Protein components of the neuronal cytoskeleton are fundamentally
important for architectural support and organelle transport in axons and dendrites.
Among them, microtubules (MTs) and microtubule-associated proteins (MAPs) are
major components involved in neuronal differentiation and neurodegenerative
diseases. Neuronal MTs are relatively stable, yet they are not static structures. A
fine-tuned balance in MT stability is required to assure proper plasticity of neuronal
processes. MAPs are key players in regulating the stability of MTs, and MAP
function is in turn regulated by phosphorylation. It was found in this study that a
dramatic increase in MAP1B phosphorylation occurred as PC12 cells developed
neurites in response to nerve growth factor. The phosphorylation of MAP1B was
identified by a monoclonal antibody MPM2, which identifies a mitosis-associated
phosphoepitope. The increase in MPM2 reactivity of MAP1B was also observed in
synchronized mitotic PC12 cells, suggesting that related kinase activities are
involved in pathways inducing cell division and the differentiation of postmitotic
neuronal cells. MPM2-reactive MAP1B was localized to neurites of PC12 cells, but
not cell bodies by indirect immunofluorescence and immunogold electron
microscopy. In order to understand the functional significance of the
phosphorylation of MAP1B, the identification of the MPM2 epitope was required.
Through proteolytic digestion and peptide microsequencing the epitope was shown
to be located in the N-terminal 40 kD of MAP1B. A potential phosphothreonine site
fitting a model of known MPM2 epitopes was identified, and peptides containing
these sequences were synthesized in both phosphorylated and dephosphorylated
forms. The selected phosphopeptide was highly reactive with the MPM2 antibody,
while the dephosphopeptide was not. Furthermore, the phosphopeptide had the
ability to compete for MPM2 antibody binding with both MAP1B and other
phosphoproteins in mitotic cells. These results indicate that the phosphopeptide
had all of the elements required for MPM2 antibody recognition. A phosphorylation
state-specific antibody PMB1 was generated by using the phosphopeptide
conjugated to keyhole limpet hemocyanin as immunogen. The purified antibody was
shown to be specific to phosphorylated MAP1B. The phosphorylation site and
antibody probe described here are expected to assist in identifying the kinase
responsible for the modification on MAP1B and understanding the functional
significance of this modification in neurogenesis.