Diacylglycerol kinase from pig brain. Purification and phospholipid dependencies.

Kanoh, Hideo; Kondoh, H; Ono, Takashi

doi:10.1016/s0021-9258(18)33053-9

Cited by 129 publications

(7 citation statements)

References 51 publications

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“…Because the specific activity of DGKα purified from pig thymus was approx. 10 µmol of PA\min per mg of protein [25,35], the increased activity represented about 21 pmol of the DGK protein per mg of cellular protein. However, Table 4 shows that the extract from COS-7 cells transfected with DGKα exhibited no detectable PDBu binding activity.…”

Section: Phorbol Ester Binding Activity Of Zinc Fingers Of Intact Dgk Isoenzymesmentioning

confidence: 98%

The C-terminal part of diacylglycerol kinase α lacking zinc fingers serves as a catalytic domain

Sakane

Kai

Wada

et al. 1996

Biochemical Journal

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All mammalian diacylglycerol kinase (DGK) isoenzymes so far cloned consist of four conserved regions, namely, C1, C2 (tandem EF-hand structures), C3 (tandem cysteine-rich zinc finger sequences) and the C-terminal C4 domains. To determine the catalytic domain we expressed in COS-7 cells various truncation mutants of pig DGK alpha and assessed their enzyme activities. We found that the C4 domain lacking the whole N-terminal region including the zinc fingers possessed DGK activity that was dependent on the concentrations of diacylglycerol and ATP very similarly, as did the wild-type DGK alpha. Furthermore the DGK activity of the wild-type DGK and that expressed by the C4 domain were similarly activated by anionic amphiphiles such as phosphatidylserine, phosphatidylinositol and deoxycholate. It was also shown that a DGK mutant consisting of the zinc fingers and the C4 domain has enzymological properties very similar to those expressed by the C4 domain alone. We also confirmed that the intact DGKs alpha, beta and gamma expressed in COS-7 cells displayed no detectable phorbol ester binding. These results show that the C4 domain of DGK is the catalytic region that is responsible for the enzyme activities sensitive to different activators. We cannot exclude the possibility that the N-terminal portion including the zinc fingers can still interact with diacylglycerol and activators without affecting the enzyme activity measured in vitro. However, it is quite likely that the DGK zinc fingers do not serve as diacylglycerol-binding sites, in contrast with those present in other proteins such as protein kinases C and n-chimaerin. Site-directed mutagenesis of all six putative ATP binding sites (Lys248, Lys383, Lys395, Lys483, Lys492, and Lys554) did not significantly affect the enzyme activity. We therefore suggest that DGK does not contain a typical P-loop of ATP binding sites.

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Section: Phorbol Ester Binding Activity Of Zinc Fingers Of Intact Dgk Isoenzymesmentioning

confidence: 98%

The C-terminal part of diacylglycerol kinase α lacking zinc fingers serves as a catalytic domain

Sakane

Kai

Wada

et al. 1996

Biochemical Journal

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“…PAs are phospholipids important for signalling and activation of lipid-gated ion channels [134] and have long been linked to neurite outgrowth [135,136]. PAs are synthesised by phospholipase D1 (PLD1) and 2 (PLD2) which hydrolyse phosphatidylcholine to form PA and choline [137] and diacylglycerol kinase (DGK) [138] which phosphorylates diacylglycerol (DAG) to produce PA. This process is important for both astrocytes and neurons; in neurons DGK knockout attenuates synaptic vesicle recovery at the presynaptic terminal [139], PLD1 dysfunction is linked to impaired neurite outgrowth in Alzheimer's [140] and PLD2 ablation rescues synaptic function [141]; and in astrocytes, knockout of PLD1 and 2 reduces astrocyte proliferation in culture [142].…”

Section: Neuronal Morphologymentioning

confidence: 99%

Lipid metabolism in astrocytic structure and function

Lee

Hall

Allsop

et al. 2021

Seminars in Cell & Developmental Biology

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Astrocytes are the most abundant glial cell in the central nervous system and are involved in multiple processes including metabolic homeostasis, blood brain barrier regulation and neuronal crosstalk. Astrocytes are the main storage point of glycogen in the brain and it is well established that astrocyte uptake of glutamate and release of lactate prevents neuronal excitability and supports neuronal metabolic function. However, the role of lipid metabolism in astrocytes in relation to neuronal support has been until recently, unclear. Lipids play a fundamental role in astrocyte function, including energy generation, membrane fluidity and cell to cell signaling. There is now emerging evidence that astrocyte storage of lipids in droplets has a crucial physiological and protective role in the central nervous system. This pathway links β-oxidation in astrocytes to inflammation, signalling, oxidative stress and mitochondrial energy generation in neurons.Disruption in lipid metabolism, structure and signalling in astrocytes can lead to pathogenic mechanisms associated with a range of neurological disorders.

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“…However, it has been shown that when the enzyme is truncated by mutation and the E-F hand removed, it becomes constitutively active, independent of the presence of calcium (12,13). DGKR is also activated in a calcium-independent fashion by sonicated dispersions of several lipids (14). However, the significance of these findings is not clear since calcium is required for activity of this enzyme using other assay methods.…”

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confidence: 99%

Lipid Modulation of the Activity of Diacylglycerol Kinase α- and ζ-Isoforms: Activation by Phosphatidylethanolamine and Cholesterol

et al. 2004

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Diacylglycerol kinase (DGK) isoforms alpha and zeta were extracted from transfected cells that overexpressed these enzymes. We determined the lipid dependence of the binding of these isoforms to liposomes. The modulation by lipid of the rate of phosphorylation of diacylglycerol by these enzymes was also measured. Incorporation of phosphatidylethanolamine into the liposomes resulted in an increased partitioning of both isoforms of DGK to the membrane as well as an increased catalytic rate. We demonstrate that the increased catalytic rate is a consequence of both increased portioning of the enzyme to the membrane and increased catalytic activity of the membrane-bound form. DGKalpha, a calcium-dependent isoform, can be activated in a calcium-independent fashion in the presence of phosphatidylethanolamine. Similar effects are observed with cholesterol. In contrast, sphingomyelin inhibits the activity of both isoforms of DGK. Our results demonstrate that the translocation to membranes and activity of DGKalpha and DGKzeta are modulated by the composition and properties of the membrane. The enzymes are activated by the presence of lipids that promote the formation of inverted phases. However, the promotion of negative curvature is not the sole factor contributing to the lipid effects on enzyme binding and activity. A truncated form of DGKalphalacking both the E-F hand and the recoverin homology domain is constitutively active and is not further activated by any of the lipids tested or by calcium. However, a truncated form lacking only the recoverin homology domain is partially activated by either calcium or certain lipids.

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Diacylglycerol kinase from pig brain. Purification and phospholipid dependencies.

Cited by 129 publications

References 51 publications

The C-terminal part of diacylglycerol kinase α lacking zinc fingers serves as a catalytic domain

The C-terminal part of diacylglycerol kinase α lacking zinc fingers serves as a catalytic domain

Lipid metabolism in astrocytic structure and function

Lipid Modulation of the Activity of Diacylglycerol Kinase α- and ζ-Isoforms: Activation by Phosphatidylethanolamine and Cholesterol

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