Scaffolding Adaptor Protein Research Articles

Erratum: Growth Factor Receptor Bound Protein 2–Associated Binder 2, a Scaffolding Adaptor Protein, Negatively Regulates Host Immunity against Tuberculosis

Erratum: Growth Factor Receptor Bound Protein 2–Associated Binder 2, a Scaffolding Adaptor Protein, Negatively Regulates Host Immunity against Tuberculosis

Cooperativity between Flt3/Syk and Proteasome Inhibitors in Flt3mutant/Wildtype AML, By Acting on the Oxphos and Wnt Endpoints of Syk and p62SQSTM1 Pathways Prohibits Protective Autophagy; While Downregulation of NRF2, NQO1, Jun, and b-Catenin Bypasses Interfering RAS or WT1 Co-Mutations

Recent attempts at single agent targeted therapy of AMLs described by mutation of Flt3 or nuclear epigenetic effectors, has led to the conclusion that combination targeted approaches will be required (CM McMahon et al. Cancer Discov 2019). The simplest combination therapy would involve inhibitors directed at mutant drivers at each level (receptor, nuclear). However, the number of those inhibitors is limited. A broader strategy would target common endpoints for converging pathways such as Wnt/beta-catenin activation elicited by mutation of IDH1/2, TET2, DNMT3A. We found marked cytoplasmic accumulation of ubiquitinated protein (especially inactive b-catenin excluded from the nucleus) by treatment with proteasome inhibitor(PI) to be an efficient, dose-dependent inducer of endoplasmic reticulum (ER) stress apoptosis in mutant Flt3/Wnt effector AML's, requiring concentrations =/>100nM Ixazomib, or =/>20nM Bortezomib, when used alone on cultured blasts. Indeed, a compensatory pathway to protective autophagic escape from PI in poor-risk AML is linked to the levels of NRF2, a major transcriptional activator of NADPH quinone reductase1 (NQO1) that buffers oxidative stress. However, the Flt3/Syk inhibitor (FSI) in clinical trial, TAK-659, at =/<250 nM, significantly broadened the ED30-66 concentrations for these PIs when acting on AML blasts annotated for the presence of mutations Flt3, DNMT3A, IDH1/2, NPM1, where inhibitory activity has been reported for all other Flt3 inhibitors (JW Tyner et al, 2018). We identified that activity involved reduction by the combination of PI with TAK-659, of p62SQSTM1, the scaffolding adaptor protein linked to both sequestration/disposal of ubiquitinated and damaged proteins, as well as to mitochondrial remodeling for OXPHOS. P62, jun, NRF2, NQO1, and b-catenin were reduced by the combination, to levels that equaled or exceeded effects by TAK-659 alone, with heightened impact on blast cell extinction by visible apoptosis and thymidine uptake. However, the combination of PI Ixazomib/Bortezomib with TAK-659 demonstrated unexpected heightened activity also for Flt3mutant and Flt3WT AML blasts with RAS or WT1 mutations, resulting in significant shifts in the ED concentrations for blasts in response to PI alone or the combination with TAK-659 at the clinically relevant dose of 250nM. In addition, blasts annotated for RAS mutation, without or with WT1 mutation (which abrogates expression of the nuclear chaperone for b-catenin, TBL1X) had heightened nuclear content for RAC1, which can participate in b-catenin chaperoning. TAK/PI combinations reduced nuclear RAC content as a measure of the antileukemic cooperativity, while also diminishing levels of nuclear b-catenin and its activating (PAK-dependent) phosphorylation on S675. The combination was also active for AML blasts with MLL-PTD, without or with additional RAS mutation. Further, genetic epistasis experiments were performed with siRNA transduced into MV-411 cells to establish the functional relationship of the individual effectors. P62 knockdown had a dominant effect on reducing NRF2 and NQO1 content, as well reducing nuclear/active expressions of c-jun, and b-catenin, thus establishing its importance in both OXPHOS and Wnt/b-catenin pathways. On the other hand, siRNA knockdown of Syk supported its role as a signaling substation for b-catenin abundance, also dependent on c-jun. Whether the consistent ability for TAK-659 to inhibit p62 expression in primary blasts depends upon it's inhibition of Syk vs. Flt3 is under further investigation. However, both NRF2 and c-jun are known to transactivate p62 gene, and p62 product can serve as scaffolding for JNK1. Indeed, an OXPHOS phenotype is dependent upon jun as well as NRF2, and jun also affects b-catenin en route to HOXA9/10, where overlapping expression of HOXA's and OXPHOS species is described. On the other hand, RAS mutations have been found to occupy an AML signaling space that is OXPHOS independent (I Baccelli, et al. Cancer Cell, 2019). In summary, we have identified common intracellular effectors for OXPHOS as well as Wnt/b-catenin-> HOXA in AML, as compared to the RAS and WT1 pathways, and have established a combination therapy (TAK-659 plus PI) that affects the inhibitory effectors elicited by these co-mutational states, which are responsible for negating activity for most Flt3 selective targeted agents, so as to allow antileukemic response. Disclosures Roodman: Amgen: Membership on an entity's Board of Directors or advisory committees. Konig:Agios: Consultancy; Amgen: Honoraria.

Up-regulation of Grb2-associated binder 1 promotes hepatocyte growth factor-induced endothelial progenitor cell proliferation and migration.

ObjectivesGrb2-associated binder 1 (Gab1), a scaffolding adaptor protein, plays an important role in transmitting key signals that control cell growth, migration, and function from multiple tyrosine kinase receptors. This study was designed to investigate the influence of upregulation of Gab1 in endothelial progenitor cells (EPCs) stimulated with hepatocyte growth factor (HGF), and the underlying molecular mechanisms.Materials and MethodsEndothelial progenitor cells isolated from human umbilical cord blood were identified and divided into four groups. EPCs in the Control group were cultured normally; those in the Control+HGF group were treated with HGF stimulation; those in the AD-Gab1 group were transfected with adenovirus containing the Gab1 gene but not treated with HGF stimulation; and, those in the AD-Gab1+HGF group were treated with both HGF stimulation and transfection with adenovirus containing the Gab1 gene. Subsequently, Gab1 expression and proliferation and migration ability were compared for EPCs grown under different conditions. Furthermore, we measured phosphorylation levels of three key proteins Gab1, SHP2, and ERK1/2.ResultsThe AD-Gab1+HGF group had the highest expression of Gab1 and higher proliferation and migration than the other three groups.ConclusionsUpregulation of Gab1 promoted HGF-induced EPC proliferation and migration. Mechanistically, HGF stimulated Gab1 tyrosine phosphorylation in EPCs, thus leading to activation of extracellular regulated MAP kinase 1/2, which is involved in proliferation and migration signaling.

Regulation of G protein-coupled receptor signaling by plasma membrane organization and endocytosis.

The trafficking of G protein coupled-receptors (GPCRs) is one of the most exciting areas in cell biology because of recent advances demonstrating that GPCR signaling is spatially encoded. GPCRs, acting in a diverse array of physiological systems, can have differential signaling consequences depending on their subcellular localization. At the plasma membrane, GPCR organization could fine-tune the initial stages of receptor signaling by determining the magnitude of signaling and the type of effectors to which receptors can couple. This organization is mediated by the lipid composition of the plasma membrane, receptor-receptor interactions, and receptor interactions with intracellular scaffolding proteins. GPCR organization is subsequently changed by ligand binding and the regulated endocytosis of these receptors. Activated GPCRs can modulate the dynamics of their own endocytosis through changing clathrin-coated pit dynamics, and through the scaffolding adaptor protein β-arrestin. This endocytic regulation has signaling consequences, predominantly through modulation of the MAPK cascade. This review explores what is known about receptor sorting at the plasma membrane, protein partners that control receptor endocytosis, and the ways in which receptor sorting at the plasma membrane regulates downstream trafficking and signaling.

Extracellular Signal-Regulated Kinases 1 and 2 Phosphorylate Gab2 To Promote a Negative-Feedback Loop That Attenuates Phosphoinositide 3-Kinase/Akt Signaling.

The scaffolding adapter protein Gab2 (Grb2-associated binder) promotes cell proliferation, survival, and motility by engaging several signaling pathways downstream of growth factor and cytokine receptors. In particular, Gab2 plays essential roles in mast cells, as it is required for phosphoinositide 3-kinase (PI3K) activation in response to Kit and the high-affinity IgE receptor. While the positive role of Gab2 in PI3K signaling is well documented, very little is known about the mechanisms that attenuate its function. Here we show that Gab2 becomes phosphorylated on multiple proline-directed sites upon stimulation of the Ras/extracellular signal-regulated kinase (ERK) signaling pathway. We demonstrate that ERK1 and ERK2 interact with Gab2 via a novel docking motif, which is required for subsequent Gab2 phosphorylation in response to ERK1/2 activation. We identified four ERK1/2-dependent phosphorylation sites in Gab2 that prevent the recruitment of the p85 regulatory subunit of PI3K. Using bone marrow-derived mast cells to study Gab2-dependent signaling, we found that the inhibition of ERK1/2 activity promotes Akt signaling in response to Kit and the high-affinity IgE receptor. Together, our results indicate that ERK1/2 participates in a negative-feedback loop that attenuates PI3K/Akt signaling in response to various agonists.

Gab2 facilitates epithelial-to-mesenchymal transition via the MEK/ERK/MMP signaling in colorectal cancer

BackgroundGrb2-associated binder 2 (Gab2), a scaffolding adaptor protein, has recently been implicated in cancer progression. However, the role of Gab2 in the progression and metastasis of colorectal cancer (CRC) remains unclear.MethodsGab2 expression was assessed in CRC patient specimens as well as in CRC cell lines. Recombinant lentivirus vector containing Gab2 gene and its small interfering RNAs were constructed and introduced into CRC cells. Cell migration and invasion ability were evaluated by transwell assays in vitro, and in vivo metastasis was performed on nude mice model. Moreover, the expression of Gab2 and epithelial-to-mesenchymal transition (EMT)-associated proteins (E-cadherin and vimentin) were assessed by western blot and qRT-PCR in CRC cells to evaluate the correlation between Gab2 and EMT. Finally, we evaluated the impact of Gab2 on the activation of its downstream signaling effectors, and furthermore the effects of these pathways on Gab2 induced-EMT were also detected.ResultsWe confirmed that increased Gab2 expression correlated with higher tumor node metastasis stage and highly invasive CRC cell lines. Ectopic expression of Gab2 promoted metastasis of CRC cells, whereas silencing of Gab2 resulted in inhibited metastasis both in vitro and in vivo. Overexpression of Gab2 in CRC cells induced EMT, whereas knockdown of Gab2 had the opposite effect. Furthermore, upregulation of Gab2 expression obviously stimulated the activation of extracellular signal-regulated kinase-1/2 (ERK1/2), and increased the expression of matrix metalloproteinase-7 (MMP7) and matrix metalloproteinase-9 (MMP9) in CRC cells. Conversely, downregulation of Gab2 expression significantly decreased the activation of ERK1/2, and inhibited MMP7 and MMP9 expression. U0126, an inhibitor of mitogen-activated protein kinase (MEK), can reverse the effects of Gab2 on EMT.ConclusionsOur work highlights that Gab2 induces EMT through the MEK/ERK/MMP pathway, which in turn promotes intestinal tumor metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0280-0) contains supplementary material, which is available to authorized users.

Cardiac Gab1 deletion leads to dilated cardiomyopathy associated with mitochondrial damage and cardiomyocyte apoptosis.

A vital step in the development of heart failure is the transition from compensatory cardiac hypertrophy to decompensated dilated cardiomyopathy (DCM) during cardiac remodeling under mechanical or pathological stress. However, the molecular mechanisms underlying the development of DCM and heart failure remain incompletely understood. In the present study, we investigate whether Gab1, a scaffolding adaptor protein, protects against hemodynamic stress-induced DCM and heat failure. We first observed that the protein levels of Gab1 were markedly reduced in hearts from human patients with DCM and from mice with experimental viral myocarditis in which DCM developed. Next, we generated cardiac-specific Gab1 knockout mice (Gab1-cKO) and found that Gab-cKO mice developed DCM in hemodynamic stress-dependent and age-dependent manners. Under transverse aorta constriction (TAC), Gab1-cKO mice rapidly developed decompensated DCM and heart failure, whereas Gab1 wild-type littermates exhibited adaptive left ventricular hypertrophy without changes in cardiac function. Mechanistically, we showed that Gab1-cKO mouse hearts displayed severe mitochondrial damages and increased cardiomyocyte apoptosis. Loss of cardiac Gab1 in mice impaired Gab1 downstream MAPK signaling pathways in the heart under TAC. Gene profiles further revealed that ablation of Gab1 in heart disrupts the balance of anti- and pro-apoptotic genes in cardiomyocytes. These results demonstrate that cardiomyocyte Gab1 is a critical regulator of the compensatory cardiac response to aging and hemodynamic stress. These findings may provide new mechanistic insights and potential therapeutic target for DCM and heart failure.

