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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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RPTPα is phosphorylated by CaMKIIα on Ser180 and Ser204. (A) Recombinant RPTPα-ID-GST or GST coupled to beads were incubated with CaMKIIα. The gel stained with Coomassie blue shows that approximately equal amounts of RPTPα-ID-GST and GST were bound to beads. The beads, treated and nontreated with CaMKIIα, were then subjected to the analysis of serine phosphorylation by alkaline hydrolysis (bottom left) or phosphatase activity (bottom right). Values for GST were subtracted as background, and values of serine phosphorylation (bottom left) or phosphatase activity (bottom right) for RPTPα-ID-GST not treated with CaMKIIα were set to 100%. (B) RPTPα from NCAM−/− brain lysates was incubated with CaMKIIα in the presence or absence of KN62 and Bis1. RPTPα was then subjected to the analysis of serine phosphorylation by alkaline hydrolysis. Values of phosphate released by alkaline from RPTPα not treated with CaMKIIα were set to 100%. (C) RPTPα immunoprecipitated from lysates of RPTPα-negative fibroblasts transfected with RPTPαWT, RPTPαS180A, RPTPαS204A, or RPTPαS180/204A was incubated with CaMKIIα. The immunoprecipitates, treated and untreated with CaMKIIα, were then subjected to the analysis of serine phosphorylation by alkaline hydrolysis (left) or phosphatase activity (right). Mean values of serine phosphorylation (left) or phosphatase activity (right) of RPTPαWT not treated with CaMKIIα were set to 100%. Mean values ± SEM are shown (A, n = 6; B, n = 11; C, n = 9). *, P < 0.05 (paired t test).
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fig4: RPTPα is phosphorylated by CaMKIIα on Ser180 and Ser204. (A) Recombinant RPTPα-ID-GST or GST coupled to beads were incubated with CaMKIIα. The gel stained with Coomassie blue shows that approximately equal amounts of RPTPα-ID-GST and GST were bound to beads. The beads, treated and nontreated with CaMKIIα, were then subjected to the analysis of serine phosphorylation by alkaline hydrolysis (bottom left) or phosphatase activity (bottom right). Values for GST were subtracted as background, and values of serine phosphorylation (bottom left) or phosphatase activity (bottom right) for RPTPα-ID-GST not treated with CaMKIIα were set to 100%. (B) RPTPα from NCAM−/− brain lysates was incubated with CaMKIIα in the presence or absence of KN62 and Bis1. RPTPα was then subjected to the analysis of serine phosphorylation by alkaline hydrolysis. Values of phosphate released by alkaline from RPTPα not treated with CaMKIIα were set to 100%. (C) RPTPα immunoprecipitated from lysates of RPTPα-negative fibroblasts transfected with RPTPαWT, RPTPαS180A, RPTPαS204A, or RPTPαS180/204A was incubated with CaMKIIα. The immunoprecipitates, treated and untreated with CaMKIIα, were then subjected to the analysis of serine phosphorylation by alkaline hydrolysis (left) or phosphatase activity (right). Mean values of serine phosphorylation (left) or phosphatase activity (right) of RPTPαWT not treated with CaMKIIα were set to 100%. Mean values ± SEM are shown (A, n = 6; B, n = 11; C, n = 9). *, P < 0.05 (paired t test).

Mentions: To directly analyze the CaMKIIα-mediated serine phosphorylation of RPTPα, recombinant intracellular domains of RPTPα (RPTPα-ID) were used in an in vitro phosphorylation assay: incubation of RPTPα-ID with recombinant CaMKIIα resulted in a pronounced increase in serine phosphorylation and phosphatase activity of RPTPα-ID (Fig. 4 A).


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)

RPTPα is phosphorylated by CaMKIIα on Ser180 and Ser204. (A) Recombinant RPTPα-ID-GST or GST coupled to beads were incubated with CaMKIIα. The gel stained with Coomassie blue shows that approximately equal amounts of RPTPα-ID-GST and GST were bound to beads. The beads, treated and nontreated with CaMKIIα, were then subjected to the analysis of serine phosphorylation by alkaline hydrolysis (bottom left) or phosphatase activity (bottom right). Values for GST were subtracted as background, and values of serine phosphorylation (bottom left) or phosphatase activity (bottom right) for RPTPα-ID-GST not treated with CaMKIIα were set to 100%. (B) RPTPα from NCAM−/− brain lysates was incubated with CaMKIIα in the presence or absence of KN62 and Bis1. RPTPα was then subjected to the analysis of serine phosphorylation by alkaline hydrolysis. Values of phosphate released by alkaline from RPTPα not treated with CaMKIIα were set to 100%. (C) RPTPα immunoprecipitated from lysates of RPTPα-negative fibroblasts transfected with RPTPαWT, RPTPαS180A, RPTPαS204A, or RPTPαS180/204A was incubated with CaMKIIα. The immunoprecipitates, treated and untreated with CaMKIIα, were then subjected to the analysis of serine phosphorylation by alkaline hydrolysis (left) or phosphatase activity (right). Mean values of serine phosphorylation (left) or phosphatase activity (right) of RPTPαWT not treated with CaMKIIα were set to 100%. Mean values ± SEM are shown (A, n = 6; B, n = 11; C, n = 9). *, P < 0.05 (paired t test).
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fig4: RPTPα is phosphorylated by CaMKIIα on Ser180 and Ser204. (A) Recombinant RPTPα-ID-GST or GST coupled to beads were incubated with CaMKIIα. The gel stained with Coomassie blue shows that approximately equal amounts of RPTPα-ID-GST and GST were bound to beads. The beads, treated and nontreated with CaMKIIα, were then subjected to the analysis of serine phosphorylation by alkaline hydrolysis (bottom left) or phosphatase activity (bottom right). Values for GST were subtracted as background, and values of serine phosphorylation (bottom left) or phosphatase activity (bottom right) for RPTPα-ID-GST not treated with CaMKIIα were set to 100%. (B) RPTPα from NCAM−/− brain lysates was incubated with CaMKIIα in the presence or absence of KN62 and Bis1. RPTPα was then subjected to the analysis of serine phosphorylation by alkaline hydrolysis. Values of phosphate released by alkaline from RPTPα not treated with CaMKIIα were set to 100%. (C) RPTPα immunoprecipitated from lysates of RPTPα-negative fibroblasts transfected with RPTPαWT, RPTPαS180A, RPTPαS204A, or RPTPαS180/204A was incubated with CaMKIIα. The immunoprecipitates, treated and untreated with CaMKIIα, were then subjected to the analysis of serine phosphorylation by alkaline hydrolysis (left) or phosphatase activity (right). Mean values of serine phosphorylation (left) or phosphatase activity (right) of RPTPαWT not treated with CaMKIIα were set to 100%. Mean values ± SEM are shown (A, n = 6; B, n = 11; C, n = 9). *, P < 0.05 (paired t test).
Mentions: To directly analyze the CaMKIIα-mediated serine phosphorylation of RPTPα, recombinant intracellular domains of RPTPα (RPTPα-ID) were used in an in vitro phosphorylation assay: incubation of RPTPα-ID with recombinant CaMKIIα resulted in a pronounced increase in serine phosphorylation and phosphatase activity of RPTPα-ID (Fig. 4 A).

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