Promotion of skeletal muscle differentiation by K252a with tyrosine phosphorylation of focal adhesion: A possible involvement of small GTPase Rho

K252a, a protein kinase inhibitor, acts as a neurotrophic factor in several neuronal cells. In this study we show that K252a enhanced the differentiat ion of C2C12 myoblasts as well as tyrosine phosphorylation of several focal adhesion-associated proteins including p130(Cas), focal adhesion kinase, a nd paxillin. The tyrosine phosphorylation of these proteins, reaching a max imum at 30 min after K252a treatment, closely correlated with the colocaliz ation of these proteins in focal adhesion com plexes and the coimmunoprecip itation of these proteins with p130(Cas). In addition, K252a stimulated lon gitudinal development of stress fiber-like structures and cell-matrix inter action in postmitotic myoblasts and eventually formation of well-developed myofibrils in multinucleated myotubes. Herbimycin A, a potent inhibitor of Src family kinases, and cytochalasin D, a selective disrupting-agent of act in filament, completely inhibited K252a-induced tyrosine phosphorylation as well as myoblast differentiation. Similar inhibitory effect was observed i n the cells scrape loaded with a Rho inhibitor, C3 transferase, and the tre atment of K252a induced a rapid translocation of Rho. These results are con sistent with the model that Rho-dependent tyrosine phosphorylation of focal adhesion-associated proteins plays an important role in skeletal muscle di fferentiation. (C) 1999 Academic Press.