A link between the light and gibberellin signaling cascades.

In a recent issue of Cell, Salomé Prat and colleagues describe the characterization of PHOR1, an armadillo-related protein involved in gibberellin signaling and also responsive to light.     

Kainate receptor activation induces mixed lineage kinase-mediated cellular signaling cascades via post-synaptic density protein 95

Kainate receptor glutamate receptor 6 (GluR6) subunit-deficient and c-Jun N-terminal kinase 3 (JNK3)-null mice share similar phenotypes including resistance to kainite-induced epileptic seizures and neuronal toxicity (Yang, D. D., Kuan, C-Y., Whitmarsh, A. J., Rincon, M., Zheng, T. S., Davis, R. J., Rakis, P., and Flavell, R. (1997) Nature 389, 865-869; Mulle, C., Seiler, A., Perez-Otano, I., Dickinson-Anson, H., Castillo, P. E., Bureau, I., Maron, C., Gage, F. H., Mann, J. R., Bettler, B., and Heinemmann, S. F. (1998) Nature 392, 601-605). This suggests that JNK activation may be involved in GluR6-mediated excitotoxicity. We provide evidence that post-synaptic density protein (PSD-95) links GluR6 to JNK activation by anchoring mixed lineage kinase (MLK) 2 or MLK3, upstream activators of JNKs, to the receptor complex. Association of MLK2 and MLK3 with PSD-95 in HN33 cells and rat brain preparations is dependent upon the SH3 domain of PSD-95, and expression of GluR6 in HN33 cells activated JNKs and induced neuronal apoptosis. Deletion of the PSD-95-binding site of GluR6 reduced both JNK activation and neuronal toxicity. Co-expression of dominant negative MLK2, MLK3, or mitogen-activated kinase kinase (MKK) 4 and MKK7 also significantly attenuated JNK activation and neuronal toxicity mediated by GluR6, and co-expression of PSD-95 with a deficient Src homology 3 domain also inhibited GluR6-induced JNK activation and neuronal toxicity. Our results suggest that PSD-95 plays a critical role in GluR6-mediated JNK activation and excitotoxicity by anchoring MLK to the receptor complex.     

A generic constitutive model for the passive porcine coronary artery

Constitutive models describing the arterial mechanical behavior are important in the development of catheterization products, to be used in arteries with a specific radius. To prove the possible existence of a constitutive model that, provided with a generic set of material and geometric parameters, is able to predict the radius-specific mechanical behavior of a coronary artery, the passive pressure–inner radius (P–r _i ) and pressure–axial force change (P–ΔF _z ) relations of seven porcine left anterior descending coronary arteries were measured in an in-vitro set-up and fitted with the model of Driessen et al. in J Biomech Eng 127(3):494–503 (2005), Biomech Model Mechanobiol 7(2):93–103 (2008). Additionally, the collagen volume fraction, physiological axial pre-stretch, and wall thickness to inner radius ratio at physiological loading were determined for each artery. From this, two generic parameter sets, each comprising four material and three geometric parameters, were obtained. These generic sets were used to compute the deformation of each tested artery using a single radius measurement at physiological loading as an artery-specific input. Artery-specific P–r _i and P–ΔF _z relations were predicted with an accuracy of 32 μm (2.3%) and 6 mN (29% relative to ΔF _z -range) on average compared to the relations measured in-vitro. It was concluded that the constitutive model provided with the generic parameters found in this study can well predict artery-specific mechanical behavior.     

Primary platelet signaling cascades and integrin-mediated signaling control ADP-ribosylation factor (Arf) 6-GTP levels during platelet activation and aggregation

