In the treatment of autoimmune diseases, intravenous Igs (IVIg) are assumed to modulate immune cells through the binding of surface receptors. IVIg act upon definite human B cell populations to modulate Ig repertoire, and such modulation might proceed through intracellular signaling. However, the heterogeneity of human B cell populations complicates investigations of the intracellular pathways involved in IVIg-induced B cell modulation. The aim of this study was to establish a model allowing the screening of IVIg signal transduction in human B cell lines and to attempt transposing observations made in cell lines to normal human B lymphocytes. Nine human B cell lines were treated with IVIg with the goal of selecting the most suitable model for human B lymphocytes. The IgG(+) DB cell line, whose response was similar to that of human B lymphocytes, showed reduced IVIg modulation following addition of PD98059, an inhibitor of extracellular signal-regulated protein kinase 1/2 (ERK1/2). The IVIg-induced ERK1/2 phosphorylation was indeed proportional to the dosage of monomeric IVIg used when tested on DB cells as well as Pfeiffer cells, another IgG(+) cell line. In addition, two other intermediates, Grb2-associated binder 1 (Gab1) and Akt, showed increased phosphorylation in IVIg-treated DB cells. IVIg induction of ERK1/2 phosphorylation was finally observed in peripheral human B lymphocytes, specifically within the IgG(+) B cell population. In conclusion, IVIg immunomodulation of human B cells can thus be linked to intracellular transduction pathways involving the phosphorylation of ERK1/2, which in combination with Gab1 and Akt, may be related to B cell antigen receptor signaling.

Mitogen-activated protein kinase (MAPK) signaling pathways play an important role in controlling embryonic proliferation and differentiation. It has been demonstrated that sequential lipophilic signal transduction mediators that participate in the MAPK pathway are null post-implantation lethal. It is not clear why the lethality of these null mutants arises after implantation and not before. One hypothesis is that the gene product of these post-implantation lethal null mutants are not present before implantation in normal embryos and do not have function until after implantation. To test this hypothesis, we selected a set of lipophilic genes mediating MAPK signal transduction pathways whose null mutants result in early peri-implantation or placental lethality. These included FRS2alpha, GAB1, GRB2, SOS1, Raf-B, and Raf1. Products of these selected genes were detected and their locations and functions indicated by indirect immunocytochemistry and Western blotting for proteins and RT-polymerase chain reaction (PCR) for mRNA transcription. We report here that all six signal mediators are detected at the protein level in preimplantation mouse embryo, placental trophoblasts, and in cultured trophoblast stem cells (TSC). Proteins are all detected in E3.5 embryos at a time when the first known mitogenic intercellular communication has been documented. mRNA transcripts of two post-implantation null mutant genes are expressed in mouse preimplantation embryos and unfertilized eggs. These mRNA transcripts were detected as maternal mRNA in unfertilized eggs that could delay the lethality of null mutants. All of the proteins were detected in the cytoplasm or in the cell membrane. This study of spatial and temporal expression revealed that all of these six null mutants post-implantation genes in MAPK pathway are expressed and, where tested, phosphorylated/activated proteins are detected in the blastocyst. Studies on RNA expression using RT-PCR suggest that maternal RNA could play an important role in delaying the presence of the lethal phenotype of null mutations.

We have shown recently that endothelial Grb-2-associated binder 1 (Gab1), an intracellular scaffolding adaptor, has a protective effect against limb ischemia via mediating angiogenic signaling pathways. However, the role of Gab1 in cardiac ischemia/reperfusion (I/R) injury remains unknown. In this study, we show that Gab1 is required for cardioprotection against I/R injury. I/R injury led to remarkable phosphorylation of Gab1 in cardiomyocytes. Compared with controls, the mice with cardiomyocyte-specific deletion of Gab1 gene (CGKO mice) exhibited an increase in infarct size and a decrease in cardiac function after I/R injury. Consistently, in hearts of CGKO mice subjected to I/R, the activation of caspase 3 and myocardial apoptosis was markedly enhanced whereas the activation of protein kinase B (Akt) and mitogen-activated protein kinase (MAPK), which are critical for cardiomyocyte survival, was attenuated. Oxidative stress is regarded as a major contributor to myocardial I/R injury. To examine the role of Gab1 in oxidative stress directly, isolated adult cardiomyocytes were subject to oxidant hydrogen peroxide and the cardioprotective effects of Gab1 were confirmed. Furthermore, we found that the phosphorylation of Gab1 and Gab1-mediated activation of Akt and MAPK by oxidative stress was suppressed by ErbB receptor and Src kinase inhibitors, accompanied by an increase in apoptotic cell death. In conclusion, our results suggest that Gab1 is essential for cardioprotection against I/R oxidative injury via mediating survival signaling.

