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High pigment1 mutation negatively regulates phototropic signal transduction in tomato seedlings.

Phototropins and phytochromes are the major photosensory receptors in plants and they regulate distinct photomorphogenic responses. The molecular mechanisms underlying functional interactions of phototropins and phytochromes remain largely unclear. We show that the tomato (Lycopersicon esculentum) phytochrome A deficient mutant fri lacks phototropic curvature to low fluence blue light, indicating requirement for phytochrome A for expression of phototropic response. The hp1 mutant that exhibits hypersensitive responses to blue light and red light reverses the impairment of second-positive phototropic response in tomato in phytochrome A-deficient background. Physiological analyses indicate that HP1 functions as a negative regulator of phototropic signal transduction pathway, which is removed via action of phytochrome A. The loss of HP1 gene product in frihp1 double mutant allows the unhindered operation of phototropic signal transduction chain, obviating the need for the phytochrome action. Our results also indicate that the role of phytochrome in regulating phototropism is restricted to low fluence blue light only, and at high fluence blue light, the phytochrome A-deficient fri mutant shows the normal phototropic response.

Chloroplasts↗

Hsp90 recognizes a common surface on client kinases.

Hsp90 is a highly abundant chaperone whose clientele includes hundreds of cellular proteins, many of which are central players in key signal transduction pathways and the majority of which are protein kinases. In light of the variety of Hsp90 clientele, the mechanism of selectivity of the chaperone toward its client proteins is a major open question. Focusing on human kinases, we have demonstrated that the chaperone recognizes a common surface in the amino-terminal lobe of kinases from diverse families, including two newly identified clients, NFkappaB-inducing kinase and death-associated protein kinase, and the oncoprotein HER2/ErbB-2. Surface electrostatics determine the interaction with the Hsp90 chaperone complex such that introduction of a negative charge within this region disrupts recognition. Compiling information on the Hsp90 dependence of 105 protein kinases, including 16 kinases whose relationship to Hsp90 is first examined in this study, reveals that surface features, rather than a contiguous amino acid sequence, define the capacity of the Hsp90 chaperone machine to recognize client kinases. Analyzing Hsp90 regulation of two major signaling cascades, the mitogen-activated protein kinase and phosphatidylinositol 3-kinase, leads us to propose that the selectivity of the chaperone to specific kinases is functional, namely that Hsp90 controls kinases that function as hubs integrating multiple inputs. These lessons bear significance to pharmacological attempts to target the chaperone in human pathologies, such as cancer.

Amino Acid Sequence↗

Protein-tyrosine phosphatases: structure, mechanism, and inhibitor discovery.

Protein-tyrosine kinases (PTKs) and their associated signaling pathways are crucial for the regulation of numerous cell functions including growth, mitogenesis, motility, cell-cell interactions, metabolism, gene transcription, and the immune response. Since tyrosine phosphorylation is reversible and dynamic in vivo, the phosphorylation states of proteins are governed by the opposing actions of PTKs and protein-tyrosine phosphatases (PTPs). In this light, both PTKs and PTPs play equally important roles in signal transduction in eukaryotic cells, and comprehension of mechanisms behind the reversible pTyr-dependent modulation of protein function and cell physiology must necessarily encompass the characterization of PTPs as well as PTKs. In spite of the large number of PTPs identified to date and the emerging role played by PTPs in disease, a detailed understanding of the role played by PTPs in signaling pathways has been hampered by the absence of PTP-specific agents. Such PTP-specific inhibitors could potentially serve as useful tools in determining the physiological significance of protein tyrosine phosphorylation in complex cellular signal transduction pathways and may constitute valuable therapeutics in the treatment of several human diseases. The goal of this review is therefore to summarize current understandings of PTP structure and mechanism of catalysis and the relationship of these to PTP inhibitor development. The review is organized such that enzyme structure is covered first, followed by mechanisms of catalysis then PTP inhibitor development. In discussing PTP inhibitor development, nonspecific inhibitors and those obtained by screening methods are initially presented with the focus then shifting to inhibitors that utilize a more structure-based rationale.

Animals↗

Regulation of TNF-alpha-induced reorganization of the actin cytoskeleton and cell-cell junctions by Rho, Rac, and Cdc42 in human endothelial cells.

