Distinct regions of Praja-1 E3 ubiquitin-protein ligase selectively bind to docosahexaenoic acid-containing phosphatidic acid and diacylglycerol kinase δ

1-Stearoyl-2-docosahexaenoyl (18:0/22:6)-phosphatidic acid (PA) interacts with and activates Praja-1 E3 ubiquitin-protein ligase (full length: 615 aa) to ubiquitinate and degrade the serotonin transporter (SERT). SERT modulates serotonergic system activity and is a therapeutic target for depression, autism, obsessive-compulsive disorder, schizophrenia and Alzheimer's disease. Moreover, diacylglycerol kinase (DGK) δ2 (full length: 1214 aa) interacts with Praja-1 in addition to SERT and generates 18:0/22:6-PA, which binds and activates Praja-1. In the present study, we investigated the interaction of Praja-1 with 18:0/22:6-PA and DGKδ2 in more detail. We first found that the N-terminal one-third region (aa 1–224) of Praja-1 bound to 18:0/22:6-PA and that Lys141 in the region was critical for binding to 18:0/22:6-PA. In contrast, the C-terminal catalytic domain of Praja-1 (aa 446–615) interacted with DGKδ2. Additionally, the N-terminal half of the catalytic domain (aa 309–466) of DGKδ2 intensely bound to Praja-1. Moreover, the N-terminal region containing the pleckstrin homology and C1 domains (aa 1–308) and the C-terminal half of the catalytic domain (aa 762–939) of DGKδ2 weakly associated with Praja-1. Taken together, these results reveal new functions of the N-terminal (aa 1–224) and C-terminal (aa 446–615) regions of Praja-1 and the N-terminal half of the catalytic region (aa 309–466) of DGKδ2 as regulatory domains. Moreover, it is likely that the DGKδ2–Praja-1–SERT heterotrimer proximally arranges the 18:0/22:6-PA-producing catalytic domain of DGKδ2, the 18:0/22:6-PA-binding regulatory domain of Praja-1, the ubiquitin-protein ligase catalytic domain of Praja-1 and the ubiquitination acceptor site-containing SERT C-terminal region. •18:0/22:6-phosphatidic acid activates Praja-1 ubiquitinating serotonin transporter.•N-terminal one-third (aa 1–224) of Praja-1 bound to 18:0/22:6-phosphatidic acid.•C-terminal catalytic domain of Praja-1 interacted with diacylglycerol kinase (DGK) δ.•N-terminal half of catalytic domain of DGKδ bound to Praja-1 with high affinity.•Complex formation of Praja-1–DGKδ–18:0/22:6-phosphatidic acid was revealed in detail.