The adaptor protein Grb2-associated binder-1 (Gab1) is known to bind to the SHP-2 tyrosine phosphatase on epidermal growth factor (EGF) receptor stimulation. To clarify the roles of these two proteins in EGF receptor (EGFR) signaling and determine their possible alteration during neoplastic cell progression, we studied these proteins in a Syrian hamster embryo (SHE) cell line model of neoplastic progression. Specifically, we used asbestos-transformed SHE fibroblasts: the 10W+8 clone, which is immortal but nontumorigenic; and the 10W2T clone, which is tumorigenic. Gab1 was detected, and the EGF-dependent formation of the EGFR-Gab1-SHP-2 complex was observed in 10W+8 cells. After cloning hamster Gab1 cDNA, exogenous expression of Gab1 significantly enhanced EGF-dependent mitogenic activity in 10W+8 cells. On the other hand, Gab1 was not detected in 10W2T cells, and the EGF-dependent association of SHP-2 with EGFR was also absent. Exogenous Gab1 expression in transfected 10W2T cells restored the EGF-dependent association of SHP-2 with EGFR, although it only showed a marginal effect on EGF-dependent mitogenic activity. Thus, Gab1 plays a pivotal role in the EGFR signaling pathway via the formation of the EGFR-Gab1-SHP-2 complex, and alteration in the expression and function of Gab1 is implicated in the neoplastic progression of SHE cells.

Acetaminophen (APAP) overdose is the leading cause of drug-induced acute liver failure. In APAP-induced acute liver failure, hepatocyte death and subsequent liver regeneration determines the prognosis of patients, making it necessary to identify suitable therapeutic targets based on detailed molecular mechanisms. Grb2-associated binder 1 (Gab1) adaptor protein plays a crucial role in transmitting signals from growth factor and cytokine receptors to downstream effectors. In this study, we hypothesized that Gab1 is involved in APAP-induced acute liver failure. Hepatocyte-specific Gab1 conditional knockout (Gab1CKO) and control mice were treated with 250 mg/kg of APAP. After APAP treatment, Gab1CKO mice had significantly higher mortality and elevated serum alanine aminotransferase levels compared to control mice. Gab1CKO mice had increased hepatocyte death and increased serum levels of high mobility group box 1, a marker of hepatocyte necrosis. In addition, Gab1CKO mice had reduced hepatocyte proliferation. The enhanced hepatotoxicity in Gab1CKO mice was associated with increased activation of stress-related c-Jun N-terminal kinase (JNK) and reduced activation of extracellular signal-regulated kinase and AKT. Furthermore, Gab1CKO mice showed enhanced mitochondrial translocation of JNK accompanied by an increase in the release of mitochondrial enzymes into the cytosol, which is indicative of increased mitochondrial dysfunction and subsequent nuclear DNA fragmentation. Finally, in vitro experiments showed that Gab1-deficient hepatocytes were more susceptible to APAP-induced mitochondrial dysfunction and cell death, suggesting that hepatocyte Gab1 is a direct target of APAP-induced hepatotoxicity.

CONCLUSIONS

Our current data demonstrate that hepatocyte Gab1 plays a critical role in controlling the balance between hepatocyte death and compensatory hepatocyte proliferation during APAP-induced liver injury.

Despite extensive study of the EGF receptor (EGFR) signaling network, the immediate posttranslational changes that occur in response to growth factor stimulation remain poorly characterized; as a result, the biological mechanisms underlying signaling initiation remain obscured. To address this deficiency, we have used a mass spectrometry-based approach to measure system-wide phosphorylation changes throughout the network with 10-s resolution in the 80 s after stimulation in response to a range of eight growth factor concentrations. Significant changes were observed on proteins far downstream in the network as early as 10 s after stimulation, indicating a system capable of transmitting information quickly. Meanwhile, canonical members of the EGFR signaling network fall into clusters with distinct activation patterns. Src homology 2 domain containing transforming protein (Shc) and phosphoinositol 3-kinase (PI3K) phosphorylation levels increase rapidly, but equilibrate within 20 s, whereas proteins such as Grb2-associated binder-1 (Gab1) and SH2-containing tyrosine phosphatase (SHP2) show slower, sustained increases. Proximity ligation assays reveal that Shc and Gab1 phosphorylation patterns are representative of separate timescales for physical association with the receptor. Inhibition of phosphatases with vanadate reveals site-specific regulatory mechanisms and also uncovers primed activating components in the network, including Src family kinases, whose inhibition affects only a subset of proteins within the network. The results presented highlight the complexity of signaling initiation and provide a window into exploring mechanistic hypotheses about receptor tyrosine kinase (RTK) biology.

PTEN is a tumor suppressor gene implicated in both sporadic cancers and inherited tumor-prone syndromes. Here we show that pten+/- mice display a partially penetrant embryonic lethality. This lethality is associated with defects in both neural and placental development. Notably, this lethality is completely rescued by grb2 haploinsufficiency. In contrast, grb2 heterozygosity did not alter tumorigenesis in either pten+/- or T cell-specific pten-/- mice. grb2-/hypomorph murine embryonic fibroblasts (MEFs) show decreased activation of both PKB and Erk upon stimulation with epidermal growth factor, whereas grb2-/hypomorph; pten+/- MEFs activate PKB but not Erk normally. Similarly, grb2-/hypomorph fibroblasts die in low serum, and this phenotype is rescued by pten haploinsufficiency. Activation of both PKB and Erk as well as survival in low serum-containing media are all rescued by reexpression of Grb2 containing mutations within the N-terminal Src homology 3 (SH3) domain, but not by C-terminal SH3 domain mutants. The N-terminal SH3 domain mutants fail to bind to Sos, whereas the C-terminal SH3 domain mutants fail to bind to Gab1, suggesting that Erk and PKB activation in fibroblasts in response to epidermal growth factor depends on Gab1 or other C-terminal SH3 domain-interacting proteins, but not on Sos. Thus, PTEN/phosphatidylinositol 3' kinase signaling requires Grb2 during both embryonic development and fibroblast survival, but Grb2 heterozygosity does not effect tumorigenesis in pten-deficient mice. In fibroblasts, survival signals emanating from the epidermal growth factor receptor appear to be PKB-dependent, and this activation depends on the C-terminal SH3 domain of Grb2, likely through the interaction of Grb2 with Gab1.

Ectopic activation of fibroblast growth factor receptor 3 (FGFR3) is associated with several cancers, including multiple myeloma (MM). FGFR3 inhibition in these cells inhibits proliferation and induces apoptosis, validating FGFR3 signaling as a therapeutic target in t(4;14) MM cases. We have identified the PI3K regulatory subunit, p85alpha, as a novel interactor of FGFR3 by yeast two-hybrid, and confirmed an interaction with both p85alpha and p85beta in mammalian cells. The interaction of FGFR3 with p85 is dependent upon receptor activation. In contrast to the Gab1-mediated association of FGFRs with p85, the FGFR3-p85 interaction we observed requires FGFR3 Y760, previously identified as a PLCgamma binding site. The interaction of p85 with FGFR3 does not require PLCgamma, suggesting the p85 interaction is direct and independent of PLCgamma binding. FGFR3 and p85 proteins also interact in MM cell lines which consistently express p85alpha and p85beta, but not p50 or p55 subunits. siRNA knockdown of p85beta in MM cells caused an increased ERK response to FGF2. These data suggest that an endogenous negative regulatory role for the p85-FGFR3 interaction on the Ras/ERK/MAPK pathway may exist in response to FGFR3 activity and identifies a novel therapeutic target for MM.

Activation of the extracellular signal-regulated kinase (ERK) pathway is a key factor in the regulation of cell proliferation by growth factors. Hepatocyte growth factor (HGF)-induced cell cycle arrest in the human hepatocellular carcinoma cell line HepG2 requires strong activation of the ERK pathway. In this study, we investigated the molecular mechanism of the activation. We constructed a chimeric receptor composed of the extracellular domain of the NGF receptor and the cytoplasmic domain of the HGF receptor (c-Met) and introduced a point mutation (N1358H) into the chimeric receptor, which specifically abrogates the direct binding of Grb2 to c-Met. The mutant chimeric receptor failed to mediate the strong activation of ERK, up-regulation of the expression of a Cdk inhibitor p16(INK4a) and inhibition of HepG2 cell proliferation by ligand stimulation. Moreover, the mutant receptor did not induce tyrosine phosphorylation of the docking protein Gab1. Knockdown of Gab1 using siRNA suppressed the HGF-induced strong activation of ERK and inhibition of HepG2 cell proliferation. These results suggest that coupling of Grb2 to Gab1 mediates the HGF-induced strong activation of the ERK pathway, which is required for the inhibition of HepG2 cell proliferation.

