The proposed research focuses on the posttranslational glutamylation and phosphorylation of mammalian brain tubulin. Very little is known regarding the function(s) of these covalent modifications. The fact that the glutamylated and phosphorylated residues are located within the carboxy terminal isotype-defining domains of alpha-tubulin and beta-tubulin suggests that these modifications have an essential role in regulating the assembly and disassembly of microtubules. Research initiated during the previous funding period indicates: a) that glutamylation is unique to tubulin, b) that glutamylation is the major covalent modification of brain tubulin c) that alpha-tubulin glutamylation occurs prior to beta-tubulin glutamylation during brain development, d) that glutamylation is associated predominantly with neuronal tubulin, e) that a microtubule-associated tyrosine kinase specifically phosphorylates the neuron-specific class III beta-tubulin isotype (betaIII) and its associated alpha tubulin subunit and f) that a microtubule-associated serine kinase specifically phosphorylates betaIII. To learn more about the complex regulation and function of tubulin glutamylation and phosphorylation, we propose: 1) To continue our characterization of tubulin glutamylation and phosphorylation by tandem mass spectrometry. We will: a) extend our characterization of changes in betaIII isoform composition during postnatal and embryonic brain development, b) localize the glutamylation sites in classes I (betaI), IV (betaIV), and V (betaV) beta-tubulin, c) determine the nature of the linkage connecting the glutamate residues in the polyglutamyl side chains, d) localize the glutamylation sites in three alpha-tubulin isotypes, m-alpha3, m- alpha4, m-alpha6, and e) determine whether specific alpha-tubulin and beta-tubulin subunits preferentially combine if forming tubulin dimers. 2) To use our recently produced antibodies against glutamylated tubulin and phosphorylated betaIII to immunocytochemically localize these posttranslation modifications in early and late neural development. 3) To purify and characterize: a) the enzyme(s) which glutamylate alpha-tubulin and beta-tubulin, b) the microtubule-associated tyrosine kinase which phosphorylates betaIII and its associated alpha-tubulin subunit, and c) the microtubule-associated serine kinase which phosphorylates betaIII exclusively. 4) To perform in vitro polymerization assays to determine how glutamylation and phosphorylation effect the interaction of betaIII with microtubule-associated proteins and divalent cations.