An intact zinc finger motif of the C1B domain is critical for stability and activity of diacylglycerol kinase-ε

Traczyk, Gabriela; Świątkowska, Anna; Hromada-Judycka, Aneta; Janikiewicz, Justyna; Kwiatkowska, Katarzyna

doi:10.1016/j.biocel.2022.106295

Cited by 4 publications

(32 citation statements)

References 53 publications

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“…It also cooperates with monoubiquitination to prevent proteasomal degradation of LRP6 (41). A similar mechanism can explain the observed accumulation of S-palmitoylated DGKε and hyperpalmitoylation of Cys135Ala mDGKε-Myc that is avidly degraded by the 26S proteasome (19). The consequences of the S-palmitoylation for the conformation of calnexin, an endoplasmic reticulum chaperone which promotes the folding of glycoproteins, are even more complex.…”

Section: Discussionmentioning

confidence: 84%

“…cDNA of mouse DGKε (mDGKε, NM_019505) was obtained and cloned into the pcDNA3.1/Hygro(+) vector (Invitrogen) and fused with a double Myc tag at the C-terminus, as described by Traczyk et al (19). Plasmid encoding human DGKε (hDGKε, NM_003647) was purchased from OriGene (cat.…”

Section: Methodsmentioning

confidence: 99%

“…The DGKε enzymatic activity was determined using mixed micelle assay according to our previous studies (19). For micelle preparation, 1-NBD-stearoyl- μg/ml aprotinin, and 20 μg/ml leupeptin) and sonicated, as described in (19). For lysis and immunoprecipitation, HEK293 cells 24 h after transfection were used since this time was optimal for coexpression of mDGKε-Myc and zDHHC17-HA, as described above.…”

Section: Dgkε Activity Determinationmentioning

confidence: 99%

“…For lysis and immunoprecipitation, HEK293 cells 24 h after transfection were used since this time was optimal for coexpression of mDGKε-Myc and zDHHC17-HA, as described above. Cells were lysed essentially as described in (19) at 4°C in 150 µl of lysis buffer supplemented with 1 mM DTT (150 mM NaCl, 20 mM…”

Section: Dgkε Activity Determinationmentioning

confidence: 99%

“…2). We have recently found that Cys135 is an element of a zinc finger motif crucial for mDGKε activity and stability and its substitution with Ala leads to proteasomal degradation of the protein (19); indeed, the Cys135Ala mDGKε-Myc variant was present in small quantities which made its detection difficult (Fig. 1C, F, see also Fig.…”

Section: Cys 38/cys40 Is the Major Site Of S-palmitoylation Of Mouse/...mentioning

confidence: 99%

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Diacylglycerol kinase-ε isS-palmitoylated on cysteine in the cytoplasmic end of its N-terminal transmembrane fragment

Traczyk

Hromada-Judycka

Swiatkowska

et al. 2023

Preprint

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Diacylglycerol kinase-epsilon (DGKepsilon) catalyzes phosphorylation of diacylglycerol to phosphatidic acid with a unique specificity toward 1-stearoyl-2-arachidonoyl-sn-glycerol which is a backbone of phosphatidylinositol (PI).DGKepsilon plays a crucial role in the PI cycle thereby maintaining the cellular level of phosphorylated PI derivatives of signaling activity, and in lipid metabolism. DGKepsilon dysfunction is linked with the development of atypical hemolytic uremic syndrome (aHUS) and other diseases in humans. Despite the DGKepsilon significance, data on the regulation of its functioning by possible co/post-translational modifications are scarce. Here we report that DGKepsilon is S-palmitoylated at Cys38/40 (mouse/human DGKepsilon) located at the cytoplasmic end of its N-terminal putative transmembrane fragment. The S-palmitoylation of DGKepsilon was revealed by metabolic labeling of cells with a palmitic acid analogue followed by click chemistry, and with acyl-biotin and acyl-PEG exchange assays. The zDHHC7, 17 and 6/16 S-acyltransferases were found to catalyze DGKepsilon S-palmitoylation which also increased the DGKepsilon abundance, likely by affecting its stability. Given the localization of the majority of DGKepsilon and zDHHC6 in the endoplasmic reticulum and zDHHC7 and zDHHC17 in the Golgi apparatus, trafficking of a fraction of DGKepsilon through the Golgi apparatus toward the plasma membrane is plausible. The Cys38Ala substitution moderately upregulated the activity of DGKepsilon, suggesting an inhibitory effect of the acylation in wild type DGKepsilon. In contrast to Cys38Ala, the substitution of neighboring Pro31 with Ala diminished the amount and the activity of DGKepsilon. Taken together, our data indicate that S-palmitoylation can fine-tune DGKepsilon activity and affect its abundance and cellular localization.

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Section: Discussionmentioning

confidence: 84%

Section: Methodsmentioning

confidence: 99%