Previous genetic analysis has shown that dos/soc-1/Gab1 functions positively in receptor tyrosine kinase (RTK)-stimulated Ras/Map kinase signaling through the recruitment of csw/ptp-2/Shp2. Using sensitized assays in Caenorhabditis elegans for let-23/Egfr and daf-2/InsR (insulin receptor-like) signaling, it is shown that soc-1/Gab1 inhibits phospholipase C-gamma (PLCgamma) and phosphatidylinositol 3'-kinase (PI3K)-mediated signaling. Furthermore, as well as stimulating Ras/Map kinase signaling, soc-1/Gab1 stimulates a poorly defined signaling pathway that represses class 2 daf-2 phenotypes. In addition, it is shown that SOC-1 binds the C-terminal SH3 domain of SEM-5. This binding is likely to be functional as the sem-5(n2195)G201R mutation, which disrupts SOC-1 binding, behaves in a qualitatively similar manner to a soc-1 null allele in all assays for let-23/Egfr and daf-2/InsR signaling that were examined. Further genetic analysis suggests that ptp-2/Shp2 mediates the negative function of soc-1/Gab1 in PI3K-mediated signaling, as well as the positive function in Ras/Map kinase signaling. Other effectors of soc-1/Gab1 are likely to inhibit PLCgamma-mediated signaling and stimulate the poorly defined signaling pathway that represses class 2 daf-2 phenotypes. Thus, the recruitment of soc-1/Gab1, and its effectors, into the RTK-signaling complex modifies the cellular response by enhancing Ras/Map kinase signaling while inhibiting PI3K and PLCgamma-mediated signaling.

Grb2-associated binder-1 (Gab1) is an adapter protein related to the insulin receptor substrate family. It is a substrate for the insulin receptor as well as the epidermal growth factor (EGF) receptor and other receptor-tyrosine kinases. To investigate the role of Gab1 in signaling pathways downstream of growth factor receptors, we stimulated rat aortic vascular smooth muscle cells (VSMC) with EGF and platelet-derived growth factor (PDGF). Gab1 was tyrosine-phosphorylated by EGF and PDGF within 1 min. AG1478 (an EGF receptor kinase-specific inhibitor) failed to block PDGF-induced Gab1 tyrosine phosphorylation, suggesting that transactivated EGF receptor is not responsible for this signaling event. Because Gab1 associates with phospholipase Cgamma (PLCgamma), we studied the role of the PLCgamma pathway in Gab1 tyrosine phosphorylation. Gab1 tyrosine phosphorylation by PDGF was impaired in Chinese hamster ovary cells expressing mutant PDGFbeta receptor (Y977F/Y989F: lacking the binding site for PLCgamma). Pretreatment of VSMC with (a specific PLCgamma inhibitor) inhibited Gab1 tyrosine phosphorylation as well, indicating the importance of the PLCgamma pathway. Gab1 was tyrosine-phosphorylated by phorbol ester to the same extent as PDGF stimulation. Studies using antisense protein kinase C (PKC) oligonucleotides and specific inhibitors showed that PKCalpha and PKCepsilon are required for Gab1 tyrosine phosphorylation. Binding of Gab1 to the protein-tyrosine phosphatase SHP2 and phosphatidylinositol 3-kinase was significantly decreased by PLCgamma and/or PKC inhibition, suggesting the importance of the PLCgamma/PKC-dependent Gab1 tyrosine phosphorylation for the interaction with other signaling molecules. Because PDGF-mediated ERK activation is enhanced in Chinese hamster ovary cells that overexpress Gab1, Gab1 serves as an important link between PKC and ERK activation by PDGFbeta receptors in VSMC.

