PMC

N-Ras and H-Ras are expressed on the Golgi apparatus of Jurkat cells. (A) CFP-H-Ras and CFP-N-Ras vectors were cotransfected in Jurkat cells together with a vector expressing the Golgi marker GalT tagged with YFP. CFP-Ras and YFP-GalT distributions were determined by LSM in living cells. (B) The localization of endogenous N-Ras in the Golgi apparatus was determined by immunofluorescence. As described in Materials and Methods, endogenous N-Ras and giantin (a Golgi-specific marker) were detected in fixed Jurkat T cells.

N-Ras is the only Ras isoform activated on the Golgi apparatus upon low-grade TCR engagement in Jurkat T cells. Jurkat (A) and COS-1 (B) cells were cotransfected with YFP-Raf1-RBD plus either H-Ras, N-Ras, or K-Ras tagged with CFP. Forty-eight hours later, cells were serum starved and either left untreated (a to c and j to l), stimulated with the indicated amounts of anti-CD3 plus anti-CD28 (d to i), or stimulated with EGF (m to r) at the times indicated. Cells were imaged alive by LSM, and those expressing equivalent amounts of H-Ras, K-Ras, or N-Ras were selected to analyze YFP-Raf1-RBD distribution. Arrowheads and arrows indicate Raf1-RBD redistribution to the Golgi apparatus and plasma membrane, respectively. NT, nontreated cells.

N-Ras activation upon TCR engagement in Jurkat cells is PLCγ1-dependent. (A) J gamma 1 cells, which are deficient in PLCγ1, were cotransfected with CFP-N-Ras and YFP-Raf1-RBD and, 48 h later, stimulated as indicated. Colocalization of CFP-N-Ras (red) and YFP-Raf1-RBD (green) is shown in yellow. (B) Wild-type and PLCγ1-deficient Jurkat cells (2 × 10⁷ per point) were serum starved for 2 h and left untreated (NT) or incubated with 1 μg of anti-CD3 plus anti-CD28/ml (αCD3+αCD28) or PMA plus ionomycin (PMA+ION). Proteins from stimulated and unstimulated cells were used to collect and detect GTP-bound and total N-Ras as described in Materials and Methods.

Expression levels of the three Ras isoforms in different human cell lines. (A) Jurkat, Karpas, and CEM T cells and HEK293 epithelial cells were analyzed for Ras isoform levels by immunoblotting as described in Materials and Methods (left panels). Recombinant proteins for each Ras isoform were used as positive controls (right panels). (B) For Jurkat cells, total Ras proteins were also immunopurified (IP) by using an anti-pan-Ras immunoaffinity column, and Ras isoforms were detected by using antibodies specific for each isoform. As has been previously reported (), a doublet was detected for K-Ras and N-Ras.

N-Ras does not colocalize with TCR complexes in TCR-dependent activation of Jurkat cells. Jurkat cells were transfected with expression vectors encoding CD8-GFP, GFP-H-Ras, GFP-K-Ras, and GFP-N-Ras and were imaged alive 48 h later by LSM. Untreated, transfected Jurkat cells (NT) revealed the intrinsic steady-state localization of each fusion protein that included the plasma membrane and, in the case of GFP-N-Ras and GFP-H-Ras, the Golgi apparatus (arrowheads). Jurkat cells were incubated with anti-CD3 plus anti-CD28, and then TCR complexes were visualized by adding a Texas red-conjugated goat anti-mouse antibody. Jurkat T cells activated in this fashion showed a characteristic TCR patching revealed by red fluorescence. The overlay reveals areas of colocalization (yellow) between patched TCR (red) and GFP-tagged molecules (green).

N-Ras-specific activation in Jurkat cells following low-grade TCR stimulation. (A) Jurkat cells (2 × 10⁷ per point) were serum starved for 2 h and incubated with or without the indicated amounts of anti-CD3 plus anti-CD28. Proteins from stimulated and unstimulated cells were used to collect GTP-bound and total Ras as described in Materials and Methods. (B) To quantify Ras activation, GDP-bound and GTP-bound bands were quantified by using Quantity One software. The graph shows GTP/GDP ratios relative to the nontreated cells (NT). (C) Both N-Ras and K-Ras are equally activated by strong TCR-dependent and TCR-independent stimuli. Cells were kept untreated or activated with the indicated mitogens and processed as described for panel A. ION, ionomycin.

RasGRP1 mediates TCR-dependent activation of N-Ras on the Golgi apparatus of Jurkat cells. (A) Wild-type Jurkat cells were transfected with YFP-tagged RasGRP proteins. Forty-eight hours posttransfection, cells were serum-starved for 2 h and stimulated as described for Fig. . Panels show the localization of RasGRP1 to RasGRP4 proteins before (left) and after (right) TCR stimulation. (B) To assess the role of RasGRP1 and RasGRP3 in N-Ras activation on the Golgi apparatus, wild-type Jurkat cells were cotransfected with untagged expression vectors of each RasGRP protein, CFP-N-Ras, and YFP-RBD. Forty-eight hours later, cells were serum starved, and RBD redistribution was analyzed by LSM. Arrowheads indicate the Golgi apparatus. (C) Recruitment of endogenous RasGRP1 to the Golgi apparatus upon TCR engagement was analyzed by immunofluorescence. As described in Materials and Methods, endogenous RasGRP1 and giantin (a Golgi-specific marker) were detected in nontreated (NT) and TCR-stimulated (5 μg of anti-CD3/ml plus 5 μg of anti-CD28/ml) Jurkat T cells.

A single palmitoylation event in the C-terminal region of N-Ras is crucial for its specific role in TCR-dependent signaling. (A) Wild-type and mutant H-Ras and N-Ras protein sequences corresponding to the membrane targeting domain. Cysteine palmitoylation sites are underlined. (B) Jurkat cells were cotransfected with YFP-Raf1-RBD and either CFP-N-RasL184C or CFP-H-RasC184L mutants. Forty-eight hours later, cells were serum starved and either left untreated (NT) or activated with 1 μg of anti-CD3 plus anti-CD28/ml. Ras localization and RBD redistribution were analyzed in live cells by LSM and compared with those found for wild-type H-Ras and N-Ras isoforms (Fig. and Fig. , g to i). Arrowheads indicate the Golgi apparatus. (C) Jurkat cells were transfected by using Amaxa technology with either CFP-N-Ras, CFP-N-RasL184C, CFP-H-Ras, or CFP-H-RasC184L vectors. Forty-eight hours later, cells were serum starved and either left untreated (−) or activated with 1 μg of anti-CD3 plus anti-CD28/ml (+). ERK1/2-activated proteins were detected with phospho-specific antibodies. Values between the upper panels indicate ERK1/2 activation relative to the CFP-N-Ras transfected cells and were determined by quantification of phospho-ERK1/2 bands (P-ERK1/2) and subsequent normalization with total ERK1/2 (middle panel). The bottom panel shows the levels of CFP-Ras proteins (∼52 kDa) detected for each construct. WT, wild type.