C1 domains are lipid-binding modules that regulate membrane activation of kinases, nucleotide exchange factors and other C1-containing proteins to trigger signal transduction. Despite annotation of typical C1 domains as diacylglycerol (DAG) and phorbol ester sensors, the function of atypical counterparts remains ill-defined. Here, we assign a key role for atypical C1 domains in mediating DAG fatty acyl specificity of diacylglycerol kinases (DGKs) in live cells. Activity-based proteomics mapped C1 probe binding as a principal differentiator of type 1 DGK active sites that combined with global metabolomics revealed a role for C1s in lipid substrate recognition. Protein engineering by C1 domain swapping demonstrated that exchange of typical and atypical C1s is functionally tolerated and can directly program DAG fatty acyl specificity of type 1 DGKs. Collectively, we describe a protein engineering strategy for studying metabolic specificity of lipid kinases to assign a role for atypical C1 domains in cell metabolism.
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All data produced or analyzed for this study are included in the published article (and its supplementary information files) or are available from the corresponding author upon reasonable request.
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We thank M. Ross and all members of the Hsu Laboratory for helpful discussions. We thank S. Campbell for assistance in designing DGK chimera plasmids. This work was supported by the University of Virginia (start-up funds to K.-L.H.), the National Institutes of Health (grant nos. DA035864 and DA043571 to K.-L.H.; grant no. GM801868 to T.B.W.; grant no. GM007055 to C.E.F.), the US Department of Defense (grant no. W81XWH-17-1-0487 to K.-L.H.), an NCI Cancer Center Support Grant (grant no. 5P30CA044579-27 to K.-L.H. and T.E.H.) and the Schiff Foundation (Brain Tumor Research Grant to K.-L.H. and T.E.H.). Images were acquired using the W.M. Keck Center for Cellular Imaging Zeiss 780 Confocal microscopy system at UVA (NIH OD016446).
The authors declare no competing interests.
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Supplementary Tables 1–3 and Figs. 1–31.
DGK C1 chimera sequence
Supplementary Dataset 1
a, Results from LipidSearch analysis of both positive and negative mode ddMS2 data from DGKε-overexpressed HEK293T lipidomes. Job results for identified lipid species are filtered as outlined in Supplementary Fig. 5. Data displayed were used to generate Fig. 1b. b, Abundances of identified DAG species from DGKε-overexpressed HEK293T lipidomes following a targeted PRM analysis identifying the NH4+ adduct. Normalization is accomplished using the detected SAG-d8 internal standard and protein concentration. Data displayed were used to generate Fig. 1d. c, log2(fold change) of DAG abundances from various DGK isoforms overexpressed in HEK293T cells. All DGK isoforms are the human variant. Data displayed were used to generate Fig. 2a,b and Supplementary Figs. 8, 9 and 11. d, SILAC ratios and modified peptides of soluble proteins from DMSO (light)/ATP (heavy) treatments of HEK293T cells overexpressed with type 1 rat DGK isoforms with the ATP acyl phosphate probe. Data displayed were used to generate Fig. 3. e, Abundances of identified DAG species from type 1 human DGK isoform-overexpressed HEK293T lipidomes following a targeted PRM analysis identifying the NH4+ adduct. Data displayed were used to generate Fig. 4a. f, Abundances of identified DAG species from A549 lipidomes following doxycycline-induced shRNA knockdown of DGKα. Data displayed were used to generate Fig. 4b. g, Abundances of identified DAG species from rat DGKα WT- and lysine mutant-overexpressed HEK293T lipidomes following a targeted PRM analysis identifying the NH4+ adduct. Data displayed were used to generate Fig. 4c. h, Abundances of identified DAG species from rat DGKα WT-overexpressed HEK293T lipidomes treated in situ with ritanserin and ketanserin. Data displayed were used to generate Fig. 4d. i, Abundances of identified DAG species from type 1 rat DGK WT- and C1 chimera-overexpressed HEK293T lipidomes following a targeted PRM analysis identifying the NH4+ adduct. Data displayed were used to generate Fig. 5. j, log2(fold change) of PA abundances from various DGK isoforms overexpressed in HEK293T cells. All DGK isoforms are the human variant. Data displayed were used to generate Supplementary Figs. 10 and 11. k, Phosphorylation activity studies of type 1 DGK WT and chimera isoforms overexpressed in HEK293T soluble proteomes. Formation of radiolabeled [γ32-P] PA product is used to determine in vitro DGK activity from liposome substrates. Data displayed were used to generate Supplementary Fig. 19. l, log2(fold change) of DAG abundances from type 1 DGK isoforms overexpressed in HEK293T cells. All DGK isoforms are the rat variant. Data displayed were used to generate Supplementary Fig. 20.
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Ware, T.B., Franks, C.E., Granade, M.E. et al. Reprogramming fatty acyl specificity of lipid kinases via C1 domain engineering. Nat Chem Biol 16, 170–178 (2020). https://doi.org/10.1038/s41589-019-0445-9