Binding of neuronal nitric-oxide synthase (nNOS) to carboxyl-terminal-binding protein (CtBP) changes the localization of CtBP from the nucleus to the cytosol: a novel function for targeting by the PDZ domain of nNOS.
Recent work suggests a role for PDZ domains in the targeting of binding partners to specific sites in the cell. To identify whether the PDZ domain of neuronal nitric-oxide synthase (nNOS) can play such a role, we performed affinity chromatography of brain extract with the nNOS PDZ domain. We identified the carboxyl-terminal-binding protein (CtBP), a phosphoprotein first identified as a binding partner to adenovirus E1A, as a nNOS binding partner. CtBP interacts with the PDZ domain of nNOS, and this interaction can be competed with peptide that binds to the PDZ peptide-binding site. In addition, binding of CtBP to nNOS is dependent on its carboxyl-terminal sequence -DXL, residues conserved between species that fit the canonical sequence for nNOS PDZ binding. Immunoprecipitation studies show that CtBP and nNOS associate in the brain. When CtBP is expressed in Madin-Darby canine kidney cells, its distribution is primarily nuclear; however, when CtBP is co-expressed with nNOS, its localization becomes more cytosolic. This change in CtBP localization does not occur when its carboxyl-terminal nNOS PDZ binding motif is mutated or when CtBP is co-expressed with postsynaptic density 95, another PDZ domain-containing protein. Taken together, our data suggest a new function for nNOS as a regulator of CtBP nuclear localization.
Riefler
G M
GM
Department of Cell Biology and Neuroscience, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854-8082, USA.
Firestein
B L
BL
D000429:Alcohol Oxidoreductases; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002846:Chromatography, Affinity; D003600:Cytosol / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004285:Dogs; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010750:Phosphoproteins / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats
D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D000429:Alcohol Oxidoreductases; C096794:C-terminal binding protein; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat