Engagement of immune recognition subunits on lymphocytes results in cytoskeletal reorganization, polarization of polymerized actin, conjugate formation between T cells and APC, stabilization of the immunological synapse (IS), and initiation of signaling cascades leading to T cell activation. Actin remodeling is essential for these events and is mandatory for T cell activation. We have identified RIAM, a novel adaptor molecule that interacts specifically with active GTP-bound Rap1 and with regulators of the actin cytoskeleton Evl, VASP and Profilin. Profilin associates with G-actin and promotes actin polymerization by adding actin monomers to the barbed ends of F-actin. Ena/VASP family proteins are cytoskeletal proteins and regulate actin dynamics. Via these interactions, RIAM functions as a regulator of the actin cytoskeleton. RIAM also has a mandatory role in regulating activation of integrins downstream of Rap1. The ability of a T cell to recognize and conjugate with an APC is dependent on cytoskeletal dynamics and “inside out” signaling leading to integrin-mediated adhesion. Activation of the b2 integrin LFA-1 leads to high avidity binding to ICAM proteins, which is crucial for stable T cell-APC conjugation. Because RIAM is involved both in cytoskeletal dynamics and integrin activation, we examined whether RIAM had a role in this process. Stable GFP-transfected Jurkat T cells were incubated with Raji B cells as APC, in the presence or absence of SEE. T cell:APC conjugates and formation of IS were examined by confocal microscopy. Synapse formation between GFP-Jurkat cells and Raji B cells was detected only in the presence of SEE. Under these conditions, polymerized (F) actin was highly recruited at the IS, as determined by phalloidin staining. Staining with RIAM-specific antibody indicated that in the absence of antigen, RIAM was diffusely expressed in the cytoplasm and at the plasma membrane. Impressively, upon incubation with SEE loaded Raji cells, RIAM was redistributed at the IS where it co-localized with polymerized (F) actin and ZAP-70. Knockdown of RIAM in Jurkat T cells resulted in significant defects in conjugate formation with SEE loaded B cells. Thus RIAM is involved in actin reorganization in the IS and in the formation of T cell:APC conjugates. Because appropriate actin reorganization is required for T cell activation we next explored whether RIAM knockdown leads to defects in TCR-mediated signaling events. RIAM-knockdown Jurkat T cells displayed significantly impaired IL-2 production in response to SEE loaded Raji and in decreased IL-2 promoter driven transcriptional activity. Surprisingly, despite the significant decrease in IL-2 transcription, RIAM-knockdown Jurkat cells did not show altered TCR-induced phosphorylation of PLCg1, or activation of MAP kinases including MEK, Erk1/2 and JNK, compared to control cells. Moreover, AP-1 and NF-kB-mediated gene transcription induced by CD3-plus-CD28 was unaffected. In contrast, NFAT-driven transcription was significantly impaired. Assessment of intracellular calcium by flow cytometry indicated that RIAM-knockdown cells had significantly reduced TCR-mediated intracellular calcium signal. These results indicate that RIAM is an important component of a signaling pathway that regulates calcium mobilization and NFAT mediated gene transcription in T lymphocytes.

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