The multisubstrate docking protein, growth-factor-receptor-bound protein 2-associated binder 1 (Gab1), which is phosphorylated on tyrosine residues following activation of receptor tyrosine kinases and cytokine receptors, regulates cell proliferation, survival and epithelial morphogenesis. Gab1 is also tyrosine phosphorylated following activation of G-protein-coupled receptors (GPCRs) where its function is poorly understood. To elucidate the role of Gab1 in GPCR signalling, we investigated the mechanism by which the type A endothelin-1 (ET-1) GPCR induced tyrosine phosphorylation of Gab1. Tyrosine phosphorylation of Gab1 induced by endothelin-1 was inhibited by PP1, a pharmacological inhibitor of Src-family tyrosine kinases. ET-1-induced Gab1 tyrosine phosphorylation was also inhibited by LY294002, which inhibits phosphoinositide 3-kinase (PI 3-kinase) enzymes. Inhibition of Src-family tyrosine kinases or PI 3-kinase also inhibited ET-1-induced activation of the mitogen activated protein kinase family member, extracellular signal-regulated kinase (ERK) 1. Thus we determined whether Gab1 regulated ET-1-induced ERK1 activation. Overexpression of wild-type Gab1 potentiated ET-1-induced activation of ERK1. Structure-function analyses of Gab1 indicated that mutant forms of Gab1 that do not bind the Src homology (SH) 2 domains of the p85 adapter subunit of PI 3-kinase or the SH2-domain-containing protein tyrosine phosphatase 2 (SHP-2) were impaired in their ability to potentiate ET-1-induced ERK1 activation. Taken together, our data indicate that PI 3-kinase and Src-family tyrosine kinases regulate ET-1-induced Gab1 tyrosine phosphorylation, which, in turn, induces ERK1 activation via PI 3-kinase- and SHP-2-dependent pathways.

Hepatocyte growth factor (HGF)/scatter factor is a multifunctional cytokine that induces mitogenesis, motility, and morphogenesis in epithelial, endothelial, and neuronal cells. The receptor for HGF/scatter factor was identified as c-Met tyrosine kinase, and activation of the receptor induces multiple signaling cascades. To gain further insight into c-Met-mediated multiple events at a molecular level, we isolated several signaling molecules including a novel binding partner of c-Met, SH2 domain-containing inositol 5-phosphatase 1 (SHIP-1). Western blot analysis revealed that SHIP-1 is expressed in the epithelial cell line, Madin-Darby canine kidney (MDCK) cells. SHIP-1 binds at phosphotyrosine 1356 at the multifunctional docking site. Because a number of signaling molecules such as Grb2, phosphatidylinositol 3-kinase, and Gab1 bind to the multifunctional docking site, we further performed an in vitro competition study using glutathione S-transferase- or His-tagged signaling molecules with c-Met tyrosine kinase. Our binding study revealed that SHIP-1, Grb2, and Gab1 bound preferentially over phosphatidylinositol 3-kinase. Surprisingly, MDCK cells that overexpress SHIP-1 demonstrated branching tubulogenesis within 2 days after HGF treatment, whereas wild-type MDCK cells showed tubulogenesis only after 6 days following treatment without altering cell scattering or cell growth potency. Furthermore, overexpression of a mutant SHIP-1 lacking catalytic activity impaired HGF-mediated branching tubulogenesis.

PC12 cells have been used as a model system for neuronal differentiation due to their ability to alter their phenotype to a sympathetic neuron-like cell in response to nerve growth factor or fibroblast growth factor. Under some conditions, epidermal growth factor (EGF) can also induce PC12 cells to differentiate. To study signaling from the EGF receptor without the confounding effects of endogenous EGF receptors we generated a chimeric receptor comprised of the ectodomain of platelet-derived growth factor (PDGF) receptor in-frame with the transmembrane and cytoplasmic domains of EGF receptor, termed PER. Expression of PER in PC12 cells confers the ability of PDGF to induce differentiation whereas PDGF has no effect on untransfected PC12 cells. This response is kinase activity-dependent since a kinase-deficient mutant (K721M) fails to induce differentiation in response to PDGF. Mutation of five tyrosine residues that are autophosphorylated in response to EGF either individually or in combination had minimal effects on the ability of these receptors to induce morphological PC12 cell differentiation. The PER mutant with all five autophosphorylation sites mutated to phenylalanine (5YF) was equivalently capable of interacting with several important signaling molecules, including Shc, Grb2, Gab1, phospholipase Cgamma, and Cbl. Furthermore, both the phosphatidylinositol 3-kinase (PI3K)/Akt and Ras/Erk pathways were activated in a sustained manner when PER or 5YF-expressing cells were stimulated with PDGF. Our results show that the five autophosphorylation sites in the extra-kinase C-terminal domain of EGFR are not required for the ability of EGFR to induce morphological differentiation of PC12 cells.

