Changes in levels of microtubule-associated proteins in relation to the outgrowth of neurites from PC12D cells, a forskolin- and nerve growth factor-responsive subline of PC12 pheochromocytoma cells

Abstract

Immunoblotting analysis and immunofluorescence studies of proteins that react with MAP1- and MAP2-specific antibodies in PC12 rat pheochromocytoma cells were carried out. When cells of the PC12D subline of PC12 cells, which rapidly extend neurites in response to NGF or drugs that elevate intracellular levels of cyclic AMP, were examined, they were found to contain a relatively higher level of MAP1 or of a MAP1-like protein than conventional PC12 cells. Immunoblotting study showed that levels of MAP1 and MAP2 or of MAP1 or MAP2-like proteins increased in PC12D cells, but not in conventional PC12 cells, in response to forskolin. Immunofluorescence studies also revealed increases in levels of MAP1 and MAP2 or of MAP1 or MAP2-like proteins in conjunction with the outgrowth of neurites from the cells. These results support the hypothesis that the induction of MAPs may be one of the first steps required for outgrowth of neurites from PC12 cells. Furthermore, PC12D cells may contain a sufficiently high level of MAP1 or MAP1-like protein to permit the extension of neurites in the absence of the lag period normally required by PC12 cells. The MAP1 or a MAP1-like protein was localized in the cell soma and neurites. An increase of MAP2-specific immunoreactivity in perikarya was observed in the differentiated cells. After immunostaining with a monoclonal antibody that reacted with phosphorylated MAP1, intense fluorescence was seen in the growth cones of neurites. This observation supports the hypothesis that the phosphorylation of MAP1 or of a MAP1-like protein may play a regulatory role in the formation of neurites in growth cones.