Arachidonoyl-diacylglycerol Kinase SPECIFIC IN VITRO INHIBITION BY POLYPHOSPHOINOSITIDES SUGGESTS A MECHANISM FOR REGULATION OF PHOSPHATIDYLINOSITOL BIOSYNTHESIS

Walsh, James P.; Suen, Rosa; Glomset, John A.

doi:10.1074/jbc.270.48.28647

Cited by 40 publications

(40 citation statements)

References 71 publications

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“…Activation of DGK␣ by OTAC and C16SB-We have previously shown that DGK␣ is markedly stimulated by the cationic amphiphile, OTAC (11). This agent stimulated three other Ca 2ϩ -dependent DGK activities from testis cytosol, salivary cytosol, and NIH 3T3 cells to a similar degree (data not shown).…”

Section: Fragments Of Dgk␣ Rvh/ef Hands Domainmentioning

confidence: 77%

“…This agent stimulated three other Ca 2ϩ -dependent DGK activities from testis cytosol, salivary cytosol, and NIH 3T3 cells to a similar degree (data not shown). Several Ca 2ϩ -independent DGK activities, including an arachidonoyl-DGK from testis membranes, and cytosolic activities from NIH 3T3 cells and thymus cytosol were only modestly (2-6-fold) activated by OTAC (11,23). Wild-type DGK␣ activity expressed in COS-1 cells was activated 109-fold by OTAC (Table III).…”

Section: Fragments Of Dgk␣ Rvh/ef Hands Domainmentioning

confidence: 99%

“…Diacylglycerol Kinase Assays-The standard DGK assay contained in volume of 200 l the following: 1 mM sodium deoxycholate, 50 mM triethanolamine⅐HCl, pH 7.5, 100 mM NaCl, 1 mM MgCl 2 , 1 mM EGTA, 0.1 mM [␥-32 P]ATP (100 cpm/pmol), 20 M sn-1-palmitoyl-2-oleoyl-glycerol (16:0, 18:1 DAG), 1 mM DTT, and enzyme (11,23). In a typical reaction, an appropriate volume of DAG stock solution (10 -20 mM in CHCl 3 ) was evaporated under a stream of nitrogen in a 16 ϫ 100-mm glass test tube.…”

Section: Methodsmentioning

confidence: 99%

“…To examine the effect of OTAC on proteolysis, the reaction mixture was supplemented with 0.4 mM Triton X-100, 0.4 mM Triton X-114, and 0.2 mM OTAC (20 mol %), and the trypsin concentration was halved. The Triton mixture was added together with the OTAC to simulate the conditions under which maximal activity stimulation is observed (11,23). Triton X-100 and Triton X-114 were also included in the control reactions.…”

Section: Methodsmentioning

confidence: 99%

“…Several studies have shown variation among DGKs with regard to activation by phospholipids, sphingosine, or Ca 2ϩ (11)(12)(13)(14)(15). Kanoh and coworkers (16 -18) have studied DGK␣, a Ca 2ϩ -activated isoform highly expressed in oligodendrocytes and thymocytes.…”

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confidence: 99%

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A Domain with Homology to Neuronal Calcium Sensors Is Required for Calcium-dependent Activation of Diacylglycerol Kinase α

Jiang

Qian²,

Hawes

et al. 2000

Journal of Biological Chemistry

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Hydrolysis of phosphatidylinositol 4,5-bisphosphate is a common mechanism of stimulus transduction (1). Diacylglycerol (DAG) 1 released in this reaction activates protein kinase C (PKC) and is then rapidly metabolized back to phosphatidylinositol in a series of reactions initiated by a diacylglycerol kinase (DGK). As such, DGKs attenuate DAG-mediated PKC activation (2). Recent studies indicate that DGKs are also activated by mechanisms independent of phosphoinositide turnover (3, 4). Diacylglycerol kinases catalyze the ATP-dependent phosphorylation of sn-1,2-diacylglycerol to form phosphatidic acid (PA), which is also a lipid mediator (5, 6). Several DGK isoforms have been cloned (7). All these sequences share a homologous catalytic domain and two or three C1 protein kinase C homology domains (7-9). Some DGKs contain EF hands, which are Ca 2ϩ -binding sites (7). These DGKs also have a domain at their N termini with homology to the recoverin family of neuronal calcium sensors (Fig. 1). We term this the recoverin homology (RVH) domain. In S-modulin, the frog orthologue of recoverin, this domain associates with the EF hands to mediate Ca 2ϩ -dependent inhibition of rhodopsin kinase (10).The varied structures of DGK regulatory domains suggest divergent mechanisms of regulation. Several studies have shown variation among DGKs with regard to activation by phospholipids, sphingosine, or Ca 2ϩ (11)(12)(13)(14)(15). Kanoh and coworkers (16 -18) have studied DGK␣, a Ca 2ϩ -activated isoform highly expressed in oligodendrocytes and thymocytes. They have shown that Ca 2ϩ binds the EF hand region of the enzyme and that deletion of the EF hands results in constitutive enzyme activation (19,20). We have now examined a series of DGK␣ mutants in which the RVH and EF hand domains are sequentially deleted. Our results indicate that the N-terminal RVH domain is required for Ca 2ϩ to activate this enzyme. In contrast to the constitutive activation seen with deletion of the EF hands, DGKs with deletions involving only the RVH domain expressed activity similar to that of wild-type enzyme in the absence of Ca 2ϩ . Sites within both the EF hands and RVH domain were protected from trypsin proteolysis by Ca 2ϩ , indicating that both domains participate in a Ca 2ϩ -induced conformational change. A cationic amphiphile, octadecyltrimethylammonium chloride, markedly stimulated DGK␣ activity in vitro. This effect, like Ca 2ϩ -dependent activation, was dependent on the RVH domain. The DGK␣ RVH domain does not itself bind Ca 2ϩ . However, it does appear to function together with the EF hands to couple Ca 2ϩ binding to release of EF handmediated autoinhibition of DGK␣. EXPERIMENTAL PROCEDURES Materials-Restriction