The activated c-Met receptor has potent effects on normal tissues and tumors. c-Met levels are regulated by hepatocyte growth factor (HGF); however, it is unknown if they can be regulated by gastrointestinal (GI) hormones. c-Met is found in many GI tissues/tumors that possess GI hormone receptors. We studied the effect of GI hormones on c-Met in rat pancreatic acini, which possess both receptors. CCK-8, carbachol, and bombesin, but not VIP/secretin, decreased c-Met. CCK-8 caused rapid and potent c-Met down-regulation and abolished HGF-induced c-Met and Gab1 tyrosine phosphorylation, while stimulating c-Met serine phosphorylation. The effect of cholecystokinin (CCK) was also seen in intact acini using immunofluorescence, in a biotinylated fraction representing membrane proteins, in single acinar cells, in Panc-1 tumor cells, and in vivo in rats injected with CCK. CCK-8 did not decrease cell viability or overall responsiveness. GF109203X, thapsigargin, or their combination partially reversed the effect of CCK-8. In contrast to HGF-induced c-Met down-regulation, the effect of CCK was decreased by a lysosome inhibitor (concanamycin) but not the proteasome inhibitor lactacystin. Inhibitors of clathrin-mediated endocytosis blocked the effect of CCK. HGF but not CCK-8 caused c-Met ubiquitination. These results show CCK and other GI hormones can cause rapid c-Met down-regulation, which occurs by a novel mechanism. These results could be important for c-Met regulation in normal as well as in neoplastic tissue in the GI tract.

The sequential reactive changes in Schwann cell phenotypes in transected peripheral nerves, including dedifferentiation, proliferation and migration, are essential for nerve repair. Even though the injury-induced migratory and proliferative behaviors of Schwann cells resemble epithelial and mesenchymal transition (EMT) in tumors, the molecular mechanisms underlying this phenotypic change of Schwann cells are still unclear. Here we show that the reactive Schwann cells exhibit migratory features dependent on the expression of a scaffolding oncoprotein Grb2-associated binder-2 (Gab2), which was transcriptionally induced by neuregulin 1-ErbB2 signaling following nerve injury. Injury-induced Gab2 expression was dependent on c-Jun, a transcription factor critical to a Schwann cell reprograming into a repair-type cell. Interestingly, the injury-induced activation (tyrosine phosphorylation) of Gab2 in Schwann cells was regulated by an EMT signal, the hepatocyte growth factor-c-Met signaling, but not by neuregulin 1. Gab2 knockout mice exhibited a deficit in nerve repair after nerve transection due to limited Schwann cell migration. Furthermore, Gab2 was required for the proliferation of Schwann cells following nerve injury and in vitro, and was over-expressed in human Schwann cell-derived tumors. In contrast, the tyrosine phosphorylation of Gab1 after nerve injury was principally regulated by the neuregulin 1-ErbB2 signaling and was indispensable for remyelination after crush injury, but not for the proliferation and migration of Schwann cells. Our findings indicate that Gab1 and Gab2 in Schwann cells are nonredundant and play a crucial role in peripheral nerve repair.

Resistance to tyrosine kinase inhibitors (TKIs) results in tumor relapse and poor prognosis in patients with lung adenocarcinoma. TKI resistance caused by epidermal growth factor receptor (EGFR) mutations at T790M and c-Met amplification occurs through persistent activation of the MEK/ERK and PI3K/AKT signaling pathways. We therefore expected that dual inhibitors of both signaling pathways could overcome TKI resistance in lung adenocarcinoma. Here, dioscin was selected from a product library of Chinese naturally occurring compounds and overcame TKI resistance in EGFR-mutated lung adenocarcinoma cells. Mechanistically, dioscin may down-regulate the expression of SH2 domain-containing phosphatase-2 (SHP2) at the transcription level by increasing p53 binding to the SHP2 promoter due to reactive oxygen species (ROS). Simultaneous inhibition of MEK/ERK and PI3K/AKT activation via decreased SHP2 expression and its interaction with GAB1 may be responsible for dioscin-mediated TKI sensitivity. A higher unfavorable response to TKI therapy occurred more commonly in patients with high SHP2 mRNA expression than in patients with low SHP2 mRNA expression. Therefore, we suggest that dioscin may act as a dual inhibitor of the MEK/ERK and PI3K/AKT signaling pathways to overcome TKI resistance via dysregulation of SHP2 expression in lung adenocarcinoma.

