T cells are a central component of the adaptive immune response. They are activated through the recognition of ligands by their T-cell receptors (TCR) leading to proliferation; however, T cells also have a multitude of other receptors that mediate different functions, including chemotaxis and differentiation. One of the essential signaling pathways downstream of not only the TCR but also other receptors on the surface of T cells is the canonical mitogen-activated protein kinase (MAPK) cascade, composed of Raf (MAP3K), MEK (MAP2K), and ERK (MAPK) (22
). When activated, MAPK modules can generate both graded and digital outputs in vivo (11
) (Fig. ). In a graded system, the pathway transmits continuous information that is proportional to the input stimulus. In contrast, the all-or-none 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. These different signal outputs can be used to drive discrete cell fate decisions within a single cell, a principle that is strikingly illustrated in PC12 cells, in which a graded MAPK output drives cells to proliferate, whereas a digital MAPK output directs differentiation (37
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 (more ...)
The MAPK pathway is thought to regulate both positive and negative selection during T-cell development, with activation of the MAPK module from the Golgi correlating with positive selection and activation from the plasma membrane with negative selection (7
). Recent results have demonstrated that activation of the MAPK module from the Golgi generates a graded output, whereas ERK activation from the plasma membrane is digital (16
). In addition, mature T cells use a digital MAPK signal output downstream of TCR engagement to commit to T-cell activation (2
). Taken together, these results suggest that T cells utilize different system outputs from the MAPK module to regulate multiple biological responses in vivo. However, whether T cells can generate multiple outputs from the MAPK cascade has not been formally assessed.
It is currently unclear how diverse MAPK system dynamics, such as graded and digital outputs, as well as different sensitivity and durations of signaling, are generated within the same cell. The use of scaffold proteins as signal-processing hubs may provide a solution to this question. Scaffold proteins act as docking platforms that bind to two or more components of the MAPK module together in a protein complex (4
). There are at least two ways scaffolds can modulate the system output of MAPK cascades. First, scaffolds could set the sensitivity of the system by bringing the three kinases of the MAPK module into close proximity to increase the efficiency of signal transfer, a hypothesis supported by in silico modeling studies (24
). Second, scaffolds could change the fundamental system output of the MAPK module. In an elegant series of experiments, Lim and coworkers engineered scaffold-specific feedback loops by regulating recruitment of positive and negative regulators to the yeast MAPK scaffold protein Ste5 (3
). The resulting synthetic circuits yielded diverse MAPK outputs, providing the first critical proof-of-principle experiments that scaffold proteins themselves can be used to rewire MAPK modules to generate different outputs (3
). Recent data revealed that the yeast scaffold protein Ste5 converts the inherent switch-like signal output of the yeast mating MAPK module into a graded output, confirming that scaffolds are indeed used to modulate MAPK system output in vivo (42
). Since Ste5 is expressed only in yeast, it is important determine whether mammalian MAPK scaffolds perform a similar role given the lack of homology between Ste5 and mammalian scaffold proteins.
The best-characterized mammalian MAPK scaffold protein is Kinase Suppressor of Ras (KSR). It binds to Raf, MEK, and ERK to facilitate ERK activation at the plasma membrane (31
). Genetic and biochemical studies in nematodes, flies, and mammals confirm that KSR1 is essential for proper MAPK signal transmission in vivo (34
). KSR1 binds to protein phosphatase 2A (17
) and casein kinase 2 (35
), positive regulators of Raf activity (17
). KSR1 is also regulated by a positive feed-forward loop from Ras through IMP (28
). Thus, KSR1 coordinates multiple MAPK positive regulatory loops, placing KSR1 in a prime position to regulate MAPK system sensitivity, output, or both.
We examined here activation of the MAPK cascade in single T cells. We show that engagement of TCR with superantigen (SAg) results in a digital ERK response, whereas stimulation through a G-protein coupled receptor (GPCR), CXCR4, generates a graded ERK output, formally demonstrating that T cells generate multiple system outputs from the MAPK module. We then show that the MAPK scaffold KSR1 does not rewire the MAPK pathway to generate digital or graded outputs. Instead, the primary function of KSR1 is to modulate MAPK system sensitivity. Finally, we demonstrate that KSR1 protein levels are regulated during T-cell activation, revealing KSR1 as a likely control point for T-cell responsiveness. These findings have important implications for our understanding of T-cell regulation by the MAPK pathway in vivo.