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J. Biol. Chem., Vol. 283, Issue 26, 18323-18330, June 27, 2008

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

Genaro Patino-Lopez¹, Xiaoyun Dong¹, Khadija Ben-Aissa, Kelsie M. Bernot, Takashi Itoh^¶||, Mitsunori Fukuda^¶||, Michael J. Kruhlak, Lawrence E. Samelson, and Stephen Shaw²

From the Experimental Immunology Branch and Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, ^¶Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, and the ^||Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan

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.

Received for publication, January 3, 2008 , and in revised form, April 11, 2008.

^* This work was supported, in whole or in part, by the National Institutes of Health NCI Intramural Research Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S8 and a movie.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: National Institutes of Health, Bldg. 10, Rm. 4B36, 10 Center Dr., MSC 1360, Bethesda, MD 20892-1360. Tel.: 301-435-6499; Fax: 301-496-0887; E-mail: sshaw{at}nih.gov.

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