Interaction of scaffolding adaptor protein Gab1 with tyrosine phosphatase SHP2 negatively regulates IGF-I-dependent myogenic differentiation via the ERK1/2 signaling pathway.
ABSTRACT: Grb2-associated binder 1 (Gab1) coordinates various receptor tyrosine kinase signaling pathways. Although skeletal muscle differentiation is regulated by some growth factors, it remains elusive whether Gab1 coordinates myogenic signals. Here, we examined the molecular mechanism of insulin-like growth factor-I (IGF-I)-mediated myogenic differentiation, focusing on Gab1 and its downstream signaling. Gab1 underwent tyrosine phosphorylation and subsequent complex formation with protein-tyrosine phosphatase SHP2 upon IGF-I stimulation in C2C12 myoblasts. On the other hand, Gab1 constitutively associated with phosphatidylinositol 3-kinase regulatory subunit p85. To delineate the role of Gab1 in IGF-I-dependent signaling, we examined the effect of adenovirus-mediated forced expression of wild-type Gab1 (Gab1(WT)), mutated Gab1 that is unable to bind SHP2 (Gab1(DeltaSHP2)), or mutated Gab1 that is unable to bind p85 (Gab1(Deltap85)), on the differentiation of C2C12 myoblasts. IGF-I-induced myogenic differentiation was enhanced in myoblasts overexpressing Gab1(DeltaSHP2), but inhibited in those overexpressing either Gab1(WT) or Gab1(Deltap85). Conversely, IGF-I-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation was significantly repressed in myoblasts overexpressing Gab1(DeltaSHP2) but enhanced in those overexpressing either Gab1(WT) or Gab1(Deltap85). Furthermore, small interference RNA-mediated Gab1 knockdown enhanced myogenic differentiation. Overexpression of catalytic-inactive SHP2 modulated IGF-I-induced myogenic differentiation and ERK1/2 activation similarly to that of Gab1(DeltaSHP2), suggesting that Gab1-SHP2 complex inhibits IGF-I-dependent myogenesis through ERK1/2. Consistently, the blockade of ERK1/2 pathway reversed the inhibitory effect of Gab1(WT) overexpression on myogenic differentiation, and constitutive activation of the ERK1/2 pathway suppressed the enhanced myogenic differentiation by overexpression of Gab1(DeltaSHP2). Collectively, these data suggest that the Gab1-SHP2-ERK1/2 signaling pathway comprises an inhibitory axis for IGF-I-dependent myogenic differentiation.
Project description:Insulin-like growth factor-I (IGF-I) activates not only the phosphatidylinositol 3-kinase (PI3K)-AKT cascade that is essential for myogenic differentiation but also the extracellular signal-regulated kinase (ERK) 1/2 cascade that inhibits myogenesis. We hypothesized that there must be a signal that inhibits ERK1/2 upon cell-cell contact required for skeletal myogenesis. Cell-cell contact-induced engagement of ephrin ligands and Eph receptors leads to downregulation of the Ras-ERK1/2 pathway through p120 Ras GTPase-activating protein (p120RasGAP). We therefore investigated the significance of the ephrin/Eph signal in IGF-I-induced myogenesis. EphrinA1-Fc suppressed IGF-I-induced activation of Ras and ERK1/2, but not that of AKT, in C2C12 myoblasts, whereas ephrinB1-Fc affected neither ERK1/2 nor AKT activated by IGF-I. IGF-I-dependent myogenic differentiation of C2C12 myoblasts was potentiated by ephrinA1-Fc. In p120RasGAP-depleted cells, ephrinA1-Fc failed to suppress the Ras-ERK1/2 cascade by IGF-I and to promote IGF-I-mediated myogenesis. EphrinA1-Fc did not promote IGF-I-dependent myogenesis when the ERK1/2 was constitutively activated. Furthermore, a dominant-negative EphA receptor blunted IGF-I-induced myogenesis in C2C12 and L6 myoblasts. However, the inhibition of IGF-I-mediated myogenesis by down-regulation of ephrinA/EphA signal was canceled by inactivation of the ERK1/2 pathway. Collectively, these findings demonstrate that the ephrinA/EphA signal facilitates IGF-I-induced myogenesis by suppressing the Ras-ERK1/2 cascade through p120RasGAP in myoblast cell lines.
