NGF-dependent neurite outgrowth in PC12 cells overexpressing the Src homology 2-domain protein Shb requires activation of the Rap1 pathway

The Src homology 2 (SH2) domain adaptor protein Shb has been shown to trans mit NGF- and FGF-2-dependent differentiation signals in PC12 cells. To stud y if this involves signaling through the small GTPase Rap1, Rap1 activity w as assessed in Shb-overexpressing PC12 cells, We demonstrate that NGF and E GF induce Rap1 activation in PC12-Shb cells, while FGF-2 fails to do so. Ho wever, PC12 cells expressing Shb with an inactivated SH2 domain do not resp ond to NGF stimulation with Rap1 activation. The CrkII SH2 domain interacts with Shb and a 130- to 135-kDa phosphotyrosine protein present mainly in P C12-Shb cells and these interactions may thus relate to the effect of Shb o n Rap1 activation. Transient expression of RalGDS-RBD or Rap1GAP to block t he Rap1 pathway reduces the NGF-dependent neurite outgrowth in PC12-Shb cel ls. These results suggest a role of Shb in NGF-dependent Rap1 signaling and this pathway may be of significance for neurite outgrowth under certain co nditions, (C) 2000 Academic Press.