BPGAP1 spatially integrates JNK/ERK signaling crosstalk in oncogenesis.

Simultaneous hyperactivation of stress-activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK) and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling cascades has been reported in carcinogenesis. However, how they are integrated to promote oncogenesis remains unknown. By analyzing breast invasive carcinoma database (The Cancer Genome Altas), we found that the mRNA expression levels of both JNK1 and ERK2 are positively correlated with the mRNA level of EEA1, an endosome associated protein, indicating the potential JNK/ERK crosstalk at endosome. Unbiased screen of different endosome-associated Rab GTPases reveals that late endosome serves as a unique platform to integrate JNK/ERK signaling. Furthermore, we identify that BPGAP1 (a BCH domain-containing, Cdc42GAP-like Rho GTPase-activating protein) promotes MEK partner 1 (MP1)-induced ERK activation on late endosome through scaffolding MP1/MEK1 complex. This regulatory function requires phosphorylation of BPGAP1 by JNK at its C terminal tail (Ser424) to unlock its autoinhibitory conformation. Consequently, phosphorylated BPGAP1 facilitates endosomal ERK signaling transduction to the nucleus, driving cell proliferation and transformation via the ERK-Myc-CyclinA axis. BPGAP1 therefore provides a crucial spatiotemporal checkpoint where JNK and MP1/MEK1 work in concert to regulate endosomal and nuclear ERK signaling in cell proliferation control.

Crosstalk of Ras and Rho: activation of RhoA abates Kras-induced liver tumorigenesis in transgenic zebrafish models.

RAS and Rho small GTPases are key molecular switches that control cell dynamics, cell growth and tissue development through their distinct signaling pathways. Although much has been learnt about their individual functions in both cell and animal models, the physiological and pathophysiological consequences of their signaling crosstalk in multi-cellular context in vivo remain largely unknown, especially in liver development and liver tumorigenesis. Furthermore, the roles of RhoA in RAS-mediated transformation and their crosstalk in vitro remain highly controversial. When challenged with carcinogens, zebrafish developed liver cancer that resembles the human liver cancer both molecularly and histopathologically. Capitalizing on the growing importance and relevance of zebrafish (Danio rerio) as an alternate cancer model, we have generated liver-specific, Tet-on-inducible transgenic lines expressing oncogenic Kras(G12V), RhoA, constitutively active RhoA(G14V) or dominant-negative RhoA(T19N). Double-transgenic lines expressing Kras(G12V) with one of the three RhoA genes were also generated. Based on quantitative bioimaging and molecular markers for genetic and signaling aberrations, we showed that the induced expression of oncogenic Kras during early development led to liver enlargement and hepatocyte proliferation, associated with elevated Erk phosphorylation, activation of Akt2 and modulation of its two downstream targets, p21Cip and S6 kinase. Such an increase in liver size and Akt2 expression was augmented by dominant-negative RhoA(T19N), but was abrogated by the constitutive-active RhoA(G14V). Consequently, induced expression of the oncogenic Kras in adult transgenic fish led to the development of hepatocellular carcinomas. Survival studies further revealed that the co-expression of dominant-negative RhoA(T19N) with oncogenic Kras increased the mortality rate compared with the other single or double-transgenic lines. This study provides evidence of the previously unappreciated signaling crosstalk between Kras and RhoA in regulating liver overgrowth and liver tumorigenesis. Our results also implicate that activating Rho could be beneficial to suppress the Kras-induced liver malignancies.

An integrated mathematical model of thrombin-, histamine-and VEGF-mediated signalling in endothelial permeability.

