Diacylglycerol Kinase γ Is One of the Specific Receptors of Tumor-Promoting Phorbol Esters

Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two different enzyme families that interact with diacylglycerol. Both enzymes contain cysteine-rich C1 domains with a zinc finger-like structure. Most of the C1 domains of PKCs show strong phorbol-12,13-dibutyrate (PDBu) binding with nanomolar dissociation constants (Kd's). However, there has been no experimental evidence that phorbol esters bind to the C1 domains of DGKs. We focused on DGKγ because its C1A domain has a high degree of sequence homology to those of PKCs, and because DGKγ translocates from the cytoplasm to the plasma membrane following 12-O-tetradecanoylphorbol-13-acetate treatment similar to PKCs. Two C1 domains of DGKγ (DGKγ-C1A and DGKγ-C1B) were synthesized and tested for their PDBu binding along with whole DGKγ (Flag-DGKγ) expressed in COS-7 cells. DGKγ-C1A and Flag-DGKγ showed strong PDBu binding affinity, while DGKγ-C1B was completely inactive. Scatchard analysis of DGKγ-C1A and Flag-DGKγ gave Kd's of 3.1 and 4.4 nM, respectively, indicating that the major PDBu binding site of DGKγ is C1A. This is the first evidence that DGKγ is a specific receptor of tumor-promoting phorbol esters.