HTLV-1 Tax protein recruitment into IKKε and TBK1 kinase complexes enhances IFN-I expression

The Tax protein expressed by human T-cell leukemia virus type 1 (HTLV-1) plays a pivotal role in the deregulation of cellular pathways involved in the immune response, inflammation, cell survival, and cancer. Many of these effects derive from Tax multiple interactions with host factors, including the subunits of the IKK-complex that are required for NF-κB activation. IKKɛ and TBK1 are two IKK-related kinases that allow the phosphorylation of interferon regulatory factors that trigger IFN type I gene expression. We observed that IKKɛ and TBK1 recruit Tax into cellular immunocomplexes. We also found that TRAF3, which regulates cell receptor signaling effectors, forms complexes with Tax. Transactivation analyses revealed that expression of Tax, in presence of IKKɛ and TBK1, enhances IFN-β promoter activity, whereas the activation of NF-κB promoter is not modified. We propose that Tax may be recruited into the TBK1/IKKɛ complexes as a scaffolding-adaptor protein that enhances IFN-I gene expression.

Growth Factor Receptor Bound Protein 2–Associated Binder 2, a Scaffolding Adaptor Protein, Negatively Regulates Host Immunity against Tuberculosis

Cell-mediated immunity is indispensable for host protection against tuberculosis (TB). Growth factor receptor bound protein 2-associated binder (Gab) 2, a scaffolding adaptor protein, negatively regulates signaling pathways critical for T cell-mediated immunity. We sought to investigate the clinical significance and immunological role of Gab2 in Mycobacterium tuberculosis infection. We evaluated Gab2 protein and messenger RNA (mRNA) expression in human patients with pulmonary TB and determined the correlation of the mRNA expression pattern with antigen-specific IFN-γ secretion. Subsequently, we carried out M. tuberculosis infection in Gab2-deficient and wild-type control mice to explore the immunological role of Gab2 by examining bacterial load, histological changes, cytokine secretion, and gene expression of immune-associated transcription factors. mRNA levels of Gab2 and its correlated family member, Gab1, were markedly decreased in untreated patients with pulmonary TB compared with healthy control subjects. Importantly, this decreased Gab2 expression to normal levels after bacterial load in the patient's sputum became undetectable under the standard anti-TB treatment, which negatively correlated with the level of M. tuberculosis antigen-specific IFN-γ secretion. In the M. tuberculosis infection mouse model, infected Gab2-deficient mice exhibited decreased bacterial load and milder lung pathological damage compared with infected wild-type mice, accompanied by decreased production of IL-2, IL-6, and granulocyte/macrophage colony-stimulating factor proinflammatory cytokines, and an increased T-cell-specific T-box transcription factor/GATA binding protein 3 expression ratio. Overall, our study indicates that down-regulation of Gab2 relates to a protective function during M. tuberculosis infection, revealing a potential negative regulatory role for Gab2 in immunity to TB.

Protein tyrosine phosphatase PTPN3 inhibits lung cancer cell proliferation and migration by promoting EGFR endocytic degradation.

