dose-dependently increased levels of [³H]AA released from cells. Pretreatment of cell cultures with quinacrine reduced the effect of SP on [³H]AA formation. After long-term treatment (24 h) of cells with TPA, the effect of TPA on [³H]AA production was not different from control, whereas SP still displayed [³H]AA-releasing abilities although not at full scale. Pretreatment of cells with thapsigargin, U 73122, methoxyverapamil, and RHC 80267, an inhibitor of diacylglycerol lipase, all resulted in reduced SP-stimulated [³H]AA liberation. Treatment of cell cultures with pertussis toxin (PTX) reduced the release of [³H]AA induced by SP, whereas PTX had no effect on SP-stimulated generation of ³H-inositol phosphates. On the basis of these results, it is concluded that (1) the PLA₂ pathways interfere with the phosphoinositide-PLC signaling system at the level of PKC isozymes α and β, the product responsible for this interaction being either AA or a metabolite produced by the action of lipoxygenase; (2) SP and TPA are able to activate the PLA₂ pathway at a level at or beyond PLA₂, and this effect is mediated, in part, through PKCα and β species and (for SP) intracellular Ca²⁺ recruited from internal stores as well as from external sources; and (3) SP also activates PLA₂ through a PTX-sensitive pathway distinct from the one coupled to phosphoinositide-PLC, which is PTX insensitive.     

Kinetic Buffering of Cross Talk between Bacterial Two-Component Sensors

Two-component systems are a class of sensors that enable bacteria to respond to environmental and cell-state signals. The canonical system consists of a membrane-bound sensor histidine kinase that autophosphorylates in response to a signal and transfers the phosphate to an intracellular response regulator. Bacteria typically have dozens of two-component systems. The key questions are whether these systems are linear and, if they are, how cross talk between systems is buffered. In this work, we studied the EnvZ/OmpR and CpxA/CpxR systems from Escherichia coli, which have been shown previously to exhibit slow cross talk in vitro. Using in vitro radiolabeling and a rapid quenched-flow apparatus, we experimentally measured 10 biochemical parameters capturing the cognate and non-cognate phosphotransfer reactions between the systems. These data were used to parameterize a mathematical model that was used to predict how cross talk is affected as different genes are knocked out. It was predicted that significant cross talk between EnvZ and CpxR only occurs for the triple mutant ΔompR ΔcpxA ΔactA-pta. All seven combinations of these knockouts were made to test this prediction and only the triple mutant demonstrated significant cross talk, where the cpxP promoter was induced 280-fold upon the activation of EnvZ. Furthermore, the behavior of the other knockouts agrees with the model predictions. These results support a kinetic model of buffering where both the cognate bifunctional phosphatase activity and the competition between regulator proteins for phosphate prevent cross talk in vivo.     

Src-family and Syk Kinases in Activating and Inhibitory Pathways in Innate Immune Cells: Signaling Cross Talk

The response of innate immune cells to growth factors, immune complexes, extracellular matrix proteins, cytokines, pathogens, cellular damage, and many other stimuli is regulated by a complex net of intracellular signal transduction pathways. The majority of these pathways are either initiated or modulated by Src-family or Syk tyrosine kinases present in innate cells. The Src-family kinases modulate the broadest range of signaling responses, including regulating immunoreceptors, C-type lectins, integrins, G-protein-coupled receptors, and many others. Src-family kinases also modulate the activity of other kinases, including the Tec-family members as well as FAK and Pyk2. Syk kinase is required for initiation of signaling involving receptors that utilize immunoreceptor tyrosine activation (ITAM) domains. This article reviews the major activating and inhibitory signaling pathways regulated by these cytoplasmic tyrosine kinases, illuminating the many examples of signaling cross talk between pathways.Innate immune cells, including macrophages, dendritic cells, granulocytes, and mast cells, function as the first line of defense against pathogens. These cells use a dizzying array of cell-surface receptors, which are connected to an equally complicated intracellular signal transduction network, to sense pathogen molecules and then orchestrate the appropriate immune response. Among the intracellular signaling molecules that are most crucial for innate immune cells are the cytoplasmic tyrosine kinases. Two major kinase families that operate in the proximal intracellular signaling pathways in innate cells are the Src-family kinases and the Syk-ZAP70 family. A third family of kinases, the Tek family, also have important roles in innate cells. They are not discussed in detail in this article, but are reviewed elsewhere in articles on the subject.There are eight members of the Src family; innate immune cells primarily express Hck, Fgr, Lyn, and to a lesser extent, Src (Lowell 2004). The Syk-ZAP70 family has only two members and only Syk is found in innate cells. Most innate cell types express the same spectrum of kinases with some specific cellular differences. For example, mast cells express a broader range of Src-family kinases than macrophages or dendritic cells (Colgan and Hankel 2010). In general, Src-family and Syk kinases tend to operate together in signaling pathways, with the Src-family being “upstream” or activated first in response to pathogen detection. These enzymes then communicate downstream to Tec-family members. The Tec-family kinases expressed in innate cells include Btk, Bmx, and Tec (Koprulu and Ellmeier 2009; Tohyama and Yamamura 2009). Additionally, Src-family kinases activate yet another family of PTKs, the FAK/Pyk2 tyrosine kinases, which play a major role in integrin signaling (Hauck et al. 2000).Though primarily studied in activating pathways, Src-family and Syk kinases also activate inhibitory signaling pathways (Nimmerjahn and Ravetch 2008). In many situations, inhibitory signaling often overrides the activating signal. Pathways can also be initiated at different times or rates. Finally, to add even more complexity, activating and inhibitory pathways often interact indirectly, for example, through the production of cytokines and growth factors and not through direct intracellular biochemical interactions; Hence the term signaling “cross talk,” which now appears commonly in the literature (O''Neill 2008; Ivashkiv 2009; Page et al. 2009).     

