Diacylglycerol kinases (DGKs) are multi-domain lipid kinases that catalyze phosphorylation of diacylglycerol
(DAG) to generate phosphatidic acid (PA). Both DAG and PA serve as potent lipid messengers to shape cellular
responses by altering subcellular localization, activation, and function of essential receptor proteins (ranging
from enzymes to transcription factors). DAG and PA also serve as building blocks for phospholipid and
triglyceride biosynthesis and integral to membrane architecture and bioenergetics. The significance of our
proposed studies is the enormous therapeutic potential of targeting individual DGKs because of their
fundamental role in sculpting the lipidome to support metabolic, structural, and signaling demands of healthy and
diseased cells. Despite their clinical value and discovery nearly 30 years ago, gaps in knowledge with regards
to ligand binding and regulation of DGK active-sites in living systems have confounded basic understanding of
how 10 mammalian DGK isoforms, which share a common catalytic domain, are capable of regulating distinct
metabolic and signaling functions. We will test our hypothesis that C1 and other non-catalytic domains, which
largely differentiate DGK isoforms, function in substrate and inhibitor recognition of DGK active sites.
The proposed research program will test whether selective blockade of DGK¿ can restore deficient DAG
signaling to overcome immunosuppression of tumor infiltrating lymphocyte activity. Genetic and clinical evidence
point to DGKs as promising targets for reversing immunosuppression of T cells although the molecular
mechanisms coupling disrupted DGK metabolism to enhanced TCR signaling are not clear. Our mechanistic
studies will establish a testable model for fundamental understanding of substrate and inhibitor recognition in
DGK active sites to guide development of new chemical strategies to perturb activity of T cell specific DGKs in
vivo for immunotherapy applications. Our long-term goals for this proposal are to functionally map novel and
druggable small molecule binding sites on DGK¿ and potentially other DGK isoforms in T cells to: 1) gain
molecular level insights into DAG fatty acyl chain recognition and specificity, 2) identify molecular features of
enzyme active sites to target lipid versus protein kinases, and 3) develop new inhibitors for selective inactivation
of DGK isoforms in live cells and animals.
We will test 2 independent yet related specific aims directed at: (Aim 1) identification of the DAG binding
site, (Aim 1) understanding how individual DGK domains couple extracellular signals to shape T cell responses,
(Aim 2) determining how DGK inhibitors amplify T cell activation, (Aim 2) understanding how DGK inhibitors
reverse T cell immunosuppression in vivo, and (Aim 2) determining if DGK inhibitors affect membrane
translocation. The overall impact of our findings will be to understand how intrinsic features of DGKs cross-talk
with extrinsic features of cellular environments to form the basis of a lipid signaling code that can be
therapeutically targeted for reversing immunosuppression of T cells.