BACKGROUND: Endothelial permeability is involved in injury, inflammation, diabetes and cancer. It is partly regulated by the thrombin-, histamine-, and VEGF-mediated myosin-light-chain (MLC) activation pathways. While these pathways have been investigated, questions such as temporal effects and the dynamics of multi-mediator regulation remain to be fully studied. Mathematical modeling of these pathways facilitates such studies. Based on the published ordinary differential equation models of the pathway components, we developed an integrated model of thrombin-, histamine-, and VEGF-mediated MLC activation pathways. RESULTS: Our model was validated against experimental data for calcium release and thrombin-, histamine-, and VEGF-mediated MLC activation. The simulated effects of PAR-1, Rho GTPase, ROCK, VEGF and VEGFR2 over-expression on MLC activation, and the collective modulation by thrombin and histamine are consistent with experimental findings. Our model was used to predict enhanced MLC activation by CPI-17 over-expression and by synergistic action of thrombin and VEGF at low mediator levels. These may have impact in endothelial permeability and metastasis in cancer patients with blood coagulation. CONCLUSION: Our model was validated against a number of experimental findings and the observed synergistic effects of low concentrations of thrombin and histamine in mediating the activation of MLC. It can be used to predict the effects of altered pathway components, collective actions of multiple mediators and the potential impact to various diseases. Similar to the published models of other pathways, our model can potentially be used to identify important disease genes through sensitivity analysis of signalling components.

Virtual screening of selective multitarget kinase inhibitors by combinatorial support vector machines.

Multitarget agents have been increasingly explored for enhancing efficacy and reducing countertarget activities and toxicities. Efficient virtual screening (VS) tools for searching selective multitarget agents are desired. Combinatorial support vector machines (C-SVM) were tested as VS tools for searching dual-inhibitors of 11 combinations of 9 anticancer kinase targets (EGFR, VEGFR, PDGFR, Src, FGFR, Lck, CDK1, CDK2, GSK3). C-SVM trained on 233-1,316 non-dual-inhibitors correctly identified 26.8%-57.3% (majority >36%) of the 56-230 intra-kinase-group dual-inhibitors (equivalent to the 50-70% yields of two independent individual target VS tools), and 12.2% of the 41 inter-kinase-group dual-inhibitors. C-SVM were fairly selective in misidentifying as dual-inhibitors 3.7%-48.1% (majority <20%) of the 233-1,316 non-dual-inhibitors of the same kinase pairs and 0.98%-4.77% of the 3,971-5,180 inhibitors of other kinases. C-SVM produced low false-hit rates in misidentifying as dual-inhibitors 1,746-4,817 (0.013%-0.036%) of the 13.56 M PubChem compounds, 12-175 (0.007%-0.104%) of the 168 K MDDR compounds, and 0-84 (0.0%-2.9%) of the 19,495-38,483 MDDR compounds similar to the known dual-inhibitors. C-SVM was compared to other VS methods Surflex-Dock, DOCK Blaster, kNN and PNN against the same sets of kinase inhibitors and the full set or subset of the 1.02 M Zinc clean-leads data set. C-SVM produced comparable dual-inhibitor yields, slightly better false-hit rates for kinase inhibitors, and significantly lower false-hit rates for the Zinc clean-leads data set. Combinatorial SVM showed promising potential for searching selective multitarget agents against intra-kinase-group kinases without explicit knowledge of multitarget agents.

Virtual screening of Abl inhibitors from large compound libraries by support vector machines.

Abl promotes cancers by regulating cell morphogenesis, motility, growth, and survival. Successes of several marketed and clinical trial Abl inhibitors against leukemia and other cancers and appearances of reduced efficacies and drug resistances have led to significant interest in and efforts for developing new Abl inhibitors. In silico methods of pharmacophore, fragment, and molecular docking have been used in some of these efforts. It is desirable to explore other in silico methods capable of searching large compound libraries at high yields and reduced false-hit rates. We evaluated support vector machines (SVM) as a virtual screening tool for searching Abl inhibitors from large compound libraries. SVM trained and tested by 708 inhibitors and 65,494 putative noninhibitors correctly identified 84.4 to 92.3% inhibitors and 99.96 to 99.99% noninhibitors in 5-fold cross validation studies. SVM trained by 708 pre-2008 inhibitors and 65 494 putative noninhibitors correctly identified 50.5% of the 91 inhibitors reported since 2008 and predicted as inhibitors 29,072 (0.21%) of 13.56M PubChem, 659 (0.39%) of 168K MDDR, and 330 (5.0%) of 6638 MDDR compounds similar to the known inhibitors. SVM showed comparable yields and substantially reduced false-hit rates against two similarity based and another machine learning VS methods based on the same training and testing data sets and molecular descriptors. These suggest that SVM is capable of searching Abl inhibitors from large compound libraries at low false-hit rates.

Evaluation of virtual screening performance of support vector machines trained by sparsely distributed active compounds.

