Interaction of γ1-syntrophin with diacylglycerol kinase-ζ -: Regulation of nuclear localization by PDZ interactions

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 gamma1-syntrophin, a recently identified brain-specific isoform, yielded overlapping clones encoding the C terminus of diacylglycerol kinase-zeta (DGK-zeta), an enzyme that converts diacylglycerol into phosphatidic acid, In biochemical assays, the C terminus of DGK-zeta, which contains a consensus PDZ-binding motif, was found to be necessary and sufficient for association with gamma1-syntrophin, When coexpressed in HeLa cells. DGK-zeta and gamma1-syntrophin formed a stable complex that partitioned between the cytoplasm and nucleus. DGK-zeta translocates from the cytosol to the nucleus, a process negatively regulated by protein kinase C phosphorylation, We found that DGK-zeta recruits gamma1-syntrophin into the nucleus and that the PDZ-binding motif is required. Disrupting the interaction altered the intracellular localization of both proteins; DGK-zeta accumulated in the nucleus, whereas gamma1-syntrophin remained in the cytoplasm, The level of endogenous syntrophins in the nucleus of HeLa cells also reflected the amount of nuclear DGK-zeta, In the brain, DGK-zeta and gamma1-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-zeta, gamma1-syntrophin, and dystrophin form a ternary complex. Collectively, our results suggest that gamma1-syntrophin participates in regulating the subcellular localization of DGK-zeta to ensure correct termination of diacylglycerol signaling.