Translocation dynamics of sorting nexin 27 in activated T cells

Abstract

SummarySorting nexin 27 (SNX27) belongs to the sorting nexin family of proteins, which participate in vesicular and protein trafficking. Similarly to all sorting nexin proteins, SNX27 has a functional PX domain that is important for endosome binding, but it is the only sorting nexin with a PDZ domain. We identified SNX27 as a partner of diacylglycerol kinase z (DGKz), a negative regulator of T cell function that metabolises diacylglycerol to yield phosphatidic acid. SNX27 interacts with the DGKz PDZ-binding motif in early/recycling endosomes in resting T cells; however, the dynamics and mechanisms underlying SNX27 subcellular localisation during T cell activation are unknown. We demonstrate that in T cells that encounter pulsed antigen-presenting cells, SNX27 in transit on early/ recycling endosomes polarise to the immunological synapse. A fraction of SNX27 accumulates at the mature immunological synapse in a process that is dependent on vesicular trafficking, binding of the PX domain to phosphatidylinositol 3-phosphate and the presence of the PDZ region. Downmodulation of expression of either SNX27 or DGKz results in enhanced basal and antigen-triggered ERK phosphorylation. These results identify SNX27 as a PDZ-containing component of the T cell immunological synapse, and demonstrate a role for this protein in the regulation of the Ras-ERK pathway, suggesting a functional relationship between SNX27 and DGKz.

A role for novel lipid interactions in the dynamic recruitment of SNX27 to the T-cell immune synapse

“…Although initial recruitment of the mutant to the IS was similar compared to that of wild type SNX27, deficiency in lipid binding by the FERM domain impaired the subsequent retrieval of the mutant into endosomal compartments following IS maturation. These results correlate with previous studies showing that PM localization of SNX27 during IS formation is absolutely PDZ domaindependent, 4 and suggest that this additional lipid binding site with a preference for the bi-and triphosphorylated PtdIns derivatives known to be involved in endocytosis, 13 favors SNX27 recycling.…”

“…During IS assembly, we observed polarization of SNX27 enriched endosomal compartments and dynamic partitioning of SNX27 between the ERC and the plasma membrane (PM). 4 In contrast to what was observed in HeLa cells, endosomal localization of the SNX27-FERM PI-binding mutant was markedly altered in the Jurkat model. Diminished vesicle association was evident in basal conditions, and this phenotype was more pronounced in Jurkat cells challenged by superantigen-loaded APC.…”

“…[1][2][3] In other polarized models such as activated T cells, the PDZ domain mediates SNX27 interaction with diacylglycerol kinase zeta (DGKz), a negative regulator of T cell activation that attenuates diacylglycerol (DAG)-mediated signals by catalyzing DAG conversion into phosphatidic acid (PA). 4 In addition, SNX27 binds ubiquitously expressed transmembrane cargos such as glucose and metal ion transporters that have a class I PDZ binding motif (PDZ-bm). 5 With SNX17 and SNX31, SNX27 is classified as a SNX-FERM protein.…”

Sorting nexin 27 (SNX27) regulates the trafficking and activity of the glutamine transporter ASCT2

“…IRdye 680 and IRdye 800 conjugated fluorescence secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE, USA). The plasmids for EGFP tagged SNX27 or SNX27ΔPDZ (SNX27-GFP, SNX27ΔPDZ-GFP) were described previously (32) , and HA-ASCT2 plasmid is the gift from Professor Shao-Cong Sun (MD Anderson Cancer Center, The University of Texas) (33). Peptide for ASCT2 (ASEKESVM) was synthesized from GenScript (Piscataway, NJ).…”

Transient PKCα shuttling to the immunological synapse is governed by (DGK)ζ and regulates L-selectin shedding

“…RNA interference of DGKf and PKCa pSUPER plasmids (Oligoengine) containing the interfering 21 nucleotide sequence of DGKf human (shRNA DGKf) or mouse (shRNA DGKa, control) in a hairpin structure were generated in our laboratory (Avila-Flores et al, 2005;Rincón et al, 2011). siRNA oligonucleotides used to silence PKCa have been described (Lønne et al, 2010) and siRNA to silence DGKf corresponds to the same region as the shRNA to DGKf.…”