Phosphorylation of diacylglycerol kinase-zeta by protein kinase C regulates its interaction with the PDZ domain of syntrophins

Abstract

Diacylglycerol kinase-zeta (DGK-zeta) attenuates diacylglycerol (DAG) signaling by converting it into phosphatidic acid (PA). DGK-zeta binds via its C-terminus, which contains a PSD-95/ Discs-Large/ZO-1 (PDZ)-binding motif, to a family of PDZ domain-containing scaffold proteins called syntrophins. Previous studies showed that some PDZ-mediated interactions are regulated by phosphorylation of the C-terminal PDZ-binding motif, however, to my knowledge, there are no published reports which demonstrate that a phosphorylation outside of this motif regulates PDZ interactions. Here, I provide evidence that protein kinase C-mediated phosphorylation of the MARCKS domain of DGK-zeta increases its association with syntrophins by a PDZ-dependent mechanism. Compared to wild-type (wt) DGK-zeta, a mutant mimicking phosphorylation of the MARCKS domain (DGK-zeta M1) bound more to recombinant syntrophin PDZ domains in in vitro binding assays. Moreover, more endogenous syntrophin co-immunoprecipitated from lysates of COS cells infected with HA-tagged DGK-zetaM1 than with wt HA-DGK-zeta. Protein kinase C (PKC) activation by phorbol myristate acetate enhanced the interaction of wt DGK-zeta and syntrophin PDZ domains, an effect that was blocked by a specific PKC inhibitor. Consistent with the idea that the MARCKS domain regulates binding, deletion of this domain decreased binding to syntrophin PDZ domains. Surprisingly, phosphorylation-mimicking mutants of extracellular signal-regulated kinase phosphorylation sites closer to the C-terminus had no detectable effect on syntrophin binding. Collectively, my findings suggest PKC-mediated phosphorylation of the MARCKS domain regulates the PDZ-dependent interaction between DGK-zeta and syntrophins.