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

Abstract

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 in¯ux 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 NShc expression were observed in several neuronal paradigms. Little is, however, known as to the signaloutput dierences between Shc and N-Shc. Here we determined the ecacy of Shc and N-Shc toward Erk activation in NGF-treated PC12 cells, and found that NShc transduced Grb2/Sos/Ras-dependent Erk activation less eciently than Shc. This was mainly because N-Shc has only one high-anity 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-speci®c 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 phosphorylationdependent interaction between N-Shc and Crk was demonstrated in vitro and in vivo. These results indicate that N-Shc has speci®c features of signal-output, and further suggest that the switching between Shc and NShc during neural development and regeneration would lead to dierentiation of downstream signalings.

The Neuron-Specific Rai (ShcC) Adaptor Protein Inhibits Apoptosis by Coupling Ret to the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway

“…4B), thereby suggesting that, as for the Shc proteins, Y231, Y232, and Y304 are the major Rai tyrosine phosphorylation sites. These results differ from those of a previous report, which showed that NGF treatment induces phosphorylation of overexpressed Rai only at the Y304 site, possibly due to differences in the experimental systems used (GDNF versus NGF and endogenous versus overexpressed Rai proteins) (23). We then measured the ability of a GST-Grb2 recombinant protein to interact with phosphorylated Rai.…”

“…Indeed, preliminary evidences suggest that, like the Shc proteins, Sli and Rai may function as cytoplasmic signaling proteins downstream of activated TK receptors. Both Rai and Sli can bind activated TK receptors, including the EGF receptor (EGFR) and Trk receptor (10,23,24,26). Whether Rai or Sli activates Ras or other intracellular pathways remains to be determined, as do the biological consequences of their activities.…”

Grit, a GTPase-Activating Protein for the Rho Family, Regulates Neurite Extension through Association with the TrkA Receptor and N-Shc and CrkL/Crk Adapter Molecules

“…The question thus arises as to whether there are any binding partners of Grit in TrkA-negative neurons. To address this issue, we studied the interaction between Grit and (11,33,34,44,50) as well as in many other phosphotyrosine-mediated signalings including the EGF pathway (7,41). By using EGF-treated COS-1 cells overexpressing FLAG-tagged Grit-CT and T7-tagged Shc family members, we found that N-Shc, a central-neuron specific adapter (34), bound efficiently to Grit-CT (Fig.…”

“…Immunoprecipitates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with the desired antibodies. The glutathione S-transferase (GST) pull-down assay was performed as previously described (33). Cell lysates were incubated with 10 to 25 g of GST fusion proteins on glutathione-Sepharose (AmershamPharmacia) for 2 h or overnight at 4°C.…”

Distinct role of ShcC docking protein in the differentiation of neuroblastoma

“…As for the difference in the downstream signaling between ShcC and ShcA, little is known so far. Regarding to this point, Nakamura et al (2002) indicated that inhibition of NGF-induced ERK activation by the expression of ShcC was due to the different Grb2-binding capacity between ShcA and ShcC in response to NGF. It was previously reported that ShcA preferentially binds to TrkA (Yamada et al, 2002), which is the key receptor against NGF due to neurite outgrowth with the sustained ERK phosphorylation, ShcC in differentiation of neuroblastoma I Miyake et al whereas ShcC associated with TrkB rather than TrkA (O'Bryan et al, 1998;Liu and Meakin, 2002).…”