ABSTRACT
<p><b>OBJECTIVE</b>To study the interaction between ShcD and TrkC and to reveal the molecular mechanism of the downstream signal transduction of TrkC.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>ShcD binds to TrkC in a kinase-activity-dependent manner through its PTB and SH2 domains.</p>
Subject(s)
Humans , Adaptor Proteins, Signal Transducing , Genetics , Metabolism , Binding Sites , Cells, Cultured , Genetic Vectors , Plasmids , Genetics , Protein Binding , Receptor, trkC , Genetics , Metabolism , Shc Signaling Adaptor Proteins , Genetics , Metabolism , Transfection , Transformation, Bacterial , Two-Hybrid System Techniques , src Homology Domains , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To study the role of adaptor protein Dok6 in neurite outgrowth in PC12 cells.</p><p><b>METHODS</b>Series of fusion clones were constructed by fusing different domains of Dok6 into mutant TrkC/Y516F. These constructs were transiently transfected into PC12 cells separately and the expression levels of fusion proteins were detected by Western blot. Neurite outgrowth in these PC12 cells was tested after stimulation of NT-3.</p><p><b>RESULTS</b>Each fusion clone was stably expressed in PC12 cells. The fusion clones that fused both TrkC/Y516F-Dok6 (PTB+C) and TrkC/Y516F-Dok6C rescued the loss of neurite outgrowth in PC12 cells resulting from the mutation in tyrosine 516, while fusion clones that fused with single TrkC/Y516F-Dok6PTB did not show such effect.</p><p><b>CONCLUSION</b>Dok6 can promote neurite outgrowth induced by NT-3 stimulation through its C-terminal in TrkC-positive PC12 cells.</p>