Ras proteins are membrane-anchored GTPases cycling between active GTP-bound and inactive GDP-bound states. Stimulation of the T-cell or B-cell antigen receptors (TCR or BCR) results in very robust Ras activation, much higher than that observed in other cell types (reviewed in reference 18
). Optimal activation of Ras is essential for thymocyte and B-lymphocyte development and remains crucial in mature lymphocytes for effector functions: e.g., proliferation and cytokine production (18
). Activated H-Ras, N-Ras, and K-Ras function as signaling branch points that couple to various effector molecules. The best-studied effectors are phosphoinositide 3′ kinase (PI3K), Ral guanine nucleotide dissociation stimulator (RalGDS), and RAF (8
). In mature T cells, activation of Ras typically leads to recruitment and activation of RAF that is capable of inducing (via MEK and extracellular signal-regulated kinase [ERK] kinases) many cellular responses critical for lymphocyte function (12
). On the cell surface, the CD69 activation marker is induced following triggering of this pathway (18
The cycle of Ras activation is under the control of guanine nucleotide exchange factors (GEFs) and Ras GTPase-activating proteins (RasGAPs). RasGEFs bind to guanine nucleotide-free Ras molecules during a process that physically dissociates GDP from Ras. Empty Ras subsequently binds to GTP, available at excess levels in the cell (8
). RasGTPases themselves demonstrate very low intrinsic GTP hydrolysis activity, but this activity is greatly enhanced by RasGAPs (28
). Tumor formation as a consequence of loss of the RasGAP neurofibromin illustrates the importance of Ras inactivation (28
). Although the mechanisms of signal-mediated RasGAP activation are an active area of research, some RasGAPs are clearly at an intersection of signaling pathways: e.g., the RasGAPs CAPRI and RASAL regulate Ras activity in a calcium-dependent fashion (23
). As such, RasGAPs control the conversion of RasGTP to RasGDP.
Son of Sevenless 1
(SOS1 and -2, respectively) are ubiquitously expressed mammalian RasGEFs that are recruited to the membrane by the adapter Grb2 following growth factor or antigen receptor stimulation which can lead to activation of membrane-anchored Ras (6
). The N-terminal SH3 domain of Grb2 plays a crucial role in this process by binding to the C-terminal proline-rich region of SOS (6
), whereas the Grb2 SH2 domain properly localizes the complex to the membrane by binding to phosphorylated tyrosines on receptors or membrane-anchored adapters. Recently, Margarit et al. reported the crystal structure of the catalytic region of SOS complexed with Ras (26
). Surprisingly, three proteins were identified in a ternary Ras-SOS-RasGTP complex. In addition to a classical GEF pocket that binds guanine nucleotide free-Ras, SOS was found to have an allosteric pocket, distal to the GEF pocket, specific for GTP-bound Ras (26
). Loading this allosteric pocket with an active Ras molecule enhances the GEF activity of SOS in vitro (26
), suggesting a possible positive feedback loop: SOS generates RasGTP, RasGTP enhances SOS GEF activity, etc. This model raises the question as to how the initial priming RasGTP in the allosteric pocket is generated.
A second family of RasGEFs, Ras guanyl nucleotide-releasing protein (RasGRP) has gained much attention. In addition to the brain, RasGRP1 and -3 are expressed predominantly in T and B lymphocytes: there are high levels of RasGRP1 and low levels of RasGRP3 in T lymphocytes, the reverse from most B lymphocytes (1
). Thus, lymphocytes have potentially redundant means, via SOS or RasGRP, of activating Ras. In order to activate Ras, RasGRP1 and -3 need to be membrane recruited via diacylglycerol (DAG) and also to be phosphorylated by protein kinase C (PKC) family members that are likewise recruited to the membrane by DAG (1
). We have recently reported that stimulation of T lymphocytes with phorbol esters, DAG mimics, or via TCR engagement leads to phosphorylation of RasGRP1 and activation of Ras-ERK (32
). Phosphorylation of both RasGRP1 as well as ERK kinases was decreased by inhibitors, such as rottlerin, of DAG-responsive novel PKC family kinases (32
). Although TCR engagement also leads to membrane recruitment of SOS, these data suggested that the DAG-PKC-RasGRP1-Ras pathway in T lymphocytes may play a more important in role in TCR-triggered Ras-ERK activation than SOS (32
). Similar findings were reported for B lymphocytes (1
). RasGRP1-deficient thymocytes are developmentally arrested when Ras-ERK-dependent positive selection normally occurs (15
), suggesting a unique role for RasGRP1 in T-lymphocyte Ras activation that cannot be compensated for by SOS proteins.
Intrigued by the apparent dominant RasGRP function, we set out to address why lymphocytes require two distinct types of GEFs that are activated via independent biochemical mechanisms, yet are regulated by the same antigen receptor. Here we demonstrate that both GEFs contribute to sensitive and robust Ras activation in stimulated T and B lymphocytes via an unusual RasGRP-RasGTP-SOS interplay that critically relies on intact RasGRP function.