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NCAM induces CaMKIIalpha-mediated RPTPalpha phosphorylation to enhance its catalytic activity and neurite outgrowth.

Bodrikov V, Sytnyk V, Leshchyns'ka I, den Hertog J, Schachner M - J. Cell Biol. (2008)

Bottom Line: NCAM associates with T- and L-type voltage-dependent Ca(2+) channels, and NCAM clustering at the cell surface results in Ca(2+) influx via these channels and activation of NCAM-associated calmodulin-dependent protein kinase IIalpha (CaMKIIalpha).Clustering of NCAM promotes its redistribution to lipid rafts and the formation of a NCAM-RPTPalpha-CaMKIIalpha complex, resulting in serine phosphorylation of RPTPalpha by CaMKIIalpha.Overexpression of RPTPalpha with mutated Ser180 and Ser204 interferes with NCAM-induced neurite outgrowth, which indicates that neurite extension depends on NCAM-induced up-regulation of RPTPalpha activity.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Molekulare Neurobiologie, Universität Hamburg, 20246 Hamburg, Germany.

ABSTRACT
Receptor protein tyrosine phosphatase alpha (RPTPalpha) phosphatase activity is required for intracellular signaling cascades that are activated in motile cells and growing neurites. Little is known, however, about mechanisms that coordinate RPTPalpha activity with cell behavior. We show that clustering of neural cell adhesion molecule (NCAM) at the cell surface is coupled to an increase in serine phosphorylation and phosphatase activity of RPTPalpha. NCAM associates with T- and L-type voltage-dependent Ca(2+) channels, and NCAM clustering at the cell surface results in Ca(2+) influx via these channels and activation of NCAM-associated calmodulin-dependent protein kinase IIalpha (CaMKIIalpha). Clustering of NCAM promotes its redistribution to lipid rafts and the formation of a NCAM-RPTPalpha-CaMKIIalpha complex, resulting in serine phosphorylation of RPTPalpha by CaMKIIalpha. Overexpression of RPTPalpha with mutated Ser180 and Ser204 interferes with NCAM-induced neurite outgrowth, which indicates that neurite extension depends on NCAM-induced up-regulation of RPTPalpha activity. Thus, we reveal a novel function for a cell adhesion molecule in coordination of cell behavior with intracellular phosphatase activity.

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NCAM associates with T- and L-type VDCC. (A) NCAM immunoprecipitates (IP) from NCAM+/+ brain lysates were analyzed by Western blotting with antibodies against NCAM and T- and L-type VDCC. Mock immunoprecipitation with nonspecific IgG served as control. Brain homogenate (BH) is shown for comparison. T- and L-type VDCC coimmunoprecipitate with NCAM. (B) A growth cone of a cultured hippocampal neuron colabeled with antibodies against NCAM, CaMKIIα, and T-type VDCC is shown. Note the colocalization of these three proteins in filopodia. (C) NCAM was clustered at the surface of cultured hippocampal neurons with NCAM monoclonal antibodies (H28live), and neurons were colabeled with antibodies against CaMKIIα and T-type VDCC. A representative neurite is shown. Note that clusters of NCAM overlap with accumulations of T-type VDCC and CaMKIIα (arrows). Bars, 5 μm.
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fig7: NCAM associates with T- and L-type VDCC. (A) NCAM immunoprecipitates (IP) from NCAM+/+ brain lysates were analyzed by Western blotting with antibodies against NCAM and T- and L-type VDCC. Mock immunoprecipitation with nonspecific IgG served as control. Brain homogenate (BH) is shown for comparison. T- and L-type VDCC coimmunoprecipitate with NCAM. (B) A growth cone of a cultured hippocampal neuron colabeled with antibodies against NCAM, CaMKIIα, and T-type VDCC is shown. Note the colocalization of these three proteins in filopodia. (C) NCAM was clustered at the surface of cultured hippocampal neurons with NCAM monoclonal antibodies (H28live), and neurons were colabeled with antibodies against CaMKIIα and T-type VDCC. A representative neurite is shown. Note that clusters of NCAM overlap with accumulations of T-type VDCC and CaMKIIα (arrows). Bars, 5 μm.

Mentions: In agreement, in cultured hippocampal neurons maintained in vitro for 24 h, CaMKIIα accumulated in growth cones of the growing neurites, where distributions of CaMKIIα and NCAM partially overlapped (see Fig. 7 B). Clustering of NCAM at the cell surface of neurites with NCAM antibodies applied to live neurons induced partial redistribution of RPTPα to NCAM clusters (Fig. 3 C; Bodrikov et al., 2005). Overlapping accumulations of NCAM and RPTPα also colocalized with CaMKIIα aggregates (Fig. 3 C). Thus, CaMKIIα is a likely candidate involved in NCAM140-induced RPTPα phosphorylation accompanying NCAM clustering.


