Abstract
Abstract 3683
Poster Board III-619
Rap1, a small GTPase of the Ras superfamily, originally identified by its ability to reverse Ras-mediated transformation, is now known to regulate cytoskeletal reorganization, cell morphology, adherens junction positioning and 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 in inside-out activation of integrins and cell adhesion. However, significant differences in the structure and interactions of these molecules indicate that they may mediate 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, multiple XPPPP motifs interacting with Profilin and multiple PXXP motifs capable of interacting with SH3 domain containing proteins. Because of these properties RIAM is a regulator of actin polymerization but also interacts with components of the T cell signaling machinery. In contrast, 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 signaling and functional events activated via the TCR. For this purpose we used RIAM-knockdown (KD) and RapL-KD Jurkat T cells in which endogenous RIAM and RapL, respectively, had been depleted by siRNA. 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 was unaltered in RIAM-KD cells but was abrogated in RapL-KD cells. Moreover, RIAM knockdown resulted in impaired activation of Ras and Rap1 due to defective activation of the calcium and diacylglycerole-dependent GEFs, RasGRP1 and CalDAG-GEFI. In contrast, RapL knockdown had no effect on these events compared to control Jurkat T cells. Strikingly, RIAM-KD cells displayed impaired IL-2 production in response to stimulation with SEE-loaded APC or to TCR/CD3-plus-CD28 crosslinking, whereas RapL-KD cells displayed a dramatic increase in IL-2 production upon stimulation under the same conditions. These results indicate that although both RIAM and RapL regulate Rap1-dependent LFA-1 activation, these molecules have distinct roles in regulating signaling and functional outcomes of T cell responses after T cell receptor triggering.
Disclosures:
No relevant conflicts of interest to declare.
The Small GTPase Rap1b: A Bidirectional Regulator of Platelet Adhesion Receptors