Ernesto Merino

Diacylglycerol kinases: at the hub of cell signalling.

DGKs (diacylglycerol kinases) are members of a unique and conserved family of intracellular lipid kinases that phosphorylate DAG (diacylglycerol), catalysing its conversion into PA (phosphatidic acid). This reaction leads to attenuation of DAG levels in the cell membrane, regulating a host of intracellular signalling proteins that have evolved the ability to bind this lipid. The product of the DGK reaction, PA, is also linked to the regulation of diverse functions, including cell growth, membrane trafficking, differentiation and migration. In multicellular eukaryotes, DGKs provide a link between lipid metabolism and signalling. Genetic experiments in Caenorhabditis elegans, Drosophila melanogaster and mice have started to unveil the role of members of this protein family as modulators of receptor-dependent responses in processes such as synaptic transmission and photoreceptor transduction, as well as acquired and innate immune responses. Recent discoveries provide new insights into the complex mechanisms controlling DGK activation and their participation in receptor-regulated processes. After more than 50 years of intense research, the DGK pathway emerges as a key player in the regulation of cell responses, offering new possibilities of therapeutic intervention in human pathologies, including cancer, heart disease, diabetes, brain afflictions and immune dysfunctions.

Type I phosphatidylinositol 4-phosphate 5-kinase controls neutrophil polarity and directional movement

Directional cell movement in response to external chemical gradients requires establishment of front–rear asymmetry, which distinguishes an up-gradient protrusive leading edge, where Rac-induced F-actin polymerization takes place, and a down-gradient retractile tail (uropod in leukocytes), where RhoA-mediated actomyosin contraction occurs. The signals that govern this spatial and functional asymmetry are not entirely understood. We show that the human type I phosphatidylinositol 4-phosphate 5-kinase isoform β (PIPKIβ) has a role in organizing signaling at the cell rear. We found that PIPKIβ polarized at the uropod of neutrophil-differentiated HL60 cells. PIPKIβ localization was independent of its lipid kinase activity, but required the 83 C-terminal amino acids, which are not homologous to other PIPKI isoforms. The PIPKIβ C terminus interacted with EBP50 (4.1-ezrin-radixin-moesin (ERM)-binding phosphoprotein 50), which enabled further interactions with ERM proteins and the Rho-GDP dissociation inhibitor (RhoGDI). Knockdown of PIPKIβ with siRNA inhibited cell polarization and impaired cell directionality during dHL60 chemotaxis, suggesting a role for PIPKIβ in these processes.

Inhibitory signaling blocks activating receptor clustering and induces cytoskeletal retraction in natural killer cells

Signaling from immunotyrosine-based inhibitory motifs (ITIMs) neutralizes activating signals by inducing a retraction of NK cells from the surface of stimulatory cells.

Importance of chroman ring and tyrosine phosphorylation in the subtype-specific translocation and activation of diacylglycerol kinase α by d-α-tocopherol

Diacylglycerol kinase (DGK) has been suggested to be a pharmacological target of d‐α‐tocopherol for diabetic renal dysfunctions. However, the DGK subtypes involved in the d‐α‐tocopherol‐induced improvement of diabetic renal dysfunctions and the activation mechanisms of DGK by d‐α‐tocopherol are still unknown. Therefore, using GFP‐tagged DGKα, β, γ, ɛ and ζ, we analyzed their response to d‐α‐tocopherol and its derivatives. Only DGKα was translocated from the cytoplasm to the plasma membrane with elevation of kinase activity. In addition, troglitazone and trolox possessing ‘chroman ring’ similarly to d‐α‐tocopherol, induced the subtype‐specific translocation of DGKα. Furthermore, the translocation of DGKα was abolished by pretreatment with tyrosine kinase inhibitors or by mutation on Tyr‐334 of the kinase (YF mutant). d‐α‐tocopheryl succinate enhanced the Src‐mediated tyrosine phosphorylation of wild‐type DGKα but the same reagent did not enhance that of the YF mutant. These results demonstrate that tyrosine phosphorylation on Tyr‐334 and chroman ring are important for the d‐α‐tocopherol‐induced translocation of DGKα.

Role of the Diacylglycerol Kinase α-Conserved Domains in Membrane Targeting in Intact T Cells

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to phosphatidic acid, modifying the cellular levels of these two lipid mediators. Ten DGK isoforms, grouped into five subtypes, are found in higher organisms. All contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. DGKα is a type I enzyme that acts as a negative modulator of diacylglycerol-based signals during T cell activation. Here we studied the functional role of the DGKα domains using mutational analysis to investigate membrane binding in intact cells. We show that the two atypical C1 domains are essential for plasma membrane targeting of the protein in intact cells but unnecessary for catalytic activity. We also identify the C-terminal sequence of the protein as essential for membrane binding in a phosphatidic acid-dependent manner. Finally we demonstrate that, in the absence of the calcium binding domain, receptor-dependent translocation of the truncated protein is regulated by phosphorylation of Tyr335. This functional study provides new insight into the role of the so-called conserved domains of this lipid kinase family and demonstrates the existence of additional domains that confer specific plasma membrane localization to this particular isoform.

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.

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