Site-directed mutagenesis of the active site of diacylglycerol kinase α: calcium and phosphatidylserine stimulate enzyme activity via distinct mechanisms

Abe, Takahiro; Lu, Xiaolan; Jiang, Ying; Boccone, Clark E; Qian, Shuiming; Vattem, Krishna M.; Wek, Ronald C.; Walsh, James P.

doi:10.1042/bj20031052

Cited by 40 publications

(59 citation statements)

References 51 publications

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“…Thus, a conformation change after Ca 2ϩ binding would be required to expose recognition motifs for this lipid. Truncation of the N-terminal region including the C1 domains does not abolish PS-dependent enzyme activation (36), confirming that PS recognition sites are found in the catalytic region. There is limited structural information on the stereospecific recognition of the PS head group by its effector proteins.…”

Section: Discussionsupporting

confidence: 54%

“…Mutation of the second Gly in the sphingosine kinase SGDG motif abolishes kinase activity as does mutation of the second Gly residue in the DGK GGDG motif (35). Recent studies comparing the catalytic domains of DGK with those of ceramide kinase and sphingosine kinase have characterized several conserved Asp residues extended along the DGK catalytic region that are essential for enzyme activity (36). One of these residues, Asp 697 , lies near the deleted C-terminal region (Fig.…”

Section: Discussionmentioning

confidence: 99%

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

Merino¹,

Sanjuán²,

Moraga

et al. 2007

Journal of Biological Chemistry

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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. DGK␣ 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 DGK␣ 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 Tyr 335. 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.The transient generation of lipids at the cell membrane by the concerted action of lipases, kinases, and phosphatases is a very effective mechanism for the localization and activation of signaling molecules. The majority of lipid-modifying enzymes are cytosolic proteins that must in turn translocate to the membrane to modify their substrates. Characterization of the mechanism that governs membrane translocation of these proteins is, thus, essential to assess precisely their role in the regulation of cell responses.The diacylglycerol kinases (DGK) 5 are a family of enzymes that phosphorylate diacylglycerol (DAG) to produce phosphatidic acid (PA); this modulates the levels of these two lipids, both of which have recognized second messenger functions. Early experiments showed that DGK activity occupies a central position in phospholipid synthesis; this enzyme attracted additional interest when it was shown to participate in intracellular signaling (1). DGK activity is found in organisms from bacteria to mammals, although the protein identified as a DGK in bacteria is an integral membrane protein (2) and has little structural homology with the DGK characterized in multicellular organisms (3). Ten DGK isoforms have been identified in mammals and grouped into five subtypes based on the presence of various domains in their primary sequences, which define distinct regulatory motifs.Translocation from one subcellular compartment to another appears to be a general theme in the regulation of DGK proteins. Many of the early data regarding DGK activity regulation refer to changes in DGK activity or localization based on enzymatic studies without specifying the isoform concerned (4, 5). Characterization of the DGK isoforms has broadened the concept that these proteins reg...

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Section: Discussionsupporting

confidence: 54%

Section: Discussionmentioning

confidence: 99%

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

Merino¹,

Sanjuán²,

Moraga

et al. 2007

Journal of Biological Chemistry

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“…The three key aspartate residues (Asp 124 , Asp 216 , and Asp 271 ) that coordinate Mg 2ϩ in domain 2 are completely conserved in the mammalian DAG kinases (19), and site-directed mutagenesis of these conserved aspartates in pig DGK␣ show that they are all required for catalysis (29). These data indicate that the mammalian DAG kinase has a structural Mg 2ϩ site like the one illustrated in Fig.…”

Section: Discussionmentioning

confidence: 50%

“…This is supported by the observation that the catalytic activities of mammalian DAG kinases are also stimulated by a variety of anionic phospholipids (25,29,(37)(38)(39)(40). Thomas and Glomset (25) studied the binding of DAG kinase to phospholipid vesicles independent of its catalysis and made two key observations.…”

Section: Discussionmentioning

confidence: 59%

“…Activation of DgkB by Anionic Phospholipid Vesicles-DAG kinase activity is typically measured by presenting DAG to the enzyme in mixed micelles with a neutral detergent such as octyl glucoside (19,28,29). The detergent mixed micelle assay is a robust in vitro biochemical tool, but in the context of the cell, DAG is embedded in the phospholipid bilayer of cell membranes.…”

Section: Resultsmentioning

confidence: 99%

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Molecular Determinants for Interfacial Binding and Conformational Change in a Soluble Diacylglycerol Kinase

