The distribution of omega-6 and omega-3 polyunsaturated fatty acid (PUFA) intake in Western diets is disproportionate, containing an overabundance of the omega-6 PUFA, linoleic acid (LA; C18:2). Increased enrichment with LA has been shown to contribute to the enhancement of tumorigenesis in several cancer models. Previous work has indicated that phosphatidylinositol 3-kinase (PI3K) may play a key role in LA-induced tumorigenesis. However, the modes by which LA affects carcinogenesis have not been fully elucidated. In this study, a mechanism for LA-induced upregulation of cancer cell growth is defined. LA treatment enhanced cellular proliferation in BT-474 human breast ductal carcinoma and A549 human lung adenocarcinoma cell lines. Enrichment of LA increased cyclooxygenase (COX) activity and led to increases in prostaglandin E2 (PGE2), followed by increases in matrix metalloproteinase (MMP) and transforming growth factor alpha (TGF-α) levels, which are all key elements involved in the enhancement of cancer cell growth. Further investigation revealed that LA supplementation in both BT-474 breast and A549 lung cancer cell lines greatly increased the association between the scaffolding protein GRB2-associated-binding protein 1 (Gab1) and epidermal growth factor receptor (EGFR), although Gab1 protein levels were significantly decreased. These LA-induced changes were associated with increases in activated Akt (pAkt), a downstream signaling component in the PI3K pathway. Treatment with inhibitors of EGFR, PI3K and Gab1-specific siRNAs reversed the upregulation of pAkt, as well as the observed increases in cell proliferation by LA in both cell lines. A549 xenograft assessment in athymic nude mice fed high levels of LA exhibited similar increases in EGFR-Gab1 association and increased levels of pAkt, while mice fed with high levels of the omega-3 PUFA, docosahexaenoic acid (DHA; C22:6), demonstrated an opposite response. The involvement of Gab1 in LA-induced tumorigenesis was further defined utilizing murine cell lines that express high levels of Gab1. Significant increases in cell proliferation were observed with the addition of increasing concentrations of LA. However, no changes in cell proliferation were detected in the murine paired cell lines expressing little or no Gab1 protein, establishing Gab1 as major target in LA-induced enhancement of tumorigenesis.

Mutations in the protein alpha-synuclein (SNCA) have been linked to Parkinson's disease. We recently reported that non-mutated SNCA enhanced glucose uptake through the Gab1-PI3 kinase-Akt pathway and elucidated its effects on glucose regulation. Here, we examined the association of SNCA with insulin resistance (IR), a condition that is characterized by decreased tissue glucose uptake. Our observations include those from a population study as well as a SNCA-deficient mouse model, which had not previously been characterized in an IR scenario. In 1,152 patients, we found that serum SNCA levels were inversely correlated with IR indicators--body mass index, homeostatic model assessment for IR (HOMA-IR) and immunoreactive insulin (IRI)--and, to a lesser extent, with blood pressure and age. Additionally, SNCA-deficient mice displayed alterations in glucose and insulin responses during diet-induced IR. Moreover, during euglycemic clamp assessments, SNCA knock-out mice fed a high-fat diet (HFD) showed severe IR in adipose tissues and skeletal muscle. These findings provide new insights into IR and diabetes and point to SNCA as a potential candidate for further research.

