Identification of an amino acid sequence motif in the cytoplasmic domain of the NCAM-140 kDa isoform essential for its neuritogenic activity.
The functions of the extracellular domains of neural cell adhesion molecule (NCAM) have been studied extensively, whereas the roles of the cytoplasmic domains of the transmembrane forms of NCAM are less elucidated. We investigated the importance of the cytoplasmic domain of the 140-kDa NCAM isoform (cytNCAM-140) and of the 180-kDa NCAM isoform (cytNCAM-180) in NCAM-induced neurite extension by estimating NCAM-dependent neurite outgrowth from PC12-E2 cells grown in coculture with NCAM-negative or NCAM-positive fibroblasts. PC12-E2 cells were transiently transfected with expression plasmids encoding cytNCAM-140, cytNCAM-180, the constitutively active form of the mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase kinase (MEK2), and the enhanced variant of the green fluorescent protein (EGFP). EGFP expression was used for identification of transfected cells. We found that expression of cytNCAM-180 had no effect on NCAM-stimulated neuritogenesis, whereas expression of cytNCAM-140 strongly inhibited this process. However, if MEK2 was expressed concomitantly with cytNCAM-140, neurite outgrowth was rescued, indicating that cytNCAM-140 is involved in signaling via the Ras-MAP kinase pathway. PC12-E2 cells were subsequently transiently transfected with constructs encoding a series of fragments of cytNCAM-140 and various full-length cytNCAM-140 mutants, and the residues Thr-Glu-Val-Lys-Thr (839-843) were identified as essential in NCAM-stimulated neuritogenesis. The combined substitution of Glu(840) and Lys(842) with Ala abrogated the effect of the construct, assigning a critical role to these two residues.
Kolkova
K
K
Protein Laboratory, Institute of Molecular Pathology, University of Copenhagen, Denmark.
Pedersen
N
N
Berezin
V
V
Bock
E
E
D000595:Amino Acid Sequence; D000818:Animals; D016384:Consensus Sequence; D003593:Cytoplasm / Q000378:metabolism; D005910:Glioma; D048370:MAP Kinase Kinase 2; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D016296:Mutagenesis; D016501:Neurites / Q000502:physiology*; D016716:PC12 Cells; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured
D020033:Protein Isoforms; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D048370:MAP Kinase Kinase 2; D020929:Mitogen-Activated Protein Kinase Kinases