Distinct spatial and temporal distribution of ZAP70 and Lck following stimulation of interferon and T-cell receptors

Abstract

T-cell receptor (TCR) stimulation results in the recruitment and activation of the proteins ZAP70 and Lck. These two proteins have been implicated in signalling derived from interferon receptors, although their precise role in this independent pathway has not been determined fully. These observations raise a fundamental question of how a given protein in a cell can be involved in more than one signalling pathway, yet each pathway is able to produce a highly specific downstream response to its own stimulant. To maintain exclusivity of response, each pathway must isolate its component molecules chemically, spatially or dynamically from other prevailing pathways. To address this question, the proteins ZAP70 and Lck were investigated following stimulation of the interferon-alpha receptor and the TCR in T cells by two different extracellular stimulants: interferon-alpha and the anti-CD3 antibody, OKT3, respectively. We first demonstrate that ZAP70 plays a pivotal role in interferon-stimulated MAPK activation, and that the tyrosine residue at position 319 of ZAP70 is important for interferon-stimulated ERK activation. Translocation of both ZAP70 and Lck to the nucleus following interferon receptor stimulation is demonstrated for the first time. Fluorescence resonance energy transfer microscopy revealed a high degree of spatial localization of the ZAP70/Lck complex within the cell following IFNalpha stimulation, in contrast to a diffuse presence following the application of OKT3. The difference in the spatio-temporal localization of these proteins following stimulation may eliminate signal crosstalk, and could explain the differentiation of the specific downstream responses of these pathways.