Previous studies showed that ADP-ribosylation factor 6 (Arf6) is important for platelet function; however, little is known about which signaling events regulate this small GTP-binding protein. Arf6-GTP was monitored in platelets stimulated with a number of agonists (TRAP, thrombin, convulxin, collagen, PMA, thapsigargin, or A23187) and all led to a time-dependent decrease in Arf6-GTP. ADP and U46619 were without effect. Using inhibitors, it was shown that the decrease of Arf6-GTP is a direct consequence of known signaling cascades. Upon stimulation via PAR receptors, Arf6-GTP loss could be blocked by treatment with U-73122, BAPTA/AM, Ro-31-8220, or Gö6976, indicating requirements for phospholipase C, calcium, and protein kinase C (PKC) α/β, respectively. The Arf6-GTP decrease in convulxin-stimulated platelets showed similar requirements and was also sensitive to piceatannol, wortmannin, and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, indicating additional requirements for Syk and phosphatidylinositol 3-kinase. The convulxin-induced decrease was sensitive to both PKCα/β and δ inhibitors. Outside-in signaling, potentially via integrin engagement, caused a second wave of signaling that affected Arf6. Inclusion of RGDS peptides or EGTA, during activation, led to a biphasic response; Arf6-GTP levels partially recovered upon continued incubation. A similar response was seen in β3 integrin-null platelets. These data show that Arf6-GTP decreases in response to known signaling pathways associated with PAR and GPVI. They further reveal a second, aggregation-dependent, process that dampens Arf6-GTP recovery. This study demonstrates that the nucleotide state of Arf6 in platelets is regulated during the initial phases of activation and during the later stages of aggregation.Platelet activation is initiated through several classes of membrane receptors, which are stimulated by agonists produced at the vascular lesion (1–3). A second wave of signaling, caused by engagement of integrins, occurs as platelets bind to the lesion surface and aggregate (4). Together, these plasma membrane proteins initiate the platelet processes important for thrombosis (e.g. adhesion, spreading, secretion, and clot retraction). Small GTP-binding proteins, specifically members of the Ras superfamily, link signaling events from various platelet receptors to defined outcomes, such as shape change (5–7), aggregation (8, 9), and secretion (10–12). Rab proteins play roles in granule secretion, with Rab4 and Rab6 being involved in alpha granule release (10, 11) and Rab27a/b in dense core granule release (12, 13). RalA is activated in response to various stimuli (14–16) and may play a role in secretion by anchoring the exocyst complex to specific membrane sites (17). Rap1 plays a role in integrin α_IIbβ₃ activation (8, 9). Rho family GTPases (Rho, Rac, and Cdc42) play roles in platelet phosphoinositide signaling and in the regulation of the actin cytoskeleton (5–7). While these small GTP-binding proteins are clearly important to platelet function, it is equally clear that other small G proteins are present and functional in platelets (18).The ADP-ribosylation factor (Arf)² family are Ras-related, small GTPases that affect both vesicular transport and cytoskeletal dynamics (19, 20). Based on their primary sequences, this family is divided into three classes, with Arf6 as the only member of class III (19). Arf6-GTP is considered the “active state” and can interact with downstream effectors, such as phospholipase D (PLD) (21), phosphatidylinositol 4-phosphate 5-kinase type α (22), and arfaptin 2 (23, 24), resulting in the recruitment of these effectors to the plasma membrane. The Arf6 GTP/GDP cycle is mediated by interactions with guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). The large number of Arf-GEF and -GAP proteins have been discussed in recent reviews where it was noted that, unlike other small GTPases, Arf functions are generally not mediated solely by the GTP-bound state but through its cycling between states (19, 20, 25, 26).The effects that Arf6 has on the secretion and actin dynamics in nucleated cells make it an ideal candidate for function in platelets. Arf6 influences cortical actin and is important for spreading, ruffling, migration, and phagocytosis (reviewed in Ref. 19). Our previous work (27) showed that Arf6 is present on platelet membranes and is important for platelet function. Unlike other small G proteins, the Arf6 GTP-bound form is readily detectible in resting platelets and upon activation with collagen or convulxin there is a rapid conversion to the GDP-bound form. Acylated peptides, which mimic the myristoylated N terminus of Arfs have been used as isoform-specific inhibitors (28). In platelets, a myristoylated-Arf6 (myr-Arf6) peptide specifically blocks the activation-dependent loss of Arf6-GTP. This peptide also blocks aggregation, spreading on collagen, and activation of the Rho family of GTPases. Other GTPases, such as Ral and Rap, were unaffected. The simplest explanation for these data is that platelet activation stimulates the GTPase activity of Arf6, perhaps through activation of an Arf6-GAP. Alternatively, platelet activation could affect an Arf6-GEF thus reducing the production of Arf6-GTP. Regardless of mechanism, disruption of the activation-dependent loss of Arf6-GTP, with the myr-Arf6 peptide, profoundly affects the actin-based cytoskeletal rearrangements associated with platelet activation. While our initial report (27) established a role for Arf6 in platelet function, it was not clear what platelet signaling events were required to induce the loss of Arf6-GTP.In this article, we delineate the signaling cascades required for the activation-dependent loss of Arf6-GTP. We show that the Arf6-GTP to -GDP conversion was stimulated by primary agonists (thrombin, TRAP, collagen, or convulxin) but not by ADP or U46619. The decrease in Arf6-GTP, downstream of thrombin and convulxin, required PLC, and PKC activity. Loss of Arf6-GTP, via stimulation of GPVI with convulxin, additionally required Syk and PI3K activities. Pretreatment with passivators, nitric oxide (NO), and prostaglandin I₂ (PGI₂) blocked thrombin- and convulxin-induced loss of Arf6-GTP. Further experiments suggested a role for “outside-in” signaling, especially once platelet aggregates begin to form. Inclusion of RGDS peptide, EGTA, or the deletion of the β3 integrin had only minimal effects on the initial loss of Arf6-GTP but led to the partial recovery of Arf6-GTP levels. This biphasic change in Arf6-GTP levels was not seen when aggregation was allowed to occur normally. Taken together, these data show that the Arf6 nucleotide state is responsive to both initial agonist-mediated signaling and to a second wave of integrin-mediated signaling that occurs upon aggregation.     