Virtual screening performance of support vector machines (SVM) depends on the diversity of training active and inactive compounds. While diverse inactive compounds can be routinely generated, the number and diversity of known actives are typically low. We evaluated the performance of SVM trained by sparsely distributed actives in six MDDR biological target classes composed of a high number of known actives (983-1645) of high, intermediate, and low structural diversity (muscarinic M1 receptor agonists, NMDA receptor antagonists, thrombin inhibitors, HIV protease inhibitors, cephalosporins, and renin inhibitors). SVM trained by regularly sparse data sets of 100 actives show improved yields at substantially reduced false-hit rates compared to those of published studies and those of Tanimoto-based similarity searching method based on the same data sets and molecular descriptors. SVM trained by very sparse data sets of 40 actives (2.4%-4.1% of the known actives) predicted 17.5-39.5%, 23.0-48.1%, and 70.2-92.4% of the remaining 943-1605 actives in the high, intermediate, and low diversity classes, respectively, 13.8-68.7% of which are outside the training compound families. SVM predicted 99.97% and 97.1% of the 9.997 M PUBCHEM and 167K remaining MDDR compounds as inactive and 2.6%-8.3% of the 19,495-38,483 MDDR compounds similar to the known actives as active. These suggest that SVM has substantial capability in identifying novel active compounds from sparse active data sets at low false-hit rates.

RhoA prevents apoptosis during zebrafish embryogenesis through activation of Mek/Erk pathway.

RhoA small GTPase, as a key regulator for actin cytoskeletal rearrangement, plays pivotal roles during morphogenesis, cytokinesis, phagocytosis and cell migration, but little is known about its signaling mechanism that controls cell survival in vivo. Using zebrafish as a model, we show that non-overlapping antisense morpholinos that block either translation or splicing of rhoA lead to extensive apoptosis during embryogenesis, resulting in overall reduction of body size and body length. These defects are associated with reduced activation of growth-promoting Erk and decreased expression of anti-apoptotic bcl-2. Moreover, ectopic expression of rhoA, Mek or BCL-2 mRNA rescues such phenotypes. Consistently, combined suppression of RhoA and Mek/Erk or Bcl-2 pathways by sub-optimal dose of rhoA morpholino and pharmacological inhibitors for either Mek (U0126) or Bcl-2 (HA 14-1) can induce developmental abnormalities and enhanced apoptosis, similar to those caused by effective RhoA knockdown. Furthermore, U0126 abrogates the rescue by RhoA and MEK but not BCL-2. In contrast, HA 14-1 effectively abolishes all functional rescues by RhoA, MEK or BCL-2, supporting that RhoA prevents apoptosis by activation of Mek/Erk pathway and requiring Bcl-2. These findings reveal an important genetic and functional relationship between RhoA with Mek/Erk and Bcl-2 for cell survival control during embryogenesis.

BNIP-Salpha induces cell rounding and apoptosis by displacing p50RhoGAP and facilitating RhoA activation via its unique motifs in the BNIP-2 and Cdc42GAP homology domain.

Changes in cell morphology are linked to many cellular events including cytokinesis, differentiation, migration and apoptosis. We recently showed that BNIP-Salpha induced cell rounding that leads to apoptosis via its BNIP-2 and Cdc42GAP Homology (BCH) domain, but the underlying mechanism has not been determined. Here, we have identified a unique region (amino acid 133-177) of the BNIP-Salpha BCH domain that targets RhoA, but not Cdc42 or Rac1 and only the dominant-negative form of RhoA could prevent the resultant cell rounding and apoptotic effect. The RhoA-binding region consists of two parts; one region (residues 133-147) that shows some homology to part of the RhoA switch I region and an adjacent sequence (residues 148-177) that resembles the REM class I RhoA-binding motif. The sequence 133-147 is also necessary for its heterophilic interaction with the BCH domain of the Rho GTPase-activating protein, p50RhoGAP/Cdc42GAP. These overlapping motifs allow tripartite competition such that overexpression of BNIP-Salpha could reduce p50RhoGAP binding to RhoA and restore RhoA activation. Furthermore, BNIP-Salpha mutants lacking the RhoA-binding motif completely failed to induce cell rounding and apoptosis. Therefore, via unique binding motifs within its BCH domain, BNIP-Salpha could interact and activate RhoA while preventing its inhibition by p50RhoGAP. This concerted mechanism could allow effective propagation of the RhoA pathway for cell rounding and apoptosis.