NCAM induces CaMKIIalpha-mediated RPTPalpha phosphorylation to enhance its catalytic activity and neurite outgrowth.

Bodrikov V, Sytnyk V, Leshchyns'ka I, den Hertog J, Schachner M - J. Cell Biol. (2008)

NCAM associates with T- and L-type VDCC. (A) NCAM immunoprecipitates (IP) from NCAM+/+ brain lysates were analyzed by Western blotting with antibodies against NCAM and T- and L-type VDCC. Mock immunoprecipitation with nonspecific IgG served as control. Brain homogenate (BH) is shown for comparison. T- and L-type VDCC coimmunoprecipitate with NCAM. (B) A growth cone of a cultured hippocampal neuron colabeled with antibodies against NCAM, CaMKIIα, and T-type VDCC is shown. Note the colocalization of these three proteins in filopodia. (C) NCAM was clustered at the surface of cultured hippocampal neurons with NCAM monoclonal antibodies (H28live), and neurons were colabeled with antibodies against CaMKIIα and T-type VDCC. A representative neurite is shown. Note that clusters of NCAM overlap with accumulations of T-type VDCC and CaMKIIα (arrows). Bars, 5 μm.
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fig7: NCAM associates with T- and L-type VDCC. (A) NCAM immunoprecipitates (IP) from NCAM+/+ brain lysates were analyzed by Western blotting with antibodies against NCAM and T- and L-type VDCC. Mock immunoprecipitation with nonspecific IgG served as control. Brain homogenate (BH) is shown for comparison. T- and L-type VDCC coimmunoprecipitate with NCAM. (B) A growth cone of a cultured hippocampal neuron colabeled with antibodies against NCAM, CaMKIIα, and T-type VDCC is shown. Note the colocalization of these three proteins in filopodia. (C) NCAM was clustered at the surface of cultured hippocampal neurons with NCAM monoclonal antibodies (H28live), and neurons were colabeled with antibodies against CaMKIIα and T-type VDCC. A representative neurite is shown. Note that clusters of NCAM overlap with accumulations of T-type VDCC and CaMKIIα (arrows). Bars, 5 μm.
Mentions: In agreement, in cultured hippocampal neurons maintained in vitro for 24 h, CaMKIIα accumulated in growth cones of the growing neurites, where distributions of CaMKIIα and NCAM partially overlapped (see Fig. 7 B). Clustering of NCAM at the cell surface of neurites with NCAM antibodies applied to live neurons induced partial redistribution of RPTPα to NCAM clusters (Fig. 3 C; Bodrikov et al., 2005). Overlapping accumulations of NCAM and RPTPα also colocalized with CaMKIIα aggregates (Fig. 3 C). Thus, CaMKIIα is a likely candidate involved in NCAM140-induced RPTPα phosphorylation accompanying NCAM clustering.

Bottom Line: NCAM associates with T- and L-type voltage-dependent Ca(2+) channels, and NCAM clustering at the cell surface results in Ca(2+) influx via these channels and activation of NCAM-associated calmodulin-dependent protein kinase IIalpha (CaMKIIalpha).Clustering of NCAM promotes its redistribution to lipid rafts and the formation of a NCAM-RPTPalpha-CaMKIIalpha complex, resulting in serine phosphorylation of RPTPalpha by CaMKIIalpha.Overexpression of RPTPalpha with mutated Ser180 and Ser204 interferes with NCAM-induced neurite outgrowth, which indicates that neurite extension depends on NCAM-induced up-regulation of RPTPalpha activity.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Molekulare Neurobiologie, Universität Hamburg, 20246 Hamburg, Germany.

ABSTRACT
Receptor protein tyrosine phosphatase alpha (RPTPalpha) phosphatase activity is required for intracellular signaling cascades that are activated in motile cells and growing neurites. Little is known, however, about mechanisms that coordinate RPTPalpha activity with cell behavior. We show that clustering of neural cell adhesion molecule (NCAM) at the cell surface is coupled to an increase in serine phosphorylation and phosphatase activity of RPTPalpha. NCAM associates with T- and L-type voltage-dependent Ca(2+) channels, and NCAM clustering at the cell surface results in Ca(2+) influx via these channels and activation of NCAM-associated calmodulin-dependent protein kinase IIalpha (CaMKIIalpha). Clustering of NCAM promotes its redistribution to lipid rafts and the formation of a NCAM-RPTPalpha-CaMKIIalpha complex, resulting in serine phosphorylation of RPTPalpha by CaMKIIalpha. Overexpression of RPTPalpha with mutated Ser180 and Ser204 interferes with NCAM-induced neurite outgrowth, which indicates that neurite extension depends on NCAM-induced up-regulation of RPTPalpha activity. Thus, we reveal a novel function for a cell adhesion molecule in coordination of cell behavior with intracellular phosphatase activity.

Show MeSH
Related in: MedlinePlus