Ligation of erythropoietin (EPO) receptor (EPOR) JAK2 kinase complexes propagates signals within erythroid progenitor cells (EPCs) that are essential for red blood cell production. To reveal hypothesized novel EPOR/JAK2 targets, a phosphotyrosine (PY) phosphoproteomics approach was applied. Beyond known signal transduction factors, 32 new targets of EPO-modulated tyrosine phosphorylation were defined. Molecular adaptors comprised one major set including growth factor receptor-bound protein 2 (GRB2)-associated binding proteins 1-3 (GAB1-3), insulin receptor substrate 2 (IRS2), docking protein 1 (DOK1), Src homology 2 domain containing transforming protein 1 (SHC1), and sprouty homologue 1 (SPRY1) as validating targets, and SPRY2, SH2 domain containing 2A (SH2D2A), and signal transducing adaptor molecule 2 (STAM2) as novel candidate adaptors together with an ORF factor designated as regulator of human erythroid cell expansion (RHEX). RHEX is well conserved in Homo sapiens and primates but absent from mouse, rat, and lower vertebrate genomes. Among tissues and lineages, RHEX was elevated in EPCs, occurred as a plasma membrane protein, was rapidly PY-phosphorylated >20-fold upon EPO exposure, and coimmunoprecipitated with the EPOR. In UT7epo cells, knockdown of RHEX inhibited EPO-dependent growth. This was associated with extracellular signal-regulated kinase 1,2 (ERK1,2) modulation, and RHEX coupling to GRB2. In primary human EPCs, shRNA knockdown studies confirmed RHEX regulation of erythroid progenitor expansion and further revealed roles in promoting the formation of hemoglobinizing erythroblasts. RHEX therefore comprises a new EPO/EPOR target and regulator of human erythroid cell expansion that additionally acts to support late-stage erythroblast development.

This review will provide insight on the current understanding of the intracellular signaling mechanisms by which hyperosmolarity mimics insulin responses such as Glut 4 translocation and glucose transport but also antagonizes insulin effects. Glucose uptake induced by insulin is largely dependent on the PI 3-kinase/PKB pathway. In both adipocyte and muscle cells, hyperosmolarity promotes glucose uptake by multiple mechanisms which do not require PI 3-kinase/PKB pathway but are dependent on the cell type. In muscle, osmotic stress induces glucose uptake by stimulation of AMP-Kinase and/or inhibition of Glut 4 endocytosis. In adipocytes, activation of Gab1-dependent signaling pathway plays an important role in osmotic stress-mediated glucose uptake. Apart of its insulin-like effects, hyperosmolarity can lead to cellular insulin resistance mediated by both prevention of PKB activation and inhibition of the Insulin Receptor Substrate-1 (IRS1) function. Serine phosphorylation and degradation of IRS1 negatively regulate its functions. Understanding how osmotic stress induces glucose transport or mediates insulin resistance may provide novel targets for strategies to enhance glucose transport or to prevent insulin resistance.

The Grb2-associated binder 1 (Gab1), which serves as a scaffolding adaptor protein, plays a crucial role in transmitting key signals that control cell growth, differentiation and function from multiple receptors. However, its biological role in osteoblast activity and postnatal bone metabolism remains unclear. To elucidate the in vivo function of Gab1 in postnatal bone remodeling, we generated osteoblast-specific Gab1 knockout mice. Disruption of Gab1 expression in osteoblasts led to decreased trabecular bone mass with a reduced bone formation rate and a decreased bone resorption. Bones from Gab1 mutants also exhibited inferior mechanical properties. Moreover, primary osteoblasts from Gab1 mutant mice demonstrated markedly suppressed osteoblast mineralization, increased susceptibility to apoptosis and decreased expression of receptor activator of NF-kappaB ligand (RANKL). Activation of serine-threonine Akt kinase and extracellular signal-regulated kinase in response to insulin and insulin-like growth factor 1 was attenuated in Gab1 mutant osteoblasts. Our results show that Gab1-mediated signals in osteoblasts are crucial for normal postnatal bone homeostasis.

Functions of signaling mediators Grb10 or Gab1 have been described in mitogenesis but remained disconnected. Here, we report the peptide hormone-dependent direct association between Grb10 and Gab1 and their functional connection in mitogenic signaling via MAP kinase using cultured fibroblasts as a model. In response to PDGF-, IGF-I, or insulin increased levels of Grb10 potentiated cell proliferation or survival whereas dominant-negative, domain-specific Grb10 peptide mimetics attenuated cell proliferation. This response was sensitive to p44/42 MAPK inhibitor but not to p38 MAPK inhibitor. In response to IGF-I or insulin Raf-1, MEK 1/2, and p44/42 MAPK were regulated by Grb10 but not Ras or p38 MAPK. In response to PDGF MEK 1/2, p44/42 MAPK and p38 MAPK were regulated by Grb10 but not Ras or Raf-1. Peptide hormone-dependent co-immunoprecipitation of Grb10 and Gab1 was demonstrated and specifically blocked by a Grb10 SH2 domain peptide mimetic. This domain was sufficient for direct, peptide hormone-dependent association with Gab1 via the Crk binding region. In response to PDGF, IGF-I, or insulin, in a direct comparison, elevated levels of mouse Grb10 delta, or human Grb10 beta or zeta equally potentiated fibroblast proliferation. Proliferation was severely reduced by Gab1 gene disruption whereas an elevated Gab1 gene dose proportionally stimulated Grb10-potentiated cell proliferation. In conclusion, Gab1 and Grb10 function as direct binding partners in the regulation of the mitogenic MAP kinase signal. In cultured fibroblasts, elevated levels of human Grb10 beta, zeta or mouse Grb10 delta comparably potentiate mitogenesis in response to PDGF, IGF-I, or insulin.

