Antonia Ávila-Flores

Modulation of the Mammalian Target of Rapamycin Pathway by Diacylglycerol Kinase-produced Phosphatidic Acid

The protein known as mammalian target of rapamycin (mTOR) regulates cell growth by integrating different stimuli, such as available nutrients and mitogenic factors. The lipid messenger phosphatidic acid (PA) binds and positively regulates the mitogenic response of mTOR. PA generator enzymes are consequently potential regulators of mTOR. Here we explored the contribution to this pathway of the enzyme diacylglycerol kinase (DGK), which produces PA through phosphorylation of diacylglycerol. We found that overexpression of the DGKζ, but not of the α isoform, in serum-deprived HEK293 cells induced mTOR-dependent phosphorylation of p70S6 kinase (p70S6K). After serum addition, p70S6K phosphorylation was higher and more resistant to rapamycin treatment in cells overexpressing DGKζ. The effect of this DGK isoform on p70S6K hyperphosphorylation required the mTOR PA binding region. Down-regulation of endogenous DGKζ by small interfering RNA in HEK293 cells diminished serum-induced p70S6K phosphorylation, highlighting the role of this isoform in the mTOR pathway. Our results confirm a role for PA in mTOR regulation and describe a novel pathway in which DGKζ-derived PA acts as a mediator of mTOR signaling.

Proteomics Identification of Sorting Nexin 27 as a Diacylglycerol Kinase ζ-associated Protein New Diacylglycerol Kinase Roles in Endocytic Recycling

Diacylglycerol kinase ζ 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 ζ 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 ζ 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 ζ-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 ζ. Because diacylglycerol kinase ζ 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 ζ regulates SNX27 localization. Analysis of cells with RNA interference-mediated knockdown of diacylglycerol kinase ζ showed accelerated transferrin receptor exit from the lymphocyte endocytic recycling compartment back to the plasma membrane, further confirming diacylglycerol kinase ζ-dependent control of vesicle trafficking. These data support a previously unreported role for diacylglycerol kinase ζ in the modulation of membrane trafficking, which may also help to define SNX27 function.

Diacylglycerol kinase ζ controls diacylglycerol metabolism at the immunological synapse.

DGKα and DGKζ negatively regulate the DAG/RasGRP1/Ras pathway in T cells. Study of the specific contribution of each isoform to DAG metabolism during immune synapse formation by use of a combination of RNAi and videomicroscopy techniques identifies DGKζ as mainly responsible for DAG consumption at the immunological synapse.

Lck-dependent tyrosine phosphorylation of diacylglycerol kinase alpha regulates its membrane association in T cells.

TCR engagement triggers phospholipase Cγ1 activation through the Lck-ZAP70-linker of activated T cell adaptor protein pathway. This leads to generation of diacylglycerol (DAG) and mobilization of intracellular Ca2+, both essential for TCR-dependent transcriptional responses. TCR ligation also elicits transient recruitment of DAG kinase α (DGKα) to the lymphocyte plasma membrane to phosphorylate DAG, facilitating termination of DAG-regulated signals. The precise mechanisms governing dynamic recruitment of DGKα to the membrane have not been fully elucidated, although Ca2+ influx and tyrosine kinase activation were proposed to be required. We show that DGKα is tyrosine phosphorylated, and identify tyrosine 335 (Y335), at the hinge between the atypical C1 domains and the catalytic region, as essential for membrane localization. Generation of an Ab that recognizes phosphorylated Y335 demonstrates Lck-dependent phosphorylation of endogenous DGKα during TCR activation and shows that pY335DGKα is a minor pool located exclusively at the plasma membrane. Our results identify Y335 as a residue critical for DGKα function and suggest a mechanism by which Lck-dependent phosphorylation and Ca2+ elevation regulate DGKα membrane localization. The concerted action of these two signals results in transient, receptor-regulated DGKα relocalization to the site at which it exerts its function as a negative modulator of DAG-dependent signals.