We have investigated the role of the small guanosine-trisphosphate (GTP)-binding proteins, Rho, Rac, and Cdc42, in the early responses of human umbilical vein endothelial cells (HUVECs) to TNF-alpha (tumor necrosis factor-alpha). Quiescent confluent HUVECs incubated with TNF-alpha for 5-30 min showed an increased formation of membrane ruffles, filopodia, and actin stress fibres followed by cell retraction and formation of intercellular gaps. This process was accompanied by the dispersion of cadherin-5 from intercellular junctions. TNF-alpha also induced a transient increase in polymerized F-actin, as determined both by measuring G-actin content and by quantifying fluorescent emission from fluorescein isothiocyanate (FITC)-phalloidin-labelled F-actin. Microinjection of cells with activated RhoA protein led to an increase in polymerized actin, formation of stress fibres, cell retraction as well as dispersion of cadherin-5. The proteins Cdc42 and Rac induced qualitatively similar effects to Rho, although not as dramatic and in addition induced formation of filopodia and lamellipodia. Microinjection of cells with a Rho inhibitor, C3 transferase, prevented gap formation caused by TNF-alpha. Similar effects were observed in cells microinjected with the dominant inhibitory proteins N17Cdc42 and N17Rac1. Cell retraction and gap formation were also prevented by inhibitors of myosin light chain kinase (MLCK). Our data suggest that Cdc42, Rac, and Rho are activated in a hierarchical cascade following stimulation with TNF-alpha leading to actomyosin-mediated cell retraction and formation of intercellular gaps.

ADP Ribose Transferases↗

Subcellular, cellular, and circuit mechanisms underlying classical conditioning in Hermissenda crassicornis.

A breakthrough for studying the neuronal basis of learning emerged when invertebrates with simple nervous systems, such as the sea slug Hermissenda crassicornis, were shown to exhibit classical conditioning. Hermissenda learns to associate light with turbulence: prior to learning, naive animals move toward light (phototaxis) and contract their foot in response to turbulence; after learning, conditioned animals delay phototaxis in response to light. The photoreceptors of the eye, which receive monosynaptic inputs from statocyst hair cells, are both sensory neurons and the first site of sensory convergence. The memory of light associated with turbulence is stored as changes in intrinsic and synaptic currents in these photoreceptors. The subcellular mechanisms producing these changes include activation of protein kinase C and MAP kinase, which act as coincidence detectors because they are activated by convergent signaling pathways. Pathways of interneurons and motorneurons, where additional changes in excitability and synaptic connections are found, contribute to delayed phototaxis. Bursting activity recorded at several points suggest the existence of small networks that produce complex spatiotemporal firing patterns. Thus, the change in behavior may be produced by a nonlinear transformation of spatiotemporal firing patterns caused by plasticity of synaptic and intrinsic channels. The change in currents and the activation of PKC and MAPK produced by associative learning are similar to those observed in hippocampal and cerebellar neurons after rabbit classical conditioning, suggesting that these represent general mechanisms of memory storage. Thus, the knowledge gained from further study of Hermissenda will continue to illuminate mechanisms of mammalian learning.

Animals↗

Control of basement membrane remodeling and epithelial branching morphogenesis in embryonic lung by Rho and cytoskeletal tension.

Local alterations in the mechanical compliance of the basement membrane that alter the level of isometric tension in the cell have been postulated to influence tissue morphogenesis. To explore whether cell tension contributes to tissue pattern formation in vivo, we modulated cytoskeletal force generation in embryonic mouse lung (embryonic days 12-14) rudiments using inhibitors of Rho-associated kinase (ROCK), myosin light chain kinase, myosin ATPase, and microfilament integrity, or a Rho stimulator (cytotoxic necrotizing factor-1). Tension inhibition resulted in loss of normal differentials in basement membrane thickness, inhibition of new terminal bud formation, and disorganization of epithelial growth patterns as well as disruption of capillary blood vessels. In contrast, increasing cell tension through Rho activation, as confirmed by quantitation of myosin light chain phosphorylation and immunohistocytochemical analysis of actin organization, accelerated lung branching and increase capillary elongation. These data suggest that changes in cytoskeletal tension mediated by Rho signaling through ROCK may play an important role in the establishment of the spatial differentials in cell growth and extracellular matrix remodeling that drive embryonic lung development.