Grb2-associated binder 1 (GAB1) is a docking protein found to associate with the activated c-MET receptor via the MET-binding domain (MBD) and appears to be critical for the tubulogenic actions of this receptor. Pull-down experiments with bacterially expressed MBD and full-length GAB1 revealed the presence of c-MET as well as phosphorylated ERK2 (pERK2). By using purified pERK2 and non-pERK2, we found that GAB1 associates exclusively with the phosphorylated form of the enzyme and that this association does not require mediation by a third protein. When epitope-tagged GAB1 was co-transfected with constitutively active MEK1 into A293 cells, co-immunoprecipitation of GAB1 and pERK2 was observed, demonstrating that this interaction can occur in intact cells. In vitro, both the MBD and full-length GAB1 were found to be substrates for activated ERK2. In intact cells, epitope-tagged GAB1 was found to be basally phosphorylated on serine with an increase following co-transfection with constitutively active MEK1 and the appearance of novel phosphorylation sites detected by phosphopeptide mapping. Thus, it appears that GAB1 can associate directly with phosphorylated ERK2 via the MET-binding domain and that GAB1 then acts as a substrate for the enzyme.

OBJECTIVE

Atherosclerosis preferentially occurs in areas of turbulent flow, whereas laminar flow is atheroprotective. Inflammatory cytokines have been shown to stimulate adhesion molecule expression in endothelial cells that may promote atherosclerosis, in part, by stimulating c-Jun N-terminal kinase (JNK) and nuclear factor (NF)-kappaB transcriptional activity.

RESULTS

Because Src kinase family and Src homology region 2-domain phosphatase-2 (SHP-2) may regulate JNK activation, we studied the effect of shear stress on endothelial inflammation and JNK. Human umbilical vein endothelial cells preexposed to flow showed decreased tumor necrosis factor (TNF)-alpha-induced c-Jun and NF-kappaB transcriptional activation. TNF-alpha-mediated JNK, c-Jun, and NF-kappaB activation required Src and SHP-2 activity. Shear stress significantly inhibited SHP-2 phosphatase activity without affecting TNF-alpha-induced Src family kinase activation. Because MEKK3 and Gab1 are critical for TNF-alpha-induced c-Jun and NF-kappaB activation, we determined the role of SHP-2 phosphatase activity in MEKK3 signaling. A catalytically inactive form of SHP-2 increased MEKK3/Gab1 interaction and inhibited MEKK3 (but not MEKK1)-mediated c-Jun and NF-kappaB activation.

CONCLUSIONS

These results suggest that SHP-2 is a key mediator for the inhibitory effects of shear stress on TNF-alpha signaling in part via regulating MEKK3/Gab1 interaction, MEKK3 signaling, and subsequent adhesion molecule expression.

