Role of the localization of Vav1 during T cell activation.
Doctoral thesis, UCL (University College London).
Vav1 is a haematopoietic-specific guanine nucleotide exchange factor for Rho-family GTPases that acts downstream of antigen receptors and regulates events such as lymphocyte proliferation, cytoskeletal reorganization, calcium flux and the activation and clustering of integrins. It is well established that localization of signalling molecules is important for their function. Here, we investigated the subcellular distribution of Vav1 in primary CD4+ and CD8+ T cells following T cell receptor (TCR) stimulation using the combination of two imaging methods: confocal and total internal reflection microscopy. This study also aimed to examine the relationship between the localization of Vav1 and its function. We found that Vav1 polarizes to the immunological synapse (IS) and localizes to TCR-induced microclusters. Analysis of Vav1 mutants showed that loss-of-function mutations in the DH, PH and N-terminal SH3 domain did not affect TCR-induced localization. However a point mutation in the SH2 domain (R696A) completely abrogates polarization of Vav1 to the IS and its localization to microclusters. Furthermore, this mutant fails to undergo TCRinduced phosphorylation and does not rescue Vav1-deficient T cells from impaired calcium flux. Interestingly, the C-terminal SH3 domain mutant exhibited a nuclear localization. This mutant also showed slightly decreased ability to undergo movement toward IS, diminished TCR-induced phosphorylation and could only partially rescue Vav1-deficient T cells from impaired calcium flux. Taken together, these data show that the SH2 domain of Vav1 is required for its correct localization at the IS, phosphorylation and function. Furthermore, the C-terminal SH3 domain of Vav1 seems to play a role in the retention of Vav1 in the cytoplasm.
|Title:||Role of the localization of Vav1 during T cell activation|
|Additional information:||Authorisation for digitisation not received|
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