Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-(DGK-), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-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-MARCKS domain. DGK-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-and regulating its activity. Consistent with a role in actin organization, DGK-and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGKregulated its association with the cytoskeleton. In adult muscle, DGK-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-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-. Together, our findings suggest syntrophins localize DGK-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-may cause abnormal cytoskeletal changes that contribute to disease pathogenesis.

Syntrophins are scaffold proteins of the dystrophin glycoprotein complex (DGC), which target ion channels, receptors, and signaling proteins to specialized subcellular domains. A yeast two-hybrid screen of a human brain cDNA library with the PSD-95, Discs-large, ZO-1 (PDZ) domain of ␥1-syntrophin yielded overlapping clones encoding the C terminus of TAPP1, a pleckstrin homology (PH) domain-containing adapter protein that interacts specifically with phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2 ). In biochemical assays, the C terminus of TAPP1 bound specifically to the PDZ domains of ␥1-, ␣1-, and ␤2-syntrophin and was required for syntrophin binding and for the correct subcellular localization of TAPP1. TAPP1 is recruited to the plasma membrane of cells stimulated with platelet-derived growth factor (PDGF), a motogen that produces PI(3,4)P 2 . Cell migration in response to PDGF stimulation is characterized by a rapid reorganization of the actin cytoskeleton, which gives rise to plasma membrane specializations including peripheral and dorsal circular ruffles. Both TAPP1 and syntrophins were localized to PDGF-induced circular membrane ruffles in NIH-3T3 cells. Ectopic expression of TAPP1 potently blocked PDGF-induced formation of dorsal circular ruffles, but did not affect peripheral ruffling. Interestingly, coexpression of ␣1-or ␥1-syntrophin with TAPP1 prevented the blockade of circular ruffling. In addition to syntrophins, several other proteins of the DGC were enriched in circular ruffles. Collectively, our results suggest syntrophins regulate the localization of TAPP1, which may be important for remodeling the actin cytoskeleton in response to growth factor stimulation.

Syntrophins are modular adapter proteins that link ion channels and signaling proteins to dystrophin and its homologues. A yeast two-hybrid screen of a human brain cDNA library using the PDZ domain of ␥1-syntrophin, a recently identified brain-specific isoform, yielded overlapping clones encoding the C terminus of diacylglycerol kinase-(DGK-), an enzyme that converts diacylglycerol into phosphatidic acid. In biochemical assays, the C terminus of DGK-, which contains a consensus PDZ-binding motif, was found to be necessary and sufficient for association with ␥1-syntrophin. When coexpressed in HeLa cells, DGK-and ␥1-syntrophin formed a stable complex that partitioned between the cytoplasm and nucleus. DGK-translocates from the cytosol to the nucleus, a process negatively regulated by protein kinase C phosphorylation. We found that DGKrecruits ␥1-syntrophin into the nucleus and that the PDZ-binding motif is required. Disrupting the interaction altered the intracellular localization of both proteins; DGK-accumulated in the nucleus, whereas ␥1-syntrophin remained in the cytoplasm. The level of endogenous syntrophins in the nucleus of HeLa cells also reflected the amount of nuclear DGK-. In the brain, DGK-and ␥1-syntrophin were colocalized in cell bodies and dendrites of cerebellar Purkinjie neurons and other neuronal cell types, suggesting that their interaction is physiologically relevant. Moreover, coimmunoprecipitation and pull-down experiments from brain extracts and cells suggest that DGK-, ␥1-syntrophin, and dystrophin form a ternary complex. Collectively, our results suggest that ␥1-syntrophin participates in regulating the subcellular localization of DGK-to ensure correct termination of diacylglycerol signaling.

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