c-Met is the receptor for hepatocyte growth factor/scatter factor (HGF/SF). It mediates multiple cellular responses in development and adult life, and c-Met hyperactivity is associated with malignant transformation of cells and the acquisition of metastatic properties. Signal transducer and activator of transcription 3 (STAT3) has been shown to contribute to c-Met-mediated cell motility and is, thus, potentially involved in the control of invasive cell behavior. We have functionally reconstituted c-Met-dependent signal transduction in fibroblasts with the aim of studying Met-driven cell invasiveness and the role of STAT3 in this phenomenon. Activation of the system was achieved by means of a hybrid receptor comprising the extracellular domain of the nerve growth factor (NGF) receptor TrkA, the cytoplasmic part of c-Met and a C-terminally fused blue fluorescent protein (BFP). In addition, a GFP-tagged derivative of adaptor protein Gab1 was expressed. NGF-stimulation of mouse fibroblasts expressing tagged versions of both Trk-Met and Gab1 with NGF resulted in anchorage-independent growth and enhanced invasiveness. By freeze-fracture cytochemistry and electron microscopy, we were able to visualize the ligand-induced formation of multivalent receptor complex assemblies within the cell membrane. NGF-stimulation of the heterologous receptor system evoked activation of STAT3 as evidenced by tyrosine phosphorylation and the formation of STAT3 clusters at the cell membrane. siRNA-mediated ablation of STAT3 expression resulted in a drastic reduction of c-Met-driven invasiveness, indicating an important role of STAT3 in the control of this particularly relevant property of transformed cells.

Medulloblastoma (MB) is composed of four molecular subgroups viz. WNT, SHH, groups 3 and 4, identified using various high-throughput methods. Translation of this molecular data into pathologist-friendly techniques that would be applicable in laboratories all over the world is a major challenge. Ninety-two MBs were analyzed using a panel of 10 IHC markers, real-time PCR for mRNA and miRNA expression, and FISH for MYC amplification. β-catenin, GAB1 and YAP1 were the only IHC markers of utility in classification of MBs into three subgroups viz. WNT (9.8%), SHH (45.6%) and non-WNT/SHH (44.6%). mRNA expression could further classify some non-WNT/SHH tumors into groups 3 and 4. This, however, was dependent on integrity of RNA extracted from FFPE tissue. MYC amplification was seen in 20% of non-WNT/SHH cases and was associated with the worst prognosis. For routine diagnostic practice, we recommend classification of MBs into three subgroups: WNT, SHH and non-WNT/SHH, with supplementation by prognostic markers like MYC for non-WNT/SHH tumors. Using this panel, we propose a new three-tier risk stratification system for MBs. Molecular subgrouping with this limited panel is rapid, economical, works well on FFPE tissue and is reliable as it correlates significantly with clinicopathological parameters and patient survival.

Growth factors and their downstream receptor tyrosine kinases (RTKs) mediate a number of biological processes controlling cell function. Adaptor (docking) proteins, which consist exclusively of domains and motifs that mediate molecular interactions, link receptor activation to downstream effectors. Recent studies have revealed that Grb2-associated-binders (Gab) family members (including Gab1, Gab2, and Gab3), when phosphorylated on tyrosine residues, provide binding sites for multiple effector proteins, such as Src homology-2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) and phosphatidylinositol 3-kinase (PI3K) regulatory subunit p85, thereby playing important roles in transducing RTKs-mediated signals into pathways with diversified biological functions. Here, we provide an up-to-date overview on the domain structure and biological functions of Gab1, the most intensively studied Gab family protein, in growth factor signaling and biological functions, with a special focus on angiogenesis.

The docking proteins of the Grb2-associated binder (Gab) family have emerged as crucial signaling compartments in metazoans. In mammals, the Gab proteins, consisting of Gab1, Gab2, and Gab3, are involved in the amplification and integration of signal transduction evoked by a variety of extracellular stimuli, including growth factors, cytokines, antigens, and other molecules. Gab proteins lack the enzymatic activity themselves; however, when phosphorylated on tyrosine residues, they provide binding sites for multiple Src homology-2 (SH2) domain-containing proteins, such as SH2-containing protein tyrosine phosphatase 2 (SHP2), phosphatidylinositol 3-kinase regulatory subunit p85, phospholipase Cγ, Crk, and GC-GAP. Through these interactions, the Gab proteins transduce signals from activated receptors into pathways with distinct biological functions, thereby contributing to signal diversification. They are known to play crucial roles in numerous physiological processes through their associations with SHP2 and p85. In addition, abnormal Gab protein signaling has been linked to human diseases including cancer, cardiovascular disease, and inflammatory disorders. In this paper, we provide an overview of the structure, effector functions, and regulation of the Gab docking proteins, with a special focus on their associations with cardiovascular disease, cancer, and inflammation.

