USE OF A 2-HYBRID SYSTEM TO INVESTIGATE MOLECULAR-INTERACTIONS OF GAP-43

We used the 'interaction trap' (two-hybrid system) to identify polypep tides that interact with the neuronal phosphoprotein, GAP-43, in an in tracellular environment. GAP-43 (neuromodulin, B-50, F1), a protein ki nase C (PKC) substrate important for the growth and plasticity of neur onal connections, has been implicated in vitro in several signal trans duction pathways. In the yeast-based cloning system, the only strong i nteraction that was detected was between GAP-43 and the calcium effect or protein, calmodulin (CaM), PKC phosphorylates GAP-43 on serine 41. Where we changed this serine to an aspartate residue to mimic constitu tive phosphorylation, the interaction with CaM was blocked. Surprising ly, the N-terminal third of GAP-43 alone bound CaM more strongly than did intact GAP-43, suggesting that the protein's C-terminus may play a role in modulating the interaction with CaM. These results, along wit h other recent findings, suggest a novel role for the interaction betw een GAP-43 and CaM.