Microtubule-associated protein-2 located in growth regions of rat hippocampal neurons is highly phosphorylated at its proline-rich region

Abstract

Neuronal morphogenesis is regulated, among other factors, by microtubule-associated proteins (MAPs). A family of these proteins, MAP2, which is very abundant in the mammalian nervous system, has been associated with the formation of neurites at early developmental stages and with the dendritic scaffold upon maturation. The function of MAP2 is regulated by its phosphorylation state. One of the phosphorylation sites that has been described is located in the proline-rich region of the protein. It comprises of the residues 1616–1626 and is specifically recognized by the antibody 305. However, little is known about the functional consequences of its modification in vivo. To gain insight into this, we have analysed the expression levels and intracellular distribution of MAP2 phosphorylated at this site (MAP2-P), in primary cultures of rat hippocampal neurons at different developmental stages. Western blot analysis of hippocampal neuron protein extracts revealed that the ratio of MAP2-P:MAP2 was 4:1 at early developmental stages and became 1:4 at later developmental stages, suggesting a role of such phosphorylated forms of the protein in neuritogenesis. Consistent with this view, immunofluorescence microscopy analysis showed that the ratio MAP2-P:MAP2 was 2 in the neurite growth cones, sites where net elongation takes place. A higher presence of phosphorylated MAP2 was observed in growth regions with higher levels of microfilaments, which may be related with the growth region stability. Indeed, when growth-cone collapse was induced in hippocampal neurons after cytochalasin D treatment, which depolymerizes microfilaments, the ratio MAP2-P:MAP2 in these growing regions decreased down to 1. Finally, acceleration of neuronal maturation induced by the activation of glutamate-receptors triggered a dramatic decrease in the phosphorylation of MAP2 at the site recognized by antibody 305. From these results we suggest that the phosphorylation of MAP2 at its proline-rich region is an important event during neuritogenesis.