Diacylglycerol Kinase-ζ Localization in Skeletal Muscle Is Regulated by Phosphorylation and Interaction with Syntrophins

Abstract

Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-ζ (DGK-ζ), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-ζ form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-ζ MARCKS domain. DGK-ζ mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-ζ and regulating its activity. Consistent with a role in actin organization, DGK-ζ and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-ζ regulated its association with the cytoskeleton. In adult muscle, DGK-ζ was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-ζ was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-ζ. Together, our findings suggest syntrophins localize DGK-ζ signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-ζ may cause abnormal cytoskeletal changes that contribute to disease pathogenesis.