Epidermal growth factor receptor (EGFR) regulates multiple signaling cascades essential for cell proliferation, growth and differentiation. Using a genetic approach, we found that Drosophila FERM and PDZ domain-containing protein tyrosine phosphatase, dPtpmeg, negatively regulates border cell migration and inhibits the EGFR/Ras/mitogen-activated protein kinase signaling pathway during wing morphogenesis. We further identified EGFR pathway substrate 15 (Eps15) as a target of dPtpmeg and its human homolog PTPN3. Eps15 is a scaffolding adaptor protein known to be involved in EGFR endocytosis and trafficking. Interestingly, PTPN3-mediated tyrosine dephosphorylation of Eps15 promotes EGFR for lipid raft-mediated endocytosis and lysosomal degradation. PTPN3 and the Eps15 tyrosine phosphorylation-deficient mutant suppress non-small-cell lung cancer cell growth and migration in vitro and reduce lung tumor xenograft growth in vivo. Moreover, depletion of PTPN3 impairs the degradation of EGFR and enhances proliferation and tumorigenicity of lung cancer cells. Taken together, these results indicate that PTPN3 may act as a tumor suppressor in lung cancer through its modulation of EGFR signaling.

Clinical significance of GAB2, a scaffolding/docking protein acting downstream of EGFR in human colorectal cancer.

Recent studies indicated that the scaffolding adaptor protein GAB2 (GRB2-associated binding protein 2) plays a critical role in the proliferation and migration of various cancers. This study aimed to determine the role of aberrant GAB2 expression in human colorectal cancer (CRC). Quantitative real-time reverse transcription polymerase chain reaction was used to evaluate GAB2 mRNA expression in 152 CRC tissues samples to determine the clinicopathological significance of GAB2 expression. We also performed in vitro proliferation assays using siGAB2-transfected CRC cells. GAB2 expression in tumor colorectal tissues was significantly higher than in normal colorectal tissues (p = 0.0212). High GAB2 expression levels were associated with malignant clinicopathologic potential factors, including lymphatic invasion (p = 0.0003), venous invasion (p = 0.0170), and liver metastasis (p = 0.0144). The survival rate of patients with high GAB2 expression levels was significantly lower than that of patients with low GAB2 expression (p = 0.0074). Multivariate analysis indicated that GAB2 expression was a factor affecting lymph node metastasis. Cell proliferation was significantly suppressed by siGAB2 expression in CRC cells in vitro. GAB2 expression was associated with lymph node metastasis and may play a role in the growth and metastasis of CRC. These results suggest that GAB2 is a potential therapeutic target in CRC.

Decreased Mitogen Inducible Gene 6 (MIG-6) Associated with Symptom Severity in Children with Autism.

BACKGROUNDIndividuals with autism spectrum disorders (ASDs) demonstrate impairment in social interactions and problems in verbal and nonverbal communication. Autism spectrum disorders are thought to affect 1 in 88 children in the US. Recent research has shown that epidermal growth factor receptor (EGFR) activation is associated with nerve cell development and repair. Mitogen inducible gene 6 (MIG-6) is a 58-kDa non-kinase scaffolding adaptor protein consisting of 462 amino-acids, which has been shown to be a negative feedback regulator of EGFR and Met receptor tyrosine kinase (RTK) signaling.SUBJECTS AND METHODSIn this study, we determined plasma levels of MIG-6, which suppresses the EGFR RTK pathway in autistic children, and compared MIG-6 levels with the EGFR ligand, epidermal growth factor (EGF), and the cMET ligand, hepatocyte growth factor (HGF). MIG-6 levels were also compared to the symptom severity of 19 different autistic behaviors.Plasma MIG-6 concentration was measured in 40 autistic children and 39 neurotypical, age, and gender similar controls using an enzyme linked immunosorbent assay (ELISA). Plasma MIG-6 levels were compared to putative biomarkers known to be associated with EGFR and cMET and severity levels of 19 autism related symptoms [awareness, expressive language, receptive language, (conversational) pragmatic language, focus/attention, hyperactivity, impulsivity, perseveration, fine motor skills, gross motor skills, hypotonia (low muscle tone), tip toeing, rocking/pacing, stimming, obsessions/fixations, eye contact, sound sensitivity, light sensitivity, and tactile sensitivity].RESULTSIn this study, we found that plasma MIG-6 levels in autistic children (182.41 ± 24.3 pg/ml) were significantly lower than neurotypical controls (1779.76 ± 352.5; P = 1.76E − 5). Decreased MIG-6 levels correlated with serotonin, dopamine, Tumor necrosis factor alpha (TNF-alpha), and urokinase receptor (uPAR) concentration, but not with other tested putative biomarkers. MIG-6 levels also correlated significantly with severity of expressive language, receptive language, tip toeing, rocking/pacing, and hand flapping/stimming.CONCLUSIONSThese results suggest a relationship between decreased plasma MIG-6 levels, biomarkers associated with the EGFR pathway, and symptom severity in autism. A strong correlation between plasma MIG-6 and dopamine and serotonin levels suggest that decreased MIG-6 levels may be associated with abnormal neurotransmitter synthesis and/or action. A strong correlation between MIG-6 and uPAR and the inflammatory marker TNF-alpha suggests that low MIG-6 levels may be associated with the HGF/Met signaling pathway, as well as inflammation in autistic children.

