TY - JOUR
T1 - Studying the dynamics of SLP-76, Nck, and Vav1 multimolecular complex formation in live human cells with triple-color FRET
AU - Pauker, Maor H.
AU - Hassan, Nirit
AU - Noy, Elad
AU - Reicher, Barak
AU - Barda-Saad, Mira
PY - 2012/4/24
Y1 - 2012/4/24
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84860246467&partnerID=8YFLogxK
U2 - https://doi.org/10.1126/scisignal.2002423
DO - https://doi.org/10.1126/scisignal.2002423
M3 - مقالة
C2 - 22534133
SN - 1945-0877
VL - 5
JO - Science Signaling
JF - Science Signaling
IS - 221
M1 - rs3
ER -