Cross-talk between olfactory second messenger pathways.

The second messengers 3'-5'-cyclic-monophosphate (cAMP) and inositol 1,4,5-trisphosphate (InsP3) have been implicated in olfactory signal transduction in various species. The results of the present study provide evidence that the two olfactory second messenger pathways in rat olfactory neurons do not work independently but rather show a functional antagonism: whereas inhibition of phospholipase C (PLC) in isolated olfactory cilia by U-73122 led to an augmentation of odor-induced cAMP signaling, activation of the phosphoinositol pathway resulted in attenuation of odor-induced cAMP formation. Furthermore, this study indicates that elevated cAMP levels cause suppression of odor-induced InsP3 signaling, whereas inhibition of adenylate cyclase (AC) by cisN-(2-phenylcyclopentyl)azacylotridec-1-en-2-amine (MDL-12,330 A) results in potentiation of odor-induced InsP3 formation. Concerning the molecular mechanism involved in cross-interaction, the experimental data indicate that the observed antagonism of elevated cAMP is based on inhibition of PLC activation rather than on stimulation of InsP3 degradation. As blockage of the endogenous protein kinase A (PKA) prevented the inhibitory effect of cAMP, the suppression of odor-induced InsP3 signaling by cAMP may be mediated by a PKA-controlled reaction.     

Involvement of caveolae and caveolae-like domains in signalling, cell survival and angiogenesis.

Caveolae, the flask-shaped membrane invaginations abundant in endothelial cells, have acquired a prominent role in signal transduction. Evidence, that events occurring in caveolae participate in cell survival and angiogenesis, has been recently substantiated by the identification of two novel caveolar constituents: prostacyclin synthase (PGIS) and the cellular form of prion protein (PrP(c)). We have shown that PGIS, previously described as an endoplasmic reticulum component, is bound to caveolin-1 (cav-1) and localized in caveolae in human endothelial cells. By generating prostacyclin, PGIS is involved in angiogenesis. Previous observations regarding the localization of PrP(c) in caveolae-like membrane domains (CLDs) have been recently confirmed and extended. It has been demonstrated that PrP(c) is bound to cav-1 and, by recruiting Fyn kinase, can participate in signal transduction events connected to cell survival and differentiation. The new entries of PGIS and PrP(c) in caveolar components place caveolae and CLDs at the centre of a network, where cells decide whether to proliferate or differentiate and whether to survive or to suicide by apoptosis.     

Cross-talk between RNA and prions

As concepts evolve in mammalian and yeast prion biology, rather preliminary research investigating the interplay between prion and RNA processes are gaining momentum. The yeast prion [PSI+] represents an aggregated state of the translation termination factor Sup35 resulting in the tendency of ribosomes to readthrough stop codons. This "nonsense suppression" activity is investigated for its possible physiological role to engender on Saccharomyces cerevisiae the ability to respond to stress or variable growth conditions and thereby act as a capacitor to evolve. The interaction between prion and RNA is a two way street--the cell may have adopted RNA processes in translation to govern the presence of prions and the [PSI+] prion's nonsense suppressor phenotype may exhibit different growth phenotypes by its control of translation termination. RNA processes in the mammalian cell also effect and are affected by prions.     

Cross-talk between snurportin1 subdomains

The initial steps of spliceosomal small nuclear ribonucleoprotein (snRNP) maturation take place in the cytoplasm. After formation of an Sm-core and a trimethylguanosine (TMG) cap, the RNPs are transported into the nucleus via the import adaptor snurportin1 (SPN) and the import receptor importin-beta. To better understand this process, we identified SPN residues that are required to mediate interactions with TMG caps, importin-beta, and the export receptor, exportin1 (Xpo1/Crm1). Mutation of a single arginine residue within the importin-beta binding domain (IBB) disrupted the interaction with importin-beta, but preserved the ability of SPN to bind Xpo1 or TMG caps. Nuclear transport assays showed that this IBB mutant is deficient for snRNP import but that import can be rescued by addition of purified survival of motor neurons (SMN) protein complexes. Conserved tryptophan residues outside of the IBB are required for TMG binding. However, SPN can be imported into the nucleus without cargo. Interestingly, SPN targets to Cajal bodies when U2 but not U1 snRNPs are imported as cargo. SPN also relocalizes to Cajal bodies upon treatment with leptomycin B. Finally, we uncovered an interaction between the N- and C-terminal domains of SPN, suggesting an autoregulatory function similar to that of importin-alpha.     

