PC12 cells have provided a useful model system to investigate signal transduction pathways involved in nerve growth factor (NGF)-induced neuronal differentiation. Earlier studies have implicated src- and ras family proto-oncogenes as critical components in NGF-stimulated neurite outgrowth in PC12 cells. One of the major objectives of the projects proposed in this application is to define role of Src- and Ras-family proteins in the early signaling events that regulate NGF-induced neurite outgrowth. We will focus on three specific aspects of NGF-induced signaling: i) the activation of Src family kinases, their binding to the NGF receptor, and their role in NGF-induced neurite outgrowth, 2) the role of Shc and Grb2/Sem5 in the activation of Ras and pathways regulated by Ras, and 3) the identification of Ras targets that are involved in the activation of MAP kinases and other cellular events. The primary strategies involved in these projects involve the use of vectors that allow inducible expression of high levels of constitutively activated or dominant-interfering molecules of Src, Ras, Shc, Grb2, GAP, and Raf, and the use of affinity purification strategies to identify previously unidentified components of these pathways. In addition, a second objective, is to identify cellular proteins that specifically interact with the SH3 domain of Src+, the variant form of Src that is specifically expressed in neurons. The goal of these latter studies is to understand how the six amino acid insert within the SH3 domain of this protein regulates its functional activity in neurons.
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