MOLECULAR BIOLOGY OF NEURAL FUNCTION

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We are interested in proteins that facilitate the transport of synaptic vesicles along axons and subsequent fusion at the pre-synaptic membrane. Included among these are intracellular motors, kinases, and cytoskeletal proteins. Proteins currently being studied in the lab include: kinesin light chains (klc), nsec-1, and acidic calponin (AC). Previously, we identified a family of klc from the nervous system of the squid in an effort to determine its role in the kinesin function. It is unclear whether klc plays a regulatory role in kinesin function or a targeting role to membrane bound organelles. We reported that at least 18 different isoforms arose as a result of alternative splicing occurring almost exclusively in the carboxyl terminal domain of the molecule thought to be the sight for kinesin targeting to membrane bound organelles. Furthermore, we found that klc transcript number and levels differed in various tissues examined further supporting the hypothesis that klc provides a targeting function to different membrane bound organelles in disparate cell types. Recently, however, we reported the identification and characterization of klc in a eubacterium, Plectonema boryanum, and failed to identify a corresponding heavy chain suggesting that klc may have some function unrelated to vesicle transport. Acidic calponin is an actin-binding protein with an unique carboxyl terminal tail domain endowed with at least three regulatory domains: a consensus tyrosine phosphorylation site, two putative P-loop elements, and the entire tail is a PEST domain. In the past year, we have focused on the biochemical characterization of C- terminal tail domain in an effort to determine if and how modification affects AC function and how this, in turn, relates to its role in neurite outgrowth. In vitro, AC is a substrate for tyrosine phosphorylation by src which has been shown to play a crucial role in neurite outgrowth. AC immunoprecipitated from neural cells treated with NGF is also phosphotyrosinated. In an assay to determine if AC inhibited Mg-dependent ATPase activity (AMA) like the well-characterized basic calponin isoform, we observed that AC stimulated it, instead. Additional experiments designed to address the effects of AC on actin polymerization have shown that AC stimulates actin polymerization and the formation of actin bundles. Similar assays that exclude AC but include src have shown that actin polymerization is impeded. This inhibition by src is completely reversible by the addition of AC. These results indicate that tyrosine phosphorylation of AC is not required for the stimulation of AMA or actin polymerization. We have observed in vitro that AC is abundant in the growth cones of very young neurons and that levels diminish as neurons terminally differentiate. We have also shown that AC turnover is mediated by the ubiquitin proteolytic pathway. Perhaps tyrosine phosphorylation of AC either targets or protects the protein from proteolytic degradation. Either way, it is clear that AC is an important molecule involved in the dynamics of actin polymerization. Our goal is to elucidate the role it plays in the growth cones of developing neurons.