Filling the GAPs in cell dynamics control: BPGAP1 promotes cortactin translocation to the cell periphery for enhanced cell migration.

Cells undergo dynamic changes in morphology or motility during cellular division and proliferation, differentiation, neuronal pathfinding, wound healing, apoptosis, host defense and organ development. These processes are controlled by signalling events relayed through cascades of protein interactions leading to the establishment and maintenance of cytoskeletal networks of microtubules and actin. Various regulators, including the Rho small GTPases (guanine nucleotide triphosphatases), serve as master switches to fine-tune the amplitude, duration as well as the integration of such circuitry responses. Rho GTPases are activated by guanine nucleotide-exchange factors and inactivated by GAPs (GTPase-activating proteins). Although normally down-regulating signalling pathways by catalysing their GTPase activity, many GAPs exist with various protein modules, the functions of which still largely remain unknown. BPGAP1 is a novel RhoGAP that co-ordinately regulates pseudopodia and cell migration through the interplay of its BNIP-2 and Cdc42GAP homology domains serving as a homophilic/heterophilic interaction device, an enzymic RhoGAP domain that inactivates RhoA and a proline-rich region that binds the Src homology-3 domain of cortactin. Both proteins co-localize to cell periphery and enhance cell migration. As a molecular scaffold in cortical actin assembly and organization, cortactin and its interaction with small GTPases, GAPs and tyrosine kinases seems set to provide further insights to the multiplicity and complexity of cell dynamics control. Elucidating how these processes might be individually or co-ordinately regulated through cortactin remains an exciting future challenge.

Evidence for direct interaction between Sprouty and Cbl.

Sprouty (SPRY) was first identified in a genetic screen in Drosophila as an antagonist of fibroblast and epidermal growth factor receptors and Sevenless signaling, seemingly by inhibiting the receptor tyrosine kinase (RTK)/Ras/MAPK pathway. To date, four mammalian Sprouty genes have been identified; the primary sequences of the gene products share a well conserved cysteine-rich C-terminal domain with their Drosophila counterpart. The N-terminal regions do not, however, exhibit a large degree of homology. This study was aimed at identifying proteins with which human SPRY2 (hSPRY2) interacts in an attempt to understand the mechanism by which Sprouty proteins exert their down-regulatory effects. Here, we demonstrate that hSPRY2 associates directly with c-Cbl, a known down-regulator of RTK signaling. A short sequence in the N terminus of hSPRY2 was found to bind directly to the Ring finger domain of c-Cbl. Parallel binding was apparent between the Drosophila homologs of Sprouty and Cbl, with cross-species associations occurring at least in vitro. Coexpression of hSPRY2 abrogated an increase in the rate of epidermal growth factor receptor internalization induced by c-Cbl, whereas a mutant hSPRY2 protein unable to bind c-Cbl showed no such effect. Our results suggest that one function of hSPRY2 in signaling processes downstream of RTKs may be to modulate c-Cbl physiological function such as that seen with receptor-mediated endocytosis.

The BNIP-2 and Cdc42GAP homology domain of BNIP-2 mediates its homophilic association and heterophilic interaction with Cdc42GAP.

We recently showed that BNIP-2 is a putative substrate of the fibroblast growth factor receptor tyrosine kinase and it possesses GTPase-activating activity toward the small GTPase, Cdc42. The carboxyl terminus of BNIP-2 shares high homology to the non-catalytic domain of Cdc42GAP, termed BCH (for BNIP-2 and Cdc42GAP homology) domain. Despite the lack of obvious homology to any known catalytic domains of GTPase-activating proteins (GAPs), the BCH domain of BNIP-2 bound Cdc42 and stimulated the GTPase activity via a novel arginine-patch motif similar to that employed by one contributing partner in a Cdc42 homodimer. In contrast, the BCH domain of Cdc42GAP, although it can bind Cdc42, is catalytically inactive. This raises the possibility that these domains might have other roles in the cell. Using glutathione S-transferase recombinant proteins, immunoprecipitation studies, and yeast two-hybrid assays, it was found that BNIP-2 and Cdc42GAP could form homo and hetero complexes via their conserved BCH domains. Molecular modeling of the BNIP-2 BCH homodimer complex and subsequent deletion mutagenesis helped to identify the region (217)RRKMP(221) as the major BCH interaction site within BNIP-2. In comparison, deletion of either the arginine-patch (235)RRLRK(239) (necessary for GAP activity) or region (288)EYV(290) (a Cdc42 binding sequence) had no effect on BCH-BCH interaction. Extensive data base searches showed that the BCH domain is highly conserved across species. The results suggest that BCH domains of BNIP-2 and Cdc42GAP represent a novel protein-protein interaction domain that could potentially determine and/or modify the physiological roles of these molecules.

Sprouty proteins are targeted to membrane ruffles upon growth factor receptor tyrosine kinase activation. Identification of a novel translocation domain.

Sprouty (Spry) was first identified in a genetic screen in Drosophila to be an antagonist of fibroblast growth factor and epidermal growth factor (EGF) signaling, seemingly by inhibiting the Ras/MAP kinase pathway. Data base searches lead to the identification and cloning of, to date, four mammalian sprouty genes. The primary sequences of the mammalian sprouty gene products share a well conserved cysteine-rich C-terminal domain with the Drosophila protein. The N-terminal regions, however, do not exhibit significant homology. This study aimed at determining the disposition of Spry proteins in intact cells before and after stimulation of the EGF receptor tyrosine kinase. Full-length or deletion mutants of Spry, tagged at the N termini with the FLAG-epitope, were expressed in COS-1 cells by transient transfection and analyzed by immunofluorescence microscopy before and after EGF stimulation of the cells. In unstimulated cells, the Spry proteins were distributed throughout the cytosol except for human Sprouty2 (hSpry2), which, although generally located in the cytosol, co-localized with microtubules. In all cases, the Spry proteins underwent rapid translocation to membrane ruffles following EGF stimulation. The optimal translocation domain was identified by deletion and immunofluorescence analysis to be a highly conserved 105-amino acid domain in the C-terminal half of the hSpry2 protein. The translocation of this conserved domain, based on hSpry2 data, was independent of the activation of phosphatidylinositol-3 kinase.

Evidence for a novel Cdc42GAP domain at the carboxyl terminus of BNIP-2.

We recently identified BNIP-2, a previously cloned Bcl-2- and E1B-associated protein, as a putative substrate of the FGF receptor tyrosine kinase and showed that it possesses GTPase-activating activity toward Cdc42 despite the lack of homology to previously described catalytic domains of GTPase-activating proteins (GAPs). BNIP-2 contains many arginine residues at the carboxyl terminus, which includes the region of homology to the noncatalytic domain of Cdc42GAP, termed BNIP-2 and Cdc42GAP homology (BCH) domain. Using BNIP-2 glutathione S-transferase recombinants, it was found that its BCH bound Cdc42, and contributed the GAP activity. This domain was predicted to fold into alpha-helical bundles similar to the topology of the catalytic GAP domain of Cdc42GAP. Alignment of exposed arginine residues in this domain helped to identify Arg-235 and Arg-238 as good candidates for catalysis. Arg-238 matched well to the arginine "finger" required for enhanced GTP hydrolysis in homodimerized Cdc42. Site-directed mutagenesis confirmed that an R235K or R238K mutation severely impaired the BNIP-2 GAP activity without affecting its binding to Cdc42. From deletion studies, a region adjacent to the arginine patch ((288)EYV(290) on BNIP-2) and the Switch I and Rho family-specific "Insert" region on Cdc42 are involved in the binding. The results indicate that the BCH domain of BNIP-2 represents a novel GAP domain that employs an arginine patch motif similar to that of the Cdc42-homodimer.

Tyrosine phosphorylation of the Bcl-2-associated protein BNIP-2 by fibroblast growth factor receptor-1 prevents its binding to Cdc42GAP and Cdc42.

