title: Studying the dynamics of SLP-76, Nck, and Vav1 multimolecular complex formation in live human cells with triple-color FRET.
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.
authors.affiliation: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.
authors.forename: Maor H
authors.initials: MH
authors.lastname: Pauker
authors.forename: Nirit
authors.initials: N
authors.lastname: Hassan
authors.forename: Elad
authors.initials: E
authors.lastname: Noy
authors.forename: Barak
authors.initials: B
authors.lastname: Reicher
authors.forename: Mira
authors.initials: M
authors.lastname: Barda-Saad
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000502:physiology; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D051567:Proto-Oncogene Proteins c-vav / Q000235:genetics / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013601:T-Lymphocytes / Q000378:metabolism*
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D046912:Multiprotein Complexes; C078332:Nck protein; D015513:Oncogene Proteins; D010750:Phosphoproteins; D051567:Proto-Oncogene Proteins c-vav; D011948:Receptors, Antigen, T-Cell; C092947:SLP-76 signal Transducing adaptor proteins; C496618:VAV1 protein, human