Neurons are polarized cells having extended processes, the neurites, which radiate from the cell body. The elaboration of these neuritic processes is a characteristic event associated with neuronal differentiation. Both the elaboration and maintenance of neurites involves changes in the dynamics of microtubules, which are a component of the neuronal cytoskeleton. A heterogeneous group of proteins, the microtubule associated proteins (MAPs) function in part to regulate the assembly and stability of microtubules. In turn, the functional properties of the MAPs appear to be regulated by their state of phosphorylation. Using the rat PC12 pheochromocytoma cell line as a model for neuronal differentiation, neuritic outgrowth has been shown to be associated with the synthesis and phosphorylation of two MAPs, tau and MAP 1B. Phosphorylated forms of MAP 1B are also associated with developing or regenerating axons in the brain, and have recently been identified as a component of neurofibrillary tangles associated with Alzheimer disease. The phosphorylation of MAPs will be examined using a series of clonal variants of the PC12 cell line, and correlated with the different morphological responses observed in these variants as a result of nerve growth factor induced differentiation. The MAPs phosphorylated during neurite outgrowth will be characterized by immunoblotting, and compared to previously identified MAPs phosphorylated during mitosis. Preliminary results indicate that an epitope phosphorylated during mitosis is related to an epitope phosphorylated during neurite outgrowth on MAP 1B. Thus, the biochemical nature of this shared phosphorylated epitope will be determined using MAP 1B as a model. The kinase responsible for the phosphorylation of the shared epitope on MAP 1B will be characterized in differentiating PC12 cells as well as neonatal rat brain tissue. Once identified, the presence of related kinase activities will be examined in mitotic cells and adult brain tissue. Efforts to determine the functional consequences of MAP 1B phosphorylation will focus on alterations in microtubule dynamics which result from changes in the phosphorylation state of MAP 1B. The regulation of MAP 1B phosphorylation may play an important role in the regenerative capacity of axons, the differentiative state of neuroblastoma cells, and the etiology of Alzheimer disease. Thus, the characterization of specific MAP 1B phosphorylation sites and the enzymes responsible for regulating their state of phosphorylation will be important in elucidating mechanisms involved in controlling neuronal morphogenesis.
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