Fibroblast growth factor (FGF) receptor tyrosine kinases are involved in the regulation of cell growth, development, and differentiation in a variety of tissues. To isolate potential signaling molecules in the FGF signaling pathway, we have initiated a yeast two-hybrid screening using the cytosolic domain of FGF receptor-1 (Flg). Here we report the identification of BNIP-2, a previously cloned Bcl-2- and adenovirus E1B-associated protein, as a putative substrate of the receptor. When cotransfected in 293T cells, BNIP-2 was tyrosine-phosphorylated via Flg, but their interaction was transient and could only be seen by "capture" experiments with catalytically inert kinase mutants. When responsive cells were challenged with basic FGF, endogenous tyrosine-phosphorylated BNIP-2 could be precipitated with a BNIP-2 antibody. In addition, the recombinant BNIP-2 expressed in bacteria could be phosphorylated by active Flg in vitro. BNIP-2 shares a region of homology with the noncatalytic domain of Cdc42GAP, a GTPase-activating protein for the small GTP-binding molecule, Cdc42. We show here that BNIP-2 and Cdc42GAP could directly bind to each other and they also compete for the binding to the same target, Cdc42. Unexpectedly, BNIP-2, either produced as a bacterial recombinant protein or expressed in 293T cells, could stimulate the intrinsic GTPase activity of Cdc42. In all cases, tyrosine phosphorylation of BNIP-2 severely impaired its association with Cdc42GAP and its induced GTPase-activating protein-like activity toward Cdc42. These findings should allow us to further characterize the integration of signaling between receptor tyrosine kinases, GTP-binding molecules, and apoptotic pathways.

Association of atypical protein kinase C isotypes with the docker protein FRS2 in fibroblast growth factor signaling.

FRS2 is a docker protein that recruits signaling proteins to the plasma membrane in fibroblast growth factor signal transduction. We report here that FRS2 was associated with PKC lambda when Swiss 3T3 cells were stimulated with basic fibroblast growth factor. PKC zeta, the other member of the atypical PKC subfamily, could also bind FRS2. The association between FRS2 and PKC lambda is likely to be direct as shown by yeast two-hybrid analysis. The C-terminal fragments of FRS2 (amino acid residues 300-508) and SNT2 (amino acids 281-492), an isoform bearing 50% identity to FRS2, interacted with PKC lambda at a region (amino acids 240-562) that encompasses the catalytic domain. In vitro kinase assays revealed neither FRS2 nor SNT2 was a substrate of PKC lambda or zeta. Mutation of the alanine residue (Ala-120) to glutamate in the pseudo-substrate region of PKC lambda results in a constitutively active kinase that exhibited more than 2-fold greater binding to FRS2 in vitro than its "closed" wild-type counterpart. Tyrosine phosphorylation of FRS2 did not affect its binding to the constitutively active PKC lambda mutant, suggesting that the activation of PKC lambda is necessary and sufficient for its association with FRS2. It is likely that FRS2 serves as an anchoring protein for targeting activated atypical PKCs to the cell plasma membrane in signaling pathways.

Growth factors stimulate tyrosine dephosphorylation of p75 and its dissociation from the SH2 domain of Grb2.

The growth factor receptor-binding protein (Grb2) has a key role in initiating the mitogen-activated protein kinase signaling cascade in major cell regulatory pathways. The binding of proteins to the SH2 domain of Grb2 has been reported to occur mainly after they are tyrosine-phosphorylated following receptor activation. Using an in vitro binding assay, immunoprecipitation, and Far Western techniques, we report that in quiescent cells a 75-kDa protein binds directly to the SH2 domain of Grb2. All of the tyrosine-phosphorylated p75 protein co-localizes with Grb2.Sos complex in the cytosolic fraction of the cell in vivo and undergoes tyrosine dephosphorylation when cells are treated with mitogenic ligands such as epidermal, platelet-derived, and fibroblast growth factors, endothelin-1, and bombesin but not tumor necrosis factor-alpha, interferon-alpha and -gamma, interleukein-6, and leukemic inhibitory factor, which are either cell growth inhibitory or not significantly mitogenic. The dephosphorylation of p75 and the ensuing dissociation from Grb2 is rapid, occurring within 30 s following mitogenic stimulation by ligands such as epidermal growth factor, suggesting p75 to be an early component in the signal transduction pathways involving Grb2.

SHP2 associates directly with tyrosine phosphorylated p90 (SNT) protein in FGF-stimulated cells.

