Myogenic differentiation depends on the interplay of Grb2 and N-WASP

Myogenesis requires a well-coordinated withdrawal from cell cycle, morphological changes and cell fusion mediated by actin cytoskeleton. Grb2 is an adaptor protein whose central SH2 domain binds to phosphorylated tyrosine residues of activated receptors and activates intracellular signaling pathway, while its N-terminal and C-terminal SH3 domains bind to proline rich proteins such as N-WASP (Neural-Wiskott Aldrich Syndrome Protein). We found that the expression of Grb2 was increased at the beginning of differentiation and remained constant during differentiation in C2C12 myoblasts. Knocking down endogenous Grb2 expression caused a significant increase in the fusion index and expression of MyHC, a terminal differentiation marker when compared with the control. Over expression of Grb2 in C2C12 (C2C12^Grb2-Myc) reduced myotube formation and expression of MyHC. Similarly over expression of Grb2^P49L-Myc (N-terminal SH3 domain mutant) or Grb2^R86K-Myc (SH2 domain mutant) inhibited myogenic differentiation of C2C12 cells. However, the expression of Grb2^P206L-Myc (C-terminal SH3 domain mutant) did not inhibit myotube formation and expression of MyHC. This suggests that the C-terminal SH3 domain of Grb2 is critical for the inhibition of myogenic differentiation. The C2C12^Grb2-Myc cells have reduced phalloidin staining at late stages of differentiation. Expression of N-WASP in C2C12^Grb2-Myc cells rescued the myogenic defect and increased phalloidin staining (increased F-actin) in these cells. Thus our results suggest that Grb2 is a negative regulator of myogenesis and reduces myogenic differentiation by inhibiting actin polymerization/remodeling through its C-terminal SH3 domain.