Rab35 and its GAP EPI64C in T cells regulate receptor recycling and immunological synapse formation.
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.
Patino-Lopez
Genaro
G
Experimental Immunology Branch and Laboratory of Cellular and Molecular Biology, NCI, NIH, Bethesda, MD 20892, USA.
Dong
Xiaoyun
X
Ben-Aissa
Khadija
K
Bernot
Kelsie M
KM
Itoh
Takashi
T
Fukuda
Mitsunori
M
Kruhlak
Michael J
MJ
Samelson
Lawrence E
LE
Shaw
Stephen
S
D011992:Endosomes / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry*; D005786:Gene Expression Regulation; D006801:Humans; D007107:Immune System; D019169:Jurkat Cells; D018613:Microscopy, Confocal; D008954:Models, Biological; D034622:RNA Interference; D015398:Signal Transduction; D013569:Synapses / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism*; D014162:Transfection; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000502:physiology*
D020690:GTPase-Activating Proteins; C425805:TBC1D10A protein, human; C511183:RAB35 protein, human; D020691:rab GTP-Binding Proteins