Protein kinase Cα phosphorylates and negatively regulates diacylglycerol kinase ζ

Diacylglycerol kinase (DGK) terminates diacylglycerol (DAG) signaling by phosphorylating DAG to produce phosphatidic acid, which also has signaling properties. Thus, precise control of DGK activity is essential for proper signal transduction. We demonstrated previously that a peptide corresponding to the myristoylated alanine-rich C kinase substrate ( MARCKS) phosphorylation site domain (PSD) in DGKzeta was phosphorylated in vitro by an active fragment of protein kinase C (PKC). In the present study, we tested full-length DGKzeta and found that PKCalpha phosphorylated DGKzeta on serines within the MARCKS PSD in vitro and in vivo. DGKzeta also coimmunoprecipitated with PKCalpha, suggesting that they reside in a regulated signaling complex. We then tested whether phosphorylation affected DAG kinase activity. We found that a mutant ( DGKzeta S/D) in which serines within the MARCKS PSD were altered to aspartates ( to mimic phosphorylation) had lower activity compared with wild-type DGKzeta or a control mutant (DGKzeta S/N) in which the same serines were changed to asparagines. Furthermore, activation of PKCalpha by phorbol 12-myristate 13-acetate inhibited the activity of wild-type DGKzeta, but not DGKzeta S/D, in human embryonic kidney 293 cells. These results suggest that by phosphorylating the MARCKS PSD, PKCalpha attenuates DGKzeta activity. Supporting this, we found that cells expressing DGKzeta S/D had higher DAG levels and grew more rapidly compared with cells expressing DGKzeta S/N that could not be phosphorylated. Taken together, these results indicate that PKCalpha phosphorylates DGKzeta in cells, and this phosphorylation inhibits its kinase activity to remove cellular DAG, thereby affecting cell growth.