Lycopene Triggers Nrf2-AMPK Cross Talk to Alleviate Atrazine-Induced Nephrotoxicity in Mice.

Atrazine (ATR), an environmental persistent and bioaccumulative herbicide, has been associated with environmental nephrosis. Lycopene (LYC) exhibits important properties of nephroprotection, but there are limited data on the specific underlying mechanism. The primary objective of this study was to explore the therapeutic effect of LYC on ATR-induced nephrotoxicity in mice. The mice were divided randomly into 6 groups and treated as follows: control group (C), 5 mg/kg LYC group (L), 50 mg/kg ATR group (A1), 200 mg/kg ATR group (A2), 50 mg/kg ATR plus 5 mg/kg LYC group (A1+L), and 200 mg/kg ATR plus 5 mg/kg LYC group (A2+L) by oral gavage administration for 21 days. We found that pretreatment with LYC significantly suppressed the ATR-induced renal tubular epithelial cell swelling. Furthermore, LYC mitigated ATR-induced dysregulation of oxidative stress markers by reducing MDA, H2O2 levels, and increasing SOD, GPx, CAT concentration, and Nrf2 activation. Moreover, LYC activated the autophagic flux by a detectable change in autophagy-related genes (Beclin-1 and ATGs) and proteins (p62/SQSTM) and by the formation of autophagic vacuole (AV) and LC3 aggregation, in parallel with AMPK activation (pAMPK/AMPK). Herein, ATR-up-regulated nuclear factor erythroid 2-related factor 2 (Nrf2) expression and Nrf2-regulated redox genes, including quinoneoxidoreductase-1 (NQO1) and heme oxidase-1 (HO1), whereas LYC down-regulated those of the above genes. In addition, LYC suppressed ATR-induced activation of autophagy (increased LC3II/LC3I, ATGs, Beclin1, and p62, in parallel with increased AMPK activation). Collectively, our findings identified a cross talk between AMPK-activated autophagy and the Nrf2 signaling pathway in LYC-mediated nephroprotection against ATR-induced toxicity in mice kidney.

Dynamic Scaling Analysis of Molecular Motion within the LAT:Grb2:SOS Protein Network on Membranes.

Biochemical signaling pathways often involve proteins with multiple, modular interaction domains. Signaling activates binding sites, such as by tyrosine phosphorylation, which enables protein recruitment and growth of networked protein assemblies. Although widely observed, the physical properties of the assemblies, as well as the mechanisms by which they function, remain largely unknown. Here we examine molecular mobility within LAT:Grb2:SOS assemblies on supported membranes by single-molecule tracking. Trajectory analysis reveals a discrete temporal transition to subdiffusive motion below a characteristic timescale, indicating that the LAT:Grb2:SOS assembly has the dynamical structure of a loosely entangled polymer. Such dynamical analysis is also applicable in living cells, where it offers another dimension on the characteristics of cellular signaling assemblies.

Feedback activation of neurofibromin terminates growth factor-induced Ras activation.

BACKGROUND: Growth factors induce a characteristically short-lived Ras activation in cells emerging from quiescence. Extensive work has shown that transient as opposed to sustained Ras activation is critical for the induction of mitogenic programs. Mitogen-induced accumulation of active Ras-GTP results from increased nucleotide exchange driven by the nucleotide exchange factor Sos. In contrast, the mechanism accounting for signal termination and prompt restoration of basal Ras-GTP levels is unclear, but has been inferred to involve feedback inhibition of Sos. Remarkably, how GTP-hydrolase activating proteins (GAPs) participate in controlling the rise and fall of Ras-GTP levels is unknown. RESULTS: Monitoring nucleotide exchange of Ras in permeabilized cells we find, unexpectedly, that the decline of growth factor-induced Ras-GTP levels proceeds in the presence of unabated high nucleotide exchange, pointing to GAP activation as a major mechanism of signal termination. Experiments with non-hydrolysable GTP analogues and mathematical modeling confirmed and rationalized the presence of high GAP activity as Ras-GTP levels decline in a background of high nucleotide exchange. Using pharmacological and genetic approaches we document a raised activity of the neurofibromatosis type I tumor suppressor Ras-GAP neurofibromin and an involvement of Rsk1 and Rsk2 in the down-regulation of Ras-GTP levels. CONCLUSIONS: Our findings show that, in addition to feedback inhibition of Sos, feedback stimulation of the RasGAP neurofibromin enforces termination of the Ras signal in the context of growth-factor signaling. These findings ascribe a precise role to neurofibromin in growth factor-dependent control of Ras activity and illustrate how, by engaging Ras-GAP activity, mitogen-challenged cells play safe to ensure a timely termination of the Ras signal irrespectively of the reigning rate of nucleotide exchange.

Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence.

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly (15)N-labeled Ras as well as [(13)C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions.

Slit-Dependent Endocytic Trafficking of the Robo Receptor Is Required for Son of Sevenless Recruitment and Midline Axon Repulsion.

Understanding how axon guidance receptors are activated by their extracellular ligands to regulate growth cone motility is critical to learning how proper wiring is established during development. Roundabout (Robo) is one such guidance receptor that mediates repulsion from its ligand Slit in both invertebrates and vertebrates. Here we show that endocytic trafficking of the Robo receptor in response to Slit-binding is necessary for its repulsive signaling output. Dose-dependent genetic interactions and in vitro Robo activation assays support a role for Clathrin-dependent endocytosis, and entry into both the early and late endosomes as positive regulators of Slit-Robo signaling. We identify two conserved motifs in Robo's cytoplasmic domain that are required for its Clathrin-dependent endocytosis and activation in vitro; gain of function and genetic rescue experiments provide strong evidence that these trafficking events are required for Robo repulsive guidance activity in vivo. Our data support a model in which Robo's ligand-dependent internalization from the cell surface to the late endosome is essential for receptor activation and proper repulsive guidance at the midline by allowing recruitment of the downstream effector Son of Sevenless in a spatially constrained endocytic trafficking compartment.

Functional evaluation of circulating hematopoietic progenitors in Noonan syndrome.

Noonan syndrome (NS) is an autosomal dominant disorder, characterized by short stature, multiple dysmorphisms and congenital heart defects. A myeloproliferative disorder (NS/MPD), resembling juvenile myelomonocytic leukemia (JMML), is occasionally diagnosed in infants with NS. In the present study, we performed a functional evaluation of the circulating hematopoietic progenitors in a series of NS, NS/MPD and JMML patients. The different functional patterns were compared with the aim to identify a possible NS subgroup worthy of stringent hematological follow-up for an increased risk of MPD development. We studied 27 NS and 5 JMML patients fulfilling EWOG-MDS criteria. The more frequent molecular defects observed in NS were mutations in the PTPN11 and SOS genes. The absolute count of monocytes, circulating CD34+ hematopoietic progenitors, their apoptotic rate and the number of circulating CFU-GMs cultured in the presence of decreasing concentrations or in the absence of granulocyte-macrophage colony-stimulating factor (GM-CSF) were evaluated. All JMML patients showed monocytosis>1,000/µl. Ten out of the 27 NS patients showed monocytosis>1,000/µl, which included the 3 NS/MPD patients. In JMML patients, circulating CD34+ cells were significantly increased (median, 109.8/µl; range, 44-232) with a low rate of apoptosis (median, 2.1%; range, 0.4-12.1%), and circulating CFU-GMs were hyper-responsive to GM-CSF. NS/MPD patients showed the same flow cytometric pattern as the JMML patients (median, CD34+ cells/µl, 205.7; range, 58-1374; median apoptotic rate, 1.4%; range, 0.2-2.4%) and their circulating CFU-GMs were hyper-responsive to GM-CSF. These functional alterations appeared 10 months before the typical clinical manifestations in 1 NS/MPD patient. In NS, the CD34+ absolute cell count and circulating CFU-GMs showed a normal pattern (median CD34+ cells/µl, 4.9; range, 1.3-17.5), whereas the CD34+ cell apoptotic rate was significantly decreased in comparison with the controls (median, 8.6%; range, 0-27.7% vs. median, 17.6%; range, 2.8-49.6%), suggesting an increased CD34+ cell survival. The functional evaluation of circulating hematopoietic progenitors showed specific patterns in NS and NS/MPD. These tests are a reliable integrative tool that, together with clinical data and other hematological parameters, could help detect NS patients with a high risk for a myeloproliferative evolution.

Sos-mediated cross-activation of wild-type Ras by oncogenic Ras is essential for tumorigenesis.

Mammalian cells contain three closely related ras genes, H-ras, K-ras and N-ras. Although in a given tumour type, oncogenic mutations are selectively observed in only one of the ras genes, the acquisition of the transformed phenotype has been shown to require the contribution of the normal products of the other ras genes. Here we demonstrate that oncogenic K-Ras promotes the activation of wild-type H- and N-Ras. This activation is mediated by oncogenic K-Ras-dependent allosteric stimulation of Sos and confers a growth advantage to oncogenic K-Ras harbouring cancer cells. These findings underscore the complementary functions of oncogenic and wild-type Ras in tumour cells and identify a potential new targeting strategy for Ras-driven tumours.

Activated O2(•−) and H2O2 mediated cell survival in SU11274-treated non-small-cell lung cancer A549 cells via c-Met-PI3K-Akt and c-Met-Grb2/SOS-Ras-p38 pathways.

The pharmacological activity of SU11274 is primarily due to its inhibition of hepotocyte growth factor receptor (c-Met) kinase overexpression. In this study, we demonstrated that the pathway involved in SU11274-induced autophagy was presumably through inhibition of c-Met and its down-stream pathways, including phosphatidylinositol 3-kinases ­ Akt (PI3K­Akt) and the growth factor receptor bound protein-2 / son of sevenless ­ Ras ­ p38 MAPK (Grb2/SOS­Ras­p38) pathway. SU11274 time-dependently induced the generation of superoxide anion (O2(•−)) and hydrogen peroxide (H2O2). There is a negative feedback loop between reactive oxygen species (ROS) induction and SU11274. Then, we investigated the role of ROS in protecting cells against SU11274-induced autophagic cell death in A549 cells. O2(•−) and H2O2 generation activated c-Met­PI3K­Akt and c-Met­Grb2/SOS­Ras­p38 signaling pathways, which were suppressed by O2(•−) scavenger superoxide dismutase (SOD) and H2O2 scavenger catalase. In conclusion, O2(•−) and H2O2 evoked cell resistance to SU11274 via activating c-Met­PI3K­Akt and c-Met­Grb2/SOS­Ras­p38 pathways in A549 cells. SU11274 also induced ROS generation in Caenorhabditis elegans.

Simultaneous learning of instantaneous and time-delayed genetic interactions using novel information theoretic scoring technique.

BACKGROUND: Understanding gene interactions is a fundamental question in systems biology. Currently, modeling of gene regulations using the Bayesian Network (BN) formalism assumes that genes interact either instantaneously or with a certain amount of time delay. However in reality, biological regulations, both instantaneous and time-delayed, occur simultaneously. A framework that can detect and model both these two types of interactions simultaneously would represent gene regulatory networks more accurately. RESULTS: In this paper, we introduce a framework based on the Bayesian Network (BN) formalism that can represent both instantaneous and time-delayed interactions between genes simultaneously. A novel scoring metric having firm mathematical underpinnings is also proposed that, unlike other recent methods, can score both interactions concurrently and takes into account the reality that multiple regulators can regulate a gene jointly, rather than in an isolated pair-wise manner. Further, a gene regulatory network (GRN) inference method employing an evolutionary search that makes use of the framework and the scoring metric is also presented. CONCLUSION: By taking into consideration the biological fact that both instantaneous and time-delayed regulations can occur among genes, our approach models gene interactions with greater accuracy. The proposed framework is efficient and can be used to infer gene networks having multiple orders of instantaneous and time-delayed regulations simultaneously. Experiments are carried out using three different synthetic networks (with three different mechanisms for generating synthetic data) as well as real life networks of Saccharomyces cerevisiae, E. coli and cyanobacteria gene expression data. The results show the effectiveness of our approach.

Complexity reduction preserving dynamical behavior of biochemical networks.

The complexity of biochemical systems, stemming from both the large number of components and the intricate interactions between these components, may hinder us in understanding the behavior of these systems. Therefore, effective methods are required to capture their key components and interactions. Here, we present a novel and efficient reduction method to simplify mathematical models of biochemical systems. Our method is based on the exploration of the so-called admissible region, that is the set of parameters for which the mathematical model yields some required output. From the shape of the admissible region, parameters that are really required in generating the output of the system can be identified and hence retained in the model, whereas the rest is removed. To describe the idea, first the admissible region of a very small artificial network with only three nodes and three parameters is determined. Despite its simplicity, this network reveals all the basic ingredients of our reduction method. The method is then applied to an epidermal growth factor receptor (EGFR) network model. It turns out that only about 34% of the network components are required to yield the correct response to the epidermal growth factor (EGF) that was measured in the experiments, whereas the rest could be considered as redundant for this purpose. Furthermore, it is shown that parameter sensitivity on its own is not a reliable tool for model reduction, because highly sensitive parameters are not always retained, whereas slightly sensitive parameters are not always removable.

