KSR1 modulates the sensitivity of mitogen-activated protein kinase pathway activation in T cells without altering fundamental system outputs

Abstract

Mitogen-activated protein kinase (MAPK) cascades are evolutionarily conserved signaling pathways that regulate cell fate decisions. They generate a wide range of signal outputs, including graded and digital responses. In T cells, MAPK activation is digital in response to T-cell-receptor stimulation; however, whether other receptors on T cells that lead to MAPK activation are graded or digital is unknown. Here we evaluate MAPK activation in T cells at the single-cell level. We show that T cells responded digitally to stimulation with superantigen-loaded antigen-presenting cells, whereas they responded in a graded manner to the chemokine SDF-1, demonstrating that the system output of the MAPK module is highly plastic and determined by components upstream of the MAPK module. These findings also confirm that different MAPK system outputs are used by T cells to control discrete biological functions. Scaffold proteins are essential for proper MAPK signaling and function as they physically assemble multiple components and regulators of MAPK cascades. We found that the scaffold protein KSR1 regulated the threshold required for MAPK activation in T cells without affecting the nature of the response. We conclude that KSR1 plays a central role in determining the sensitivity of T-cell responses and is thus well positioned as a key control point.

FIG. 1.

Graded versus digital signaling. Hypothetical curves representing the relationship between input (stimuli) and output (pERK) at the single-cell level for graded compared to digital signaling. On the right of each curve are hypothetical flow-cytometric histograms depicting increasing pERK levels in relation to increasing stimuli for the two systems. In a graded system, the pathway transmits continuous information that is proportional to the input stimulus. In contrast, the all-or-nothing digital output can switch between two steady states but cannot rest in intermediate states, thereby functioning as a digital switch with the low and high steady states representing “off” and “on,” respectively (11). These different signal outputs can be used to drive discrete cell fate decisions within a single cell (37).

FIG. 2.

Activation of the MAPK pathway in Jurkat T cells is rapid and sustained. Jurkat T cells were stimulated with SEE-coated (1 μg/ml) APCs for the indicated time points. Cells were then fixed and stained for pERK and analyzed by flow cytometry. Samples were also stained with anti-human immunoglobulin to exclude APCs during analysis.

FIG. 3.

Jurkat T cells can generate both digital and graded responses. (A) SEE was serially diluted twofold starting at 250 ng/ml prior to incubation with APCs. Cells were then stimulated for 3 min, fixed, and stained with an anti-pERK monoclonal antibody and the appropriate secondary antibody, followed by analysis by flow cytometry. (B) Jurkat T cells were stimulated with twofold serially diluted concentrations of anti-TCR monoclonal antibody starting at a 1:500 dilution of ascitic fluid for 3 min and analyzed with an anti-pERK polyclonal antibody. (C) Jurkat T cells were stimulated with twofold serially diluted concentrations of SDF-1 starting at 12.5 nM for 3 min and analyzed as described for panel A. (D) Cells were stimulated with 2-fold serial dilutions starting at 25 ng of PMA/ml for 3 min and analyzed as described for panel A.

FIG. 4.

KSR1 knockdown increases the threshold required to activate ERK at a per-cell basis. (A) Control T cells or KSR1 knockdown cells were stimulated for 3 min with serially diluted SEE-coated APCs followed by staining with an anti-pERK monoclonal antibody and the appropriate secondary antibody. Cells were then analyzed by flow cytometry. Shown are fourfold serial dilutions starting at 250 ng of SEE/ml. (B) Control T cells or KSR1 knockdown cells were stimulated for 3 min with serially diluted SDF-1, followed by staining and analysis as described for panel A. Shown are twofold serial dilutions starting at 5 nM SDF-1. Inset numbers represent the mean fluorescence intensity. The panels on the right show the overlay of all of the stimulation points for the individual control and KSR1 knockdown cell lines. The lower panels show the overlay between control and KSR1 knockdown cells for each individual stimulation point.

FIG. 5.

Overexpression of KSR1 inhibits ERK activation. (A) Jurkat T cells were transfected with either a control GFP or KSR1-GFP fusion and, 18 h later, the cells were stimulated for 3 min with either SEE-coated (1 μg/ml) APCs or 1 μM SDF-1. Cells were then stained with anti-pERK and analyzed by flow cytometry. A narrow gate was drawn to show the cells at the inflection point of low to high pERK staining. Note that the gate is slightly shifted to the right in the SDF-1 stimulation to maintain approximately equal numbers of low and high cells in the histogram. (B) Control GFP or KSR1-GFP transfected cells were stimulated for 3 min with serially diluted SEE-coated APCs, followed by staining for pERK. Cells were then analyzed by flow cytometry. Shown are fourfold serial dilutions starting at 250 ng of SEE/ml. (C) Jurkat T cells were transfected with either a control GFP or KSR1-GFP fusion and, 18 h later, the cells were stimulated for 3 min with twofold serially diluted SDF-1 starting at 16 nM. Cells were then stained for pERK and analyzed by flow cytometry.

FIG. 6.

KSR1 inhibition requires MEK binding. (A) Jurkat T cells were transfected with vector control, wild-type (wt) KSR1-FLAG, or KSR (C809Y)-FLAG and then left unstimulated or stimulated for 3 min with anti-TCR. Cells were then lysed and immunoprecipitated with an anti-FLAG monoclonal antibody. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by immunoblotting with anti-FLAG and anti-MEK1 antibodies. (B) Jurkat T cells were transfected with control GFP, KSR1-IRES-GFP, or KSR (C809Y)-IRES-GFP. After 18 h, the cells were stimulated for 3 min, as before, with SEE-coated APC (1 μg/ml). Cells were fixed and stained as described above. (C) Jurkat T cells were transfected with control GFP or a dominant-negative MEK (DN-MEK). Cells were then stimulated with SEE-coated APC (1 μg/ml), fixed, and stained as described above.

FIG. 7.

KSR1-deficient primary T cells fail to activate ERK in response to TCR or SDF-1. (A) Primary CD4⁺ T cells were isolated from wild-type (wt) or KSR1 knockout mice to >85% purity and stimulated with either an anti-TCR antibody or 100 nM SDF-1 for 3 min. Cells were then lysed, and ERK activation was measured by pERK blotting. (B) Chemotaxis of purified primary CD4⁺ T cells from either wild-type (wt) or KSR1 knockout mice was measured by a Transwell migration assay. The migration index is a measure of the number of cells that migrated into the bottom chamber, in the presence of the indicated SDF-1 concentration, divided by the number of cells that migrated with no chemokine. The bars represent the averages, and the error bars indicate the standard deviations of samples evaluated in triplicate.

FIG. 8.

KSR1 levels are regulated in primary T cells after stimulation. Primary CD4⁺ T cells were isolated from wt mice and stimulated with plate-bound anti-CD3 and anti-CD28 antibody for 5 days. Cells were then lysed, and proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. KSR1, c-Raf, MEK1, and ERK2 were visualized by immunoblotting with their respective antibodies.