Discrimination between phosphotyrosine-mediated signaling properties of conventional and neuronal Shc adapter molecules

The phosphotyrosine (pTyr) adapter Shc/ShcA is a major connector in various tyrosine kinase signalings following a variety of stimulation such as growth factor/neurotrophin, as well as in those following calcium influx and integrin activation. As in other tissues, Shc has been implicated in neuronal signalings; however, recent evidence suggests that N-Shc/ShcC and Sck/ShcB would take over most of the roles of Shc in mature central neurons, and switching phenomena between Shc and N-Shc expression were observed in several neuronal paradigms. Little is, however, known as to the signal-output differences between Shc and N-Shc. Here we determined the efficacy of Shc and N-Shc toward Erk activation in NGF-treated PC12 cells, and found that N-Shc transduced Grb2/Sos/Ras-dependent Erk activation less efficiently than Shc. This was mainly because N-Shc has only one high-affinity Grb2-binding site, whereas Shc has two such sites. Phosphopeptide mapping revealed that N-Shc has novel tyrosine-phosphorylation sites at Y259/Y260 and Y286; in vivo-phosphorylation of these tyrosines was demonstrated by site-specific anti-pTyr antibodies. Phosphorylated Y286 bound to several proteins, of which one was Crk. The pY221/pY222 site, corresponding to one of the Grb2-binding sites of Shc, also preferentially bound to Crk. The phosphorylation-dependent interaction between N-Shc and Crk was demonstrated in vitro and in vivo. These results indicate that N-Shc has specific features of signal-output, and further suggest that the switching between Shc and N-Shc during neural development and regeneration would lead to differentiation of downstream signalings.