In a number of cell lines responsive to basic fibroblast growth factor (bFGF), two major tyrosine phosphorylated proteins, of molecular weights around 120kDa and 90kDa, are precipitated along with the tyrosine phosphatase SHP2 from the lysates of stimulated cells. The docker protein Gab-1 represents at least part of the 120kDa protein(s). The p90 protein was identified as the SNT protein. The two SH2 domains of SHP2 bind directly and synergistically to tyrosine phosphorylated SNT. Tyrosine phosphorylated SNT does not bind SHP1 and does not appear to be an in vivo substrate of SHP2 but is likely to function as an adapter protein in FGF-signalling.

Interaction of interferon-gamma and epidermal growth factor in the regulation of nitric oxide production and cellular proliferation in a cultured murine mammary cell line, COMMA-D.

The regulation of nitric oxide synthase (NOS) activity and gene expression by cytokines and growth factors has been studied using the murine mammary epithelial cell line, COMMA-D. NOS activity was stimulated by exposure to interferon-gamma (IFN-gamma) and could be further stimulated by tumour necrosis factor-alpha (TNF-alpha) and epidermal growth factor (EGF) although neither was affective alone. The maximal activity observed in the presence of IFN-gamma and EGF was not affected by the order in which cells were exposed. Messenger RNA levels for the inducible NOS isoform were increased by IFN-gamma and TNF-alpha in a manner consistent with the elevation of NOS activity. EGF also stimulated thymidine incorporation into DNA which was attenuated by coexposure with IFN-gamma in a manner that appeared to be largely NO-independent.

Suc1-associated neurotrophic factor target (SNT) protein is a major FGF-stimulated tyrosine phosphorylated 90-kDa protein which binds to the SH2 domain of GRB2.

In Swiss 3T3 fibroblasts, basic fibroblast growth factor (bFGF) stimulates the unique tyrosine phosphorylation of a protein complex around 90 kDa, as ascertained by high resolution 2-D PAGE and anti-phosphotyrosine blotting. The majority of this complex consists of the protein(s) designated previously as SNT (suc1-associated neurotrophic factor target). Tyrosine phosphorylated SNT binds to both p13suc1 protein and the SH2 domain of Grb2. Binding of SNT to Grb2 is likely to be mediated through the consensus binding motif, pYXN, on SNT. The binding of SNT to p13suc1 is independent of the pYXN motif. Tyrosine phosphorylated SNT is localised in the plasma membrane where it could form a complex with Grb2 and Sos, enabling the initiation of a novel FGF-specific signalling pathway.

Interleukin-1 beta and tumor necrosis factor-alpha stimulate the cat-2 gene of the L-arginine transporter in cultured vascular smooth muscle cells.

The production of nitric oxide (NO) from L-arginine by nitric oxide synthase (NOS) in cytokine-stimulated vascular smooth muscle cells (VSMC) is thought to play an important role in the pathophysiology of several vascular disease states including septic shock. This study examines the relationship between cytokine-stimulated NO production and L-arginine transport in cultured VSMC. Cultured VSMC from rat aorta were stimulated with interleukin-1 beta, tumor necrosis factor-alpha, and/or angiotensin II (Ang II); and the accumulation of nitrite, a stable product of NO metabolism, in the culture media and the rates of net L-arginine uptake were measured. Interleukin-1 beta and tumor necrosis factor-alpha, alone or in combination, stimulated both the uptake of L-arginine and the accumulation of nitrite in the culture media in a dose-dependent manner. Inhibition of NOS activity by substituted analogues of L-arginine had no effect on cytokine-stimulated L-arginine transport. Ang II in the presence of cytokines up-regulated L-arginine transport while inhibiting nitrite accumulation. Two forms of the L-arginine transporter, cat-1b and cat-2, are expressed in VSMC. Northern analysis revealed that the cytokine-stimulated increase in L-arginine transport coincided with increased levels of cat-2 mRNA. In contrast, cat-1b does not appear to be regulated by cytokines at the mRNA level, although significant increases in response to Ang II were observed. These results show that, while cytokines can stimulate both NOS activity and L-arginine uptake, NO production is not required to signal the increase in L-arginine transport. Furthermore, Ang II and cytokine stimulation of L-arginine uptake involves the differential regulation of the cationic amino acid transporter (cat) genes.