Proteomics Identification of Sorting Nexin 27 as a Diacylglycerol Kinase ζ-associated Protein

Abstract

Diacylglycerol kinase is a member of the diacylglycerol kinase family of enzymes, which generate phosphatidic acid through diacylglycerol phosphorylation. In addition to the catalytic and cysteine-rich domains found in all diacylglycerol kinases, diacylglycerol kinase has a MARCKS domain as well as a C-terminal region containing four ankyrin repeats and a PDZ-binding motif. Previous reports demonstrated that diacylglycerol kinase interaction with several proteins is an important mechanism for modulating the localization and activity of this enzyme. Here we used a proteomics approach to search for novel diacylglycerol kinase -interacting proteins and identified sorting nexin 27 (SNX27), a recently described member of a protein family involved in intracellular trafficking, which has a PDZ domain in addition to the phox homology domain characteristic of SNX proteins. Co-immunoprecipitation studies and two-hybrid analysis confirmed physical, PDZ-dependent association between SNX27 and diacylglycerol kinase . Because diacylglycerol kinase is expressed abundantly in T lymphocytes, we characterized SNX27 expression and subcellular localization in these cells. SNX27 co-localized with transferrin receptor-positive vesicles, pointing to its participation in T cell endocytic recycling. Expression of deletion mutants revealed that in addition to the phox homology domain the SNX27 PDZ domain contributed to vesicle localization of this protein, suggesting that interaction with diacylglycerol kinase regulates SNX27 localization. Analysis of cells with RNA interference-mediated knockdown of diacylglycerol kinase showed accelerated transferrin receptor exit from the lymphocyte endocytic recycling compartment back to the plasma membrane, further confirming diacylglycerol kinase -dependent control of vesicle trafficking. These data support a previously unreported role for diacylglycerol kinase in the modulation of membrane trafficking, which may also help to define SNX27 function.

Translocation dynamics of sorting nexin 27 in activated T cells

“…We showed colocalisation of DGKz and SXN27 at microsomal compartments, which suggests a role for PDZ interaction at this site (Rincon et al, 2007). The characterisation of a SNX27-DGKz complex provided a putative role for DGKz as a negative regulator of recycling endosomal compartments, as DGKz knockdown cells showed enhanced transferrin receptor recycling (Rincon et al, 2007).…”

“…Over 33 mammalian sorting nexins have been identified and are thought to regulate distinct steps in vesicular trafficking, generally through spatial association with lipid or protein partners (Carlton et al, 2005;Cullen, 2008;Worby and Dixon, 2002). We recently identified SNX27 in early and recycling endosomes in T cells (Rincon et al, 2007). This protein has a specific phox homology (PX) region that is the signature of all sorting nexin family members (Worby and Dixon, 2002).…”

“…Our previous experiments identified SNX27 as a protein that interacts with diacylglycerol kinase z (DGKz); this interaction is dependent on the SNX27 PDZ domain (Rincon et al, 2007). DGKz is a member of the extensive DGK family of enzymes that metabolise diacylglycerol (DAG) into phosphatidic acid.…”

Diacylglycerol kinases in membrane trafficking

“…DGKz-siRNA accelerated the recycling of transferrin receptor from the endocytic recycling compartment to the plasma membrane in T lymphocytes. 52 Although the reason why DGKzknock-down in T lymphocytes has such a dramatically different impact than DGKa-knock-down in other cell types remains unknown, it is possible that compensation by other DGKs and/ or other PA-generating pathways in T cells is more robust.…”

“…The mechanisms by which DAG regulates membrane trafficking are diverse: 1) serving as a second messenger to activate PKC, PKD, and downstream signaling cascades; [44][45][46] 2) involvement in PI cycling regulation of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) abundance, inositol 1,4,5-trisphosphate (IP 3 ) 47 and subsequent Ca 2C influx. 48 On the other hand, the local concentration of PA is an important regulator of trafficking, possibly because: 1) PA-enriched membranes with higher curvature tend to undergo fission; 49 2) it serves as a docking site for recruiting specific proteins such as Rab coupling proteins (RCP), 50 Molecules Interacting withCAsL-Like1 (MICAL-L1), 51 and Sorting nexin 27 (SNX27) 52 to the membrane; 3) it is an intermediate for PtdIns re-synthesis. 24 Given the increasing awareness of the relevance of lipid metabolites such as DAG and PA in membrane trafficking, understanding the significance of DGK function in regulating the fine balance of DAG-to-PA levels is becoming an increasingly important research goal.…”