TCR-mediated Erk activation does not depend on Sos and Grb2 in peripheral human T cells.

Sos proteins are ubiquitously expressed activators of Ras. Lymphoid cells also express RasGRP1, another Ras activator. Sos and RasGRP1 are thought to cooperatively control full Ras activation upon T-cell receptor triggering. Using RNA interference, we evaluated whether this mechanism operates in primary human T cells. We found that T-cell antigen receptor (TCR)-mediated Erk activation requires RasGRP1, but not Grb2/Sos. Conversely, Grb2/Sos­but not RasGRP1­are required for IL2-mediated Erk activation. Thus, RasGRP1 and Grb2/Sos are insulators of signals that lead to Ras activation induced by different stimuli, rather than cooperating downstream of the TCR.

Regulation in the targeting of TRAIL receptor 1 to cell surface via GODZ for TRAIL sensitivity in tumor cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5), promote the selective clearing of various malignancies by inducing apoptosis, holding the promise as a potent therapeutic agent for anticancer. Though DR4 and DR5 have high sequence similarity, differential regulation of both receptors in human tumor cells remains largely unexplored. Here, we repot that golgi-specific Asp-His-His-Cys (DHHC) zinc finger protein (GODZ) regulates TRAIL/DR4-mediated apoptosis. Using the SOS protein recruitment-yeast two-hybrid screening, we isolated GODZ that interacted with the death domain of DR4. GODZ binds to DR4, but not to DR5, through the DHHC and the C-terminal transmembrane domain. Expression level of GODZ affects apoptosis of tumor cells triggered by TRAIL, but not that induced by TNF-α/cycloheximide (CHX) or DNA-damaging drugs. In parallel, GODZ functions to localize DR4 to the plasma membrane (PM) via DHHC motif. Also, introduction of mutation into the cysteine-rich motif of DR4 results in its mistargeting and attenuates TRAIL- or GODZ-mediated apoptosis. Interestingly, GODZ expression is highly downregulated in Hep-3B tumor cells, which show resistance to TRAIL. However, reconstitution of GODZ expression enhances the targeting of DR4 to cell surface and sensitizes Hep-3B cells to TRAIL. Taken together, these data establish that GODZ is a novel DR4-selective regulator responsible for targeting of DR4 to the PM, and thereby for TRAIL-induced apoptosis.

The exquisite regulation of PLD2 by a wealth of interacting proteins: S6K, Grb2, Sos, WASp and Rac2 (and a surprise discovery: PLD2 is a GEF).

Phospholipase D (PLD) catalyzes the conversion of the membrane phospholipid phosphatidylcholine to choline and phosphatidic acid (PA). PLD's mission in the cell is two-fold: phospholipid turnover with maintenance of the structural integrity of cellular/intracellular membranes and cell signaling through PA and its metabolites. Precisely, through its product of the reaction, PA, PLD has been implicated in a variety of physiological cellular functions, such as intracellular protein trafficking, cytoskeletal dynamics, chemotaxis of leukocytes and cell proliferation. The catalytic (HKD) and regulatory (PH and PX) domains were studied in detail in the PLD1 isoform, but PLD2 was traditionally studied in lesser detail and much less was known about its regulation. Our laboratory has been focusing on the study of PLD2 regulation in mammalian cells. Over the past few years, we have reported, in regards to the catalytic action of PLD, that PA is a chemoattractant agent that binds to and signals inside the cell through the ribosomal S6 kinases (S6K). Regarding the regulatory domains of PLD2, we have reported the discovery of the PLD2 interaction with Grb2 via Y169 in the PX domain, and further association to Sos, which results in an increase of de novo DNA synthesis and an interaction (also with Grb2) via the adjacent residue Y179, leading to the regulation of cell ruffling, chemotaxis and phagocytosis of leukocytes. We also present the complex regulation by tyrosine phosphorylation by epidermal growth factor receptor (EGF-R), Janus Kinase 3 (JAK3) and Src and the role of phosphatases. Recently, there is evidence supporting a new level of regulation of PLD2 at the PH domain, by the discovery of CRIB domains and a Rac2-PLD2 interaction that leads to a dual (positive and negative) effect on its enzymatic activity. Lastly, we review the surprising finding of PLD2 acting as a GEF. A phospholipase such as PLD that exists already in the cell membrane that acts directly on Rac allows a quick response of the cell without intermediary signaling molecules. This provides only the latest level of PLD2 regulation in a field that promises newer and exciting advances in the next few years.

