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.
    Full-text · Article · Sep 2013 · PLoS ONE
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    • "A second small GTPase necessary for efficient recycling of cargo from endosomes is Rab35. Once activated by its specific GEFs, the connecdenn/DENND1 family of proteins (Allaire et al., 2010;Marat and McPherson, 2010), Rab35 drives the recycling of a wide variety of cargo including MHC class I (Allaire et al., 2010) and MHC class II (Walseng et al., 2008), T-cell receptor (Patino-Lopez et al., 2008), the calcium activated potassium channel KCa 2+ 2.3 (Gao et al., 2010), exosomes () and synaptic vesicles (Uytterhoeven et al., 2011). Rab35 also functions during cell division to recycle lipid and protein components necessary for furrow ingression (Kouranti et al., 2006;Dambournet et al., 2011). "
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    ABSTRACT: Cells inversely adjust the plasma membrane levels of integrins and cadherins during cell migration and cell-cell adhesion but the regulatory mechanisms that coordinate these trafficking events remain unknown. Here, we demonstrate that the small GTPase Rab35 maintains cadherins at the cell surface to promote cell-cell adhesion. Simultaneously, Rab35 supresses the activity of the GTPase Arf6 to down regulate an Arf6-dependent recycling pathway for β1-integrin and EGF receptors, resulting in inhibition of cell migration and attenuation of signaling downstream of these receptors. Importantly, the phenotypes observed following Rab35 knock down are consistent with the epithelial-mesenchymal transition, a feature of invasive cancer cells, and we show that Rab35 expression is suppressed in a subset of cancers characterized by Arf6 hyperactivity. Our data thus identify a key molecular mechanism that efficiently coordinates the inverse intracellular sorting and cell surface levels of cadherin and integrin receptors for cell migration and differentiation.
    Preview · Article · Dec 2012 · Journal of Cell Science
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    • "To investigate the Rab35 dynamics during the early stage of phagosome formation, the internalization of IgG-opsonized erythrocytes (IgG-Es) was observed by time-lapse fluorescence microscopy in live RAW264 macrophages expressing GFP–Rab35. Before to the onset of phagocytosis, GFP–Rab35 was largely observed in the cytosol, and was also associated with the plasma membrane and some intracellular vesicles in some degree, as previously shown in other cell types (Kouranti et al., 2006; Patino-Lopez et al., 2008). After adding IgG-Es to the cells, more GFP–Rab35 appeared to be recruited to the sites of IgG-E binding. "
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    ABSTRACT: Phagosome formation and subsequent maturation are complex sequences of events that involve actin cytoskeleton remodeling and membrane trafficking. Here, we demonstrate that the Ras-related protein Rab35 is involved in the early stage of FcγR-mediated phagocytosis in macrophages. Live-cell image analysis revealed that Rab35 was markedly concentrated at the membrane where IgG-opsonized erythrocytes (IgG-Es) are bound. Rab35 silencing by RNA interference (RNAi) or the expression of GDP- or GTP-locked Rab35 mutant drastically reduced the rate of phagocytosis of IgG-Es. Actin-mediated pseudopod extension to form phagocytic cups was disturbed by the Rab35 silencing or the expression of GDP-Rab35, although initial actin assembly at the IgG-E binding sites was not inhibited. Furthermore, GTP-Rab35-dependent recruitment of ACAP2, an ARF6 GTPase-activating protein, was shown in the phagocytic cup formation. Concomitantly, overexpression of ACAP2 along with GTP-locked Rab35 showed a synergistic inhibitory effect on phagocytosis. It is likely that Rab35 regulates actin-dependent phagosome formation by recruiting ACAP2, which might control actin remodeling and membrane traffic through ARF6.
    Full-text · Article · Nov 2011 · Journal of Cell Science
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