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.

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.

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.

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.

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.

Angiotensin II stimulates system y+ and cationic amino acid transporter gene expression in cultured vascular smooth muscle cells.

The effect of angiotensin II (Ang II) on the transport of cationic amino acids has been examined in vascular smooth muscle cells (VSMC) isolated from rat aortae. Ang II stimulated the uptake rates of radiolabeled arginine and lysine in a time- and concentration-dependent manner. The stimulated arginine uptake could be blocked by pretreatments with cycloheximide and actinomycin D or co-treatment with valsartan, an antagonist specific for Ang II receptor subtype-1. The modulation by Ang II was bidirectional as the efflux of arginine was also stimulated, 5-fold over basal. Using reverse transcription-coupled polymerase chain reaction methodology, a partial cDNA with 94% sequence identity to that of cationic amino acid transporter subtype-1 (CAT-1) of mouse fibroblasts was obtained from VSMC. This sequence also exhibited 14 base changes compared with the sequence of ecotropic retrovirus receptor (ERR)/CAT-1 from rat hepatoma. Northern analyses with this partial CAT-1 cDNA and CAT-2 cDNA of mouse T-lymphocytes showed that Ang II rapidly stimulated the expression of both CAT-1 and CAT-2 in VSMC. Both signals peaked at 2 h after exposure to Ang II. The CAT-1 signal decayed over the next 6 h to levels 3-fold above basal, which are maintained up until 24 h. The induced CAT-2 mRNA concentration also decayed rapidly but increased again between 16 and 24 h to levels comparable with those observed at 2 h.

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.

Who stays? who leaves? an examination of sex differences in staying and leaving.

This article examines some of the major variables associated with the "staying" and "leaving" process, particularly as it operates similarly or differentially for male and female adolescents. Sixty attitude statements about aspects of school, parents, peers, teachers, and value orientations were obtained from 796 adolescents and factor analyzed. This subset of independent variables, together with a set of personality and ability measures, was then used to predict "staying" or "leaving" status. The discriminant analysis was undertaken using "leaving" status and sex (LVSEX) as the dependent variable. Using a stepwise solution method of discriminant analysis, two significant functions were identified-the first suggesting a sex dimension, the second a stayer-leaver dimension.

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.

Angiotensin II stimulates glucose transport activity in cultured vascular smooth muscle cells.

The glucose transport system in cultured rat vascular smooth muscle cells has been examined by measuring the uptake of 2-deoxyglucose. Angiotensin II (Ang II) stimulated 2-deoxyglucose uptake in cells made quiescent by removing serum from the culture medium in a dose- and time-dependent manner that was shown to be receptor-mediated. Epidermal growth factor (EGF), fetal calf serum, thrombin, and arginine vasopressin also stimulated glucose transport. Cycloheximide did not affect the immediate-early (30 min) activation by either Ang II or EGF, but abolished any further increase. This suggested that, whereas the initial activation of glucose transport was independent of protein synthesis, the sustained increase required the synthesis of new glucose transporters. This was supported by 4-fold and 2-fold accumulations of GLUT-1 mRNA 4 h after exposure to Ang II and EGF, respectively. The induction of GLUT-1 mRNA was preceded by rapid and transient expression of c-fos and c-jun protooncogenes. In nuclear run-on assays, nuclei from Ang II-treated cells showed increased synthesis of GLUT-1 mRNA at 30 min and 1 h after hormone treatment. In contrast, in cells exposed to actinomycin D, pretreatment with Ang II had no effect on the turnover rates of GLUT-1 mRNa. These results are consistent with Ang II acting to stimulate the rate of transcription of the GLUT-1 gene leading to increased production of GLUT-1 protein and glucose transport.

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.

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.

Characterization of system L and system y+ amino acid transport activity in cultured vascular smooth muscle cells.

The uptake of L-leucine and L-lysine into vascular smooth muscle cells cultured from the aortas of rats has been investigated. Both amino acids are taken up by saturable systems that are independent of the presence of a Na+ gradient and can be stimulated in trans by neutral bulky amino acids for leucine and cationic amino acids for lysine. Leucine uptake is inhibited competitively in cis by several neutral amino acids, whereas lysine uptake is inhibited strongly by other cationic amino acids but also significantly by neutral amino acids such as leucine. The leucine inhibition is noncompetitive. Cells preloaded with leucine and lysine could also export these amino acids and the rate of efflux was stimulated by the presence of appropriate amino acids in trans. These data are all consistent with leucine being transported largely if not entirely by System L and lysine by the System y+ transporter.

Glucose deprivation and acute cycloheximide treatment stimulate system L amino acid transport in cultured vascular smooth muscle cells.

The effect of glucose deprivation on the uptake of leucine has been examined in cultured vascular smooth muscle cells isolated from rat aortae. Equimolar substitution of sucrose or fructose for glucose in the culture medium enhanced the uptake of leucine in a time- and concentration-dependent manner. The effect was first detectable after 12 h and reached the maximum, 2-fold, after 48 h with an apparent half-maximal effect at 1 mM glucose and could be reversed after 48 h of glucose refeeding. The enhanced leucine uptake was completely inhibited by 2-amino-2-norbornane-carboxylic acid, a specific substrate for System L, but not by alpha-(methylamino)isobutyric acid or lysine. Kinetic analyses indicated that this stimulation was mediated via a homogenous system with a 1.7-fold increase in the Vmax without any change in the Km (0.15 mM). Prolonged treatments with cycloheximide (10 micrograms/ml) or actinomycin D (10 micrograms/ml) blocked this glucose deprivation effect and its reversal. However, cycloheximide also very rapidly stimulated leucine uptake, reaching the maximum, 2.5-fold over the basal at 1 h. This effect occurred at concentrations that matched its inhibition on protein synthesis (half-maximal at 0.1 micrograms/ml) and could be reproduced with puromycin as well as actinomycin D. The stimulatory effect of cycloheximide was also accompanied by an increase in the Vmax but not in the Km, being sensitive to 2-amino-2-norbornane-carboxylic acid inhibition only, and appeared to occur in an additive manner to that of glucose deprivation. Although the uptake of leucine was stimulated by glucose deprivation and brief exposure to cycloheximide, these treatments had no effect on the efflux of the substrate. These results are all consistent with the System L amino acids transport activity in cultured rat vascular smooth muscle cells being under the control of at least two non-hormonal regulatory mechanisms, one that is likely to involve a labile repressor molecule and the other involving de novo protein synthesis as a result of chronic glucose deprivation.

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.

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.

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.