Regulation of clathrin-dependent endocytosis by diacylglycerol kinase δ:: importance of kinase activity and binding to AP2α

DGK delta (diacylglycerol kinase 3), which phosphorylates DAG (diacylglycerol) and converts it into PA (phosphatidic acid), has an important role in signal transduction. In the present study, we have demonstrated the molecular mechanism of DGK delta-mediated regulation of clathrin-dependent endocytosis that controls the internalization, recycling and degradation of receptors. Involvement of DGK6 in the regulation of clathrin-dependent endocytosis was previously proposed following genome-wide RNAi (RNA interference) screening. Clathrin-coated pits are mainly formed by clathrin and AP-2 (adaptor protein 2) complex. These proteins assemble a polyhedral lattice at the membrane and gather several endocytic accessory proteins. As the intracellular localization of DGK delta 2 overlapped with clathrin-coated pits, we predicted the possible regulation of clathrin-dependent endocytosis by DGK delta 2 and its interaction with some endocytosis-regulatory proteins. DGK delta 2 contained the DXF-type binding motifs, and DGK delta 2 bound to AP2 alpha, a subunit of the AP-2 complex. DGK delta 2 interacted with the platform subdomain in the AP2 alpha ear domain via (FDTFRIL)-D-369 and (DPF)-P-746 sequences in the catalytic domain of DGK delta 2. For further insight into the role for DGK delta 2 in clathrin-dependent endocytosis, we measured the transferrin and EGF (epidermal growth factor) uptakeexpressing wild-type or mutant DGK delta 2 under knockdown of endogenous DGK delta. Mutants lacking binding ability to AP2 alpha as well as kinase-negative mutants could not compensate for the uptake of transferrin inhibited by siRNA (small interfering RNA) treatment, whereas overexpression of wild-type DGK delta 2 completely recovered the transferrin uptake. These results demonstrate that binding between DGK delta 2 and AP2 alpha is involved in the transferrin internalization and that DGK activity is also necessary for the regulation of the endocytic process.