| Description |
Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DAG) to form phosphatidic acid (PA). Both DAG and PA are lipid second messengers and participate in a broad array of cellular functions through the activation of lipid-responsive proteins. Nine mammalian DGK isoforms have been identified and they are divided into five subtypes based on their structural motifs. Accumulating evidence suggests that each DGK isoform may regulate a distinct subset of lipid signals. DGK[zeta], a type IV DGK, contains several potential PKC phosphorylation sites within its MARCKS motif. We tested whether PKC phosphorylates and regulates DGK[zeta] activity and found that PKC? phosphorylated DGK[zeta] on serines within the MARCKS motif in vitro and in vivo and that this phosphorylation inhibited DGK[zeta] activity in cells. Additionally, a phosphorylation-mimicking DGK[zeta] mutant had lower DAG kinase activity compared to wild type DGK[zeta], indicating that PKC? negatively regulates DGK[zeta] by phosphorylating its MARCKS motif. Activation of PKC depends on the availability of DAG. We demonstrated that DGK[zeta] associated with and inhibited PKC? activity, and DGK activity was required for this inhibition. Interestingly, phosphorylation of the MARCKS motif in DGK[zeta] inhibited their association. Furthermore, a phosphorylation-mimicking DGK[zeta] mutant could not bind to and inhibit PKC?, suggesting that DGK[zeta] spatially regulates PKC? activity by attenuating local accumulation of signaling DAG, and that this regulation is impaired by PKC?-mediated DGK[zeta] phosphorylation. PA, the product of the PGK reaction, stimulates phosphatidylinositol 4-phosphate 5-kinase (PIP5K) in vitro. We verified that endogenous PA can activate PIP5K? in vivo. Expression of wild type DGK[zeta], but not a kinase-dead DGK[zeta] mutant, stimulated inhibited PIP5K? activity. Additionally, DGK[zeta] associated with PIP5K? activity by generating PA. Together, these data demonstrate that DGK[zeta] can function by lowering the local level of DAG as an off single for DAG-activated events, or DGK[zeta] can act as a source of PA that then activates proteins such as PIP5K. Some of these events appear to be reciprocally regulated as in the case where PKC? negatively regulates DGK[zeta] activity |