M2-polarized macrophages, also known as alternatively activated macrophages, have long been associated with pulmonary fibrosis; however, the mechanism has not been fully defined. Gab1 and Gab2 proteins belong to the Gab family of adaptors and are integral components of the signal specificity in response to various extracellular stimuli. In this report, we found that levels of both Gab1 and Gab2 were elevated in M2-polarized macrophages isolated from bleomycin-induced fibrotic lungs. In vitro Gab1/2 deficiency in bone marrow-derived macrophages abrogated IL-4-mediated M2 polarization. Furthermore, in vivo conditional removal of Gab1 (Gab1MyKO) and germ line knock-out of Gab2 (Gab2-/-) in macrophages prevented a bias toward the M2 phenotype and attenuated bleomycin-induced fibrotic lung remodeling. In support of these observations, Gab1/2 were involved in responses predominated by IL-4 signaling, an essential determinant for macrophage M2 polarization. Further investigation revealed that both Gab1 and -2 are recruited to the IL-4 receptor, synergistically enhancing downstream signal amplification but conferring IL-4 signal preference. Mechanistically, the loss of Gab1 attenuated AKT activation, whereas the absence of Gab2 suppressed STAT6 activation in response to IL-4 stimulation, both of which are commonly attributed to M2-driven pulmonary fibrosis in mice. Taken together, these observations define a non-redundant role of Gab docking proteins in M2 polarization, adding critical insights into the pathogenesis of idiopathic pulmonary fibrosis.

Epithelial-mesenchymal transition (EMT) is thought to contribute to metastasis and chemoresistance in patients with hepatocellular carcinoma (HCC), leading to their poor prognosis. The genes driving EMT in HCC are not yet fully understood, however. Here, we show that mobilization of Sleeping Beauty (SB) transposons in immortalized mouse hepatoblasts induces mesenchymal liver tumors on transplantation to nude mice. These tumors show significant down-regulation of epithelial markers, along with up-regulation of mesenchymal markers and EMT-related transcription factors (EMT-TFs). Sequencing of transposon insertion sites from tumors identified 233 candidate cancer genes (CCGs) that were enriched for genes and cellular processes driving EMT. Subsequent trunk driver analysis identified 23 CCGs that are predicted to function early in tumorigenesis and whose mutation or alteration in patients with HCC is correlated with poor patient survival. Validation of the top trunk drivers identified in the screen, including MET (MET proto-oncogene, receptor tyrosine kinase), GRB2-associated binding protein 1 (GAB1), HECT, UBA, and WWE domain containing 1 (HUWE1), lysine-specific demethylase 6A (KDM6A), and protein-tyrosine phosphatase, nonreceptor-type 12 (PTPN12), showed that deregulation of these genes activates an EMT program in human HCC cells that enhances tumor cell migration. Finally, deregulation of these genes in human HCC was found to confer sorafenib resistance through apoptotic tolerance and reduced proliferation, consistent with recent studies showing that EMT contributes to the chemoresistance of tumor cells. Our unique cell-based transposon mutagenesis screen appears to be an excellent resource for discovering genes involved in EMT in human HCC and potentially for identifying new drug targets.

The insulin receptor (IR) is an important hub in insulin signaling and its activation is tightly regulated. Upon insulin stimulation, IR is activated through autophosphorylation, and consequently phosphorylates several insulin receptor substrate (IRS) proteins, including IRS1-6, Shc and Gab1. Certain adipokines have also been found to activate IR. On the contrary, PTP, Grb and SOCS proteins, which are responsible for the negative regulation of IR, are characterized as IR inhibitors. Additionally, many other proteins have been identified as IR substrates and participate in the insulin signaling pathway. To provide a more comprehensive understanding of the signals mediated through IR, we reviewed the upstream and downstream signal molecules of IR, summarized the positive and negative modulators of IR, and discussed the IR substrates and interacting adaptor proteins. We propose that the molecular events associated with IR should be integrated to obtain a better understanding of the insulin signaling pathway and diabetes.

