[ShcD interacts with TrkC through its PTB and SH2 domains].
OBJECTIVE
To study the interaction between ShcD and TrkC and to reveal the molecular mechanism of the downstream signal transduction of TrkC.


METHODS
Yeast two-hybrid assay was used. The intracellular domains of TrkC and TrkC mutants were cloned into pAS2-1, and ShcD and its four domains (CH2, PTB, CH1, and SH2 domains) were cloned into pACT2 vector respectively. The constructs were separately cotransformed into yeast. beta-galactosidase activity was measured to detect their interactions. TrkC was cloned into pmRFP (carrying red fluorescent protein), and ShcD was cloned into pEGFP (carrying green fluorescent protein). pmRFP-TrkC and pEGFP-ShcD were co-transfected into 293T cells, and then the cells were fixed and subjected to confocal analysis to study their subcellular localization.


RESULTS
ShcD interacted with TrkC but not with kinase dead mutant TrkCM1(K572A). Both PTB and SH2 domains were capable of binding to TrkC, and PTB domain bound NPQY motif of TrkC. ShcD colocalized with TrkC throughout the cytoplasm and in the plasma membrane in 293T cells.


CONCLUSIONS
ShcD binds to TrkC in a kinase-activity-dependent manner through its PTB and SH2 domains.
You
Yuan-gang
YG
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Beijing 100005, China.
Li
Wei-qi
WQ
Yin
Bin
B
Peng
Xiao-zhong
XZ
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002478:Cells, Cultured; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D020812:Receptor, trkC / Q000235:genetics / Q000378:metabolism*; D055765:Shc Signaling Adaptor Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014169:Transformation, Bacterial; D020798:Two-Hybrid System Techniques; D018909:src Homology Domains / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; C519335:SHC4 protein, human; D055765:Shc Signaling Adaptor Proteins; D020812:Receptor, trkC