Rab35 and Its GAP EPI64C in T Cells Regulate Receptor Recycling and Immunological Synapse Formation

Experimental Immunology Branch and Laboratory of Cellular and Molecular Biology, NCI, NIH, Bethesda, MD 20892, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 07/2008; 283(26):18323-30. DOI: 10.1074/jbc.M800056200
Source: PubMed


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.

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    • "For Rab11a, the expression level is somewhat similar to Rab5a, with a 50% increase within 4-8 hours followed by a return to initial levels (Figure 3 B). As Arf6, Rab8a and Rab35 have also been shown to function in recycling [45,56,57], and the expression of these Rabs exhibit a similar pattern (Figure 2 B–D), this indicates that elevated expression of recycling Rabs might facilitate the transient increase in macropinocytosis and phagocytosis early after DC activation. "
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    ABSTRACT: The regulation of Rab expression to modulate cellular function has recently been proposed. Dendritic cells are a prototypic example of cells that drastically alter their function in response to environmental cues by reducing endocytosis, secreting cytokines, changing surface protein repertoires and altering morphology and migration. This is not a binary event, but is subject to fluctuations through the activation process, termed maturation. Consequently, DCs transiently increase endocytosis and production of major histocompatibility complex class II molecules, and secrete inflammatory cytokines in infected tissues before migrating to secondary lymph nodes and releasing T cell polarizing factors. All these cellular processes rely on intracellular membrane transport, which is regulated by Rab family GTPases and their diverse effectors. Here we examine how the Rabs likely to be involved in these functions are regulated throughout DC maturation. We find that Rab expression is altered upon lipopolysaccharide-induced activation, and discuss how this correlates to the reported functions of these cells during maturation.
    PLoS ONE 09/2013; 8(9):e73538. DOI:10.1371/journal.pone.0073538 · 3.23 Impact Factor
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    • "al., 2010). In immune cells, Rab35 is implicated in Tcell receptor recycling, immunological synapse formation (Patino-Lopez et al., 2008), and major histocompatibility complex (MHC) class II molecule recycling (Walseng et al., 2008). Connecdenn/DENND1A, a guanine nucleotide exchange factor (GEF) for Rab35, plays a role in synaptic vesicle endocytosis/recycling (Allaire et al., 2006) and cargo-specific exit from early endosomes (Allaire et al., 2010). "
    Biosensors - Emerging Materials and Applications, 07/2011; , ISBN: 978-953-307-328-6
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    • "Rab35, a recently characterized Rab functions in endosomal trafficking (Kouranti et al., 2006; Patino-Lopez et al., 2008; Sato et al., 2008; Walseng et al., 2008). Rab35 localizes to the PM and to internal vesicles and tubules and is required for endosomal secretion during immunological synapse formation and for stabilization and successful abscission of the cytokinesis bridge (Kouranti et al., 2006; Patino-Lopez et al., 2008). Additionally, Rab35 is linked to actin dynamics during neurite outgrowth and to actin bundling during bristle formation in Drosophila (Chevallier et al., 2009; Zhang et al., 2009). "
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    ABSTRACT: The DENN domain is an evolutionarily ancient protein module. Mutations in the DENN domain cause developmental defects in plants and human diseases, yet the function of this common module is unknown. We now demonstrate that the connecdenn/DENND1A DENN domain functions as a guanine nucleotide exchange factor (GEF) for Rab35 to regulate endosomal membrane trafficking. Loss of Rab35 activity causes an enlargement of early endosomes and inhibits MHC class I recycling. Moreover, it prevents early endosomal recruitment of EHD1, a common component of tubules involved in endosomal cargo recycling. Our data reveal an enzymatic activity for a DENN domain and demonstrate that distinct Rab GTPases can recruit a common protein machinery to various sites within the endosomal network to establish cargo-selective recycling pathways.
    Molecular cell 02/2010; 37(3):370-82. DOI:10.1016/j.molcel.2009.12.037 · 14.02 Impact Factor
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