The platelet-derived growth factor beta receptor (PDGFbetaR) plays an important role in proliferation and motility of fibroblasts. We have been investigating the effects of sustained PDGFbetaR activation in mortal human diploid fibroblasts (HDFs), which are typically difficult to transform. We have previously shown that the bovine papillomavirus E5 protein, through its ability to crosslink and constitutively activate the PDGFbetaR, induces morphological transformation, enhanced growth and loss of contact inhibition (focus formation) in HDFs. Here, we characterized two E5 mutants as being severely defective for focus formation but still competent for enhanced growth, suggesting that proliferation is insufficient for loss of contact inhibition. These E5 mutants were then used in a comparative study to distinguish the PDGFbetaR signaling intermediates required for the enhanced growth phenotype from those required for focus formation. Our data suggested that a PI 3-kinase (PI3K)-AKT-cyclin D3 pathway, a Grb2-Gab1-SHP2 complex and JNK played a role in the enhanced growth phenotype. However, a SHP2-p66Shc-p190BRhoGAP complex and ROCK were implicated exclusively in focus formation. We speculate that a SHP2-p66Shc-p190BRhoGAP signaling complex recruited to the activated PDGFbetaR promotes a distinct Rho-dependent process required for focus formation but not growth of HDFs.

The essential GAB1 gene, which encodes an endoplasmic reticulum (ER)-membrane protein, was identified in a screen for mutants defective in cellular morphogenesis. A temperature-sensitive gab1 mutant accumulates complete glycosylphosphatidylinositol (GPI) precursors, and its temperature sensitivity is suppressed differentially by overexpression of different subunits of the GPI transamidase, from strong suppression by Gpi8p and Gpi17p, to weak suppression by Gaa1p, and to no suppression by Gpi16p. In addition, both Gab1p and Gpi17p localize to the ER and are in the same protein complex in vivo. These findings suggest that Gab1p is a subunit of the GPI transamidase with distinct relationships to other subunits in the same complex. We also show that depletion of Gab1p or Gpi8p, but not Gpi17p, Gpi16p, or Gaa1p causes accumulation of cofilin-decorated actin bars that are closely associated with the perinuclear ER, which highlights a functional interaction between the ER network and the actin cytoskeleton.

The normal human breast epithelial cell line, MCF10A, was used to investigate the mechanism by which high-density inhibits EGF-dependent cell cycle progression. EGF-dependent Akt activation was found to be transient in high-density cells and sustained in low-density cells. High-density cells also showed decreased EGF receptor (EGFR) autophosphorylation, decreased retinoblastoma protein phosphorylation, and increased p27 protein expression. Although EGFR activation was decreased in the high-density cells, the activation was sufficient to stimulate EGFR substrates comparable to low-density cells. EGF-dependent activation of the Erk1/2 pathway and the upstream activators of Akt (Gab1, erbB3, PI3 kinase, and PDK1) showed no density dependency. Antagonists of Akt activity provided further evidence that regulation of Akt activation is the critical signal transduction step controlling EGF-dependent cell cycle progression. Both adenovirus-mediated expression of dominant-negative Akt and inhibition of PI3 kinase-mediated Akt activation with LY294002 blocked cell cycle progression of low-density cells. In summary, we report the novel finding that high-density blocks EGF-dependent cell cycle progression by inhibiting EGF signaling at the level of EGF-dependent Akt activation rather than at the level of EGFR activation.

The epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of head and neck squamous cell carcinoma (HNSCC). Despite the high expression of EGFR in HNSCC, EGFR inhibitors have only limited success as monotherapy. The Grb2-associated binder (GAB) family of adaptor proteins acts as docking/scaffolding molecules downstream of tyrosine kinase receptors. We hypothesized that GAB1 may amplify EGFR-induced signaling in HNSCCs and therefore could play a role in the reduced sensitivity of HNSCC to EGFR inhibitors. We used representative human HNSCC cell lines overexpressing wild type EGFR, and expressing GAB1 but not GAB2. We demonstrated that baseline Akt and MAPK signaling were reduced in HNSCC cells in which GAB1 expression was reduced. Furthermore, the maximal EGF-induced activation of the Akt and MAPK pathway was reduced and delayed, and the duration of the EGF-induced activation of these pathways was reduced in cells with GAB1 knock-down. In agreement with this, HNSCC cells in which GAB1 levels were reduced showed an increased sensitivity to the EGFR inhibitor gefitinib. Our work demonstrates that GAB1 plays an important role as part of the mechanism of by which EGFR induces induced activation of the MAPK and AKT pathway. Our results identify GAB1 as an amplifier of the EGFR-initiated signaling, which may also interfere with EGFR degradation. These findings support the emerging notion that reducing GAB1 function may sensitize HNSCC to EGFR inhibitors, hence representing a new therapeutic target HNSCC treatment in combination with EGFR targeting agents.

Infection of erythroid progenitor cells by Friend spleen focus-forming virus (SFFV) leads to acute erythroid hyperplasia and eventually to erythroleukemia in susceptible strains of mice. The viral envelope protein, SFFV gp55, forms a complex with the erythropoietin receptor (EpoR) and a short form of the receptor tyrosine kinase Stk (sf-Stk), activating both and inducing Epo-independent proliferation. Recently, we discovered that coexpression of SFFV gp55 and sf-Stk is sufficient to transform NIH 3T3 and primary fibroblasts. In the current study, we demonstrate that sf-Stk and its downstream effectors are critical to this transformation. Unlike SFFV-derived erythroleukemia cells, which depend on PU.1 expression for maintenance of the transformed phenotype, SFFV gp55-sf-Stk-transformed fibroblasts are negative for PU.1. Underscoring the importance of sf-Stk to fibroblast transformation, knockdown of sf-Stk abolished the ability of these cells to form anchorage-independent colonies. Like SFFV-infected erythroid cells, SFFV gp55-sf-Stk-transformed fibroblasts express high levels of phosphorylated MEK, ERK, phosphatidylinositol 3-kinase (PI3K), Gab1/2, Akt, Jun kinase (JNK), and STAT3, but unlike virus-infected erythroid cells they fail to express phosphorylated STATs 1 and 5, which may require involvement of the EpoR. In addition, the p38 mitogen-activated protein kinase (MAPK) stress response is suppressed in the transformed fibroblasts. Inhibition of either JNK or the PI3K pathway decreases both monolayer proliferation and anchorage-independent growth of the transformed fibroblasts as does the putative kinase inhibitor luteolin, but inhibition of p38 MAPK has no effect. Our results indicate that sf-Stk is a molecular endpoint of transformation that could be targeted directly or with agents against its downstream effectors.

Protein-protein interactions play critical regulatory roles in mediating signal transduction. Previous studies have identified an unconventional, small-molecule, Src signal transduction inhibitor, UCS15A. UCS15A differed from conventional Src-inhibitors in that it did not alter the levels or the tyrosine kinase activity of Src. Our studies suggested that UCS15A exerted its Src-inhibitory effects by a novel mechanism that involved the disruption of protein-protein interactions mediated by Src. In the present study we have examined the ability of UCS15A to disrupt the interaction of Src-SH3 with Sam68, both in vivo and in vitro. This ability of UCS15A was not restricted to Src-SH3 mediated protein-protein interactions, since the drug was capable of disrupting the in vivo interactions of Sam68 with other SH3 domain containing proteins such as Grb2 and PLCgamma. In addition, UCS15A was capable of disrupting other typical SH3-mediated protein-protein interactions such as Grb2-Sos1, cortactin-ZO1, as well as atypical SH3-mediated protein-protein interactions such as Grb2-Gab1. However, UCS15A was unable to disrupt the non-SH3-mediated protein-protein interactions of beta-catenin, with E-cadherin and alpha-catenin. In addition, UCS15A had no effect on the SH2-mediated interaction between Grb2 and activated Epidermal Growth Factor receptor. Thus, the ability of UCS15A, to disrupt protein-protein interactions appeared to be restricted to SH3-mediated protein-protein interactions. In this regard, UCS15A represents the first example of a non-peptide, small molecule agent capable of disrupting SH3-mediated protein-protein interactions. In vitro analyses suggested that UCS15A did not bind to the SH3 domain itself but rather may interact directly with the target proline-rich domains.