Cross-talk between Diverse Serine Integrases

Phage-encoded serine integrases are large serine recombinases that mediate integrative and excisive site-specific recombination of temperate phage genomes. They are well suited for use in heterologous systems and for synthetic genetic circuits as the attP and attB attachment sites are small (< 50 bp), there are no host factor or DNA supercoiling requirements, and they are strongly directional, doing only excisive recombination in the presence of a recombination directionality factor. Combining different recombinases that function independently and without cross-talk to construct complex synthetic circuits is desirable, and several different serine integrases are available. However, we show here that these functions are not reliably predictable, and we describe a pair of serine integrases encoded by mycobacteriophages Bxz2 and Peaches with unusual and unpredictable specificities. The integrases share only 59% amino acid sequence identity and the attP sites have fewer than 50% shared bases, but they use the same attB site and there is non-reciprocal cross-talk between the two systems. The DNA binding specificities do not result from differences in specific DNA contacts but from the constraints imposed by the configuration of the component half-sites within each of the attachment site DNAs.     

Cross-talk between enteric pathogens and the intestine

Enteric pathogens finely regulate the expression of virulence genes in reply to stimuli generated by the intestinal environment. This minireview focuses on recently discovered strategies developed by enteric bacteria to cause intestinal secretion through the elaboration of factors that share structure and function with specific host counterparts. Such bacterial antigens appear to interfere largely with the epithelial cell signalling that physiologically regulates the numerous and, as yet not fully elucidated, mechanisms controlling both the transcellular and the paracellular secretion pathways. Heat-stable enterotoxins (STs) elaborated by enterotoxigenic Escherichia coli and the enteroaggregative E. coli enterotoxin (EAST1) are both typical examples of enteric toxins that activate the transcellular secretion pathway by mimicking guanylin, the endogenous modulator of cGMP signalling. Alternative strategies have been developed by Salmonella to induce intestinal secretion through the elaboration of a factor (SopB) that resembles at least two of the host cell 4-phosphatases, enzymes that activate the Ca-dependent transcellular secretion pathway. Finally, Vibrio cholerae has developed innovative tactics to activate the paracellular secretion pathway through the elaboration of Zonula occludens toxin (Zot), a factor that mimics a recently described physiological modulator of intercellular tight junctions.     

Cross-talk between IGF-I and TGF-beta signaling pathways

Insulin-like growth factor-I (IGF-I) has gained broad recognition as an important survival factor for epithelial cells in numerous tissues. The IGF-I receptor signaling pathway is deregulated in the majority of carcinomas, and such deregulation has also been reported to be tightly associated with enhanced tumor progression and metastasis. One of the key proteins that transduces IGF-I signals and is phospho-activated downstream of the IGF-I receptor, is the non-receptor serine/threonine kinase proto-oncogene protein kinase B (PKB, also known as Akt). This kinase serves as a major molecular node to control the function of many cell survival and death proteins through phosphorylation-mediated protein modification. The end result of the activation of Akt is enhanced cell survival and proliferation, pre-requisites for malignant transformation. Recent studies show that IGF-I signals cross-talk at multiple levels with various components of the TGF-beta signaling pathway, which depending on context may function either as tumor suppressor or as tumor promoter. Thus, a better understanding of how the IGF-I and TGF-beta signaling pathways are mutually interconnected is likely to unveil novel targets for the therapeutic intervention of many cancers.     

Cross-talk between hematopoiesis and angiogenesis signaling pathways

The relationship between hematopoietic cells and endothelial cells has been seen as an indication that a common progenitor, the hemangioblast, gives rise to both cell types in the yolk sac, the initial site of hematopoiesis and blood vessel formation during mammalian development. The existence of angioblast-like circulating endothelial precursor cells in adults humans has recently been suggested. In this review, we have summarized the principle mechanisms involved in the cross-talk signaling pathway between hematopoiesis and angiogenesis in order to further understand how the hematopoietic and vascular systems are established during the development.     

Structure and cholesterol dynamics of caveolae/raft and nonraft plasma membrane domains

Despite recognition that the plasma membrane (PM) is comprised of lipid raft domains that are key organizing sites of multiple signaling pathways and other cell functions, limited information is available regarding the structure and function in sterol dynamics of these microdomains. To begin to resolve these issues, MDCK membranes were subfractionated by three different techniques to produce (i) detergent-resistant membranes (DRM) and detergent-soluble membranes (DSM), (ii) nondetergent caveolae/rafts (NDCR), and (iii) nondetergent, affinity-purified caveolae/rafts (ACR) and noncaveolae/nonrafts (NR). ACR exhibited the least cross contamination with other PM domains or intracellular membranes, in marked contrast to DRM that contained the highest level of cross contaminants. Spectral properties of dehydroergosterol (DHE), a naturally occurring fluorescent sterol, showed that ACR, NDCR, and NR did not contain crystalline sterol, consistent with the lack of crystalline sterol in PM of intact cells. In contrast, DRM contained significant levels of crystalline sterol. Fluorescence polarization of membrane probes showed that ACR were the least fluid and had the highest transbilayer fluidity gradient, the most liquid ordered phase, and the sterol dynamics most responsive to sterol carrier protein-2 (SCP-2). In contrast, DRM had structural properties similar to those of NR, anomalous (very fast) spontaneous sterol dynamics, and sterol dynamics that were unresponsive to SCP-2. Differences between the structural and functional properties of DRM and those of the nondetergent preparations (ACR and NDCR) were not due to the presence of detergent. A nondetergent, affinity-purified (ACR) lipid domain fraction isolated from MDCK cells for the first time revealed unique structural (noncrystalline sterol, liquid-ordered, high transbilayer fluidity gradient) and functional (cholesterol dynamics) properties of lipid rafts as compared to nonrafts (NR). In summary, this study showed membrane microdomains (rafts/caveolae) isolated by three different methodologies have unique structural, functional, and organizational characteristics.     

