title: Proteomics identification of sorting nexin 27 as a diacylglycerol kinase zeta-associated protein: new diacylglycerol kinase roles in endocytic recycling.
abstract: Diacylglycerol kinase zeta 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 zeta 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 zeta 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 zeta-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 zeta. Because diacylglycerol kinase zeta 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 zeta regulates SNX27 localization. Analysis of cells with RNA interference-mediated knockdown of diacylglycerol kinase zeta showed accelerated transferrin receptor exit from the lymphocyte endocytic recycling compartment back to the plasma membrane, further confirming diacylglycerol kinase zeta-dependent control of vesicle trafficking. These data support a previously unreported role for diacylglycerol kinase zeta in the modulation of membrane trafficking, which may also help to define SNX27 function.
authors.affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, E-28049 Madrid, Spain.
authors.forename: Esther
authors.initials: E
authors.lastname: Rincón
authors.forename: Teresa
authors.initials: T
authors.lastname: Santos
authors.forename: Antonia
authors.initials: A
authors.lastname: Avila-Flores
authors.forename: Juan P
authors.initials: JP
authors.lastname: Albar
authors.forename: Vasiliki
authors.initials: V
authors.lastname: Lalioti
authors.forename: Cai
authors.initials: C
authors.lastname: Lei
authors.forename: Wanjin
authors.initials: W
authors.lastname: Hong
authors.forename: Isabel
authors.initials: I
authors.lastname: Mérida
mesh_terms: D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D019852:Diacylglycerol Kinase / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D006413:Hematopoietic System / Q000166:cytology / Q000187:drug effects; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D040901:Proteomics; D051381:Rats; D011990:Receptors, Transferrin / Q000378:metabolism; D058305:Sorting Nexins; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D002352:Carrier Proteins; D011990:Receptors, Transferrin; C522885:SNX27 protein, human; C500393:Snx27 protein, mouse; D058305:Sorting Nexins; D033921:Vesicular Transport Proteins; D019852:Diacylglycerol Kinase; D000077191:Wortmannin