MAP-2a and MAP-2b are high-molecular-weight neuronal microtubule- associated proteins that interact with dendritic microtubules (MTs), and MAP-2c is a corresponding lower-molecular-weight MAP abundant in developing axons. Both proteins share highly conserved N-terminal and C-terminal regions of about 150 and 310 amino acids, respectively, with the latter containing the three 18-amino-acid repeated sequences thought to constitute the MT-binding domain. We propose to further define microtubule binding interactions of the MT-binding fragment, relying on new procedures for preparing the fragment and building on our studies of the three non-identical repeated amino-acid regions. We also plan to characterize m2-peptide displacement of intact MAP-2 from assembled microtubules. To define the amino acid side-chains responsible for binding of peptide-m2 to microtubules and to assess the minimal sequence needed to promote tubule assembly and/or MAP-2 displacement. We plan to study microtubule length redistribution kinetics of microtubules assembled from pure tubulin in the absence or presence of the peptide-m2. We propose to complete the determination of the amino acid sequence of the bovine MT-binding fragment using PCR-derived bovine brain cDNA clones. In a parallel effort, we will investigate phosphorylation of tubule-binding fragment of bovine MAP-2, including: (a) evaluation of the affinity of phosphorylated-tubule-binding fragment for microtubules with the peptide displacement assay; (b) characterization of m2-peptide phosphorylation effects using direct synthesis of m2 analogues containing p-Ser and p-Thr residues; and (c) localization of phosphorylated sites in the MT-binding fragment using enzymatic and chemical cleavage, followed by the diagonal electrophoresis with intervening alkaline phosphatase treatment. Finally, we will carry out microinjection experiments using the MT-binding fragment and synthetic peptides to investigate their efficacy in displacing MAP-2 and tau proteins, and their action in altering cellular distribution of MAP-2 and tau, as well as any changes in cell morphology.
