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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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NCAM140 clustering induces phosphorylation of RPTPα on Ser180 and Ser204 and enhances the phosphatase activity of RPTPα. (A) Graph shows phosphatase activity of RPTPα from NCAM+/+ and NCAM−/− brain lysates. (B–D) RPTPα immunoprecipitates were probed by Western blotting with antibodies against RPTPα and phospho serines (B and C) or subjected to serine phosphorylation estimation by the alkaline hydrolysis (D). Note that similar levels of RPTPα were immunoprecipitated from NCAM+/+ and NCAM−/− brain lysates (B). Mock immunoprecipitation with nonspecific IgG served as a control. Phosphatase activity and serine phosphorylation of RPTPα are reduced in NCAM−/− brains. Graph in C shows quantitation of the blots in B with optical density for NCAM+/+ brains set to 100%. In A and D, mean values of phosphatase activity (A) or phosphate released by alkaline (D) in RPTPα immunoprecipitates from NCAM+/+ brains were set to 100%. (E) RPTPα-negative fibroblasts transfected with RPTPαWT alone or cotransfected with NCAM140 and RPTPαWT, RPTPαS180A, RPTPαS204A, or RPTPαS180/204A were treated with NCAM polyclonal antibodies or nonspecific rabbit IgG. Note that similar levels of NCAM140 were expressed in cells (lysates) and that similar levels of RPTPα were immunoprecipitated from the lysates (IP: HA-tag). Mutation of Ser180 and/or Ser204 does not influence coimmunoprecipitation of NCAM140 with RPTPα. Immunoprecipitated RPTPα was then subjected to the serine phosphorylation estimation by the alkaline hydrolysis (top) and phosphatase activity analysis (bottom). Application of NCAM antibodies but not IgG increased serine phosphorylation and phosphatase activity of RPTPαWT in NCAM140–RPTPαWT–cotransfected cells. Phosphorylation and activation of RPTPα mutants were inhibited. Mean values of phosphate released by alkaline (top) or phosphatase activity (bottom) in RPTPα immunoprecipitates from IgG-treated NCAM140–RPTPαWT–cotransfected cells were set to 100%. Mean values ± SEM are shown (A and D, n ≥ 8; C, n = 6; E, n ≥ 6). *, P < 0.05 (paired t test).
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fig1: NCAM140 clustering induces phosphorylation of RPTPα on Ser180 and Ser204 and enhances the phosphatase activity of RPTPα. (A) Graph shows phosphatase activity of RPTPα from NCAM+/+ and NCAM−/− brain lysates. (B–D) RPTPα immunoprecipitates were probed by Western blotting with antibodies against RPTPα and phospho serines (B and C) or subjected to serine phosphorylation estimation by the alkaline hydrolysis (D). Note that similar levels of RPTPα were immunoprecipitated from NCAM+/+ and NCAM−/− brain lysates (B). Mock immunoprecipitation with nonspecific IgG served as a control. Phosphatase activity and serine phosphorylation of RPTPα are reduced in NCAM−/− brains. Graph in C shows quantitation of the blots in B with optical density for NCAM+/+ brains set to 100%. In A and D, mean values of phosphatase activity (A) or phosphate released by alkaline (D) in RPTPα immunoprecipitates from NCAM+/+ brains were set to 100%. (E) RPTPα-negative fibroblasts transfected with RPTPαWT alone or cotransfected with NCAM140 and RPTPαWT, RPTPαS180A, RPTPαS204A, or RPTPαS180/204A were treated with NCAM polyclonal antibodies or nonspecific rabbit IgG. Note that similar levels of NCAM140 were expressed in cells (lysates) and that similar levels of RPTPα were immunoprecipitated from the lysates (IP: HA-tag). Mutation of Ser180 and/or Ser204 does not influence coimmunoprecipitation of NCAM140 with RPTPα. Immunoprecipitated RPTPα was then subjected to the serine phosphorylation estimation by the alkaline hydrolysis (top) and phosphatase activity analysis (bottom). Application of NCAM antibodies but not IgG increased serine phosphorylation and phosphatase activity of RPTPαWT in NCAM140–RPTPαWT–cotransfected cells. Phosphorylation and activation of RPTPα mutants were inhibited. Mean values of phosphate released by alkaline (top) or phosphatase activity (bottom) in RPTPα immunoprecipitates from IgG-treated NCAM140–RPTPαWT–cotransfected cells were set to 100%. Mean values ± SEM are shown (A and D, n ≥ 8; C, n = 6; E, n ≥ 6). *, P < 0.05 (paired t test).

