Interaction of neuronal nitric-oxide synthase and phosphofructokinase-M.
Neurons that express neuronal nitric-oxide synthase (nNOS) are resistant to NO-induced neurotoxicity; however, the mechanism by which these neurons are protected is not clear. To identify proteins possibly involved in this process, we performed affinity chromatography with the nNOS PDZ domain, a N-terminal motif that mediates protein interactions. Using this method to fractionate soluble tissue extracts, we identified the muscle isoform of phosphofructokinase (PFK-M) as a protein that binds to nNOS both in brain and skeletal muscle. PFK-M interacts with the PDZ domain of nNOS, and nNOS-PFK-M binding can be competed by peptides that bind to the PDZ domain of nNOS. We found that nNOS is significantly associated with PFK-M in skeletal muscle because nNOS can be immunodepleted from cytosolic skeletal muscle extracts using an antibody directed against PFK-M. In brain, nNOS and PFK-M are both enriched in synaptosomes, and specifically, in the synaptic vesicle fraction, where they can interact. At the cellular level, PFK-M is enriched in neurons that express nNOS protein. As fructose-1, 6-bisphosphate, the product of PFK activity, is neuroprotective, the interaction of nNOS and PFK may contribute to neuroprotection of nNOS positive cells.
Firestein
B L
BL
Department of Physiology, UCSF, San Francisco, California 94143-0444, USA. bonfire@itsa.ucsf.edu
Bredt
D S
DS
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology; D002384:Catalysis; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D009474:Neurons / Q000201:enzymology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats
D007527:Isoenzymes; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D010732:Phosphofructokinase-1