The role of 14-3-3 proteins in regulating T cell adhesion and migration

We are studying how white blood cells move and bind to other cells. These processes are important when white blood cells monitor the body for presence of disease-causing pathogens, as well as during the immune response when white blood cells destroy or neutralize pathogens. Without attachment and movement, cells cannot be in the right place at the right time to mount an immune response to a pathogen. We are trying to understand the molecular basis of white blood cell attachment and movement. We focus on a family of cell adhesion proteins called integrins. We are interested in finding out how the integrins become activated to achieve cell binding and movement. In particular, we are interested in the role of a group of proteins called 14-3-3 proteins in these processes. 14-3-3 proteins bind to the 'tails' of the integrins inside the cell, as well as to many other proteins in activated immune cells. We now aim to understand if these interactions are important for the regulation of immune cell adhesion and movement. We will use transgenic technology as well as novel biochemical methods for these studies. The results will increase our understanding of how immune cells move and attach to other cells during an immune response.

The main objective for this proposal is to investigate the role of 14-3-3 proteins in regulating T cell adhesion and migration mediated by integrins and changes in actin cytoskeleton dynamics. These processes are important for T cell recirculation, recruitment into inflamed tissue, T cell activation and effector functions. 14-3-3 proteins bind to serine/threonine phosphorylated sequences in proteins, including many proteins involved in regulating actin cytoskeleton dynamics. We have shown that Thr-phosphorylation of the LFA-1 integrin leads to 14-3-3 recruitment in T cell receptor-activated T cells. This interaction regulates actin cytoskeleton rearrangements and adhesion in vitro. We have also shown that 14-3-3s bind many other proteins in activated T cells. Thus, 14-3-3s can be used as a 'phospho-fingerprinting device' to investigate cell signaling. We now aim to investigate the role of the 14-3-3-integrin complex in vivo, as well as search for novel 14-3-3 interactors in primary human T cells. Aim 1. Investigate the role of the 14-3-3-binding site of LFA-1 (beta2-integrin Thr758-760) in T cell adhesion and migration in vivo. We will produce a transgenic mouse model with a triple Thr758-760-beta2-integrin mutation and study T cell functions ex vivo and in vivo, including adhesion, migration, trafficking of cells in vivo and T cell effector functions. Aim 2. Identify novel 14-3-3 binding partners and phosphopeptides interacting with 14-3-3 proteins in resting, primary human T cells, as well as in T cells activated through the T cell receptor. We will isolate human T cells from buffy coats and use 14-3-3 affinity pulldowns and quantitative proteomics techniques to identify 14-3-3 binding partners and novel phosphorylation sites in resting and activated T cells. Investigations of the regulation of integrins and cell motility are fundamental for our understanding of T cell biology and for enhancing our knowledge about immune system regulation.