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Detection of Homo- or Hetero-Association of Doks by Fluorescence Resonance Energy Transfer in Living Cells

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Abstract

Purpose

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.

Procedure

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.

Conclusion

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.

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Acknowledgments

The authors thank Dr. Luo Jian-hong for generously providing CFP-YFP vector and technical assistance on FRET determination. This work was supported by National Key Basic Research Program (2002CB713808, 2006CB500702, and 2007CB947100), National Natural Science Foundation (30291705, 30770657, and 30530240), Program for Changjiang Scholars and Innovative Research Team in University (IRT0528), National 863 program (2006 AA02A114) and Shanghai Metropolitan Fund for Research and Development (07DJ14005).

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Authors and Affiliations

  1. Department of Neurobiology, Institute of Neuroscience, Second Military Medical University, Shanghai, 200433, China

    Feng Zhang, Guo Fu, Li Cao, Hua-yan Yang, Yi-zhang Chen & Cheng He

  2. State Key Laboratory of High Field Laser Physics, Shanghai Institute of Optics and Fine Mechanics, Chinese Academy of Sciences, Shanghai, 201800, China

    Guo Fu, Chen Wang & Gui-ying Wang

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  1. Feng Zhang
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  2. Guo Fu
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  3. Chen Wang
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  4. Li Cao
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  7. Yi-zhang Chen
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  8. Cheng He
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Correspondence to Cheng He.

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Zhang, F., Fu, G., Wang, C. et al. Detection of Homo- or Hetero-Association of Doks by Fluorescence Resonance Energy Transfer in Living Cells. Mol Imaging Biol 11, 188–194 (2009). https://doi.org/10.1007/s11307-008-0189-5

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  • DOI: https://doi.org/10.1007/s11307-008-0189-5

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