DGKδ (diacylglycerol kinase δ), 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 DGKδ-mediated regulation of clathrin-dependent endocytosis that controls the internalization, recycling and degradation of receptors. Involvement of DGKδ 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 DGKδ2 overlapped with clathrin-coated pits, we predicted the possible regulation of clathrin-dependent endocytosis by DGKδ2 and its interaction with some endocytosis-regulatory proteins. DGKδ2 contained the DXF-type binding motifs, and DGKδ2 bound to AP2α, a subunit of the AP-2 complex. DGKδ2 interacted with the platform subdomain in the AP2α ear domain via F369DTFRIL and D746PF sequences in the catalytic domain of DGKδ2. For further insight into the role for DGKδ2 in clathrin-dependent endocytosis, we measured the transferrin and EGF (epidermal growth factor) uptake-expressing wild-type or mutant DGKδ2 under knockdown of endogenous DGKδ. Mutants lacking binding ability to AP2α 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 DGKδ2 completely recovered the transferrin uptake. These results demonstrate that binding between DGKδ2 and AP2α is involved in the transferrin internalization and that DGK activity is also necessary for the regulation of the endocytic process.
Regulation of clathrin-dependent endocytosis by diacylglycerol kinase δ: importance of kinase activity and binding to AP2α
Takumi Kawasaki, Takeshi Kobayashi, Takehiko Ueyama, Yasuhito Shirai, Naoaki Saito; Regulation of clathrin-dependent endocytosis by diacylglycerol kinase δ: importance of kinase activity and binding to AP2α. Biochem J 15 January 2008; 409 (2): 471–479. doi: https://doi.org/10.1042/BJ20070755
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