Upon antigen recognition, T-cell receptor (TCR/CD3) and other signaling molecules become enriched in a specialized contact site between the T cell and antigen-presenting cell, i.e. the immunological synapse (IS). Enrichment occurs via mechanisms that include polarized secretion from recycling endosomes, but the Rabs and RabGAPs that regulate this are unknown. EPI64C (TBC1D10C) is an uncharacterized candidate RabGAP we identified by mass spectrometry as abundant in human peripheral blood T cells that is preferentially expressed in hematopoietic cells. EPI64C is a Rab35-GAP based both on in vitro Rab35-specific GAP activity and findings in transfection assays. EPI64C and Rab35 dominant negative (DN) constructs each impaired transferrin export from a recycling pathway in Jurkat T-cells and induced large vacuoles marked by transferrin receptor, TCR, and SNAREs implicated in TCR-polarized secretion. Rab35 localized to the plasma membrane and to intracellular vesicles where it substantially colocalized with TfR and with TCR. Rab35 was strongly recruited to the IS. Conjugate formation was impaired by transfection with Rab35-DN or EPI64C and by EPI64C knock down. TCR enrichment at the IS was impaired by Rab35-DN. Thus, EPI64C and Rab35 regulate a recycling pathway in T cells and contribute to IS formation, most likely by participating in TCR transport to the IS. The immunological synapse (IS)3 is a specialized contact between T lymphocytes and antigen-presenting cells (APC) into which accumulate T-cell antigen receptor (TCR), coreceptors, and signaling and cytoskeletal components (1-7). Enrichment of proteins in the IS occurs in part by diffusion and retention at the contact region (i.e."mutual co-capping") (8, 9). Furthermore, submembranous flow of the actomyosin cortex contributes to molecular concentration in the IS (10, 11). Recent data indicate that polarized exocytosis from a rapid recycling pathway is particularly important for the enrichment of some synapse components, including TCR (12-16).Rab proteins are major intracellular transport regulators in eukaryotes; they function in vesicle formation, motility, docking, and fusion (17, 18). Over 60 mammalian Rab proteins have been identified, and each is thought to regulate distinct intracellular transport steps through its temporal and spatial association with various interacting proteins. Guanine exchange factors and GTPase-activating proteins (GAPs) control the switch between active GTP-bound and inactive GDP-bound Rab proteins. Although the TBC (Tre/Bub2/Cdc16) domain is a hallmark of RabGAPs (19), few of the more than 50 putative TBC domain-containing proteins present in the human genome have been paired with their target Rab. One recent notable success was identification of EPI64 (EBP50-PDZ interactor of 64kD) as a GAP specific for Rab27a (20).EPI64 is a broadly expressed TBC domain-containing protein first identified in placental microvilli (21). Recent studies have implicated it in regulating microvillus architecture (22). EPI64 has two paralogs in mouse and...

The proteins STIM1 and Orai1 are the long sought components of the store-operated channels required in T-cell activation. However, little is known about the interaction of these proteins in T-cells after engagement of the T-cell receptor. We found that T-cell receptor engagement caused STIM1 and Orai1 to colocalize in puncta near the site of stimulation and accumulate in a dense structure on the opposite side of the T-cell. FRET measurements showed a close interaction between STIM1 and Orai1 both in the puncta and in the dense cap-like structure. The formation of cap-like structures did not entail rearrangement of the entire endoplasmic reticulum. Cap formation depended on TCR engagement and tyrosine phosphorylation, but not on channel activity or Ca 2؉ influx. These caps were very dynamic in T-cells activated by contact with superantigen pulsed B-cells and could move from the distal pole to an existing or a newly forming immunological synapse. One function of this cap may be to provide preassembled Ca 2؉ channel components to existing and newly forming immunological synapses. INTRODUCTIONT-cell activation in response to antigen induces and requires the elevation of intracellular Ca 2ϩ (Hogan et al., 2003;Quintana et al., 2005;Feske, 2007). T-cell receptor (TCR) engagement leads to activation of well-documented signal transduction pathways that cause a rapid release of Ca 2ϩ from the endoplasmic reticulum (ER; Samelson, 2002;Panyi et al., 2004;Gwack et al., 2007a). The depletion of Ca 2ϩ from this internal store then leads to the opening of ion channels in the plasma membrane (PM) allowing an influx of Ca 2ϩ from outside the cell. The store-operated channel responsible for Ca 2ϩ influx in T-cells is known as the Ca 2ϩ release-activated Ca 2ϩ (CRAC) channel, which has been studied by electrophysiological techniques for many years (Lewis, 2001;Prakriya and Lewis, 2003;Cahalan et al., 2007;Hewavitharana et al., 2007;Hogan and Rao, 2007;Putney, 2007a).Sustained elevation of cytosolic Ca 2ϩ is required for complete T-cell activation (Iezzi et al., 1998;Lewis, 2001;Hogan et al., 2003;Feske, 2007). High levels of intracellular Ca 2ϩ are necessary to maintain the interaction between a T-cell and antigen-presenting cell (APC) that leads to formation of the specialized contact surface known as the immunological synapse (IS). Increased Ca 2ϩ levels are also required for the activation of transcription factors. In particular, elevated Ca 2ϩ maintains prolonged nuclear accumulation of nuclear factor of activated T-cells (NFAT) by activating the phosphatase calcineurin that dephosphorylates NFAT, allowing it to translocate to the nucleus and activate genes such as IL2 (Hogan et al., 2003;Macian, 2005). Several hours of Ca 2ϩ influx are required to complete the T-cell activation program, which involves expression of a large number of activation-associated genes (Lewis, 2001;Macian et al., 2002;Hogan et al., 2003).Although sustained Ca 2ϩ influx in T-cells has been studied for decades, the proteins involved have only been ident...

T-cell antigen receptor engagement causes the rapid assembly of signaling complexes. The adapter protein SLP-76, detected as SLP-yellow fluorescent protein, initially clustered with the TCR and other proteins, then translocated medially on microtubules. As shown by total internal reflection fluorescence microscopy and the inhibition of SLP-76 movement at 168C, this movement required endocytosis. Immunoelectron microscopy showed SLP-76 staining of smooth pits and tubules. Cholesterol depletion decreased the movement of SLP-76 clusters, as did coexpression of the ubiquitin-interacting motif domain from eps15. These data are consistent with the internalization of SLP-76 via a lipid raft-dependent pathway that requires interaction of the endocytic machinery with ubiquitinylated proteins. The endocytosed SLP-76 clusters contained phosphorylated SLP-76 and phosphorylated LAT. The raft-associated, transmembrane protein LAT likely targets SLP-76 to endocytic vesicles. The endocytosis of active SLP-76 and LAT complexes suggests a possible mechanism for downregulation of signaling complexes induced by TCR activation.