Actins↗

Illuminating the clock: circadian photobiology.

Light and circadian (daily) clocks are intimately related. Undoubtedly, the daily light/dark cycle has played a key role in the evolution of these oscillators. Now, light and dark signals synchronize these internal timekeepers to the precisely 24 h environmental cycle. Light also affects the period and amplitude of circadian rhythms. The expression of some 'light-responsive' genes are regulated by an intertwining of clock and light control mechanisms. These genes may help us to understand the evolution of circadian rhythmicity and can be employed as tools to identify and clone other genes which encode components of clock mechanisms or of their entrainment pathways.

Circadian Rhythm↗

Different retina-lamina projections in mosquitoes with fused and open rhabdoms.

Anopheles gambiae and Toxorhynchites brevipalpis represent the nocturnal and diurnal extremes of the mosquito light intensity range, and their eyes are structurally very different. A. gambiae has fused rhabdoms with huge acceptance angles, whereas T. brevipalpis has open rhabdoms with rhabdomere acceptance angles comparable with those of advanced (brachyceran) flies. Here, we show that the retina-lamina projections are consistent with these differences. The short receptor axons from each ommatidium in A. gambiae insert as a group between four lamina monopolar cell clusters. In T. brevipalpis axon bundles from each ommatidium undergo a twist in their passage through the nuclear layer of the lamina, and then fan out into a space the diameter of which is about twice the separation of the monopolar cell clusters. This arrangement is consistent with a neural superposition mechanism closely similar to that found in higher Diptera, but which must have evolved independently.

Animals↗

Photosensitivity of 8BrcGMP-induced conductance in ROS-excised patches.

Inside-out patches from ROS plasma membranes contain the basic enzymes of the phototransduction cascade. Similar to a native photoreceptor cell, such patches are capable of responding to light, the effect of which suppresses the cGMP-activated current. Photoresponses are observed only in the presence of GTP, whereas ATP essentially accelerates the current recovery to a dark level. Photoresponses are also observed in the presence of 8BrcGMP. Phosphodiesterase (PDE) hydrolyzes 8BrcGMP two orders of magnitude slower than cGMP, so the light inhibition of the 8BrcGMP-induced current cannot be accounted for by PDE activation. It seems that activity of cGMP-gated channels depends not only on cGMP concentration, but is additionally controlled by some other regulatory mechanisms.

1-Methyl-3-isobutylxanthine↗

Bioactivity and protective effects of natural carotenoids.

Carotenoids comprise a class of natural fat-soluble pigments which are found in numerous fruits and vegetables. The consumption of a diet rich in carotenoids has been epidemiologically correlated with a lower risk for several diseases. The antioxidant activity of carotenoids and biochemical properties influencing signaling pathways have been discussed as basic mechanisms of prevention. Conflicting data from intervention studies with beta-carotene to prevent cancers and cardiovascular disorders have challenged the concept. However, there is convincing evidence that carotenoids are important components of the antioxidant network. Photooxidative damage is suggested to be involved in the pathobiochemistry of several diseases affecting the skin and the eye, and carotenoids may protect light-exposed tissues. Lutein and zeaxanthin are the predominant carotenoids of the retina and are considered to act as photoprotectants preventing retinal degeneration. The unique distribution, localization and high levels of both carotenoids within the macula lutea as well as their physicochemical properties make them suitable candidates for photoprotection. beta-Carotene is used as an oral sun protectant for the prevention of sunburn and has been shown to be effective either alone or in combination with other carotenoids or antioxidant vitamins. Protective effects are also achieved with a diet rich in lycopene.

Animals↗

Possible role of light in the maintenance of EIN3/EIL1 stability in Arabidopsis seedlings.

To examine the mechanism of EIN3-mediated gene expression by ethylene, the expression patterns of ethylene-inducible genes by ethylene were monitored in Col-0 and ethylene signaling mutants. In Col-0, the inducibility of ACC oxidase by ethylene in light-grown seedlings was much higher than in dark-grown seedlings. While the expression of ACC oxidase was highly increased by ethylene not only in Col-0 but in ein3-1 under light treatment, this pattern was completely abrogated in etiolated ein3-1 seedlings, suggesting the expression of EIN3-mediated ACC oxidase genes could be affected by light. To check if the level of EIN3 and EIL1 was regulated by light, cell-free degradation assays were performed. This resulted in the rapid degradation of these proteins within 1h after adding dark-grown cell extracts and this degradation was retarded by light-grown extracts. Here, we propose that light may act as a negative regulator in the destabilization of EIN3/EIL1.

