Research will be conducted on the pathway and mechanism of assembly of mammalian neurofilaments from their constituent proteins and the role of phosphorylation in regulating the assembly process and the properties of the filaments. Neurofilaments (NL) are those members of the family of intermediate filaments which form part of the cytoskeleton in neurons of the central nervous system. They are composed of three """"""""NF triplet"""""""" proteins, termed NF-H(high), NF-M(middle) and NF-L(low) according to their molecular weights. A number of diseases of motor neurons involve abnormal NF, and overexpression of either NF-L or NF-H in transgenic mice leads to pathology very similar to amyotrophic lateral sclerosis. In addition, in this and other neuropathologies abnormally phosphorylated NF accumulate in different parts of the neuron. The goals of the project are: 1) to gain information about the arrangement of the three proteins in NF, their structural roles, and the pathway by which they assemble into filaments and by which they become incorporated into preexisting filaments. Early intermediates in NF assembly which have been demonstrated by """"""""native gel"""""""" electrophoresis will be further characterized by electron microscopy and cross-linking experiments. Proteins produced by recombinant DNA techniques, which combine in a single polypeptide chain domains derived from the different NF proteins, will be used to determine the nature of the specific chemical interactions which determine the rules by which the three proteins combine. 2) to investigate the role of phosphorylation in regulating assembly and in molecular conformation of the proteins. The effect of phosphorylation of the head domains of NF-M and NF-H on assembly will be determined by adding phosphates with the use of enzymes known to phosphorylate these domains and then conducting centrifugation experiments to examine the ability of the phosphorylated proteins to form pelletable filaments. The effect of tail domain phosphorylation on protein conformation will be investigated by using kinase and phosphatase enzymes to prepare NF in different states of phosphorylation and then to measure their circular dichroism and infrared spectra. 3) to gain information on the protein kinase enzymes which regulate NF phosphorylation. Protein kinases which catalyze NF phosphorylation will be identified by first separating neuronal extracts in gels in which NF proteins and fragments of NF proteins are entrapped, and then looking at the ability of the protein bands separated in the gel to incorporate radioactive phosphate into the entrapped NF proteins when the gel is incubated with radioactive ALP, the source of phosphate int eh phosphorylation reaction. Finally, the action of the enzyme glycogen synthase kinase 3, shown in previous work of phosphorylate NF in vitro, will be further characterized.
