RIAM and RapL, Two Structurally Divergent Rap1 Effectors, Have Distinct Signaling and Functional Roles in TCR-Mediated Activation

Abstract 1118

Rap1, a small GTPase of the Ras superfamily, is a regulator of cytoskeletal reorganization, adherens junctions positioning and cell adhesion. The best-studied function of Rap1 is inside-out activation of integrins and cell adhesion. Two newly identified Rap1 effectors, RapL and RIAM, have been implicated in Rap1-mediated inside-out activation of integrins and cell adhesion. However, significant differences in the structure of these molecules indicate that they may regulate distinct signaling events. RIAM has a N-terminal coiled-coil region, central RA and pleckstrin homology (PH) domains, and proline-rich N-terminal and C-terminal regions, with multiple FPPPP motifs capable of interacting with the EVH1 domains of the actin regulatory proteins Ena/VASP and multiple XPPPP motifs interacting with Profilin. RIAM interacts with PLC-γ1 and regulates PLC-γ1 spatiotemporal distribution and activation during TCR-mediated stimulation. RapL has an RBD (Ras binding domain-structurally similar to RA domain) and a C-terminal coiled-coil region and interacts with Rap1 via its RBD domain and its N-terminal region and with the aL subunit of LFA-1 via its C-terminal domain. In the present study we investigated the role of RIAM and RapL in regulating TCR-mediated signaling and IL-2 production. We generated RIAM-knockdown (KD) and RapL-KD Jurkat T cells in which endogenous RIAM and RapL, respectively, were depleted by shRNA. Whereas activation of the extracellular signal regulated kinases MEK1/2 and Erk1/2 was impaired by depletion of RIAM, activation of these kinases was unaffected by depletion of RapL. In contrast, activation of p38 and JNK was unaltered in RIAM-KD cells but was abrogated in RapL-KD cells. RIAM knockdown resulted in impaired activation of PLC-γ1, leading to impaired calcium release and activation of calcium and diacylglycerol-dependent GEFs, RasGRP1 and CalDAG-GEFI, thereby inhibiting activation of Ras and Rap1 and abrogating IL-2 production. In contrast, RapL knockdown did not affect PLC-γ1 activation but resulted in dramatic increase in IL-2 production. Because activation of p38 and JNK has been associated with suppression of IL-2, we examined whether inhibition of these pathways in RapL-KD cells might have a causative role in the increased IL-2 production. Forced expression of enzymatically active p38, JNK, or their combination suppressed IL-2 transcription and protein production to the levels of control T cells. To examine whether these two distinct Rap1 effectors display signal hierarchy or independently mediate their opposing effects on MAPKs and IL-2 transcription, we examined whether elimination of RapL in RIAM-KD T cells might restore their ability to produce IL-2. Elimination of RapL did not restore the ability of RIAM-KD cells to produce IL-2. Taken together our results indicate that although RIAM and RapL can mediate LFA-1 activation downstream of Rap1, these Rap1 effectors regulate distinct signaling events and have opposing roles on IL-2 production upon TCR triggering. Moreover, RIAM-mediated signaling is a pre-requisite for RapL to exert its inhibitory effect on IL-2 production. These results indicate that the selective and temporally regulated interaction of Rap1 with these two distinct effectors might have significant implications on the outcome of TCR triggering by inducing initial activation and subsequent suppression of IL-2 production.