Abstract
T cell receptor (TCR) ligation induces rapid polarization of the actin cytoskeleton resulting in the formation of the immunological synapse (IS), recruitment of signaling molecules, and initiation of signaling cascades leading to T cell activation. Specific recruitment, redistribution and organization of signaling molecules in the IS is facilitated by lipid raft microdomains, which provide a scaffold for focal protein assembly. Fyn and ZAP-70 are the most proximal TCR signaling molecules that localize in the IS and are redistributed in the lipid rafts during T cell activation. Currently, it is poorly understood how signals originating from the TCR are linked to specific mechanisms that regulate T cell activation. We have identified RIAM, an adaptor molecule that contains a RA (Ras Association) domain, a PH (Plekstrin Homology) domain and proline-rich motifs. RIAM interacts with active GTP-bound Rap1 and with regulators of the actin cytoskeleton Evl, VASP and Profilin. RIAM also interacts with ADAP/SKAP-55 and, thereby, is recruited to the plasma membrane during T cell activation. We have previously determined that, during TCR ligation by antigen, RIAM localizes at the IS and the lipid rafts and serves as a substrate for Fyn and ZAP-70. Because of these properties, we examined whether RIAM might be involved in regulating the molecular and functional outcome of T cell activation. Using RIAM-knock down (KD) T cells in which endogenous RIAM was depleted by siRNA, we determined that RIAM was necessary for IL-2 transcription and RIAM-KD cells had impaired capacity for IL-2 production in response to stimulation with SEE-loaded APC or to TCR/CD3-plus-CD28 crosslinking. However, despite the impaired IL-2 production, analysis of TCR-proximal signaling events did not show impairment of ZAP-70 phosphorylation or formation of the LAT signalosome comprised of phosphorylated PLC-γ1, SLP-76 and Vav1. TCR triggering of both control and RIAM-KD cells also resulted in similar phosphorylation of PLC-γ1. Activation of PLC-γ1 leads to the generation of InsP3 and diacylglycerol from phosphatidylinositol-4,5-bisphosphate (PtdIns (4,5)P2). InsP3 binds to InsP3 receptors and triggers Ca2+ release from intracellular stores. Strikingly, TCR triggering of RIAM-KD cells resulted in markedly reduced upregulation of InsP3 compared to that in control T cells. Consistent with the defective upregulation of InsP3, calcium flux of RIAM-KD cells was dramatically impaired. This event was due to the impaired InsP3-mediated calcium release from the endoplasmic reticulum and not due to impaired store content or impaired calcium release-activated calcium (CRAC) channel entry as determined by using the Ca2+ ATPase blocker thapsigargin, which resulted in abundant calcium release in RIAM-KD cells. To analyze the consequences of deregulated InsP3 production and to investigate whether RIAM is specifically involved in PLC-γ1-mediated processes we evaluated activation of several signaling events on which PLC-γ1 activation has distinct effects. Whereas activation of the extracellular signal regulated kinases MEK1/2 and Erk1/2 that are PLC-γ1 and Ca2+-dependent, was impaired in the absence of RIAM, activation of p38 and IKK was unaltered compared to control T cells. These results are consistent with a specific role of RIAM in InsP3-mediated Ca2+ release and indicate that deletion of RIAM does not result in a generalized defect in TCR-mediated signaling. Activation of PLC-γ1 at the cell membrane for proper generation of InsP3 requires appropriate docking and positioning of PLC-γ1. For this reason, we examined whether RIAM interacted with PLC-γ1 and regulated its subcellular localization after T cell activation. Detailed analysis by in vivo co-precipitation experiments in cell lysates and by in vitro association assays of purified proteins revealed a direct RIAM-PLC-γ1 interaction that was mediated via the SH3 domain of PLC-γ1. Furthermore, subcellular fractionation into cytoplasmic and cytoskeletal fractions revealed that PLC-γ1 translocated to the cytoskeleton upon T cell activation and this event was abrogated in RIAM-KD cells. These results indicate a novel and unexpected role of RIAM in T cell responses that involves regulation of spatio-temporal distribution and activation of PLC-γ1, leading to generation of InsP3 and Ca2+ mobilization after T cell receptor triggering.
Coordination of asparagine uptake and asparagine synthetase expression is required for T cell activation