MCP-1-stimulated chemotaxis of monocytic and endothelial cells is dependent on activation of different signaling cascades

Monocyte chemoattractant protein-1 (MCP-1) is important in attracting monocytes to sites of inflammation. Besides induction of monocyte recruitment, MCP-1 can also affect chemotactic response of endothelial cells. The molecular mechanisms involved in MCP-1-induced cell migration are poorly understood. In the current investigation, we demonstrate activation of p42/44(ERK1/2) and p38 mitogen-activated protein kinases (MAPKs), phosphatydilinositol-3-kinase (PI3K) and Src-kinases in both monocytes and endothelial cells stimulated with MCP-1 in vitro. The response was rapid and time-dependent, detectable within 3 min of MCP-1 stimulation. MCP-1-induced phosphorylation of p42/44(ERK1/2) MAPKs was partially blocked by inhibitor of PI3K LY294002, while phosphorylation of p38 MAPK was diminished to a greater extent in presence of Src-kinase inhibitor PP2. There was a substantial inhibition of monocyte migration upon treatment with inhibitors of p38 MAPK, at the same time inhibition of p42/44(ERK1/2) MAPK activation had no effect. On the contrary, the MCP-1-stimulated chemotaxis of endothelial cells was completely abolished by inhibitors of PI3K and p42/44(ERK1/2), but not by p38 MAPK inhibitors. These results suggest that parallel signal transduction pathways are activated by MCP-1, and that depending on the cell type these pathways differentially contribute to cell chemotactic activity.     

A generic theoretical model for biological control of foliar plant diseases

We have developed a generic modelling framework to understand the dynamics of foliar pathogen and biocontrol agent (BCA) populations in order to predict the likelihood of successful biocontrol in relation to the mechanisms involved. The model considers biocontrol systems for foliar pathogens only and, although it is most applicable to fungal BCA systems, does not address a specific biocontrol system. Four biocontrol mechanisms (competition, antibiosis, mycoparasitism and induced resistance) were included within the model rubric. Because of the wide range of mechanisms involved we use Trichoderma/Botrytis as an exemplar system. Qualitative analysis of the model showed that the rates of a BCA colonising diseased and/or healthy plant tissues and the time that the BCA remains active are two of the more important factors in determining the final outcome of a biocontrol system. Further evaluation of the model indicated that the dynamic path to the steady-state population levels also depends critically on other parameters such as the host-pathogen infection rate. In principle, the model can be extended to include other potential mechanisms, including spatio-temporal heterogeneity, fungicide effects, non-fungal BCA and strategies for BCA application, although with a cost in model tractability and ease of interpretation.     