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Section: Fragments Of Dgk␣ Rvh/ef Hands Domainmentioning

confidence: 77%

Section: Fragments Of Dgk␣ Rvh/ef Hands Domainmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

A Domain with Homology to Neuronal Calcium Sensors Is Required for Calcium-dependent Activation of Diacylglycerol Kinase α

Jiang

Qian²,

Hawes

et al. 2000

Journal of Biological Chemistry

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Septin‐based readout of PI(4,5)P2 incorporation into membranes of giant unilamellar vesicles

et al. 2018

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Septins constitute a novel class of cytoskeletal proteins. Budding yeast septins self-assemble into non-polar filaments bound to the inner plasma membrane through specific interactions with l-α-phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Biomimetic in vitro assays using giant unilamellar vesicles (GUVs) are relevant tools to dissect and reveal insights in proteins-lipids interactions, membrane mechanics and curvature sensitivity. GUVs doped with PI(4,5)P2 are challenging to prepare. This report is dedicated to optimize the incorporation of PI(4,5)P2 lipids into GUVs by probing the proteins-PI(4,5)P2 GUVs interactions. We show that the interaction between budding yeast septins and PI(4,5)P2 is more specific than using usual reporters (phospholipase Cδ1). Septins have thus been chosen as reporters to probe the proper incorporation of PI(4,5)P2 into giant vesicles. We have shown that electro-formation on platinum wires is the most appropriate method to achieve an optimal septin-lipid interaction resulting from an optimal PI(4,5)P2 incorporation for which, we have optimized the growth conditions. Finally, we have shown that PI(4,5)P2 GUVs have to be used within a few hours after their preparation. Indeed, over time, PI(4,5)P2 is expelled from the GUV membrane and the PI(4,5)P2 concentration in the bilayer decreases.

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Analysis of hormone‐stimulated phosphatidylinositol synthesis

Monaco

Moldover

Walden

2002

Journal Cellular Physiology

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Agonist-stimulated phosphoinositide turnover is accompanied by compensatory resynthesis of these lipids. Several lines of evidence suggest that resynthesis of phosphatidylinositol (PtdIns) involves phosphorylation of diacylglycerol (DG) (salvage pathway) rather than acylation of glycerol phosphate (de novo pathway), although a contribution from the de novo pathway has not been ruled out. To determine the relative contribution of the de novo and salvage pathways in stimulated PtdIns resynthesis, an inhibitor of de novo synthesis (Triacsin C) was incubated simultaneously with the hormone agonist. Results indicate that at early times (90 min), hormone-stimulated PtdIns synthesis proceeds predominantly via the salvage pathway, although some de novo synthesis is also taking place. At later times (24 h), stimulated synthesis is solely via the de novo pathway. Increasing cellular DG content by either adding exogenous DG or treating cells with bacterial phospholipase C (bPLC) results in deacylation of the DG rather than phosphorylation; however, inhibition of this deacylation fails to stimulate phosphorylation by DG kinase (DGK), suggesting channeling of the DG substrate between PLC and DG kinase. Receptor activation is not required for activation of DGK, since treatment with a calcium ionophore induces the same Triacsin C-insensitive PtdIns synthesis. Depletion of the polyphosphoinositide pools by treatment with wortmannin prevents both hormone and A23187-induced polyphosphoinositide hydrolysis; however, A23187 is still able to induce hydrolysis of PtdIns and subsequent compensatory resynthesis. The inability of R59949 to inhibit either hormone-induced or ionophore-induced PtdIns resynthesis suggests that the alpha isoform is not involved; however, its possible that the channeling phenomenon prevents the inhibitor from gaining access to the diacylglycerol kinase enzyme. Further study will be required to determine which isoform catalyzes hormone-induced resynthesis of PtdIns.

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Arachidonoyl-diacylglycerol Kinase SPECIFIC IN VITRO INHIBITION BY POLYPHOSPHOINOSITIDES SUGGESTS A MECHANISM FOR REGULATION OF PHOSPHATIDYLINOSITOL BIOSYNTHESIS

Cited by 40 publications

References 71 publications

A Domain with Homology to Neuronal Calcium Sensors Is Required for Calcium-dependent Activation of Diacylglycerol Kinase α

A Domain with Homology to Neuronal Calcium Sensors Is Required for Calcium-dependent Activation of Diacylglycerol Kinase α

Septin‐based readout of PI(4,5)P2 incorporation into membranes of giant unilamellar vesicles

Analysis of hormone‐stimulated phosphatidylinositol synthesis

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