Amino Acid Oxidoreductases↗

Involvement of Ca2+ channels in endothelin-1-induced MAP kinase phosphorylation, myosin light chain phosphorylation and contraction in rabbit iris sphincter smooth muscle.

The purpose of the present study was to investigate the role and type of Ca2+ channels involved in the stimulatory effects of endothelin-1 (ET-1) on the Ca2+-dependent functional responses, p42/p44 MAP kinase phosphorylation, 20-kDa myosin light chain (MLC) phosphorylation and contraction, in rabbit iris sphincter, a nonvascular smooth muscle. ET-1 induced inositol phosphates production, MAP kinase phosphorylation, MLC phosphorylation (MLC20-P plus MLC20-2P) and contraction in a concentration-dependent manner with EC50 values of 71, 8, 6 and 25 nM, respectively. ET-1-induced MAP kinase phosphorylation, MLC phosphorylation and contraction were not significantly affected by nifedipine (1-60 microM), an L-type Ca2+ channel blocker, or by LOE 908 (1-100 microM), a blocker of Ca2+-permeable nonselective cation channels. However, SKF96365, a receptor-operated Ca2+ channel (ROCC) blocker, inhibited MAP kinase phosphorylation, MLC phosphorylation and contraction in a concentration-dependent manner with IC50 values of 28, 30 and 42 microM, respectively. 2-APB, a store-operated Ca2+ channel (SOCC) blocker, inhibited ET-1-induced MLC phosphorylation and contraction in a concentration-dependent manner with IC50 values of 12.7 and 19 microM, respectively, but was without effect on MAP kinase phosphorylation. The combined effects of submaximal concentrations of SKF96365 and 2-APB on ET-1-induced MLC phosphorylation and contraction were not additive, implying that their inhibitory actions could be mediated through a common Ca2+ entry channel. PD98059, a MAP kinase inhibitor, had no effect on ET-1-induced MLC phosphorylation and contraction, suggesting that these ET-1 effects in the rabbit iris muscle are MAP kinase-independent. In conclusion, the present study demonstrated for the first time that in rabbit iris sphincter (a) ET-1, through the ETA receptor, stimulates MAP kinase phosphorylation, MLC phosphorylation and contraction in a concentration-dependent manner, (b) that these Ca2+-dependent functional responses are not significantly affected by nifedipine or LOE908, and (c) that ET-1-induced MLC phosphorylation and contraction are inhibited by SKF96365 and 2-APB, suggesting that these effects are mainly due to store- and/or receptor Ca2+ entry.

Animals↗

Altered NF-kappaB gene expression and collagen formation induced by polyunsaturated fatty acids.

Inability to control collagen formation in vital organs (e.g., fibrosis) or stimulate healthy collagen production (CP) in connective tissues (e.g., ligaments) is a major cause of death and disability. This study tested the hypothesis that arachidonic acid (AA) and eicosapentaenoic acid (EPA) influenced CP in 3T3-Swiss fibroblasts by altering gene expression in the nuclear factor-kappa B (NF-kappaB) pathway. 3T3-Swiss fibroblasts were grown in medium containing either AA or EPA. Lipopolysaccharide (LPS) was used to activate NF-kappaB, and parthenolide was used to block it. Cells treated with EPA had increased expression of genes in the NF-kappaB pathway when exposed to LPS and also produced more collagen. Parthenolide blocked NF-kappaB activation to a greater extent in EPA-treated cells and also decreased CP induced by NF-kappaB activation. Genes in the NF-kappaB signaling pathway that had increased expression in EPA-treated cells included the toll-like receptor 4 (Tlr4), adaptor proteins [TNF receptor-associated factor 6 (Traf6), myeloid differentiation primary response gene 88], signal transduction kinases (NF-kappaB-inducing kinase, inhibitors of kappa light polypeptide gene enhancer isoforms), inhibitor protein (I-kappaB alpha chain), transcription factors (nuclear factor of kappa light chain gene enhancer, (p)105 and NF-kappaB subunit p100), DNA binding proteins (cAMP response element binding protein) and response genes known to affect CP [interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), monocyte chemotactic protein-1]. This study raises the possibility that fatty acids may be used as adjuvants in combination with other therapies (e.g., selective targeting of the NF-kappaB pathway) to control collagen formation.

