NAP-22 (CAP-23, BASP1) is a neuron-enriched membrane protein localized in the presynaptic membrane regions such as the synaptic plasma membrane and the synaptic vesicles.
This protein is also a major component of the neuronal lipid raft.
Recent studies however showed that some amount of NAP-22 is present in the cytoplasm and in the nucleus.
In order to know NAP-22 interacting proteins in the soluble fraction of rat brain, a pull-down assay using anti-NAP-22 antibody bound to Sepharose-beads charged with NAP-22 was performed and calcineurin (CaN) was identified.
CaN is a Ca²⁺-calmodulin dependent Ser/Thr-phosphatase and shows broad distribution within neurons.
The participation of CaN in the process of the synaptic plasticity is well studied and the protective effects of CaN on neurons in Alzheimer's disease are well recognized.
Identification of the CaN binding proteins is hence of primary importance.
Previously, we reported that bacterially expressed CaN and brain-derived NAP-22 bound in vitro and co-localized at the presynaptic region of cultured neuron.
Furthermore, NAP-22 decreased the phosphatase activity of CaN on artificial substrate, pNPP.
On the other hand, bacterially expressed NAP-22 did not bind to CaN in vitro and did not affect the phosphatase activity of CaN.
During attempts to solve this inconsistency, we found that brain-derived NAP-22 fraction contains several lipids and these lipid fractions have an inhibitory effect on CaN phosphatase activity.
Several high polarity lipid species were found in the lipid fraction with HPTLC and these lipids affected CaN phosphatase activity.
This time, we report about identification of lipid species which affect CaN activity and analyzing interaction among NAP-22, CaN and lipid.