Mentions: To analyze the role of NCAM in regulation of the phosphatase activity of RPTPα, RPTPα was immunoprecipitated from NCAM+/+ and NCAM−/− brain lysates. We then used two commercially available phosphotyrosine-containing peptides derived from the epidermal growth factor receptor, which serve as substrates for many protein tyrosine phosphatases, and estimated the efficiency of the release of phosphate from these peptides in the presence of RPTPα immunoprecipitates. This analysis showed that the phosphatase activity of RPTPα from NCAM−/− brains was reduced by ∼55% when compared with RPTPα from NCAM+/+ brains (Fig. 1 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)

NCAM140 clustering induces phosphorylation of RPTPα on Ser180 and Ser204 and enhances the phosphatase activity of RPTPα. (A) Graph shows phosphatase activity of RPTPα from NCAM+/+ and NCAM−/− brain lysates. (B–D) RPTPα immunoprecipitates were probed by Western blotting with antibodies against RPTPα and phospho serines (B and C) or subjected to serine phosphorylation estimation by the alkaline hydrolysis (D). Note that similar levels of RPTPα were immunoprecipitated from NCAM+/+ and NCAM−/− brain lysates (B). Mock immunoprecipitation with nonspecific IgG served as a control. Phosphatase activity and serine phosphorylation of RPTPα are reduced in NCAM−/− brains. Graph in C shows quantitation of the blots in B with optical density for NCAM+/+ brains set to 100%. In A and D, mean values of phosphatase activity (A) or phosphate released by alkaline (D) in RPTPα immunoprecipitates from NCAM+/+ brains were set to 100%. (E) RPTPα-negative fibroblasts transfected with RPTPαWT alone or cotransfected with NCAM140 and RPTPαWT, RPTPαS180A, RPTPαS204A, or RPTPαS180/204A were treated with NCAM polyclonal antibodies or nonspecific rabbit IgG. Note that similar levels of NCAM140 were expressed in cells (lysates) and that similar levels of RPTPα were immunoprecipitated from the lysates (IP: HA-tag). Mutation of Ser180 and/or Ser204 does not influence coimmunoprecipitation of NCAM140 with RPTPα. Immunoprecipitated RPTPα was then subjected to the serine phosphorylation estimation by the alkaline hydrolysis (top) and phosphatase activity analysis (bottom). Application of NCAM antibodies but not IgG increased serine phosphorylation and phosphatase activity of RPTPαWT in NCAM140–RPTPαWT–cotransfected cells. Phosphorylation and activation of RPTPα mutants were inhibited. Mean values of phosphate released by alkaline (top) or phosphatase activity (bottom) in RPTPα immunoprecipitates from IgG-treated NCAM140–RPTPαWT–cotransfected cells were set to 100%. Mean values ± SEM are shown (A and D, n ≥ 8; C, n = 6; E, n ≥ 6). *, P < 0.05 (paired t test).
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fig1: NCAM140 clustering induces phosphorylation of RPTPα on Ser180 and Ser204 and enhances the phosphatase activity of RPTPα. (A) Graph shows phosphatase activity of RPTPα from NCAM+/+ and NCAM−/− brain lysates. (B–D) RPTPα immunoprecipitates were probed by Western blotting with antibodies against RPTPα and phospho serines (B and C) or subjected to serine phosphorylation estimation by the alkaline hydrolysis (D). Note that similar levels of RPTPα were immunoprecipitated from NCAM+/+ and NCAM−/− brain lysates (B). Mock immunoprecipitation with nonspecific IgG served as a control. Phosphatase activity and serine phosphorylation of RPTPα are reduced in NCAM−/− brains. Graph in C shows quantitation of the blots in B with optical density for NCAM+/+ brains set to 100%. In A and D, mean values of phosphatase activity (A) or phosphate released by alkaline (D) in RPTPα immunoprecipitates from NCAM+/+ brains were set to 100%. (E) RPTPα-negative fibroblasts transfected with RPTPαWT alone or cotransfected with NCAM140 and RPTPαWT, RPTPαS180A, RPTPαS204A, or RPTPαS180/204A were treated with NCAM polyclonal antibodies or nonspecific rabbit IgG. Note that similar levels of NCAM140 were expressed in cells (lysates) and that similar levels of RPTPα were immunoprecipitated from the lysates (IP: HA-tag). Mutation of Ser180 and/or Ser204 does not influence coimmunoprecipitation of NCAM140 with RPTPα. Immunoprecipitated RPTPα was then subjected to the serine phosphorylation estimation by the alkaline hydrolysis (top) and phosphatase activity analysis (bottom). Application of NCAM antibodies but not IgG increased serine phosphorylation and phosphatase activity of RPTPαWT in NCAM140–RPTPαWT–cotransfected cells. Phosphorylation and activation of RPTPα mutants were inhibited. Mean values of phosphate released by alkaline (top) or phosphatase activity (bottom) in RPTPα immunoprecipitates from IgG-treated NCAM140–RPTPαWT–cotransfected cells were set to 100%. Mean values ± SEM are shown (A and D, n ≥ 8; C, n = 6; E, n ≥ 6). *, P < 0.05 (paired t test).
Mentions: To analyze the role of NCAM in regulation of the phosphatase activity of RPTPα, RPTPα was immunoprecipitated from NCAM+/+ and NCAM−/− brain lysates. We then used two commercially available phosphotyrosine-containing peptides derived from the epidermal growth factor receptor, which serve as substrates for many protein tyrosine phosphatases, and estimated the efficiency of the release of phosphate from these peptides in the presence of RPTPα immunoprecipitates. This analysis showed that the phosphatase activity of RPTPα from NCAM−/− brains was reduced by ∼55% when compared with RPTPα from NCAM+/+ brains (Fig. 1 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