Project description:Noonan syndrome (NS), a genetic disease caused in half of cases by activating mutations of the tyrosine phosphatase SHP2 (PTPN11), is characterized by congenital cardiopathies, facial dysmorphic features, and short stature. How mutated SHP2 induces growth retardation remains poorly understood. We report here that early postnatal growth delay is associated with low levels of insulin-like growth factor 1 (IGF-1) in a mouse model of NS expressing the D61G mutant of SHP2. Conversely, inhibition of SHP2 expression in growth hormone (GH)-responsive cell lines results in increased IGF-1 release upon GH stimulation. SHP2-deficient cells display decreased ERK1/2 phosphorylation and rat sarcoma (RAS) activation in response to GH, whereas expression of NS-associated SHP2 mutants results in ERK1/2 hyperactivation in vitro and in vivo. RAS/ERK1/2 inhibition in SHP2-deficient cells correlates with impaired dephosphorylation of the adaptor Grb2-associated binder-1 (GAB1) on its RAS GTPase-activating protein (RASGAP) binding sites and is rescued by interfering with RASGAP recruitment or function. We demonstrate that inhibition of ERK1/2 activation results in an increase of IGF-1 levels in vitro and in vivo, which is associated with significant growth improvement in NS mice. In conclusion, NS-causing SHP2 mutants inhibit GH-induced IGF-1 release through RAS/ERK1/2 hyperactivation, a mechanism that could contribute to growth retardation. This finding suggests that, in addition to its previously shown beneficial effect on NS-linked cardiac and craniofacial defects, RAS/ERK1/2 modulation could also alleviate the short stature phenotype in NS caused by PTPN11 mutations.
Project description:Phosphoinositide 3-kinase (PI3K) participates in extracellular signal-regulated kinase 1 and 2 (ERK1-2) activation according to signal strength, through unknown mechanisms. We report herein that Gab1/Shp2 constitutes a PI3K-dependent checkpoint of ERK1-2 activation regulated according to signal intensity. Indeed, by up- and down-regulation of signal strength in different cell lines and through different methods, we observed that Gab1/Shp2 and Ras/ERK1-2 in concert become independent of PI3K upon strong epidermal growth factor receptor (EGFR) stimulation and dependent on PI3K upon limited EGFR activation. Using Gab1 mutants, we observed that this conditional role of PI3K is dictated by the EGFR capability of recruiting Gab1 through Grb2 or through the PI3K lipid product PIP(3), according to a high or weak level of receptor stimulation, respectively. In agreement, Grb2 siRNA generates, in cells with maximal EGFR stimulation, a strong dependence on PI3K for both Gab1/Shp2 and ERK1-2 activation. Therefore, Ras/ERK1-2 depends on PI3K only when PIP(3) is required to recruit Gab1/Shp2, which occurs only under weak EGFR mobilization. Finally, we show that, in glioblastoma cells displaying residual EGFR activation, this compensatory mechanism becomes necessary to efficiently activate ERK1-2, which could probably contribute to tumor resistance to EGFR inhibitors.
Project description:In a classic model, G(i)? proteins including G(i1)?, G(i2)? and G(i3)? are important for transducing signals from G(i)? protein-coupled receptors (G(i)?PCRs) to their downstream cascades in response to hormones and neurotransmitters. Our previous study has suggested that G(i1)?, G(i2)? and G(i3)? are also important for the activation of the PI3K/Akt/mTORC1 pathway by epidermal growth factor (EGF) and its family members. However, a genetic role of these G(i)? proteins in the activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) by EGF is largely unknown. Further, it is not clear whether these G(i)? proteins are also engaged in the activation of both the Akt/mTORC1 and ERK1/2 pathways by other growth factor family members. Additionally, a role of these G(i)? proteins in breast cancer remains to be elucidated.We found that Gi1/3 deficient MEFs with the low expression level of G(i2)? showed defective ERK1/2 activation by EGFs, IGF-1 and insulin, and Akt and mTORC1 activation by EGFs and FGFs. Gi1/2/3 knockdown breast cancer cells exhibited a similar defect in the activations and a defect in in vitro growth and invasion. The G(i)? proteins associated with RTKs, Gab1, FRS2 and Shp2 in breast cancer cells and their ablation impaired Gab1's interactions with Shp2 in response to EGF and IGF-1, or with FRS2 and Grb2 in response to bFGF.G(i)? proteins differentially regulate the activation of Akt, mTORC1 and ERK1/2 by different families of growth factors. G(i)? proteins are important for breast cancer cell growth and invasion.