Cross Talk between 2,4-Diacetylphloroglucinol-Producing Biocontrol Pseudomonads on Wheat Roots

The performance of Pseudomonas biocontrol agents may be improved by applying mixtures of strains which are complementary in their capacity to suppress plant diseases. Here, we have chosen the combination of Pseudomonas fluorescens CHA0 with another well-characterized biocontrol agent, P. fluorescens Q2-87, as a model to study how these strains affect each other's expression of a biocontrol trait. In both strains, production of the antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a crucial factor contributing to the suppression of root diseases. DAPG acts as a signaling compound inducing the expression of its own biosynthetic genes. Experimental setups were developed to investigate whether, when combining strains CHA0 and Q2-87, DAPG excreted by one strain may influence expression of DAPG-biosynthetic genes in the other strain in vitro and on the roots of wheat. DAPG production was monitored by observing the expression of lacZ fused to the biosynthetic gene phlA of the respective strain. Dual-culture assays in which the two strains were grown in liquid medium physically separated by a membrane revealed that Q2-87 but not its DAPG-negative mutant Q2-87::Tn5-1 strongly induced phlA expression in a ΔphlA mutant of strain CHA0. In the same way, phlA expression in a Q2-87 background was induced by DAPG produced by CHA0. When coinoculated onto the roots of wheat seedlings grown under gnotobiotic conditions, strains Q2-87 and CHA0, but not their respective DAPG-negative mutants, were able to enhance phlA expression in each other. In summary, we have established that two nonrelated pseudomonads may stimulate each other in the expression of an antimicrobial compound important for biocontrol. This interpopulation communication occurs in the rhizosphere, i.e., at the site of pathogen inhibition, and is mediated by the antimicrobial compound itself acting as a signal exchanged between the two pseudomonads.     

茉莉酸信号转导突变体ber15的分离和基因克隆表明油菜素内酯的合成影响茉莉酸信号转导

bestatin是一种氨肽酶抑制剂,能够激活茉莉酸信号转导途径而诱导抗性相关基因的表达,从而为用化学遗传学手段解析茉莉酸途径提供了一个有效的工具。ber15是我们鉴定到的一个对bestatin不敏感的拟南芥突变体,随后的研究表明该突变体对外源茉莉酸的敏感性也明显降低,表明相应的野生型基因BER15在茉莉酸信号转导中起重要作用。图位克隆结果表明BER15编码一个细胞色素P450单加氧酶,是植物激素油菜素内酯合成途径中的一个关键酶。对BER15基因功能的深入研究将会为了解油菜素内酯的合成与茉莉酸信号途径间的互作关系提供证据。     

Host Cell-Virus Cross Talk: Phosphorylation of a Hepatitis B Virus Envelope Protein Mediates Intracellular Signaling

Phosphorylation of cytosolic pre-S domains of the duck hepatitis B virus (DHBV) large envelope protein (L) was identified as a regulatory modification involved in intracellular signaling. By using biochemical and mass spectrometric analyses of phosphopeptides obtained from metabolically radiolabeled L protein, a single phosphorylation site was identified at serine 118 as part of a PX(S/T)P motif, which is strongly preferred by ERK-type mitogen-activated protein kinases (MAP kinases). ERK2 specifically phosphorylated L at serine 118 in vitro, and L phosphorylation was inhibited by a coexpressed MAP kinase-specific phosphatase. Furthermore, L phosphorylation and ERK activation were shown to be induced in parallel by various stimuli. Functional analysis with transfected cells showed that DHBV L possesses the ability to activate gene expression in trans and, by using mutations eliminating (S→A) or mimicking (S→D) serine phosphorylation, that this function correlates with L phosphorylation. These mutations had, however, no major effects on virus production in cell culture and in vivo, indicating that L phosphorylation and transactivation are not essential for hepadnavirus replication and morphogenesis. Together, these data suggest a role of the L protein in intracellular host-virus cross talk by varying the levels of pre-S phosphorylation in response to the state of the cell.     

Hypoxia-Inducible Factor 1 Regulation through Cross Talk between mTOR and MT1-MMP

Hypoxia-inducible factor 1 (HIF-1) plays a key role in the cellular adaptation to hypoxia. Although HIF-1 is usually strongly suppressed by posttranslational mechanisms during normoxia, HIF-1 is active and enhances tumorigenicity in malignant tumor cells that express the membrane protease MT1-MMP. The cytoplasmic tail of MT1-MMP, which can bind a HIF-1 suppressor protein called factor inhibiting HIF-1 (FIH-1), promotes inhibition of FIH-1 by Mint3 during normoxia. To explore possible links between HIF-1 activation by MT1-MMP/Mint3 and tumor growth signals, we surveyed a panel of 252 signaling inhibitors. The mTOR inhibitor rapamycin was identified as a possible modulator, and it inhibited the mTOR-dependent phosphorylation of Mint3 that is required for FIH-1 inhibition. A mutant Mint3 protein that cannot be phosphorylated exhibited a reduced ability to inhibit FIH-1 and promoted tumor formation in mice. These data suggest a novel molecular link between the important hub proteins MT1-MMP and mTOR that contributes to tumor malignancy.