A generic, object-oriented data model for life cycle analysis applications

Life Cycle Analysis (LCA) requires the manipulation of a large amount of both quantitative and qualitative data. It is also a continuous and iterative process in which the knowledge of previous steps is always necessary for the realization and understanding of subsequent stages. Current LCA data models are mostly organized as lists from which quantitative data can be extracted and manipulated, subsequently leading to lists of numerical results. Performing an LCA can benefit from more flexible structures that allow for complete or partial treatments and provides links between the successive transformations of various types of information. A generic, object-oriented model is presented, written in ISO/STEP EXPRESS, using the STEP principles.     

Transient activation of the MAP kinase signaling pathway by the forward signaling of EphA4 in PC12 cells

In the present study, we demonstrate that ephrin-A5 is able to induce a transient increase of MAP kinase activity in PC12 cells. However, the effects of ephrin-A5 on the MAP kinase signaling pathway are about three-fold less than that of EGF. In addition, we demonstrate that EphA4 is the only Eph member expressed in PC12 cells, and that tyrosine phosphorylation induced by ephrin-A5 treatment is consistent with the magnitude and longevity of MAP kinase activation. Experiments using the Ras dominant negative mutant N17Ras reveal that Ras plays a pivotal role in ephrin-A5-induced MAP kinase activation in PC12 cells. Importantly, we found that the EphA4 receptor is rapidly internalized by endocytosis upon engagement of ephrin-A5, leading to a subsequent reduction in the MAP kinase activation. Together, these data suggest a novel regulatory mechanism of differential Ras-MAP kinase signaling kinetics exhibited by the forward signaling of EphA4 in PC12 cells.     

A model for steady isometric muscle activation

The present model of the motoneuronal (MN) pool – muscle complex (MNPMC) is deterministic and designed for steady isometric muscle activation. Time-dependent quantities are treated as time-averages. The character of the model is continuous in the sense that the motor unit (MU) population is described by a continuous density function. In contrast to most already published models, the wiring (synaptic weight) between the input fibers to the MNPMC and the MNs (about which no detailed data are known) is deduced, whereas the input–force relation is given. As suggested by experimental data, this relation is assumed to be linear during MU recruitment, but the model allows other, nonlinear relations. The input to the MN pool is defined as the number of action potentials per second in all input fibers, and the excitatory postsynaptic potential (EPSP) conductance in MNs evoked by the input is assumed to be proportional to the input. A single compartment model with a homogeneous membrane is used for a MN. The MNs start firing after passing a constant voltage threshold. The synaptic current–frequency relation is described by a linear function and the frequency–force transformation of a MU by an exponential function. The sum of the MU contraction forces is the muscle force, and the activation of the MUs obeys the size principle. The model parameters were determined a priori, i.e., the model was not used for their estimation. The analysis of the model reveals special features of the activation curve which we define as the relation between the input normalized by the threshold input of the MN pool and the force normalized by the maximal muscle force. This curve for any muscle turned out to be completely determined by the activation factor, the slope of the linear part of the activation curve (during MU recruitment). This factor determines quantitatively the relation between MU recruitment and rate modulation. This property of the model (the only known model with this property) allows a quantification of the recruitment gain (Kernell and Hultborn 1990). The interest of the activation factor is illustrated using two human muscles, namely the first dorsal interosseus muscle, a small muscle with a relatively small force at the end of recruitment, and the medial gastrocnemius muscle, a strong muscle with a relatively large force at the end of recruitment. It is concluded that the present model allows us to reproduce the main features of muscle activation in the steady state. Its analytical character facilitates a deeper understanding of these features. Received: 24 November 1997 / Accepted in revised form: 30 November 1998     

A role of some intracellular signaling cascades in planarian regeneration activated under irradiation with low-temperature argon plasma

Using inhibitory analysis the role of some intracellular signaling pathways in activation of planarian regeneration under the influence of low-temperature argon plasma (LTAP) has been investigated. Inactivation of specific inhibitors of intracellular signaling enzymes such as the receptor tyrosine kinase (EGFR), TGF β receptor, calmodulin, adenylate cyclase, phospholipase A2, phospholipase C, cyclin-dependent protein kinase, JAK2-protein kinase, JNK-protein kinase MEK-protein kinase led to inhibition of the head growth during its regeneration in planarians. Pretreatment with LTAP irradiation provided no inhibitory action of some cascades regulating proliferation. However, the inhibitors of the key regulators of regeneration: TGF β receptor, calmodulin and MEK-protein kinase completely suppressed the activating effect of plasma. Thus, by the example of regenerating planarians it is shown, that biological activity of low-temperature argon plasma LTAP is caused by modulation of a plurality of cellular signaling systems.     

