This project contains two specific goals: 1) To study the cell biological proteins; and 2) to test the hypothesis that axonal protein synthesis occurs in the squid giant axon. For the latter study, we generated specific antibodies and cDNA probes for squid neurofilament (NF) proteins to study protein synthesis in stellate ganglia (cell bodies) and in axons. Protein biosynthesis/immunoprecipitation experiments confirmed robust biosynthesis of NF proteins in squid stellate ganglia, but we failed to detect any NF protein biosynthesis in the giant axon. This is in the face of our findings of significant NF protein mRNA in the squid axon. In the other study, we have examined the dynamic equilibrium of phosphorylation/dephosphorylation reactions mediated by multimeric complexes of kinases and phosphatases, their regulators and inhibitors. We believe these complexes are compartmentalized in axons and cell bodies and suggest that they play an important role in assembly and transport of cytoskeletal structures and organelles. In the neuronal cell bodies, there is a delicate balance between factors regulating the activity of kinases and phosphatases that modulate the incomplete phosphorylation (e.g., head domains only in NFs) of NFs and tau prior to their assembly and transport into the axon. In axons, NFs are further phosphorylated (in tail domains) and organize into a lattice that stabilizes the above hypothesis, we used P13suc1 sepharose-conjugated beads to extract the kinases that phosphorylate neurofilaments in the axoplasm from the squid giant axon. Using Western blots and in vitro kinase assays, we demonstrated the presence of an active cdc2-like kinase and its putative regulators such as cyclin E, P13 homologue and P67 in axoplasm and a (CK) I and II were also found in the P13-Ax. Western blot analysis of neurofilaments (NFs; NF 60, 70 and 220), tubulin, actin and microtubule associated proteins. NF 220 and tubulin were phosphorylated by the kinases in the P13-Ax. To determine whether NFs bound directly to the P13 beads, or bound indirectly by association with cdc2 kinase, a washed, tubulin, but no cdc2-like kinase, yielded no bound proteins after incubation with P13suc1. The wash supernatant from the neurofilament preparation, however, containing the cdc2-like kinase, did yield cytoskeletal components that bound to P13suc1. Moreover, a bacterial- expressed cdk5 associated with P13 beads, was able to complex with selected cytoskeletal components in the washed neurofilament preparation. These data indicate that direct binding of P13 beads with a cdc2-like kinase could extract active multimeric complexes composed of axonal cytoskeletal proteins and kinases.