Gab1 (Grb2 associated binder1) has been identified as an adaptor molecule downstream of many growth factors, including epidermal growth factor (EGF), fibroblast growth factor, and platelet-derived growth factor, which have been shown to play crucial roles as mitotic signals for a variety of neural progenitor cells, including stem cells, both in vitro and in vivo. Here, we show that Gab1 deficiency results in a reduction in the number of Olig2-positive (Olig2(+)) progenitor cells in the developing mouse spinal cord after embryonic day 12.5 (E12.5), when gliogenesis starts in the pMN domain where the EGF receptor (EGFR) is expressed predominantly. Our in vitro analysis further revealed that Gab1 is essential for EGF-dependent proliferation of Olig2(+) progenitor cells derived from the E12.5 ventral and E14.5 dorsal but not ventral spinal cord, whereas Gab1 is always required for the activation of Akt1 but not of ERK1/2. Moreover, we found that the action of the Gab1/Akt pathway is context-dependent, since constitutively active Akt1 could rescue the proliferation defect only in the E12.5 spinal cord of the Gab1-deficient mouse in vitro. Finally, we demonstrated that EGFR-deficient mice and Gab1-deficient mice showed a similar reduction in the number of Olig2(+) progenitor cells in the developing spinal cord. These findings indicate that EGFR-mediated signaling through Gab1/Akt contributes to the sufficient expansion of Olig2(+) progenitor cells in a spatiotemporally regulated manner, which represents the origin of glial cells in the developing spinal cord. Disclosure of potential conflicts of interest is found at the end of this article.

Based on our previous observations that active ERK associates with and phosphorylates Gab1 in response to HGF, and the prediction that the ERK phosphorylation site is adjacent to one of the phosphatidylinositol 3-kinase (PI3K) SH2 binding motifs, we examined the possibility that ERK phosphorylation can regulate the Gab1/PI3K association. The HGF-mediated association of Gab1 with either full-length GST-p85 or its isolated N- or C-terminal SH2 domains was inhibited by approximately 50% in the setting of ERK inhibition, a result confirmed by co-immunoprecipitation of the native proteins. A 14-amino acid peptide encoding (472)YVPMTP(477) (one of the major p85 binding sites in Gab1 and the predicted ERK phosphorylation site) was synthesized with either phosphotyrosine alone (pY), or phosphotyrosine + phosphothreonine (pYT). In both pull-down assays and competition assays, pYT demonstrated a higher affinity for p85 than did pY alone. Finally, examination of the phosphorylation state of Akt after HGF stimulation revealed that ERK inhibition resulted in a decrease in Akt activation at both 5 and 10 min. These results suggest that activated ERK can phosphorylate Gab1 in response to HGF stimulation and thereby potentiate the Gab1/PI3K association and subsequent PI3K activation.

The EGF receptor can bind seven different agonist ligands. Although each agonist appears to stimulate the same suite of downstream signaling proteins, different agonists are capable of inducing distinct responses in the same cell. To determine the basis for these differences, we used luciferase fragment complementation imaging to monitor the recruitment of Cbl, CrkL, Gab1, Grb2, PI3K, p52 Shc, p66 Shc, and Shp2 to the EGF receptor when stimulated by the seven EGF receptor ligands. Recruitment of all eight proteins was rapid, dose-dependent, and inhibited by erlotinib and lapatinib, although to differing extents. Comparison of the time course of recruitment of the eight proteins in response to a fixed concentration of each growth factor revealed differences among the growth factors that could contribute to their differing biological effects. Principal component analysis of the resulting data set confirmed that the recruitment of these proteins differed between agonists and also between different doses of the same agonist. Ensemble clustering of the overall response to the different growth factors suggests that these EGF receptor ligands fall into two major groups as follows: (i) EGF, amphiregulin, and EPR; and (ii) betacellulin, TGFα, and epigen. Heparin-binding EGF is distantly related to both clusters. Our data identify differences in network utilization by different EGF receptor agonists and highlight the need to characterize network interactions under conditions other than high dose EGF.