Focal adhesion kinase signaling pathway is involved in mechanotransduction in MG-63 cells.

Interstitial fluid flow, generated upon induced movement of extracellular fluid after mechanical loading, activates many signal transduction pathways in bone cells. The mechanisms of mechanobiology in bone tissue are still not clearly understood. Recently focal adhesion kinase (FAK) was shown to be involved in mechanotransduction in a number of cells. This study was designed to characterize the functional roles of FAK in mediating osteoblast response to mechanical steady-state fluid shear stress (FSS). We reported here that FSS (15 dynes/cm(2)) induced activation of FAK and formation of FAK·Grb2·Sos ternary complex in MG-63 cells, which was necessary for activation of the downstream mitogen-activated protein kinase pathway signaling molecules extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). Our results also showed that transfection of FAK (F397Y) plasmid, a negative mutant of FAK, blocked the increased expression of binding factor alpha 1, osterix, osteocalcin and alkaline phosphatase induced by FSS in MG-63 cells. These results demonstrate that FAK signaling is critical for FSS-induced activation of ERK and JNK, and for promotion of osteoblast differentiation and osteogenesis via its association with Grb2/Sos complex.

Abelson tyrosine kinase and Calmodulin interact synergistically to transduce midline guidance cues in the Drosophila embryonic CNS.

Calmodulin and Abelson tyrosine kinase are key signaling molecules transducing guidance cues at the Drosophila embryonic midline. A reduction in the signaling strength of either pathway alone induces ectopic midline crossing errors in a few segments. When Calmodulin and Abelson signaling levels are simultaneously reduced, the frequency of ectopic crossovers is synergistically enhanced as all segments exhibit crossing errors. But as the level of signaling is further reduced, commissures begin to fuse and large gaps form in the longitudinal connectives. Quantitative analysis suggests that the level of Abelson activity is particularly important. Like Calmodulin, Abelson interacts with son-of-sevenless to increase ectopic crossovers suggesting all three contribute to midline repulsive signaling. Axons cross the midline in almost every segment if Frazzled is co-overexpressed with the Calmodulin inhibitor, but the crossovers induced by the Calmodulin inhibitor itself do not require endogenous Frazzled. Thus, Calmodulin and Abelson tyrosine kinase are key signaling molecules working synergistically to transduce both midline attractive and repulsive cues. While they may function downstream of specific receptors, the emergence of commissural and longitudinal connective defects point to a novel convergence of Calmodulin and Abelson signaling during the regulation of actin and myosin dynamics underlying a guidance decision.

New insights into the mechanisms of SOS activation.

The activation of the small guanosine triphosphatase Ras is critical for many biological events. It is therefore not surprising that the ubiquitously expressed Ras guanine nucleotide exchange factor (GEF) SOS (Son of Sevenless), which couples protein tyrosine kinases to Ras activation, is under tight autoinhibitory control. Several studies have revealed how multiple regulatory domains might affect SOS activity. Most notably, a second Ras-binding site on SOS allosterically regulates the duration and amplitude of Ras activation. This allosteric Ras-GTP is produced by another GEF, Ras guanine nucleotide-releasing protein 1 (RasGRP1). SOS and RasGRP1 are both activated downstream of phospholipase D(2), and gain-of-function mutants of SOS contribute to inherited diseases. These studies not only enable us to better appreciate the complexity of the regulation of GEFs but also prompt us to reevaluate our current understanding of pathways that lead to Ras activation.

TGF-beta activates Erk MAP kinase signalling through direct phosphorylation of ShcA.

Erk1/Erk2 MAP kinases are key regulators of cell behaviour and their activation is generally associated with tyrosine kinase signalling. However, TGF-beta stimulation also activates Erk MAP kinases through an undefined mechanism, albeit to a much lower level than receptor tyrosine kinase stimulation. We report that upon TGF-beta stimulation, the activated TGF-beta type I receptor (TbetaRI) recruits and directly phosphorylates ShcA proteins on tyrosine and serine. This dual phosphorylation results from an intrinsic TbetaRI tyrosine kinase activity that complements its well-defined serine-threonine kinase function. TGF-beta-induced ShcA phosphorylation induces ShcA association with Grb2 and Sos, thereby initiating the well-characterised pathway linking receptor tyrosine kinases with Erk MAP kinases. We also found that TbetaRI is tyrosine phosphorylated in response to TGF-beta. Thus, TbetaRI, like the TGF-beta type II receptor, is a dual-specificity kinase. Recruitment of tyrosine kinase signalling pathways may account for aspects of TGF-beta biology that are independent of Smad signalling.

Prolidase-independent mechanism of camptothecin-induced inhibition of collagen biosynthesis in cultured human skin fibroblasts.

The present study was undertaken to evaluate the mechanism of campthotecin (CPT)-induced deregulation of collagen metabolism in cultured human skin fibroblast. It has been found that CPT strongly induced inhibition of collagen biosynthesis. The mechanism of this phenomenon was found to be independent of prolidase activity, an enzyme that plays an important role in enhancement of collagen biosynthesis at post-translational level. In fact, the enzyme activity was found to be stimulated by CPT. Increase in the enzyme activity was accompanied by increase in the expression of beta(1) integrin receptor and some beta(1) integrin-dependent signalling proteins, Sos, MAPK (ERK(1), ERK(2)) and transcription factor NF-kappaB. Since activation of beta(1) integrin induces NF-kappaB that inhibits collagen gene transcription, therefore the mechanism of CPT-dependent inhibition of collagen biosynthesis may be related to beta(1) integrin-dependent stimulation of NF-kappaB. Supporting evidence comes from experiments showing that specific MEK/ERK inhibitor (UO126) inhibited CPT-induced up-regulation of prolidase activity while it had no effect on CPT-induced inhibition of collagen biosynthesis and activation of NF-kappaB. The data suggest that CPT induces inhibition of collagen biosynthesis in cultured human skin fibroblasts by stimulation of NF-kappaB signalling.

Son of sevenless directly links the Robo receptor to rac activation to control axon repulsion at the midline.

Son of sevenless (Sos) is a dual specificity guanine nucleotide exchange factor (GEF) that regulates both Ras and Rho family GTPases and thus is uniquely poised to integrate signals that affect both gene expression and cytoskeletal reorganization. Here, using genetics, biochemistry, and cell biology, we demonstrate that Sos is recruited to the plasma membrane, where it forms a ternary complex with the Roundabout receptor and the SH3-SH2 adaptor protein Dreadlocks (Dock) to regulate Rac-dependent cytoskeletal rearrangement in response to the Slit ligand. Intriguingly, the Ras and Rac-GEF activities of Sos can be uncoupled during Robo-mediated axon repulsion; Sos axon guidance function depends on its Rac-GEF activity, but not its Ras-GEF activity. These results provide in vivo evidence that the Ras and RhoGEF domains of Sos are separable signaling modules and support a model in which Robo recruits Sos to the membrane via Dock to activate Rac during midline repulsion.

A role for focal adhesion kinase signaling in tumor necrosis factor-alpha-dependent matrix metalloproteinase-9 production in a cholangiocarcinoma cell line, CCKS1.

We have previously reported that tumor necrosis factor-alpha (TNF-alpha) stimulation of CCKS1, a cell line established from cholangiocarcinoma with i.p. dissemination, dramatically increased matrix metalloproteinase-9 (MMP-9) production and tumor invasion. We investigated the role of focal adhesion kinase (FAK) in TNF-alpha-dependent production of MMP-9 in CCKS1 and FAK-null mouse fibroblast cells. TNF-alpha stimulation of CCKS1 or wild-type fibroblasts substantially activated FAK phosphorylation and increased MMP-9 production. In contrast, FAK-null fibroblasts could not respond well to TNF-alpha stimulation. Conditional expression of wild-type FAK in FAK-null cells restored the TNF-alpha-dependent production of MMP-9. TNF-alpha treatment activated the kinase activity of FAK and its phosphorylation especially at Y397 and Y925. Phosphorylated FAK accumulated at focal adhesions and formed a complex with growth factor receptor binding protein 2 and SOS. In contrast, Y397F FAK and Y925F FAK, whose Y397 and Y925 were replaced with phenylalanine, respectively, as well as KD FAK, whose kinase was inactivated, could not restore the MMP-9 production. In addition, small interfering RNA against FAK drastically suppressed the TNF-alpha-dependent production of MMP-9 and inhibited the TNF-alpha-dependent invasion of CCKS1. Taken together, our results suggest the pivotal role of FAK in TNF-alpha-dependent production of MMP-9 and subsequent activation of tumor invasion.