The mitogen‐inducible gene‐6 is involved in regulation of cellular senescence in normal diploid fibroblasts

The mitogen-inducible gene-6 (Mig-6) is a non-kinase scaffolding adaptor protein. It has been shown that Mig-6 may play important roles in regulating stress response, maintaining homeostasis and functioning as a tumour suppressor. In this study, we investigated the role of Mig-6 in cellular senescence. Our results showed that Mig-6 is up-regulated during the senescence process. Functional analysis indicated that cells over-expressing Mig-6 have reduced DNA synthesis and showed the signs of senescence. Knockdown of Mig-6 delayed the initiation of Ras-induced cellular senescence. These results suggest that the increase of Mig-6 expression contributes to establishment of cellular senescence. Furthermore, our results showed that Mig-6 induction of senescence is related to its inhibition of EGF receptor (EGFR)/Erb B signalling. Subsequent analysis of the mechanism responsible for the up-regulation of its expression showed that FOXO3A transcriptionally up-regulates Mig-6 expression via directly binding to the FOXO response element in Mig-6 5'-flanking regulatory sequences. Mig-6 induces premature senescence via functioning in regulation of cellular senescence in normal diploid fibroblasts.

The AP-1 µ Adaptin is Required for KNOLLE Localization at the Cell Plate to Mediate Cytokinesis in Arabidopsis

Formation of clathrin-coated vesicles (CCVs) requires the scaffolding adaptor protein (AP) complexes, which are conserved across all eukaryotes. The Arabidopsis genome encodes five AP complexes (AP-1 to AP-5), and each complex consists of four subunits. In this study, we characterized the poorly defined AP-1 complex by using genetics, proteomics and live cell imaging. We showed that the AP-1 µ adaptin subunit (AP1M2) was localized to the trans-Golgi network (TGN) and interacted physically with the AP-1 subunits in Arabidopsis. During treatment with brefeldin A (BFA), the functional fluorophore-tagged AP1M2 relocated to the BFA compartment. The AP1M2 loss-of-function mutant ap1m2 displayed deleterious growth defects, which were particularly evident in the compromised cytokinesis that was revealed by the presence of cell wall stubs in multinucleate cells. Immunolocalization of the cytokinesis-specific syntaxin KNOLLE (KN) in ap1m2 showed that KN was mislocalized and aggregated around the division plane, while a secretory marker targeting to the cell plate remained unaffected. Taken together, we propose that the AP-1 complex is required for cell plate-targeted trafficking of KN in dividing plant cells, and that it has a common role in mediating plant and yeast/animal cytokinesis systems which are fundamentally different.

Abstract 5191: Gab2 phosphorylation by RSK inhibits Shp2 recruitment and cell motility.

Abstract The scaffolding adapter protein Gab2 (Grb2-associated binder) participates in the signaling response evoked by various growth factors and cytokines. Gab2 is overexpressed in several human malignancies, including breast cancer, and was shown to promote mammary epithelial cell migration. The role of Gab2 in the activation of different signaling pathways is well documented, but less is known regarding the feedback mechanisms responsible for its inactivation. We now demonstrate that activation of the Ras/mitogen-activated protein kinase (MAPK) pathway promotes Gab2 phosphorylation on basic consensus motifs. More specifically, we show that RSK (p90 ribosomal S6 kinase) phosphorylates Gab2 on three conserved residues, both in vivo and in vitro. Mutation of these phosphorylation sites does not alter Gab2 binding to Grb2, but instead, we show that Gab2 phosphorylation inhibits the recruitment of the tyrosine phosphatase Shp2 in response to growth factors. Expression of an unphosphorylatable Gab2 mutant in mammary epithelial cells promotes an invasive-like phenotype and increases cell motility. Taken together, these results suggest that RSK is part of a negative feedback loop that restricts Gab2-dependent epithelial cell motility. Based on the widespread role of Gab2 in receptor signaling, these findings also suggest that RSK plays a regulatory function in diverse receptor systems. Citation Format: Xiaocui Zhang, Geneviève Lavoie, Loic Fort, Edward L. Huttlin, Joseph Tcherkezian, Jacob A. Galan, Haihua Gu, Steven P. Gygi, Sebastien Carreno, Philippe P. Roux. Gab2 phosphorylation by RSK inhibits Shp2 recruitment and cell motility. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5191. doi:10.1158/1538-7445.AM2013-5191

