Detection of homo- or hetero-association of Doks by fluorescence resonance energy transfer in living cells.
OBJECTIVE
The Dok proteins represent a family of adaptor proteins serving as common substrates for protein tyrosine kinases and play an important role in regulating signal transduction in multiple cell functions. Dimerization of Dok proteins may represent a powerful and flexible regulatory mechanism that can achieve a variety of consequences. This study aims to detect the homo- or hetero-association of Doks in living cells.


METHODS
The transfection of CFP or YFP fusion protein constructs was carried out using lipofectamine 2000. FRET Measurements were performed using three-channel microscopy and Spectroscopy.


RESULTS
By using fluorescence resonance energy transfer technology, we demonstrated, for the first time to our knowledge, that Dok5 and Dok1 could form homomeric and heteromeric associations in living cells. Moreover, pleckstrin homology (PH) domain was found to be essential for homomeric associations of Dok5, while PH domain and phosphotyrosine binding domain were found to be crucial for homomeric associations of Dok1 or heteromeric associations between Dok1 and Dok5.


CONCLUSIONS
The mechanisms underlying Doks' association may benefit the further understanding of the important role of Dok proteins in regulating signal transduction activated by tyrosine kinases.
Zhang
Feng
F
Department of Neurobiology, Institute of Neuroscience, Second Military Medical University, Shanghai, 200433, China.
Fu
Guo
G
Wang
Chen
C
Cao
Li
L
Yang
Hua-Yan
HY
Wang
Gui-Ying
GY
Chen
Yi-Zhang
YZ
He
Cheng
C
D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D055503:Protein Multimerization; D016601:RNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C489560:DOK1 protein, human; C432912:DOK5 protein, human; D008164:Luminescent Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins