Syntrophin regulates the subcellular localization of diacylglycerol kinase-zeta

Abstract

Syntrophins are scaffolding proteins that link signaling molecules to the dystrophin protein complex at the plasma membrane. To further understand the roles of syntrophins a yeast two-hybrid screen of a human brain cDNA library was done using the PDZ domain of the recently identified brain-specific gamma 1-syntrophin, an isoform for which no signaling ligands had yet been identified. This screen yielded ten overlapping clones coding for the C-terminal portion of diacylglycerol kinase-zeta (DGK-zeta), a kinase that phosphorylates the membrane lipid diacylglycerol (DAG) to phosphatidic acid (PA). Biochemical experiments and binding assays confirmed that this kinase's C-terminus, containing the consensus PDZ-binding motif Q-E-T-A-V-COOH, was both necessary and sufficient for the interaction. This complex can be immunoprecipitated from co-transfected Hela cells, and the C-terminal-PDZ interaction appears to be required for the distribution of this complex between both the cytosol and nucleus. In the brain, DGK-zeta and gamma1-syntrophin colocalize in cerebellar Purkinje fibres and pyramidal neurons of the hippocampus and cortex, areas where dystrophin is expressed, and DGK-zeta can be detected in dystrophin immunoprecipitates from mouse brain extracts. Furthermore, biochemical experiments also show that DGK-zeta binds to the PDZ domain of alpha- and beta-syntrophins, and, endogenous syntrophin can be detected in DGK-zeta immunoprecipitates from cultured C2C12 skeletal muscle cell extracts. In normal skeletal muscle, DGK-zeta and syntrophin co-localizes at the NMJ, and the sarcolemma of oxidative fibres. In mdx mouse skeletal muscle, DGK-zeta's localization at the NMJ appears unaffected, while sarcolemmal levels are decreased in degenerating fibres but not in regenerating or regenerated ones. Together, our data suggest that syntrophin binds to DGK-zeta and links it to dystrophin-associated protein complexes in neurons and muscle cells, thereby playing a role in regulating the subcellular localization and function of this lipid kinase.