Gab2 Phosphorylation by RSK Inhibits Shp2 Recruitment and Cell Motility

The scaffolding adapter protein Gab2 (Grb2-associated binder) participates in the signaling response evoked by various growth factors and cytokines. Gab2 is overexpressed in several human malignancies, including breast cancer, and was shown to promote mammary epithelial cell migration. The role of Gab2 in the activation of different signaling pathways is well documented, but less is known regarding the feedback mechanisms responsible for its inactivation. We now demonstrate that activation of the Ras/mitogen-activated protein kinase (MAPK) pathway promotes Gab2 phosphorylation on basic consensus motifs. More specifically, we show that RSK (p90 ribosomal S6 kinase) phosphorylates Gab2 on three conserved residues, both in vivo and in vitro. Mutation of these phosphorylation sites does not alter Gab2 binding to Grb2, but instead, we show that Gab2 phosphorylation inhibits the recruitment of the tyrosine phosphatase Shp2 in response to growth factors. Expression of an unphosphorylatable Gab2 mutant in mammary epithelial cells promotes an invasion-like phenotype and increases cell motility. Taken together, these results suggest that RSK is part of a negative-feedback loop that restricts Gab2-dependent epithelial cell motility. On the basis of the widespread role of Gab2 in receptor signaling, these findings also suggest that RSK plays a regulatory function in diverse receptor systems.

Vac14 Protein Multimerization Is a Prerequisite Step for Fab1 Protein Complex Assembly and Function

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) helps control various endolysosome functions including organelle morphology, membrane recycling, and ion transport. Further highlighting its importance, PtdIns(3,5)P2 misregulation leads to the development of neurodegenerative diseases like Charcot-Marie-Tooth disease. The Fab1/PIKfyve lipid kinase phosphorylates PtdIns(3)P into PtdIns(3,5)P2 whereas the Fig4/Sac3 lipid phosphatase antagonizes this reaction. Interestingly, Fab1 and Fig4 form a common protein complex that coordinates synthesis and degradation of PtdIns(3,5)P2 by a poorly understood process. Assembly of the Fab1 complex requires Vac14/ArPIKfyve, a multimeric scaffolding adaptor protein that coordinates synthesis and turnover of PtdIns(3,5)P2. However, the properties and function of Vac14 multimerization remain mostly uncharacterized. Here we identify several conserved C-terminal motifs on Vac14 required for self-interaction and provide evidence that Vac14 likely forms a dimer. We also show that monomeric Vac14 mutants do not support interaction with Fab1 or Fig4, suggesting that Vac14 multimerization is likely the first molecular event in the assembly of the Fab1 complex. Finally, we show that cells expressing monomeric Vac14 mutants have enlarged vacuoles that do not fragment after hyperosmotic shock, which indicates that PtdIns(3,5)P2 levels are greatly abated. Therefore, our observations support an essential role for the Vac14 homocomplex in controlling PtdIns(3,5)P2 levels.