Project description:Insulin receptor substrate (IRS)-1 is a major substrate of insulin-like growth factor (IGF)-I receptors. It is well-known that IGF-I and II play essential roles in myogenesis progression. Herein, we report an unexpected phenomenon that IRS-1-overexpressing L6 myoblasts are eliminated from normal cell layers at the beginning of differentiation. Initially, the IRS protein level and apoptosis were examined during myogenic differentiation in L6 myoblasts. We found that the IRS-1 protein level decreased, whereas active caspase 3 increased around 1 day after induction of differentiation. The addition of a pan-caspase inhibitor, Z-VAD-FMK, inhibited differentiation-induced suppression of the IRS-1 protein level. Apoptosis was not enhanced in L6 myoblasts stably expressing high levels of IRS-1 (L6-IRS-1). However, when L6-IRS-1 was cultured with control cells (L6-mock), we observed that L6-IRS-1 was eliminated from the cell layer. We have recently reported that, in L6-IRS-1, internalization of the IGF-I receptor was delayed and IGF signal activation was sustained for a longer period than in L6-mock. When cells stably expressing IRS-1 3YA mutant, which could not maintain the IGF signals, were cultured with normal cells, elimination from the cell layer was not detected. These data suggested that the high level of IRS-1 in myoblasts induces elimination from the cell layer due to abnormal sustainment of IGF-I receptor activation.
Project description:Lung cancer is a major disease carrying heterogeneous molecular lesions and many of them remain to be analyzed functionally in vivo. Gain-of-function (GOF) SHP2 (PTPN11) mutations have been found in various types of human cancer, including lung cancer. However, the role of activating SHP2 mutants in lung cancer has not been established. We generated transgenic mice containing a doxycycline (Dox)-inducible activating SHP2 mutant (tetO-SHP2(E76K)) and analyzed the role of SHP2(E76K) in lung tumorigenesis in the Clara cell secretory protein (CCSP)-reverse tetracycline transactivator (rtTA)/tetO-SHP2(E76K) bitransgenic mice. SHP2(E76K) activated Erk1/Erk2 (Erk1/2) and Src, and upregulated c-Myc and Mdm2 in the lungs of bitransgenic mice. Atypical adenomatous hyperplasia and small adenomas were observed in CCSP-rtTA/tetO-SHP2(E76K) bitransgenic mice induced with Dox for 2-6 months and progressed to larger adenoma and adenocarcinoma by 9 months. Dox withdrawal from bitransgenic mice bearing magnetic resonance imaging-detectable lung tumors resulted in tumor regression. These results show that the activating SHP2 mutant promotes lung tumorigenesis and that the SHP2 mutant is required for tumor maintenance in this mouse model of non-small cell lung cancer. SHP2(E76K) was associated with Gab1 in the lung of transgenic mice. Elevated pGab1 was observed in the lung of Dox-induced CCSP-rtTA/tetO-SHP2(E76K) mice and in cell lines expressing SHP2(E76K), indicating that the activating SHP2 mutant autoregulates tyrosine phosphorylation of its own docking protein. Gab1 tyrosine phosphorylation is sensitive to inhibition by the Src inhibitor dasatinib in GOF SHP2-mutant-expressing cells, suggesting that Src family kinases are involved in SHP2 mutant-induced Gab1 tyrosine phosphorylation.
Project description:During the differentiation of muscle satellite cells, committed myoblasts respond to specific signalling cues by exiting the cell cycle, migrating, aligning, expressing muscle-specific genes and finally fusing to form multinucleated myotubes. The predominant foetal growth factor, IGF-2, initiates important signals in myogenesis. The aim of this study was to investigate whether ERK5 and its upstream MKK activator, MEK5, were important in the pro-myogenic actions of IGF-2. ERK5 protein levels, specific phosphorylation and kinase activity increased in differentiating C2 myoblasts. ERK5-GFP translocated from the cytoplasm to the nucleus after activation by upstream MEK5, whereas phospho-acceptor site mutated (dominant-negative) ERK5AEF-GFP remained cytoplasmic. Exogenous IGF-2 increased MHC levels, myogenic E box promoter-reporter activity, ERK5 phosphorylation and kinase activity, and rapidly induced nuclear localisation of ERK5. Transfection with antisense Igf2 decreased markers of myogenesis, and reduced ERK5 phosphorylation, kinase and transactivation activity. These negative effects of antisense Igf2 were rescued by constitutively active MEK5, whereas transfection of myoblasts with dominant-negative MEK5 blocked the pro-myogenic action of IGF-2. Our findings suggest that the MEK5-ERK5 pathway is a novel key mediator of IGF-2 action in myoblast differentiation.
