ABSTRACT
The neural cell adhesion molecule (NCAM) is the major carrier of polysialic acid (PSA) which modulates NCAM functions of neural cells at the cell surface. In previous studies, we have shown that stimulation of cultured neurons with surrogate NCAM ligands leads to the generation and nuclear import of PSA-lacking and -carrying NCAM fragments. Here, we show that the nuclear import of the PSA-carrying NCAM fragment is mediated by positive cofactor 4 and cofilin, which we identified as novel PSA-binding proteins. In the nucleus, the PSA-carrying NCAM fragment interacts via PSA with PC4 and cofilin, which are involved in RNA polymerase II-dependent transcription. Microarray analysis revealed that the nuclear PSA-carrying and -lacking NCAM fragments affect expression of different genes. By qPCR and immunoblot analysis we verified that the nuclear PSA-carrying NCAM fragment increases mRNA and protein expression of nuclear receptor subfamily 2 group F member 6, whereas the PSA-lacking NCAM fragment increases mRNA and protein expression of low density lipoprotein receptor-related protein 2 and α-synuclein. Differential gene expression evoked by nuclear NCAM fragments without and with PSA indicates that PSA-carrying and -lacking NCAM play different functional roles in the nervous system.
Subject(s)
Neural Cell Adhesion Molecules/metabolism , Sialic Acids/metabolism , Actin Depolymerizing Factors/chemistry , Actin Depolymerizing Factors/metabolism , Animals , Cell Nucleus/metabolism , Cells, Cultured , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Female , Gene Expression Regulation , Low Density Lipoprotein Receptor-Related Protein-2/genetics , Low Density Lipoprotein Receptor-Related Protein-2/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neural Cell Adhesion Molecules/deficiency , Neural Cell Adhesion Molecules/genetics , Neurons/cytology , Neurons/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Protein Binding , Transcription Factors , alpha-Synuclein/genetics , alpha-Synuclein/metabolismABSTRACT
Polysialic acid (PSA) and its major protein carrier, the neural cell adhesion molecule NCAM, play important roles in many nervous system functions during development and in adulthood. Here, we show that a PSA-carrying NCAM fragment is generated at the plasma membrane by matrix metalloproteases and transferred to the cell nucleus via endosomes and the cytoplasm. Generation and nuclear import of this fragment in cultured cerebellar neurons is induced by a function-triggering NCAM antibody and a peptide comprising the effector domain (ED) of myristoylated alanine-rich C kinase substrate (MARCKS) which interacts with PSA within the plane of the plasma membrane. These treatments lead to activation of the fibroblast growth factor (FGF) receptor, phospholipase C (PLC), protein kinase C (PKC) and phosphoinositide-3-kinase (PI3K), and subsequently to phosphorylation of MARCKS. Moreover, the NCAM antibody triggers calmodulin-dependent activation of nitric oxide synthase, nitric oxide (NO) production, NO-dependent S-nitrosylation of matrix metalloprotease 9 (MMP9) as well as activation of matrix metalloprotease 2 (MMP2) and MMP9, whereas the ED peptide activates phospholipase D (PLD) and MMP2, but not MMP9. These results indicate that the nuclear PSA-carrying NCAM fragment is generated by distinct and functionally defined signal transducing mechanisms.
Subject(s)
Cell Nucleus/metabolism , Cytoplasm/metabolism , Neural Cell Adhesion Molecules/metabolism , Peptide Fragments/metabolism , Sialic Acids/metabolism , Active Transport, Cell Nucleus , Animals , Antibodies/immunology , Antibodies/pharmacology , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , Female , Male , Mice, Inbred C57BL , Mice, Knockout , Myristoylated Alanine-Rich C Kinase Substrate/metabolism , Neural Cell Adhesion Molecules/genetics , Neural Cell Adhesion Molecules/immunology , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Phosphorylation/drug effectsABSTRACT
In the mammalian nervous system, the neural cell adhesion molecule NCAM is the major carrier of the glycan polymer polysialic acid (PSA) which confers important functions to NCAM's protein backbone. PSA attached to NCAM contributes not only to cell migration, neuritogenesis, synaptic plasticity, and behavior, but also to regulation of the circadian rhythm by yet unknown molecular mechanisms. Here, we show that a PSA-carrying transmembrane NCAM fragment enters the nucleus after stimulation of cultured neurons with surrogate NCAM ligands, a phenomenon that depends on the circadian rhythm. Enhanced nuclear import of the PSA-carrying NCAM fragment is associated with altered expression of clock-related genes, as shown by analysis of cultured neuronal cells deprived of PSA by specific enzymatic removal. In vivo, levels of nuclear PSA in different mouse brain regions depend on the circadian rhythm and clock-related gene expression in suprachiasmatic nucleus and cerebellum is affected by the presence of PSA-carrying NCAM in the cell nucleus. Our conceptually novel observations reveal that PSA attached to a transmembrane proteolytic NCAM fragment containing part of the extracellular domain enters the cell nucleus, where PSA-carrying NCAM contributes to the regulation of clock-related gene expression and of the circadian rhythm.