Smurf1 Protein Negatively Regulates Interferon-γ Signaling through Promoting STAT1 Protein Ubiquitination and Degradation

Interferons are important cytokines that mediate antiviral, antiproliferative, antitumor, and immunoregulatory activities. However, uncontrolled IFN signaling may lead to autoimmune diseases. Here we identified Smurf1 as a negative regulator for IFN-γ signaling by targeting STAT1 for ubiquitination and proteasomal degradation. Smurf1 interacted with STAT1 through the WW domains of Smurf1 and the PY motif in STAT1 and catalyzed K48-linked polyubiquitination of STAT1. Interestingly, the Smurf1-mediated ubiquitination and degradation did not require STAT1 tyrosine and serine phosphorylation. Subsequently, overexpression of Smurf1 attenuated IFN-γ-mediated STAT1 activation and antiviral immune responses, whereas knockdown of Smurf1 enhanced IFN-γ-mediated STAT1 activation, expression of STAT1 target genes, and antiviral immune responses. Furthermore, IFN-γ stimulation led to enhanced expression of Smurf1. Therefore, our results demonstrate that Smurf1 is a negative feedback regulator for IFN-γ signaling by targeting STAT1 for ubiquitination and proteasomal degradation.

ERBB receptor feedback inhibitor 1: Identification and regulation by estrogen in chickens

The ERBB receptor feedback inhibitor 1 (ERRFI1) is a scaffolding adaptor protein, that plays a pivotal role in the epidermal growth factor receptor (EGFR) cell signaling cascade as a negative regulator affecting many important physiological processes. It was recently reported that ERRFI1 is a critical regulator of the response of the endometrium to estrogen regulation of tissue homeostasis in mice. But, very little is known about ERRF11 and hormonal regulation of the ERRFI1 gene in chickens. Therefore, in the present study, ERRFI1 gene was cloned and its differential expression profile analyzed at different embryonic stages, in various adult organs, and in oviducts from estrogen-treated chickens. Chicken ERRFI1 has an open-reading frame of 2848 nucleotides that encode for a protein of 465 amino acids that has considerable homology to mammalian ERRFI1 proteins (>62% identity). Importantly, ERRFI1 mRNA is abundantly distributed in various organs from chickens. We then determined that DES (diethylstilbestrol, a synthetic nonsteroidal estrogen) induced ERRFI1 mRNA and protein predominantly in luminal and glandular epithelial cells of the oviduct. Further, we determined whether microRNAs, specifically miR-200b, miR-429 and miR-1639, influence ERRFI1 expression via its 3′UTR and found that it does not directly target the 3′UTR of ERRFI1 mRNA. Therefore, it is unlikely that post-transcriptional regulation influences ERRFI1 expression in the chicken oviduct. In conclusion, our results indicate that ERRFI1 is a novel estrogen-stimulated gene expressed in epithelial cells of the chicken oviduct that likely plays an important role in oviduct growth and differentiation during early development of the chicken.

Gab2 regulates the migratory behaviors and E-cadherin expression via activation of the PI3K pathway in ovarian cancer cells

Ovarian cancer, the most deadly gynecologic malignancy, is often diagnosed late and at the advanced stage when the cancer cells have already migrated and invaded into other tissues and organs. Better understanding of the mechanism of metastasis in ovarian cancer cells is essential to the design of effective therapy. In this study, we investigated the function of scaffolding adaptor protein Gab2 in ovarian cancer cells. Gab2 is found to be overexpressed in a subset of ovarian tumors and cancer cell lines. Gab2 expression mainly regulates the migratory behaviors of ovarian cancer cells. Overexpression of Gab2 promotes the migration and invasion, and down-regulates E-cadherin expression in ovarian cancer cells with low-Gab2 expression. Conversely, knockdown of Gab2 expression inhibits the migration and invasion, and promotes E-cadherin expression in ovarian cancer cells with high-Gab2 expression. By expressing Gab2 wild type and Gab2 mutants that are defective in activation the PI3K and Shp2-Erk pathways, we find that Gab2 inhibits E-cadherin expression and enhances the expression of Zeb1, a transcription factor involved in epithelial-to-mesenchymal transition (EMT), and cell migration and invasion through the activation of the PI3K pathway. Knockdown of Zeb1 expression blocks Gab2-induced suppression of E-cadherin expression and increase in cell invasion. LY294002 and GDC-0941, inhibitors of PI3K, or Rapamycin, an inhibitor of PI3K downstream target mTOR, can reverse the effects of Gab2 on migration and invasion. Overall, our studies reveal that Gab2 overexpression, via activation of the PI3K-Zeb1 pathway, promotes characteristics of EMT in ovarian cancer cells.