Project description:Glycerol kinase (GK) is at the interface of fat and carbohydrate metabolism and has been linked to obesity and type 2 diabetes mellitus (T2DM). The purpose of this study was to investigate the role of GK in fat metabolism and insulin signaling in skeletal muscle (an important end organ tissue in T2DM). Microarray analysis determined that there were 525 genes that were differentially expressed (1.2-fold, p value<0.05) between knockout (KO) and wild-type (WT) mice. Quantitative PCR (qPCR) confirmed the differential expression of genes including glycerol kinase (Gyk), phosphatidylinositol 3-kinase regulatory subunit, polypeptide 1 (p85 alpha) (Pik3r1), insulin-like growth factor 1 (Igf1), and growth factor receptor bound protein 2-associated protein 1 (Gab1). Network component analysis demonstrated that transcription factor activities of myogenic differentiation 1 (MYOD), myogenic regulatory factor 5 (MYF5), myogenin (MYOG), nuclear receptor subfamily 4, group A, member 1 (NUR77) are decreased in the Gyk KO whereas the activity of paired box 3 (PAX3) is increased. The activity of MYOD was confirmed using a DNA binding assay. In addition, myoblasts from Gyk KO had less ability to differentiate into myotubes compared to WT myoblasts. These findings support our previous studies in brown adipose tissue and demonstrate that the role of Gyk in muscle is due in part to its non-metabolic (moonlighting) activities.
Project description:This study was conducted to elucidate the biological effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on cell proliferation, differentiation and gene expression in C2C12 myoblasts. C2C12 were treated with various concentrations of EPA or DHA under proliferation and differentiation conditions. Cell viability was analyzed using cell counting kit-8 assays (CCK-8). The Edu assays were performed to analyze cell proliferation. To analyze cell differentiation, the expressions of myogenic marker genes were determined at the transcriptional and translational levels by qRT-PCR, immunoblotting and immunofluorescence. Global gene expression patterns were characterized using RNA-sequencing. Phosphorylation levels of ERK and Akt were examined by immunoblotting. Cell viability and proliferation was significantly inhibited after incubation with EPA (50 and 100 ?M) or DHA (100 ?M). Both EPA and DHA suppressed C2C12 myoblasts differentiation. RNA-sequencing analysis revealed that some muscle-related genes were significantly downregulated following EPA or DHA (50 ?M) treatment, including insulin-like growth factor 2 (IGF-2), troponin T3 (Tnnt3), myoglobin (Mb), myosin light chain phosphorylatable fast skeletal muscle (Mylpf) and myosin heavy polypeptide 3 (Myh3). IGF-2 was crucial for the growth and differentiation of skeletal muscle and could activate the PI3K/Akt and the MAPK/ERK cascade. We found that EPA and DHA (50 ?M) decreased the phosphorylation levels of ERK1/2 and Akt in C2C12 myoblasts. Thus, this study suggested that EPA and DHA exerted an inhibitory effect on myoblast proliferation and differentiation and downregulated muscle-related genes expression.
Project description:Insulin-like growth factors (IGFs) stimulate myoblast proliferation and differentiation. It remains elusive how these mutually exclusive cellular responses are elicited by the same growth factor. Here we report that whereas IGF promotes myoblast differentiation under normoxia, it stimulates proliferation under hypoxia. Hypoxia activates the HIF-1 transcriptional program and knockdown of HIF-1alpha changes the mitogenic action of IGF into myogenic action under hypoxia. Conversely, overexpression of HIF-1alpha abolishes the myogenic effect of IGF under normoxia. Under normoxia, IGF activates the Akt-mTOR, p38, and Erk1/2 MAPK pathways. Hypoxia suppresses basal and IGF-induced Akt-mTOR and p38 activity, whereas it enhances and prolongs IGF-induced Erk1/2 activation in a HIF-1-dependent fashion. Activation of Akt-mTOR and p38 promotes myogenesis, and p38 also inhibits proliferation. Activation of Erk stimulates myoblast proliferation but inhibits differentiation. These results suggest that hypoxia converts the myogenic action of IGFs into mitogenic action by differentially regulating multiple signaling pathways via HIF-1-dependent mechanisms. Our findings provide a mechanistic explanation for the paradoxical actions of IGFs during myogenesis and reveal a novel mechanism by which cells sense and integrate growth factor signals and oxygen availability in their microenvironments.