Jerga

Miller

White

et al. 2009

Journal of Biological Chemistry

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DgkB is a soluble diacylglycerol (DAG) kinase that is essential for membrane lipid homeostasis in many Gram-positive pathogens. Anionic phospholipids, like phosphatidylglycerol (PtdGro), were required for DgkB to recognize diacylglycerol embedded in a phospholipid bilayer. An activity-independent vesicle binding assay was used to determine the role of specific residues in DgkB-PtdGro interactions. Lys 15 and Lys 165 were required for DgkB to dock with PtdGro vesicles and flank the entrance to the DgkB active site. Mg 2؉ was required for vesicle binding. The compromised vesicle binding by mutants in the key asparate residues forming the structural Mg 2؉ -aspartatewater network within the substrate binding domain revealed that interfacial binding of DgkB required a Mg 2؉ -dependent conformational change. DgkB interaction with phospholipid vesicles was not influenced by the presence of ATP, but anionic vesicles decreased the K m of the enzyme for ATP. Arg 100 and Lys 15 are two surface residues in the ATP binding domain that were necessary for high affinity ATP binding. The key residues responsible for the structural Mg 2؉ binding site, the conformational changes that increase ATP affinity, and interfacial recognition of anionic phospholipids were identical in DgkB and the mammalian diacylglycerol kinase catalytic cores. This sequence conservation suggests that the mammalian enzymes also require a structural divalent cation and surface positively charged residues to bind phospholipid bilayers and trigger conformational changes that accelerate catalysis.The diacylglycerol (DAG) 2 kinase superfamily (Pfam00781) defines a group of soluble lipid kinases that phosphorylate membrane-associated DAG and function in regulating intracellular phospholipid metabolism and signaling. The mammalian family members share a conserved catalytic core domain that is associated with a variety of modules that confer intracellular membrane targeting specificity (for reviews, see Refs. 1 and 2). Staphylococcus aureus DgkB is a prototypical member of Pfam00781 that has the catalytic core of the DAG kinase superfamily, but lacks the ancillary domains characteristic of its mammalian cousins. DgkB proteins are essential in Gram-positive bacteria to re-introduce the large amount of DAG arising from lipoteichoic acid biosynthesis into the phospholipid biosynthetic pathway (3). DAG is exclusively found in the membrane phospholipid bilayer and the soluble DgkB must be capable of interfacial catalysis despite the absence of the membrane targeting modules present in its mammalian counterparts.Interfacial activation is a key feature of soluble proteins that act on substrates embedded in a phospholipid bilayer and has been extensively studied in phospholipid hydrolases (4, 5). These enzymes can utilize monomeric substrates, but their activity increases significantly when substrate is presented in micelles or unilamellar vesicles. Electrostatic effects are crucial to interfacial recognition by the soluble phospholipase A 2 proteins (4), and the structu...

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Crystal structure and calcium‐induced conformational changes of diacylglycerol kinase α EF‐hand domains

et al. 2019

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Diacylglycerol kinases (DGKs) are multi-domain lipid kinases that phosphorylate diacylglycerol into phosphatidic acid, modulating the levels of these key signaling lipids. Recently, increasing attention has been paid to DGKα isozyme as a potential target for cancer immunotherapy. We have previously shown that DGKα is positively regulated by Ca 2+ binding to its N-terminal EF-hand domains (DGKα-EF). However, little progress has been made for the structural biology of mammalian DGKs and the molecular mechanism underlying the Ca 2+ -triggered activation remains unclear. Here we report the first crystal structure of Ca 2+ -bound DGKα-EF and analyze the structural changes upon binding to Ca 2+ . DGKα-EF adopts a canonical EF-hand fold, but unexpectedly, has an additional α-helix (often called a ligand mimic [LM] helix), which is packed into the hydrophobic core. Biophysical and biochemical analyses reveal that DGKα-EF adopts a protease-susceptible "open" conformation without Ca 2+ that tends to form a dimer. Cooperative binding of two Ca 2+ ions dissociates the dimer into a well-folded monomer, which resists to proteolysis. Taken together, our results provide experimental evidence that Ca 2+ binding induces substantial conformational changes in DGKα-EF, which likely regulates intra-molecular interactions responsible for the activation of DGKα and suggest a possible role of the LM helix for the Ca 2+ -induced conformational changes.Significance statement: Diacylglycerol kinases (DGKs), which modulates the levels of two lipid second messengers, diacylglycerol and phosphatidic acid, is still structurally enigmatic enzymes since its first identification in 1959. We here present the first crystal structure of EF-hand domains of diacylglycerol kinase α in its Ca 2+ bound form and characterize Ca 2+ -induced conformational changes, which likely regulates intra-molecular interactions. Our study paves the way for future studies to understand the structural basis of DGK isozymes.

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Site-directed mutagenesis of the active site of diacylglycerol kinase α: calcium and phosphatidylserine stimulate enzyme activity via distinct mechanisms

Cited by 40 publications

References 51 publications

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

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

Molecular Determinants for Interfacial Binding and Conformational Change in a Soluble Diacylglycerol Kinase

Crystal structure and calcium‐induced conformational changes of diacylglycerol kinase α EF‐hand domains

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