Porin is present in the plasma membrane where it is concentrated in caveolae and caveolae-related domains.

Mitochondrial porin, or voltage-dependent anion channel, is a pore-forming protein first discovered in the outer mitochondrial membrane. Later investigations have provided indications for its presence also in other cellular membranes, including the plasma membrane, and in caveolae. This extra-mitochondrial localization is debated and no clear-cut conclusion has been reached up to now. In this work, we used biochemical and electrophysiological techniques to detect and characterize porin within isolated caveolae and caveolae-like domains (low density Triton-insoluble fractions). A new procedure was used to isolate porin from plasma membrane. The outer surface of cultured CEM cells was biotinylated by an impermeable reagent. Low density Triton-insoluble fractions were prepared from the labeled cells and used as starting material to purify a biotinylated protein with the same electrophoretic mobility and immunoreactivity of mitochondrial porin. In planar bilayers, the porin from these sources formed slightly anion-selective pores with properties indistinguishable from those of mitochondrial porin. This work thus provides a strong indication of the presence of porin in the plasma membrane, and specifically in caveolae and caveolae-like domains.     

Membrane microdomains and caveolae.

Glycosphingolipid- and cholesterol-enriched microdomains, or rafts, within the plasma membrane of eukaryotic cells have been implicated in many important cellular processes, such as polarized sorting of apical membrane proteins in epithelial cells and signal transduction. Until recently, however, the existence of such domains remained controversial. The past year has brought compelling evidence that microdomains indeed exist in living cells. In addition, several recent papers have suggested that caveolae, which are considered to be a specific form of raft, and caveolins, the major membrane proteins of caveolae, are involved in the dynamic cholesterol-dependent regulation of specific signal transduction pathways.     

Cross-talk between phospholipase D and MAP kinases activities

Phospholipase D interacts with both p38 MAP kinase and the ERK2 MAP kinase in HeLa cells. Inhibition of PLD signaling was without effect upon p38 MAP kinase activity whilst inhibition of ERK signaling was without effect upon PLD activity. Therefore there exists cross-talk between MAP kinase and PLD signaling the details of which remain to be elucidated.     

Cross-talk between IRAK-4 and the NADPH oxidase

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.     

Intracellular trafficking of raft/caveolae domains: insights from integrin signaling

Cells have a complex system for delivering and compartmentalizing proteins and lipids in order to achieve spatio-temporal coordination of signaling. Rafts/caveolae are plasma membrane microdomains that regulate signaling pathways and processes such as cell migration, polarization and proliferation. Regulation of raft/caveolae trafficking involves multiple steps regulated by different proteins to ensure coordination of signaling cascades. The best studied raft-mediated endocytic route is controlled by caveolins. Recent data suggest integrin-mediated cell adhesion is a key regulator of caveolar endocytosis. In this review we examine the regulation of caveolar trafficking and the interplay between integrins, cell adhesion and caveolae internalization.     

Cross-talk among structural domains of human DBP upon binding 25-hydroxyvitamin D

Serum vitamin D-binding protein (DBP) is structurally very similar to serum albumin (ALB); both have three distinct structural domains and high cysteine-content. Yet, functionally they are very different. DBP possesses high affinity for vitamin D metabolites and G-actin, but ALB does not. It has been suggested that there may be cross-talk among the domains so that binding of one ligand may influence the binding of others. In this study we have employed 2-p-toluidinyl-6-sulfonate (TNS), a reporter molecule that fluoresces upon binding to hydrophobic pockets of DBP. We observed that recombinant domain III possesses strong binding for TNS, which is not influenced by 25-hydroxyvitamin D₃ (25-OH-D₃), yet TNS fluorescence of the whole protein is quenched by 25-OH-D₃. These results provide a direct evidence of cross-talk among the structural domains of DBP.