Animals↗

Characterisation of Upd2, a Drosophila JAK/STAT pathway ligand.

The characterisation of ligands that activate the JAK/STAT pathway has the potential to throw light onto a comparatively poorly understood aspect of this important signal transduction cascade. Here, we describe our analysis of the only invertebrate JAK/STAT pathway ligands identified to date, the Drosophila unpaired-like family. We show that upd2 is expressed in a pattern essentially identical to that of upd and demonstrate that the proteins encoded by this region activate JAK/STAT pathway signalling. Mutational analysis demonstrates a mutual semi-redundancy that can be visualised in multiple tissues known to require JAK/STAT signalling. In order to better characterise the in vivo function of these ligands, we developed a reporter based on a natural JAK/STAT pathway responsive enhancer and show that ectopic upd2 expression can effectively activate the JAK/STAT pathway. While both Upd and Upd2 are secreted JAK/STAT pathway agonists, tissue culture assays show that the signal-sequences of Upd and Upd2 confer distinct properties, with Upd associated primarily with the extracellular matrix and Upd2 secreted into the media. The differing biophysical characteristics identified for Upd-like molecules have implications for their function in vivo and adds another aspect to our understanding of cytokine signalling in Drosophila.

Amino Acid Sequence↗

Increase in intracellular cAMP is a prerequisite signal for initiation of physiological oocyte meiotic maturation in the hydrozoan Cytaeis uchidae.

In medusae of the hydrozoan Cytaeis uchidae, oocyte meiotic maturation and spawning occur as a consequence of dark-light transition. In this study, we investigated the mechanism underlying the initiation of meiotic maturation using in vitro (isolated oocytes from ovaries) and in vivo (ovarian oocytes in medusae) systems. Injection of cAMP derivatives into isolated oocytes induced meiotic maturation in a dose-dependent manner. Meiotic maturation was also achieved in isolated oocytes preloaded with caged cAMP and exposed to UV irradiation. The caged cAMP/UV irradiation-induced meiotic maturation was completely inhibited by blockers of protein kinase A (PKA), H-89, KT5720, and Rp-cAMPS. The medusae from which most parts of the umbrella were removed (umbrella-free medusae) survived for at least 2 weeks, during which time oocyte meiotic maturation and spawning occurred. When H-89 and Rp-cAMPS were injected into ovarian oocytes of umbrella-free medusae within 3 min of dark-light stimulation, meiotic maturation was inhibited or delayed. An increase in intracellular cAMP was confirmed by FlCRhR, a fluorescent cAMP indicator, in ovarian oocytes exposed to dark-light transition as well as in isolated oocytes stimulated by caged cAMP/UV irradiation. These results indicate that the cAMP/PKA signaling pathway positively contributes to light-triggered physiological oocyte meiotic maturation in Cytaeis uchidae.

Animals↗

Antagonistic dark/light-induced SigB/SigD, group 2 sigma factors, expression through redox potential and their roles in cyanobacteria.

The expression of group 2 sigma factors is characterized in a cyanobacterium Synechocystis sp. PCC 6803 grown in culture, changing light conditions (white, red and blue light, and darkness), or the presence of drugs (rifampicin, chloramphenicol, DCMU, and DBMIB), and the roles of these sigma factors are elucidated. The expression of dark/light-induced SigB/SigD was accelerated under opposite redox (oxidation/reduction) states in an electron transport chain of photosynthesis. Expression of the dark-induced lrtA and light-induced psbA2/3 transcript was significantly reduced in the sigB and sigD knockout strains, respectively. Abundant amounts of sigB transcript and protein were observed in the sigC knockout strain, implying that SigC represses SigB expression under light. These findings clearly showed that SigB/SigD with another group 2 sigma, SigC, contribute to transcription for a subset of dark/light-responsive genes in the cyanobacterium. A possible model for SigB/SigD is presented and the potential ability for promoter recognition is also discussed.

Bacterial Proteins↗