Interaction of gamma 1-syntrophin with diacylglycerol kinase-zeta. 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 gamma 1- 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 gamma 1-syntrophin. When coexpressed in HeLa cells, DGK-zeta and gamma 1-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 gamma 1-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 gamma 1-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 gamma 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-zeta, gamma 1-syntrophin, and dystrophin form a ternary complex. Collectively, our results suggest that gamma 1-syntrophin participates in regulating the subcellular localization of DGK-zeta to ensure correct termination of diacylglycerol signaling.
Hogan
A
A
Department of Cellular and Molecular Medicine, Center for Neuromuscular Disease, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada.
Shepherd
L
L
Chabot
J
J
Quenneville
S
S
Prescott
S M
SM
Topham
M K
MK
Gee
S H
SH
D048868:Adaptor Proteins, Signal Transducing; D002467:Cell Nucleus / Q000378:metabolism; D019852:Diacylglycerol Kinase / Q000235:genetics / Q000378:metabolism*; D049029:Dystrophin-Associated Proteins; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae; D015398:Signal Transduction
D048868:Adaptor Proteins, Signal Transducing; D049029:Dystrophin-Associated Proteins; D008565:Membrane Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; C432243:SNTG1 protein, human; C091824:syntrophin; D019852:Diacylglycerol Kinase