Diacylglycerol-dependent binding recruits PKCtheta and RasGRP1 C1 domains to specific subcellular localizations in living T lymphocytes

Abstract

Diacylglycerol (DAG) signaling relies on the presence of conserved domain 1 (C1) in its target proteins. Phospholipase C-dependent generation of DAG after T cell receptor (TCR) triggering is essential for the correct immune response onset. Accordingly, two C1-containing proteins expressed in T lymphocytes, Ras guanyl nucleotide-releasing protein1 (RasGRP1) and protein kinase C (PKC), were shown to be fundamental for T-cell activation and proliferation. Although containing the same regulatory domain, they are proposed to relocate to distinct subcellular locations in response to TCR triggering. Here we studied intracellular localization of RasGRP1 and PKC C1 domains in living Jurkat T cells. The results demonstrate that, in the absence of significant primary sequence differences, the C1 domains of these proteins show specific localization within the cell and distinct responses to pharmacological stimulation and TCR triggering. These differences help explain the divergent localization and distinct functional roles of the full-length proteins, which contains them. The properties of these DAG-binding modules allow their characterization as functional markers that discriminate between DAG pools. Finally, we show that by binding to different diacylglycerol forms, overexpression of distinct C1 modules can attenuate DAG-dependent signals originating from the plasma or internal membranes. This is shown by analyzing the contribution of these two lipid pools to PLC-dependent Ras activation in response to TCR triggering.

Figure 1.

C1 domain constructs and their expression in Jurkat cells. (A) alignment of C1bPKCθ, C1β2chimerin, C1RasGRP1, and C1aPKCθ domains with C1bPKCδ, as an example of a typical C1 domain. ^*, single, fully-conserved residue,:, highly conservative changes in side groups and., low conservative changes in side groups. Alignment was done with the Clustal W program using Biology Workbench 3.2 (San Diego Supercomputer Center, University of California, San Diego, CA). (B) In vivo expression of GFP-fused C1 domains in Jurkat cells. Cells were transfected and after 24 h, pelleted and suspended in HBSS for plating on poly-d,l-lysine–coated chamber slides. Slides were mounted on a 37°C plate on the confocal microscope and analyzed. Images are representative of the field observed for each condition. (C) Immunoblot analysis of Jurkat cells transfected with indicated C1 domain constructs. Cells were transfected and processed 24 h later for Western blot (see MATERIALS AND METHODS). Analysis with anti-GFP mAb showed proteins of the predicted molecular weight. C1a+C1bPKCθ was less abundant than the other constructs in all experiments. In flow cytometry analysis, C1a+C1bPKCθ–expressing cells were less numerous and less bright than cells expressing the other constructs.

Figure 2.

Confocal microscopy analysis of C1 domain internal localization. Jurkat cells transfected with distinct GFP-fused C1 domains were fixed after 24 h and processed (see MATERIALS AND METHODS) to stain Golgi with anti-giantin-α (red, left panels) or ER with anti-PDI (red, right panels). Merged GFP and Cy3 fluorescence is shown.

Figure 3.

C1 domains change their localization in U73122- or propranolol-treated cells. (A) Cells transfected with GFPC1 constructs alone (-) or treated (+) with U73122 (1 μM, 30 min), were plated and mounted for confocal microscopy as in Figure 1B. Images are representative of a field observed for each condition. (B) Cells transfected with GFPC1 constructs were plated and mounted for confocal microscopy as in Figure 1B. Propranolol (250 μM) was added immediately after the first frame, and images captured every 5 s. Time-lapse confocal recording is shown in Supplementary Videos 1–4. Images at times indicated are shown to illustrate GFPC1 domain subcellular localization.

Figure 4.

C1 domains bind selectivity to vesicles containing distinct DAG species. DPPC and PS (4:1, mol:mol) vesicles containing no DAG (0), 1,2-dioleyl-sn-glycerol (DOG) or 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) were incubated with lysates from Jurkat cells transfected with different GFPC1 constructs or with GFP (control). Bound protein was recovered by centrifugation and detected by SDS-PAGE and western blot, (p, pellet fraction; s, supernatant fraction). Bands were quantified by densitometry; these values were used to calculate the percentage of membrane-bound protein using the formula %bound = band intensity in p/(band intensity in p + band intensity in s).

Figure 5.

C1β2chimerin, C1bPKCθ, C1a+C1bPKCθ, and C1RasGRP1, but not C1aPKCθ translocate in response to PMA. Cells transfected with GFPC1 constructs were plated and mounted for confocal microscopy as in Figure 1B. PMA (200 nM) was added immediately after the first frame, and images captured every 11 s. Time-lapse confocal recording is shown in Supplementary Videos 5–9. Images corresponding to times indicated show GFPC1 domain translocation.

Figure 6.

C1RasGRP1 associates and dissociates continuously from internal membranes. Cells transfected with GFPC1RasGRP1 were plated and mounted for confocal microscopy. (A) Perinuclear region was selected and exposed to maximum laser power. Images before and after (at indicated times) photobleaching were captured. Color range: red: maximum GFP fluorescence, blue: minimum GFP fluorescence. (B) Data from six representative experiments were plotted.

Figure 7.

Translocation of full-length RasGRP1, PKCθ, and their C1 domains in response to CD3+CD28 stimulation. Jurkat cells were transfected with (A) GFP-fused PKCθ, C1aPKCθ, C1bPKCθ or C1a+C1bPKCθ or (C) with GFPRasGRP1 or GFPC1RasGRP1 constructs. At 24 h post-transfection, cells were suspended in HBSS and CD3+CD28-coated microspheres were added at a 2:1 cell:bead proportion, mounted as in Figure 1B for confocal microscopy, and representative cells captured for each condition. (B) Images at times indicated are shown to illustrate GFPC1 domain subcellular localization; full time-lapse confocal recording is shown in Supplementary Videos 15–17.

Figure 8.

C1RasGRP and C1a+C1bPKCθ modify Ras activation in response to soluble CD3 activation. Cells transfected with GFP, C1RasGRP or C1a+C1bPKCθ were sorted to recover GFP-positive cells. GFP expression was assessed by flow cytometry after 8 h culture; cells were then serum-starved (1 h), stimulated with soluble CD3 (1 μg/ml) for the times indicated, then collected and processed (see MATERIALS AND METHODS) for Western blot. Analysis with PMAPK antibody showed Jurkat cell activation differences according to the construct transfected. Anti-MAPK antibody was used as a protein loading control. Results are representative of four independent experiments. GFP and GFPC1RasGRP cells were 95% GFP-positive; GFPC1a+C1bPKCθ cells were 78% GFP-positive. The x-fold change in activation for each condition was estimated by densitometric analysis of filters. Values (arbitrary units) were normalized taking into consideration the protein levels of each sample as determined by Western blot analysis.