SH2-B is required for nerve growth factor-induced neuronal differentiation

Nerve growth factor (NGF) is essential for the development and survival of sympathetic and sensory neurons. NGF binds to TrkA, activates the intrinsic kinase activity of TrkA, and promotes the differentiation of pheochromocyt oma (PC12) cells into sympathetic-like neurons. Several signaling molecules and pathways are known to be activated by NGF, including phospholipase C g amma, phosphatidylinositol-3 kinase, and the mitogen-activated protein kina se cascade. However, the mechanism of NGF-induced neuronal differentiation remains unclear. In this study, we examined whether SH2-B beta, a recently identified pleckstrin homology and SH2 domain-containing signaling protein, is a critical signaling protein for NGF, TrkA bound to glutathione S-trans ferase fusion proteins containing SH2-B beta, and NGF stimulation dramatica lly increased that binding. In contrast, NGF was unable to stimulate the as sociation of TrkA with a glutathione S-transferase fusion protein containin g a mutant SH2-B beta(R555E) with a defective SH2 domain. When overexpresse d in PC12 cells, SH2-B beta coimmunoprecipitated with TrkA in response to N GF. NGF stimulated tyrosyl phosphorylation of endogenous SH2-B beta as well as exogenously expressed GFP-SH2-B beta but not GFP-SH2-B beta(R555E). Ove rexpression of SH2-B beta(R555E) blocked NGF-induced neurite outgrowth of P C12 cells, whereas overexpression of wild type SH2-B beta enhanced NGF-indu ced neurite outgrowth. Overexpression of either wild type or mutant SH2-B b eta(R555E) did not alter tyrosyl phosphorylation of TrkA, Shc, or phospholi pase C gamma in response to NGF or NGF-induced activation of ERK1/2, sugges ting that SH2-B beta may initiate a previously unknown pathway(s) that is e ssential for NGF-induced neurite outgrowth. Taken together, these data indi cate that SH2-B beta is a novel signaling molecule required for NGF-induced neuronal differentiation.