A Functional Study Of ShcD In The Trk Receptor Signaling Pathway In The Nervous System

The neurotrophin family is a group of polypeptide growth factors that influence several aspects of nervous system,including survival,proliferation, differentiation, myelination, apoptosis,axonal growth, and synaptic plasticity. Neurotrophins exert their functions by binding to two types of receptors:the Trk receptor and p75NTR. Upon neurotrophin stimulation, Trk receptor's intrinsic protein-tyrosine kinase are activated and results in Trk auto-phosphorylation and recruitment of downstream adaptors containing PTB or SH2 domains. Shc proteins are a family of adaptors containing four domains:from N-terminal to C-terminal is CH2,PTB,CH1 and SH2 domains.Both PTB and SH2 domains can bind phosphorylated tyrosine residues,making ShcD a potential substrate of RTK.It is well documented that ShcA,ShcB and ShcC are downstream adaptors of Trk family members.The newly identified Shc member-ShcD,is highly expressed in nervous system.We propose that ShcD is a substrate of Trk receptors and invovled in neurotrophin-induced signaling pathways.First, yeast two-hybrid system and Co-IP assay showed that ShcD associate with ICD of Trk receptors. This interaction is dependent on Trk kinase activity since ShcD only interact with the wild type TrkA/B but not the kinase dead mutant TrkA/B.To define the region of ShcD responsible for binding TrkA/B,we performed yeast two-hybrid assay and GST pull-down assay.The results showed that both the PTB and SH2 domains of ShcD mediate binding to TrkA/B.To further characterize the site of interaction on TrkA/B for ShcD PTB domain binding, we carried out yeast two-hybrid and GST pull-down assay using a series of TrkA/B mutation constructs and found the phosphotyrosine binding (PTB) domain of ShcD recognizes activated TrkA/B at NPQY motif.Confocal analysis showed that ShcD colocalized well with TrkA/B in 293T cells,further confirming their interaction in the cellular physiological condition.To test whether ShcD is involved in TrkA/B signaling pathway,we mutated the 6 tyrosine residues localized within CHI domain of ShcD and designated this construct as ShcD 6F. Then we cotransfected ShcDWT/6F with TrkA/B into 293T cells followed by stimulation by neurotrophin. Immunoprecipitation and immunoblotting analysis indicates that neurotrophin treatment stimulates ShcD-TrkA/B interaction and the tyrosine phosphorylation of ShcD,while the phosphorylation of ShcD 6F mutant can almost not be detectable although it can still exibit the capability to associate with TrkA/B.And only the activated ShcD wild type but not the 6F mutant can recruit Grb2 adaptor in response to neurotrophins.These observations suggest that ShcD can be phosphorylated upon neurotrophin treatment and associate with Grb2 through its CHI domain.Next,we proceeded to examine whether ShcD could regulate the neurotrophin-induced signaling pathways. We showed that ShcD could sustained BDNF induced MAPK activation in 293T cells.In order to examine the role of ShcD in NGF-TrkA signaling pathway,we generated PC 12 stable cell lines expressing pEGFP-N1-ShcD. Similar to its effect on BDNF-TrkB pathway, ShcD was also involved in the activation of MAPK pathway induced by NGF in PC 12 cell lines.In summary, the results showed that ShcD can bind Trk receptors in a kinase-dependent manner and this interaction involves both the ShcD PTB and SH2 domain. Moreover,ShcD can mediate the MAPK activation induced by neurotrophins.