Redundant and specialized roles for diacylglycerol kinases α and ζ in the control of T cell functions

Two isoforms of the enzyme that attenuates signaling by the second messenger diacylglycerol regulate multiple properties of T lymphocytes. Gloss T lymphocytes patrol the body continuously, and they use antigen-specific T cell receptors (TCRs) to scan and detect foreign peptides on the surface of infected or cancerous cells. When the TCR recognizes a foreign antigen, it sends signals that activate the T lymphocytes, enabling them to help other immune cells or to directly kill those cells that are infected or have become cancerous. T lymphocytes must discriminate correctly between self and foreign antigens because any failure to do so could lead them to destroy healthy cells, as occurs in autoimmune diseases, or to allow tumors to grow. The diacylglycerol kinases (DGKs) are enzymes that inhibit some of the signals needed for T cell activation; by characterizing them, we can better understand the mechanisms by which T lymphocytes distinguish between self and foreign antigens. This Review, which contains 5 figures and 105 references, highlights how the study of DGKs and the signals that they regulate is important because it will help scientists learn how to manipulate T cell responses for the treatment of diseases. The diacylglycerol kinases (DGKs) attenuate diacylglycerol (DAG)–mediated signals by catalyzing the conversion of DAG to phosphatidic acid. In T lymphocytes, the antigen-stimulated generation of DAG links signal strength to the intensity and duration of signaling by the Ras–extracellular signal–regulated kinase (ERK) and protein kinase C (PKC)–dependent pathways. The generation of DAG at the plasma membrane of T cells lies at the core of the mechanisms that delimit T cell functions. DGKα and DGKζ are the two main isoforms that are found in T cells, and several approaches define their precise contribution to T cell responses. Each of these isoforms has specialized and redundant functions that limit the intensity of DAG-regulated signals downstream of antigenic stimulation. This ability, which in normal T cells contributes to maintaining homeostasis and function, is exploited by tumors to evade immune surveillance. Modification of DGK activity offers new perspectives for the therapeutic manipulation of T cell functions for treatment of autoimmune pathologies, or for overcoming tumor-induced T cell tolerance. Precise knowledge of the mechanisms that sustain DGK isoform–specific regulation in T lymphocytes is indispensable for the development of new tools for pharmacological intervention.

FoxO-dependent regulation of diacylglycerol kinase α gene expression.

ABSTRACT Diacylglycerol kinase α (DGKα) regulates diacylglycerol levels, catalyzing its conversion into phosphatidic acid. The α isoform is central to immune response regulation; it downmodulates Ras-dependent pathways and is necessary for establishment of the unresponsive state termed anergy. DGKα functions are regulated in part at the transcriptional level although the mechanisms involved remain poorly understood. Here, we analyzed the 5′ end structure of the mouse DGKα gene and detected three binding sites for forkhead box O (FoxO) transcription factors, whose function was confirmed using luciferase reporter constructs. FoxO1 and FoxO3 bound to the 5′ regulatory region of DGKα in quiescent T cells, as well as after interleukin-2 (IL-2) withdrawal in activated T cells. FoxO binding to this region was lost after complete T cell activation or IL-2 addition, events that correlated with FoxO phosphorylation and a sustained decrease in DGKα gene expression. These data strongly support a role for FoxO proteins in promoting high DGKα levels and indicate a mechanism by which DGKα function is downregulated during productive T cell responses. Our study establishes a basis for a causal relationship between DGKα downregulation, IL-2, and anergy avoidance.

