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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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PKCδ associates with NCAM and RPTPα but its activity is not regulated by NCAM. (A and B) PKCδ (A) or NCAM (B) immunoprecipitates (IP) from NCAM+/+ brain lysates were analyzed by Western blotting as indicated. Mock immunoprecipitation with nonspecific IgG (A) or from NCAM−/− brain lysates (B) served as a control. RPTPα and NCAM120 coimmunoprecipitate with PKCδ, and PKCδ coimmunoprecipitates with NCAM. BH, brain homogenate. (C and D) NCAM+/+ and NCAM−/− brain homogenates (C) and RPTPα immunoprecipitates from NCAM+/+ and NCAM−/− brain lysates (D) were probed by Western blotting with antibodies against total PKCδ and active Thr505-phosphorylated PKCδ. Note the similar loading (GAPDH labeling in C) and immunoprecipitation efficiency (RPTPα labeling in D). Total levels of active PKCδ and levels of active PKCδ associated with RPTPα are not changed in NCAM−/− brains.
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fig2: PKCδ associates with NCAM and RPTPα but its activity is not regulated by NCAM. (A and B) PKCδ (A) or NCAM (B) immunoprecipitates (IP) from NCAM+/+ brain lysates were analyzed by Western blotting as indicated. Mock immunoprecipitation with nonspecific IgG (A) or from NCAM−/− brain lysates (B) served as a control. RPTPα and NCAM120 coimmunoprecipitate with PKCδ, and PKCδ coimmunoprecipitates with NCAM. BH, brain homogenate. (C and D) NCAM+/+ and NCAM−/− brain homogenates (C) and RPTPα immunoprecipitates from NCAM+/+ and NCAM−/− brain lysates (D) were probed by Western blotting with antibodies against total PKCδ and active Thr505-phosphorylated PKCδ. Note the similar loading (GAPDH labeling in C) and immunoprecipitation efficiency (RPTPα labeling in D). Total levels of active PKCδ and levels of active PKCδ associated with RPTPα are not changed in NCAM−/− brains.

Mentions: Previous studies indicated that among all PKC isoforms, only PKCδ phosphorylates RPTPα on Ser180 and Ser204 in nonneuronal cells (den Hertog et al., 1995; Tracy et al., 1995; Zheng et al., 2002; Brandt et al., 2003). NCAM associates with and induces activation of several PKC isoforms (Leshchyns'ka et al., 2003; Kolkova et al., 2005), but its role in PKCδ activation has not been analyzed. We thus analyzed whether NCAM regulates RPTPα phosphorylation by inducing PKCδ activation. RPTPα coimmunoprecipitated with PKCδ (Fig. 2 A), and PKCδ coimmunoprecipitated with NCAM (Fig. 2 B) from brain lysates. However, levels of activated PKCδ phosphorylated at Thr505 in the activation loop of the kinase were not changed in NCAM−/− brain homogenates (Fig. 2 C). Furthermore, similar levels of activated PKCδ coimmunoprecipitated with RPTPα from NCAM+/+ and NCAM−/− brain lysates (Fig. 2 D). Thus, NCAM does not regulate association of RPTPα with PKCδ, nor does it regulate PKCδ activation in the brain. In agreement, the most prominent NCAM isoform that coimmunoprecipitated with PKCδ was the smallest GPI-linked NCAM isoform with a molecular mass of 120 kD (NCAM120; Fig. 2 A). NCAM120 lacks the intracellular domain and therefore probably associates indirectly with PKCδ, as has been shown for spectrin, another intracellular binding partner of NCAM120 that associates with NCAM120 via lipids (Leshchyns'ka et al., 2003).


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)

PKCδ associates with NCAM and RPTPα but its activity is not regulated by NCAM. (A and B) PKCδ (A) or NCAM (B) immunoprecipitates (IP) from NCAM+/+ brain lysates were analyzed by Western blotting as indicated. Mock immunoprecipitation with nonspecific IgG (A) or from NCAM−/− brain lysates (B) served as a control. RPTPα and NCAM120 coimmunoprecipitate with PKCδ, and PKCδ coimmunoprecipitates with NCAM. BH, brain homogenate. (C and D) NCAM+/+ and NCAM−/− brain homogenates (C) and RPTPα immunoprecipitates from NCAM+/+ and NCAM−/− brain lysates (D) were probed by Western blotting with antibodies against total PKCδ and active Thr505-phosphorylated PKCδ. Note the similar loading (GAPDH labeling in C) and immunoprecipitation efficiency (RPTPα labeling in D). Total levels of active PKCδ and levels of active PKCδ associated with RPTPα are not changed in NCAM−/− brains.
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fig2: PKCδ associates with NCAM and RPTPα but its activity is not regulated by NCAM. (A and B) PKCδ (A) or NCAM (B) immunoprecipitates (IP) from NCAM+/+ brain lysates were analyzed by Western blotting as indicated. Mock immunoprecipitation with nonspecific IgG (A) or from NCAM−/− brain lysates (B) served as a control. RPTPα and NCAM120 coimmunoprecipitate with PKCδ, and PKCδ coimmunoprecipitates with NCAM. BH, brain homogenate. (C and D) NCAM+/+ and NCAM−/− brain homogenates (C) and RPTPα immunoprecipitates from NCAM+/+ and NCAM−/− brain lysates (D) were probed by Western blotting with antibodies against total PKCδ and active Thr505-phosphorylated PKCδ. Note the similar loading (GAPDH labeling in C) and immunoprecipitation efficiency (RPTPα labeling in D). Total levels of active PKCδ and levels of active PKCδ associated with RPTPα are not changed in NCAM−/− brains.
Mentions: Previous studies indicated that among all PKC isoforms, only PKCδ phosphorylates RPTPα on Ser180 and Ser204 in nonneuronal cells (den Hertog et al., 1995; Tracy et al., 1995; Zheng et al., 2002; Brandt et al., 2003). NCAM associates with and induces activation of several PKC isoforms (Leshchyns'ka et al., 2003; Kolkova et al., 2005), but its role in PKCδ activation has not been analyzed. We thus analyzed whether NCAM regulates RPTPα phosphorylation by inducing PKCδ activation. RPTPα coimmunoprecipitated with PKCδ (Fig. 2 A), and PKCδ coimmunoprecipitated with NCAM (Fig. 2 B) from brain lysates. However, levels of activated PKCδ phosphorylated at Thr505 in the activation loop of the kinase were not changed in NCAM−/− brain homogenates (Fig. 2 C). Furthermore, similar levels of activated PKCδ coimmunoprecipitated with RPTPα from NCAM+/+ and NCAM−/− brain lysates (Fig. 2 D). Thus, NCAM does not regulate association of RPTPα with PKCδ, nor does it regulate PKCδ activation in the brain. In agreement, the most prominent NCAM isoform that coimmunoprecipitated with PKCδ was the smallest GPI-linked NCAM isoform with a molecular mass of 120 kD (NCAM120; Fig. 2 A). NCAM120 lacks the intracellular domain and therefore probably associates indirectly with PKCδ, as has been shown for spectrin, another intracellular binding partner of NCAM120 that associates with NCAM120 via lipids (Leshchyns'ka et al., 2003).

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