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ABSTRACT: Activation of Ras proteins underlies functional decisions in diverse cell types. Two molecules, Ras-GRP and SOS (Ras-guanine nucleotide-releasing protein and Son of Sevenless, respectively), catalyze Ras activation in lymphocytes. Binding of active Ras to the allosteric pocket of SOS markedly increases the activity of SOS. Thus, there is a positive feedback loop regulating SOS. Combining in silico and in vitro studies, we demonstrate that "digital" signaling in lymphocytes (cells are "on" or "off") is predicated on this allosteric regulation of SOS. The SOS feedback loop leads to hysteresis in the dose-response curve, which may enable T cells to exhibit "memory" of past encounters with antigen. Ras activation by Ras-GRP alone is "analog" (a graded increase in activation in response to an increase in the amplitude of the stimulus). We describe how the complementary analog (Ras-GRP) and digital (SOS) pathways act on Ras to efficiently convert analog input to digital output and make predictions regarding the importance of digital signaling in lymphocyte function and development.
Science Signaling 02/2009; 2(66):pt2. · 7.50 Impact Factor
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ABSTRACT: Activation of Ras proteins underlies functional decisions in diverse cell types. Two molecules, RasGRP and SOS, catalyze Ras activation in lymphocytes. Binding of active Ras to SOS' allosteric pocket markedly increases SOS' activity establishing a positive feedback loop for SOS-mediated Ras activation. Integrating in silico and in vitro studies, we demonstrate that digital signaling in lymphocytes (cells are "on" or "off") is predicated upon feedback regulation of SOS. SOS' feedback loop leads to hysteresis in the dose-response curve, which can enable a capacity to sustain Ras activation as stimuli are withdrawn and exhibit "memory" of past encounters with antigen. Ras activation via RasGRP alone is analog (graded increase in amplitude with stimulus). We describe how complementary analog (RasGRP) and digital (SOS) pathways act on Ras to efficiently convert analog input to digital output. Numerous predictions regarding the impact of our findings on lymphocyte function and development are noted.
Cell 02/2009; 136(2):337-51. · 32.40 Impact Factor
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ABSTRACT: Ras activation is crucial for lymphocyte development and effector function. Both T and B lymphocytes contain two types of Ras activators: ubiquitously expressed SOS and specifically expressed Ras guanyl nucleotide-releasing protein (RasGRP). The need for two activators is enigmatic since both are activated following antigen receptor stimulation. In addition, RasGRP1 appears to be dominant over SOS in an unknown manner. The crystal structure of SOS provides a clue: an unusual allosteric Ras-GTP binding pocket. Here, we demonstrate that RasGRP orchestrates Ras signaling in two ways: (i) by activating Ras directly and (ii) by facilitating priming of SOS with RasGTP that binds the allosteric pocket. Priming enhances SOS' in vivo activity and creates a positive RasGTP-SOS feedback loop that functions as a rheostat for Ras activity. Without RasGRP1, initiation of this loop is impaired because SOS' catalyst is its own product (RasGTP)-hence the dominance of RasGRP1. Introduction of an active Ras-like molecule (RasV12C40) in T- and B-cell lines can substitute for RasGRP function and enhance SOS' activity via its allosteric pocket. The unusual RasGRP-SOS interplay results in sensitive and robust Ras activation that cannot be achieved with either activator alone. We hypothesize that this mechanism enables lymphocytes to maximally respond to physiologically low levels of stimulation.
Molecular and Cellular Biology 05/2007; 27(7):2732-45. · 5.53 Impact Factor
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ABSTRACT: CD28 plays a critical role in T cell immune responses. Although the kinase Akt has been shown to act downstream of CD28 in T helper (Th)1 cytokine induction, it does not induce Th2 cytokines such as interleukin 4 (IL-4). We recently reported that phosphoinositide-dependent kinase 1 (PDK1) partially corrects the defect in IL-4 production present in CD28-deficient T cells, suggesting that PDK1 regulates IL-4 independently of Akt. We now describe a signaling pathway in which PDK1 targets IL-4 in the murine Th2 cell line D10. PDK1-mediated activation of this pathway is dependent on protein kinase A (PKA) and the nuclear factor of activated T cells (NFAT) P1 transcriptional element in the IL-4 promoter. PDK1 localizes to the immune synapse in a phosphatidylinositol 3-kinase-dependent manner, partially colocalizes with PKA at the synapse, and physically interacts with PKA. In RNA interference knockdown experiments, PDK1 is necessary for phosphorylation of PKA in T cells, as well as for activation of the IL-4 NFAT P1 element by the T cell receptor (TCR) and CD28. Phosphorylation of the critical PKA threonine residue is stimulated by engagement of TCR/CD28 via a PDK1-dependent mechanism. These findings together define a pathway linking the kinases PDK1 and PKA in the induction of the Th2 cytokine IL-4.
Journal of Experimental Medicine 08/2006; 203(7):1733-44. · 13.85 Impact Factor
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ABSTRACT: TRAIL receptor (TRAIL-R) signaling has been implicated in inducing apoptosis in tumor cells, but little is understood about its physiological function. Here, we report the generation and characterization of TRAIL-R(-/-) mice, which develop normal lymphocyte populations but possess enhanced innate immune responses. TRAIL-R(-/-) mice exhibited increased clearance of murine cytomegalovirus that correlated with increased levels of IL-12, IFN-alpha, and IFN-gamma. Stimulation of macrophages with Mycobacterium and Toll-like receptor (TLR)-2, -3, and -4, but not TLR9, ligands resulted in high levels of TRAIL upregulation and enhanced cytokine production in TRAIL-R(-/-) cells. The immediate-early TLR signaling events in TRAIL-R(-/-) macrophages and dendritic cells are normal, but I kappa B-alpha homeostatic regulation and NF-kappa B activity at later time points is perturbed. These data suggest that TRAIL-R negatively regulates innate immune responses.
Immunity 01/2005; 21(6):877-89. · 21.64 Impact Factor