Background -Although the role of thrombin in atherothrombosis is well studied, its role in the pathogenesis of diet-induced atherosclerosis is not known. Methods -Using a mouse model of diet-induced atherosclerosis and molecular biological approaches, here we have explored the role of thrombin and its G protein-coupled receptor (GPCR) signaling in diet-induced atherosclerosis. Results -In exploring the role of GPCR signaling in atherogenesis, we found that thrombin triggers foam cell formation via inducing CD36 expression and these events require Par1-mediated Gα12-Pyk2-Gab1-PKCθ-dependent ATF2 activation. Genetic deletion of PKCθ in ApoE-/- mice reduced western diet (WD)-induced plaque formation. Furthermore, thrombin induced Pyk2, Gab1, PKCθ and ATF2 phosphorylation, CD36 expression and foam cell formation in peritoneal macrophages of ApoE-/- mice. On the other hand, thrombin only stimulated Pyk2 and Gab1 but not ATF2 phosphorylation or its target gene CD36 expression in the peritoneal macrophages of ApoE-/-:PKCθ-/- mice and it had no effect on foam cell formation. In addition, the aortic root cross sections of WD-fed ApoE-/- mice showed increased Pyk2, Gab1, PKCθ and ATF2 phosphorylation and CD36 expression as compared to ApoE-/-:PKCθ-/- mice. Furthermore, while the monocytes from peripheral blood and aorta of WD-fed ApoE-/- mice were found to contain more of Ly6Chi cells than Ly6Clo cells, the monocytes from WD-fed ApoE-/-:PKCθ-/- mice were found to contain more of Ly6Clo cells than Ly6Chi cells. Interestingly, the Ly6Chi cells showed higher CD36 expression with enhanced capacity to form foam cells as compared to Ly6Clo cells. Conclusions -The above findings reveal for the first time that thrombin-mediated Par1-Gα12 signaling via targeting Pyk2-Gab1-PKCθ-ATF2-dependent CD36 expression might be playing a crucial role in diet-induced atherogenesis.

Shp2, an ubiquitously expressed protein tyrosine phosphatase, is essential for regulation of Ras/ERK signaling pathway and tumorigenesis. Here we report that Shp2 is modified by SUMO1 at lysine residue 590 (K590) in its C-terminus, which is reduced by SUMO1-specific protease SENP1. Analysis of wild-type Shp2 and SUMOylation-defective Shp2(K590R) mutant reveals that SUMOylation of Shp2 promotes EGF-stimulated ERK signaling pathway and increases anchorage-independent cell growth and xenografted tumor growth of hepatocellular carcinoma (HCC) cell lines. Furthermore, we find that mutant Shp2(K590R) reduces its binding with the scaffolding protein Gab1, and consistent with this, knockdown of SENP1 increased the interaction between Shp2 and Gab1. More surprisingly, we show that human Shp2 (hShp2) and mouse Shp2 (mShp2) have differential effects on ERK activation as a result of different SUMOylation level, which is due to the event of K590 at hShp2 substituted by R594 at mShp2. In summary, our data demonstrate that SUMOylation of Shp2 promotes ERK activation via facilitating the formation of Shp2-Gab1 complex and thereby accelerates HCC cell and tumor growth, which presents a novel regulatory mechanism underlying Shp2 in regulation of HCC development.

Mutations in the CdGAP/ARHGAP31 gene, which encodes a GTPase-activating protein for Rac1 and Cdc42, have been reported causative in the Adams-Oliver developmental syndrome often associated with vascular defects. However, despite its abundant expression in endothelial cells, CdGAP function in the vasculature remains unknown. Here, we show that vascular development is impaired in CdGAP-deficient mouse embryos at E15.5. This is associated with superficial vessel defects and subcutaneous edema, resulting in 44% embryonic/perinatal lethality. VEGF-driven angiogenesis is defective in CdGAP(-/-) mice, showing reduced capillary sprouting from aortic ring explants. Similarly, VEGF-dependent endothelial cell migration and capillary formation are inhibited upon CdGAP knockdown. Mechanistically, CdGAP associates with VEGF receptor-2 and controls VEGF-dependent signaling. Consequently, CdGAP depletion results in impaired VEGF-mediated Rac1 activation and reduced phosphorylation of critical intracellular mediators including Gab1, Akt, PLCγ and SHP2. These findings are the first to demonstrate the importance of CdGAP in embryonic vascular development and VEGF-induced signaling, and highlight CdGAP as a potential therapeutic target to treat pathological angiogenesis and vascular dysfunction.

