THE SRC HOMOLOGY 2 DOMAIN PROTEIN SHB TRANSMITS BASIC FIBROBLAST GROWTH FACTOR-DEPENDENT AND NERVE GROWTH FACTOR-DEPENDENT DIFFERENTIATION SIGNALS IN PC12 CELLS

To assess a possible role for the Src homology 2 (SH2) domain adaptor protein Shb in PC12 cell signal transduction and differentiation, we h ave investigated the expression of Shb in PC12 cells and found that th e differentiation factors nerve growth factor (NGF) and basic fibrobla st growth factor (bFGF), as well as the PC12 cell mitogen epidermal gr owth factor, increased Shb protein expression and Shb mRNA steady-stat e levels, Two PC12 cell clones stably overexpressing the Shb cDNA exhi bited enhanced NGF- or bFGF-induced differentiation, assessed as neuri te outgrowth, This effect showed no direct correlation to mitogen-acti vated protein kinase activation, although the mitogen-activated protei n kinase/kinase inhibitor PD-98059 caused a partial inhibition of neur ite outgrowth, Furthermore, it was found that the Shb-overexpressing c ells extended neurites in response to epidermal growth factor, The eff ects of Shb overexpression on neurite outgrowth required a functional SH2 domain because PC12 cells expressing Shb with an inactivated SH2 d omain did not differentiate more readily in response to NGF, Tyrosine phosphorylation of the p13 Suc1-associated neurotrophic factor-induced tyrosine-phosphorylated target protein in response to bFGF was strong ly inhibited by Shb overexpression, without correlating with the corre sponding rate of neurite outgrowth, NGF-induced tyrosine phosphorylati on of phospholipase C gamma, TrkA, and Shc was unaltered in the Shb-ov erexpressing cells, whereas that of Shb was greatly enhanced in these cells compared with control PC12-neo cells. In addition, an NGF-activa ted M-r 140,000 phosphotyrosine protein was found to be associated wit h Shb in immunoprecipitation experiments. The data in this study sugge st that Shb is involved in transmission of NGF- and bFGF-dependent dif ferentiation signals in PC12 cells.