Role of SHP-2 in Fibroblast Growth Factor Receptor-Mediated Suppression of Myogenesis in C2C12 Myoblasts

Abstract

Ligand activation of the fibroblast growth factor receptor (FGFR) represses myogenesis and promotes activation of extracellular signal-regulated kinases 1 and 2 (Erks). The precise mechanism through which the FGFR transmits both of these signals in myoblasts remains unclear. The SH2 domain-containing protein tyrosine phosphatase, SHP-2, has been shown to participate in the regulation of FGFR signaling. However, no role for SHP-2 in FGFR myogenic signaling is known. In this study, we show that stimulation of C2C12 myoblasts with FGF-2 induces SHP-2 complex formation with tyrosyl-phosphorylated FGFR substrate 2␣ (FRS-2␣). Both the catalytic activity and, to a much lesser extent, the Grb2 binding-tyrosyl phosphorylation sites of SHP-2 are required for maximal FGF-2-induced Erk activity and Elk-1 transactivation. When overexpressed in C2C12 myoblasts, wild-type SHP-2, but not a catalytically inactive SHP-2 mutant, potentiates the suppressive effects of FGF-2 on muscle-specific gene expression. In addition, expression of a constitutively active mutant of SHP-2 is sufficient to prevent myogenesis. The constitutively active mutant of SHP-2 induces hyper-tyrosyl phosphorylation of FRS-2␣ but fails to stimulate or potentiate either FGF-2-induced Erk activation or Elk-1 transactivation. These data suggest that in myoblasts, SHP-2 represses myogenesis via a pathway that is independent of the Erks. We propose that SHP-2 plays a pivotal role in FGFR signaling in myoblasts via both Erk-dependent and Erk-independent pathways.Cell lines of the myogenic lineage provide an excellent model for studying signaling pathways that control the decision between growth and differentiation. In culture, myogenic cell lines are inhibited in their ability to differentiate when exposed to serum, fibroblast growth factors (FGFs), and transforming growth factor ␤ (8, 29,57,63,73). It is now believed that mitogens prevent myogenesis by maintaining myoblasts in the proliferative phase, thereby suppressing muscle-specific gene expression and terminal differentiation (28,38,56). Upon mitogen withdrawal, cultured myoblasts exit the cell cycle and coordinately activate muscle regulatory factors (MRFs) in the basic helix-loop-helix (bHLH) family. These MRFs, such as MyoD, myogenin, Myf5, and MRF4, promote skeletal muscle differentiation by activating muscle-specific genes that include the myosin light and heavy chains, desmin, and troponin T (11,43,67).The intracellular signaling cascades that regulate entry into myogenesis have been the focus of several laboratories. Some of these studies suggest that extracellular signal-regulated kinases 1 and 2 (Erks) negatively regulate the initiation of myogenesis. A combination of pharmacological inhibitors, overexpression of dominant-interfering mutants of the Erk pathway, and overexpression of the mitogen-activated protein kinase (MAPK) phosphatase 1 all demonstrate that the Erks negatively regulate myogenesis (5, 9, 69, 70). One report indicates that the Erks positively regulate myogenesis (16), while others ...

Amphoterin Stimulates Myogenesis and Counteracts the Antimyogenic Factors Basic Fibroblast Growth Factor and S100B via RAGE Binding

“…5A to F were obtained using bFGF, a known inhibitor of myogenic differentiation (29), instead of S100B; bFGF caused a smaller reduction of MCK induction in L6/RAGE myoblasts than in WT myoblasts, and smaller doses of amphoterin were required to override the antimyogenic effect of bFGF on L6/RAGE myoblasts than were required for WT myoblasts (Fig. 5G and H).…”

“…Several extracellular factors have been identified that participate in the regulation of myogenesis, some of which promote myoblast differentiation and/or myotube formation, while other factors inhibit these processes. Insulin, insulin-like growth factors (IGF I and IGF II), neuregulin, and nerve growth factor belong to the first category of agents (13-15, 28, 45), while tumor necrosis factor alpha (TNF-␣), basic fibroblast growth factor (bFGF), and transforming growth factor ␤ belong to the second category (12,29,30,35,37,40,42,50,56). However, IGF I and IGF II were reported to promote or inhibit myogenic differentiation depending on the absence or presence of TNF-␣, respectively (16), and down-regulation of nerve growth factor low-affinity receptor was shown to be required for myoblast terminal differentiation (12).…”

Novel Role for SHP-2 in Nutrient-Responsive Control of S6 Kinase 1 Signaling

“…To examine whether SHP-2 signals upstream of PLC␤4 in the activation of S6K1, we knocked down PLC␤4 in the presence of a constitutively active mutant of SHP-2 (SHP-2 E76A) (25). Whereas overexpression of SHP-2 E76A enhanced leucine-induced S6K1 phosphorylation (Fig.…”

The Major Vault Protein Is a Novel Substrate for the Tyrosine Phosphatase SHP-2 and Scaffold Protein in Epidermal Growth Factor Signaling

“…These PTP domain PCR products were subcloned into pGEX-2TK. GST-SHP-2-EA was constructed by PCR amplifying SHP-2-Glu 76 to Ala 76 (EA) described previously (35) and subcloning the product into pGEX-2TK. The NH 2 -SH2 and COOH-SH2 domains of SHP-2 from amino acids 1-215 (GST-NϩC-SH2) have been described previously (36).…”