Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.


Sci. Signal., 24 April 2012
Vol. 5, Issue 221, p. rs3
[DOI: 10.1126/scisignal.2002423]


Studying the Dynamics of SLP-76, Nck, and Vav1 Multimolecular Complex Formation in Live Human Cells with Triple-Color FRET

Maor H. Pauker*, Nirit Hassan*, Elad Noy, Barak Reicher, and Mira Barda-Saad{dagger}

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.

* These authors contributed equally to this work.

Abstract: Protein-protein interactions regulate and control many cellular functions. A multimolecular complex consisting of the adaptor proteins SLP-76 (Src homology 2 domain–containing leukocyte protein of 76 kD), Nck, and the guanine nucleotide exchange factor Vav1 is recruited to the T cell side of the interface with an antigen-presenting cell during initial T cell activation. This complex is crucial for regulation of the actin machinery, antigen recognition, and signaling in T cells. We studied the interactions between these proteins as well as the dynamics of their recruitment into a complex that governs cytoskeletal reorganization. We developed a triple-color Förster resonance energy transfer (3FRET) system to observe the dynamics of the formation of this trimolecular signaling complex in live human T cells and to follow the three molecular interactions in parallel. Using the 3FRET system, we demonstrated that dimers of Nck and Vav1 were constitutively formed independently of both T cell activation and the association between SLP-76 and Nck. After T cell receptor stimulation, SLP-76 was phosphorylated, which enabled the binding of Nck. A point mutation in the proline-rich site of Vav1, which abolishes its binding to Nck, impaired actin rearrangement, suggesting that Nck-Vav1 dimers play a critical role in regulation of the actin machinery. We suggest that these findings revise the accepted model of the formation of a complex of SLP-76, Nck, and Vav1 and demonstrate the use of 3FRET as a tool to study signal transduction in live cells.

{dagger} To whom correspondence should be addressed. E-mail: Mira.Barda-Saad{at}

Citation: M. H. Pauker, N. Hassan, E. Noy, B. Reicher, M. Barda-Saad, Studying the Dynamics of SLP-76, Nck, and Vav1 Multimolecular Complex Formation in Live Human Cells with Triple-Color FRET. Sci. Signal. 5, rs3 (2012).

Read the Full Text

Triple-Color FRET Analysis Reveals Conformational Changes in the WIP-WASp Actin-Regulating Complex.
S. Fried, B. Reicher, M. H. Pauker, S. Eliyahu, O. Matalon, E. Noy, J. Chill, and M. Barda-Saad (2014)
Science Signaling 7, ra60
   Abstract »    Full Text »    PDF »
Multimolecular Analysis of Stable Immunological Synapses Reveals Sustained Recruitment and Sequential Assembly of Signaling Clusters.
L. Philipsen, T. Engels, K. Schilling, S. Gurbiel, K.-D. Fischer, K. Tedford, B. Schraven, M. Gunzer, and P. Reichardt (2013)
Mol. Cell. Proteomics 12, 2551-2567
   Abstract »    Full Text »    PDF »
Modulation of T cell signaling by the actin cytoskeleton.
Y. Yu, A. A. Smoligovets, and J. T. Groves (2013)
J. Cell Sci. 126, 1049-1058
   Abstract »    Full Text »    PDF »

To Advertise     Find Products

Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882