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 t...

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Bibliographic Details
Published in:	The Journal of biological chemistry 2003-10, Vol.278 (41), p.39542-39547
Main Authors:	Luo, Bai, Prescott, Stephen M., Topham, Matthew K.
Format:	Article
Language:	eng
Subjects:	Binding Sites Cell Division Cell Line Diacylglycerol Kinase - metabolism Diglycerides - metabolism Humans In Vitro Techniques Intracellular Signaling Peptides and Proteins Membrane Proteins Myristoylated Alanine-Rich C Kinase Substrate Phospholipase C gamma Phosphorylation Protein Kinase C - metabolism Protein Kinase C-alpha Protein Structure, Tertiary Proteins - metabolism Recombinant Proteins - metabolism Signal Transduction Tetradecanoylphorbol Acetate - pharmacology Type C Phospholipases - metabolism
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Summary:	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 DGKζ was phosphorylated in vitro by an active fragment of protein kinase C (PKC). In the present study, we tested full-length DGKζ and found that PKCα phosphorylated DGKζ on serines within the MARCKS PSD in vitro and in vivo. DGKζ also coimmunoprecipitated with PKCα, suggesting that they reside in a regulated signaling complex. We then tested whether phosphorylation affected DAG kinase activity. We found that a mutant (DGKζ S/D) in which serines within the MARCKS PSD were altered to aspartates (to mimic phosphorylation) had lower activity compared with wild-type DGKζ or a control mutant (DGKζ S/N) in which the same serines were changed to asparagines. Furthermore, activation of PKCα by phorbol 12-myristate 13-acetate inhibited the activity of wild-type DGKζ, but not DGKζ S/D, in human embryonic kidney 293 cells. These results suggest that by phosphorylating the MARCKS PSD, PKCα attenuates DGKζ activity. Supporting this, we found that cells expressing DGKζ S/D had higher DAG levels and grew more rapidly compared with cells expressing DGKζ S/N that could not be phosphorylated. Taken together, these results indicate that PKCα phosphorylates DGKζ in cells, and this phosphorylation inhibits its kinase activity to remove cellular DAG, thereby affecting cell growth.
ISSN:	0021-9258 1083-351X