Interaction of Neuronal Nitric-oxide Synthase and Phosphofructokinase-M

Abstract

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.