No abstract

The neural cell adhesion molecule (NCAM) plays a fundamental role during development and regeneration. NCAM is expressed in three major isoforms, two of them with intracellular domains of different length and one without any intracellular domain. The cytoplasmic domain of NCAM contains, depending on the isoform, up to 49 phosphorylation sites, and it has been demonstrated previously by phosphoproteomic analysis that NCAM is phosphorylated on serine 774. However, the impact of NCAM phosphorylation is unclear. Here we have analyzed the phosphorylation of serine 774 in more detail and found that phosphorylation of this site is crucial for NCAM-mediated signal transduction. A serine-to-alanine exchange at position 774 (NCAM140-S774A) resulted in decreased activation of the cAMP response element binding protein (CREB) after NCAM stimulation and, as a consequence, in decreased neurite outgrowth of NCAM140-S774A-transfected B35 neuroblastoma cells.

No abstract

Collapsin response mediator proteins (CRMPs) are involved in cell differentiation and axonal growth. In this study, we investigated neurite outgrowth and the expression of CRMP-2 in PC12 cells. Nondifferentiated PC12 cells hardly express CRMP-2, but the expression of CRMP-2 increases with neurite outgrowth. We established a stable CRMP-2-overexpressing PC12 cell line and found that PC12 cells, which constitutively overexpress CRMP-2, were unable to extend neurites after stimulation with the nerve growth factor or the neural cell adhesion molecule, a procedure that promotes neurite outgrowth in untransfected cells. In contrast, a PC12 cell line deficient in CRMP-2 extends neurites after the stimulation of nerve growth factor or neural cell adhesion molecule.

The neural cell adhesion molecule NCAM is a major adhesion receptor involved in the development and regeneration of the nervous system. It is expressed in three major isoforms of which two have large intracellular domains of different lengths (NCAM140 and NCAM180). Several intracellular ligands of NCAM have been described. One of them is the collapsin response mediator protein-2 (CRMP-2), which is known to be involved in cell differentiation and axonal growth. The cytoplasmic domains of NCAM contain up to 49 phosphorylation sites and it has been demonstrated recently that the phosphorylation of serine 774 is crucial for NCAM-mediated signal transduction and neurite outgrowth. Here we analyzed the interaction of NCAM with CRMP-2 in more detail using a biochemical approach. We found that CRMP-2 binds specifically to NCAM180 in a sequence between amino acid 788 and 819. In addition we could demonstrate that serine 774, which has been shown previously to be phosphorylated and involved in neurite outgrowth, is also important for the interaction of CRMP-2 with NCAM.