Swiatkowska, A. | bioRxiv Science

bioRxiv2023

DGKepsilon is S-palmitoylated at the cysteine located at the cytoplasmic end of its N-terminal transmembrane fragment

Diacylglycerol kinase-{varepsilon} (DGK{varepsilon}) 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). Owing to this specificity, DGK{varepsilon} is involved in the PI cycle maintaining the cellular level of phosphorylated PI derivatives of signaling activity, and was also found crucial for lipid metabolism. DGK{varepsilon} dysfunction is linked with the development of atypical hemolytic uremic syndrome and possibly other human diseases. Despite the DGK{varepsilon} significance, data on its regulation by co/posttranslational modifications are scarce. Here we report that DGK{varepsilon} is S-palmitoylated at Cys38/40 (mouse/human DGK{varepsilon}) located in the cytoplasmic end of its N-terminal putative transmembrane fragment. The S-palmitoylation of DGK{varepsilon} 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 S-acyltransferases zDHHC7 and zDHHC17, and the zDHHC6/16 tandem were found to catalyze DGK{varepsilon} S-palmitoylation which also increased the DGK{varepsilon} abundance. Mouse DGK{varepsilon}-Myc ectopically expressed in HEK293 cells localized to the endoplasmic reticulum where zDHHC6/16 reside and in small amounts also to the Golgi apparatus where zDHHC7 and zDHHC17 are present. The Cys38Ala substitution upregulated while hyperpalmitoylation of wild type DGK{varepsilon} reduced the kinase activity, indicating an inhibitory effect of the Cys38 S-palmitoylation. Additionally, the substitution of neighboring Pro31 with Ala also diminished the activity of DGK{varepsilon}. Taken together, our data indicate that S-palmitoylation can fine-tune DGK{varepsilon} activity in distinct cellular compartments, possibly by affecting the distance between the kinase and its substrate in a membrane.

cell biology↗

bioRxiv2020

A genome-wide screen in mice to identify cell-extrinsic regulators of pulmonary metastatic colonisation

Metastatic colonisation, whereby a disseminated tumour cell is able to survive and proliferate at a secondary site, involves both tumour cell-intrinsic and -extrinsic factors. To identify tumour cell-extrinsic (microenvironmental) factors that regulate the ability of metastatic tumour cells to effectively colonise a tissue, we performed a genome-wide screen utilising the experimental metastasis assay on mutant mice. Mutant and wildtype (control) mice were tail vein-dosed with murine metastatic melanoma B16-F10 cells and 10 days later the number of pulmonary metastatic colonies were counted. Of the 1,300 genes/genetic locations (1,344 alleles) assessed in the screen 34 genes were determined to significantly regulate pulmonary metastatic colonisation (15 increased and 19 decreased; P<0.005 and genotype effect <-60 or >+60). Whilst several of these genes have known roles in immune system regulation (Bach2, Cyba, Cybb, Cybc1, Id2, Igh-6, Irf1, Irf7, Ncf1, Ncf2, Ncf4 and Pik3cg) most are involved in a disparate range of biological processes, ranging from ubiquitination (Herc1) to diphthamide synthesis (Dph6) to Rho GTPase-activation (Arhgap30 and Fgd4), with no previous reports of a role in the regulation of metastasis. Thus, we have identified numerous novel regulators of pulmonary metastatic colonisation, which may represent potential therapeutic targets.

cancer biology↗