T cell receptor induced intracellular redistribution of type I protein kinase A

Abstract

The productive activation of CD4(+) T lymphocytes, leading to proliferation and cytokine secretion, requires precise temporal regulation of intracellular cyclic AMP concentrations. The major effector molecule activated by cyclic AMP in mammalian cells is the cyclic AMP-dependent protein kinase A (PKA). The type I PKA isozyme mediates the inhibitory effects of cyclic AMP on T-cell activation. Using laser scanning confocal microscopy, we demonstrated that the regulation of PKA type I activity involves spatial redistribution of PKA type I molecules following T-cell receptor (TCR) stimulation. In resting T cells, PKA type I was located in membrane proximal regions and distributed equally across the cell. Shortly after antigen engagement, T cells and antigen-presenting cells formed an area of intense contact, known as the immunological synapse. TCR concentrated at the synapse, whereas PKA type I molecules redistributed to the opposite cell pole within 10 min after T-cell stimulation. Type I PKA redistribution was solely dependent on TCR signalling, because we observed the same temporal and spatial distribution after antibody-mediated cross-linking of the TCR-associated CD3 complex. Segregation of TCR and PKA type I molecules was maintained for at least 20 min. Thirty minutes after stimulation, PKA type I partially colocalized with the TCR. After 60 min, PKA type I distribution again approached the resting state. Considering that initial TCR signals lead to increases in intracellular cyclic AMP, PKA type I molecules may be targeted towards localized cyclic AMP accumulations or transported away from these areas, depending on the requirements of the cellular response.

Figure 1

Distribution of PKA RIα in resting CD4⁺ T cells and specificity controls. The left panels show transmission micrographs of the same cells as depicted in the middle and right panels. The cells in the upper panels were incubated with anti-CD3 mAb 145-2C11 followed by secondary antibody [goat anti-hamster (GAH) IgG-Alexa Fluor® 633]. The cells were then permeabilized and incubated with anti-PKA I mAb and secondary antibody [goat anti-mouse (GAM) IgG2b-Alexa Fluor® 488]. The incubations were performed at 4° to prevent the activation of the cells. The upper right panel shows distribution of PKA RIα throughout the cell. The cells in the lower panels were incubated with irrelevant control mAbs followed by secondary antibody.

Figure 2

Distribution of PKA RIα in unstimulated CD4⁺ T cells. Cells were incubated on ice with antibodies against CD3 (green) and PKA RIα (red). CD3 staining indicates diffuse distribution of TCR/CD3 complexes on the cell surface. Images taken from sections through the centre of the cell reveal equal distribution of PKA RIα beneath the plasma membrane. Combined staining of CD3 and PKA RIα. A confocal image reconstruction from 0·5-μm sections showing simultaneously the distribution of CD3 and PKA RIα can be seen in Figure 3. The images are representative of multiple cells from five independently performed experiments.

Figure 3

Distribution of PKA RIα in antigen-activated T cells. CD4⁺ lymph node T cells from TCR transgenic DO.11.10 mice were incubated with APC loaded with Ova323 for the indicated lengths of time. The first panel shows an unstimulated T cell. After incubation, cells were stained for CD3 (green) and PKA RIα (red). During confocal microscopical examination, three or four fields containing CD3-capped T cells were randomly chosen for each slide. Image analyses were performed only on CD3-capped T cells. Confocal image reconstructions from 0·5-μm sections from one representative experiment of three independently performed experiments are shown.

Figure 4

Distribution of PKA RIα in CD3-cross-linked T cells. CD4⁺ lymph node T cells from TCR transgenic DO.11.10 mice were either left untreated or cross-linked with anti-CD3 antibody plus goat anti-hamster secondary antibody (green) for the indicated length of time. Then, T cells were stained for PKA RIα (red). During confocal microscopical examination, three or four fields containing CD3-capped T cells were randomly chosen for each slide. Image analyses were performed only on CD3-capped T cells. The images are from one representative experiment of four independently performed experiments in which two to three slides per time point were analysed.

Figure 5

Model for the kinetics of the movement of PKA I in antigen-activated T cells.