Transient PKCα shuttling to the immunological synapse is governed by DGKζ and regulates L-selectin shedding

Summary Considerable evidence indicates that diacylglycerol (DAG) generation at the immunological synapse (IS) determines T cell functions by regulating the duration and amplitude of Ras/ERK signals. The exact mechanism by which DAG regulates Ras/ERK activation downstream of the T cell receptor (TCR) nonetheless remains poorly understood. Here we characterize PKC&agr; as a previously unrecognized component of the machinery that translates cell receptor occupancy into Ras/ERK-propagated signals. We show transient translocation of PKC&agr; to the IS, mediated by DAG generation at the contact area. Diacylglycerol kinase (DGK)&zgr; negatively regulated PKC&agr; translocation kinetics, whereas PKC&agr; activity limited its own persistence at the IS. Coordinated activation of DGK&zgr; and PKC&agr; in response to antigen recognition regulated the amplitude and duration of Ras/ERK activation; this in turn mediated early processes of T cell surface proteolysis such as L-selectin shedding. Analysis of DGK&zgr;-deficient mice further showed that increased DAG signaling is translated to downstream elements of this pathway, as reflected by enhanced PKC&agr;-dependent L-selectin shedding. We propose that early activation of a DAG–PKC&agr; axis contributes to the mechanisms by which antigen affinity translates into TCR biological responses.

Diacylglycerol kinase alpha, from negative modulation of T cell activation to control of cancer progression.

IntroductionThe diacylglycerol kinases (DGK) are a family of signaling proteins that modulate diacylglycerol(DAG) levels bycatalyzing its conversion to phosphatidic acid (PA) (Merida et al., 2008). DGK belong toasuperfamilythatalsoincludestherecentlyidentifiedbacterialDgkBaswellasthesphingosinekinase(SPK) and ceramide kinase (CEK) families. Proteins in this superfamily share a common catalyticdomain (DAGKc: Pfam00781) (Marchler-Bauer et al., 2007), that encompasses a number of highlyconservedmotifs.TherecentresolutionofthebacterialDgkBstructurehasprovidedimportantinsightsinto the catalytic mechanism, showing that the essential elements that define the structure andcatalytic properties of the bacterial DGKB are conserved in the mammalian enzymes (Miller et al.,2008). The most striking homology occurs in the ATP-binding loop, which represents the signaturemotif of this superfamily. Mammalian DGKs also contain the Asp residues that bridge an Mg

ZO-2 silencing induces renal hypertrophy through a cell cycle mechanism and the activation of YAP and the mTOR pathway

The absence of ZO-2 promotes an increase in cell size by two mechanisms: an increase in cyclin D, which extends the time that the cells spend in the G1 phase of the cell cycle, and an accumulation of YAP at the nucleus, which promotes its transcriptional activity, triggering the activation of the mTORC1 complex and its target, S6K1.

Predominant contribution of DGKζ over DGKα in the control of PKC/PDK-1-regulated functions in T cells

Diacylglycerol kinase (DGK)‐mediated consumption of the diacylglycerol (DAG) generated in response to antigen recognition is an important mechanism to limit T‐cell function. Targeting DGK activity presents new opportunities for therapeutic manipulation of the immune response, but assessment of individual DGK functions is complex. T cells express two DGK isoforms, DGKα and DGKζ, and there are no isoform‐specific inhibitors. Here we used short interfering RNA‐mediated gene silencing in human T cells and DGKα‐ and DGKζ‐deficient mice to define DGK isoform‐specific regulation of key signaling pathways during T‐cell activation. Our results identify DGKζ as the predominant brake on basal/tonic conditions as well as on downstream T‐cell receptor/co‐stimulatory signals. DGKζ silencing triggers basal RasGTP activation and facilitates enhanced membrane stability of protein kinase C alpha as well as increased activity of AGC kinases. Downstream of T‐cell receptor/co‐stimulation, DGKζ silencing results in enhanced and maintained recruitment of PKC theta to the membrane, as well as phosphoinositide‐dependent protein kinase‐1 activation and scaffolding functions. Our studies identify a previously unrecognized DGKζ contribution as a negative regulator of the crosstalk between phospholipase C‐gamma‐ and phosphoinositide 3‐kinase‐regulated pathways. This DGKζ input helps to explain previous observations in DGK‐deficient mice and suggests that the development of isoform‐specific DGK inhibitors is of great interest for the manipulation of distinct aspects of T‐cell responses.