Complexes of signaling proteins that are nucleated upon activation of receptor tyrosine kinases are dynamic macromolecular assemblies held together by interactions, such as the recognition of phosphotyrosines by Src homology 2 (SH2) domains. We predicted that reversible binding and phosphatase activity enable dynamic regulation of these protein complexes, which could affect signal transduction. We explored how dynamics in the interactions among the epidermal growth factor (EGF) receptor (EGFR), GRB2-associated binder protein 1 (GAB1), and SH2 domain-containing phosphatase 2 (SHP2) affected EGFR signaling output, specifically SHP2 binding to tyrosine-phosphorylated GAB1, which relieves the autoinhibition of SHP2. Among the effects of activated SHP2 is increased extracellular signal-regulated kinase (ERK) activity. We found that in H1666 lung adenocarcinoma cells, EGFR-activated Src family kinases (SFKs) counteracted repeated GAB1 dephosphorylation events and maintained the association of SHP2 with phosphorylated GAB1 at a cytosolic site distal from EGFR. A computational model predicted that an experimentally verified delay in SFK inactivation after EGFR inactivation, combined with an amplification of GAB1 phosphorylation in cells with proteins in a specific range of concentrations, enabled GAB1 phosphorylation and GAB1-SHP2 complexes to persist longer than EGFR phosphorylation persisted in response to EGF. This SFK-dependent mechanism was specific to EGFR and did not occur in response to activation of the receptor tyrosine kinase c-MET. Thus, our results quantitatively describe a regulatory mechanism used by some receptor tyrosine kinases to remotely control the duration of a signal by regulating the persistence of a signaling protein complex.

Malignant transformation and progression in cancer is associated with the altered expression of multiple miRNAs, which are considered as post-transcriptional regulators of genes participating in various cellular processes. Although, it has been proposed that miR-23b-3p acts as a tumor suppressor in cervical cancer (CC), not all the pathways through which it alters the cellular processes have been described. The present study examines whether miR-23b-3p directly represses the c-Met expression and that consequently modifies the proliferation, migration and invasion of C33A and CaSki cells. c-Met has five microRNA response elements (MREs) for miR-23b-3p in the 3'-UTR region. The ectopic overexpression of miR-23b-3p significantly reduces c-Met expression in C33A and CaSki cells. The overexpression of miR-23b-3p reduces proliferation, migration and invasion of CaSki cells and the proliferation and invasion in C33A cells. In CaSki cells, the activation of Gab1 and Fak, downstream of c-Met, is reduced in response to the overexpression of miR-23b-3p. Together, the results in the present study indicate that miR-23b-3p is a tumor suppressor that modulates the progression of CC via post-transcriptional regulation of the c-Met oncogene.

Keloids are benign dermal fibroproliferative tumors that develop as a result of several dysregulated processes. Emerging evidence has revealed that miRNAs contribute to keloid formation. However, the molecular mechanisms of keloid pathogenesis remain unclear. In our study, we found that miR-141-3p in keloid tissues and keloid fibroblasts was significantly decreased compared with the levels in normal tissues and normal skin fibroblasts, respectively. miR-141-3p overexpression resulted in significantly decreased proliferation and migration and the promotion of apoptosis in keloid fibroblasts, whereas miR-141-3p knockdown in keloid fibroblasts yielded the opposite results. Growth factor receptor binding 2-associated binding protein 1 (GAB1) was identified and confirmed as a direct target of miR-141-3p. The expression of GAB1 was up-regulated in keloid tissues, and the restoration of GAB1 partially reversed the inhibitory effects of miR-141-3p on the proliferation and migration of keloid fibroblasts. All data suggested that miR-141-3p decreased the proliferation and migration of keloid fibroblasts by repressing GAB1 expression, providing a useful target for keloid management.

Gab1 and Gab2 are conserved scaffolding proteins that amplify and integrate signals stimulated by many growth factor receptors including the Met receptor. Gab1 acts to diversify the signal downstream from Met through the recruitment of multiple signaling proteins, and is essential for epithelial morphogenesis. However, whereas Gab1 and Gab2 are both expressed in epithelial cells, Gab2 fails to support a morphogenic response. We demonstrate that Gab1 and Gab2 are divergent in their function whereby Gab1, but not Gab2, promotes lamellipodia formation, and is localized to the membrane of lamellipodia upon Met activation. We have identified activation of ERK1/2 as a requirement for lamellipodia formation. Moreover, activated ERK1/2 are localized to lamellipodia in Gab1 expressing cells but not in cells that overexpress Gab2. By structure-function studies, we identify that enhanced membrane localization conferred through the addition of a myristoylation signal, together with the addition of the direct Met binding motif (MBM) from Gab1, are required to promote lamellipodia and confer a morphogenic signaling response to Gab2. Moreover, the morphogenesis competent myristoylated Gab2MBM promotes localization of activated ERK1/2 to the leading edge of lamellipodia in a similar manner to Gab1. Hence, subcellular localization of the Gab scaffold, as well as the ability of Gab to interact directly with the Met receptor, are both essential components of the morphogenic signaling response which involves lamellipodia formation and the localization of ERK1/2 activation in membrane ruffles.