A unified model for apical caspase activation

Apoptosis is orchestrated by the concerted action of caspases, activated in a minimal two-step proteolytic cascade. Existing data suggests that apical caspases are activated by adaptor-mediated clustering of inactive zymogens. However, the mechanism by which apical caspases achieve catalytic competence in their recruitment/activation complexes remains unresolved. We explain that proximity-induced activation of apical caspases is attributable to dimerization. Internal proteolysis does not activate these apical caspases but is a secondary event resulting in partial stabilization of activated dimers. Activation of caspases-8 and -9 occurs by dimerization that is fully recapitulated in vitro by kosmotropes, salts with the ability to stabilize the structure of proteins. Further, single amino acid substitutions at the dimer interface abrogate the activity of caspases-8 and -9 introduced into recipient mammalian cells. We propose a unified caspase activation hypothesis whereby apical caspases are activated by dimerization of monomeric zymogens.     

Restricted collision coupling of the A2A receptor revisited: evidence for physical separation of two signaling cascades

The A(2A)-adenosine receptor is a prototypical G(s) protein-coupled receptor but stimulates MAPK/ERK in a G(s)-independent way. The A(2A) receptor has long been known to undergo restricted collision coupling with G(s); the mechanistic basis for this mode of coupling has remained elusive. Here we visualized agonist-induced changes in mobility of the yellow fluorescent protein-tagged receptor by fluorescence recovery after photobleaching microscopy. Stimulation with a specific A(2A) receptor agonist did not affect receptor mobility. In contrast, stimulation with dopamine decreased the mobility of the D(2) receptor. When coexpressed in the same cell, the A(2A) receptor precluded the agonist-induced change in D(2) receptor mobility. Thus, the A(2A) receptor did not only undergo restricted collision coupling, but it also restricted the mobility of the D(2) receptor. Restricted mobility was not due to tethering to the actin cytoskeleton but was, in part, related to the cholesterol content of the membrane. Depletion of cholesterol increased receptor mobility but blunted activation of adenylyl cyclase, which was accounted for by impaired formation of the ternary complex of agonist, receptor, and G protein. These observations support the conclusion that the A(2A) receptor engages G(s) and thus signals to adenylyl cyclase in cholesterol-rich domains of the membrane. In contrast, stimulation of MAPK by the A(2A) receptor was not impaired. These findings are consistent with a model where the recruitment of these two pathways occurs in physically segregated membrane microdomains. Thus, the A(2A) receptor is the first example of a G protein-coupled receptor documented to select signaling pathways in a manner dependent on the lipid microenvironment of the membrane.     

Ectodomain shedding of TGF-alpha and other transmembrane proteins is induced by receptor tyrosine kinase activation and MAP kinase signaling cascades

A variety of transmembrane proteins, such as transforming growth factor-alpha (TGF-alpha), tumor necrosis factor-alpha (TNF-alpha) and L-selectin, undergo shedding, i.e. cleavage of the ectodomain, resulting in release of a soluble protein. Although the physiological relevance of ectodomain shedding is well recognized, little is known about the signaling mechanisms activating this process. We show that growth factor activation of cell surface tyrosine kinase receptors induces ectodomain cleavage of transmembrane TGF-alpha through activation of the Erk MAP kinase signaling cascade without the need for new protein synthesis. In addition, expression of constitutively activated MEK1 or its downstream target Erk2 MAP kinase was sufficient to stimulate TGF-alpha shedding. The basal cleavage level in the absence of exogenous growth factor stimulation was due to p38 MAP kinase signaling. Accordingly, a constitutively activated MKK6, a p38 activator, activated TGF-alpha shedding in the absence of exogenous stimuli. In addition to TGF-alpha shedding, these mechanisms also mediate L-selectin and TNF-alpha cleavage. Thus, L-selectin shedding by neutrophils, induced by N-formylmethionyl-leucyl-phenylalanine, was strongly inhibited by inhibitors of Erk MAP kinase or p38 MAP kinase signaling. Our results indicate that activation of Erk and p38 signaling pathways may represent a general physiological mechanism to induce shedding of a variety of transmembrane proteins.