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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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A schematic model of molecular interactions induced by NCAM140 clustering and resulting in fyn activation. (A) Nonclustered NCAM140 resides outside of lipid rafts and does not associate with RPTPα, CaMKIIα (shown as a holoenzyme), and fyn, which are present in inactive forms in the plasma membrane outside of lipid rafts, in the cytosol and in lipid rafts, respectively. (B) NCAM140 clustering induces palmitoylation of the intracellular domain of NCAM140 that promotes NCAM140 redistribution to lipid rafts, where it binds to prion protein (PrP). In parallel, NCAM140 clustering induces FGFR activation, promoting arachidonic acid production (not depicted), which results in arachidonic acid–dependent Ca2+ influx (Williams et al., 1994) via T- and L-type VDCC. An increase in Ca2+ concentration promotes binding of Ca2+/CaM to CaMKIIα, releasing the catalytic domain of CaMKIIα from inhibition by autoregulatory sequences proximal to the CaM binding site (not depicted). By associating with T- and L-type VDCC, NCAM anchors CaMKIIα, which is bound to NCAM140 via spectrin, near the Ca2+ influx sites. NCAM-induced aggregation of CaMKIIα in lipid rafts promotes transautophosphorylation of the CaMKIIα holoenzymes at Thr286, resulting in the constitutive activation of CaMKIIα. (C) In parallel to CaMKIIα activation, NCAM140 promotes redistribution of RPTPα to lipid rafts. In lipid rafts, activated CaMKIIα (only one CaMKIIα molecule is shown for simplicity) phosphorylates RPTPα at Ser180 and Ser204, which changes the conformation of RPTPα, resulting in enzyme activation. (D) Activated RPTPα binds and dephosphorylates fyn at Tyr531, activating the enzyme. Downstream targets of active fyn include the Ras–MAP (MAP) kinase pathway, the sustained activity of which is required for neuronal differentiation (not depicted).
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fig10: A schematic model of molecular interactions induced by NCAM140 clustering and resulting in fyn activation. (A) Nonclustered NCAM140 resides outside of lipid rafts and does not associate with RPTPα, CaMKIIα (shown as a holoenzyme), and fyn, which are present in inactive forms in the plasma membrane outside of lipid rafts, in the cytosol and in lipid rafts, respectively. (B) NCAM140 clustering induces palmitoylation of the intracellular domain of NCAM140 that promotes NCAM140 redistribution to lipid rafts, where it binds to prion protein (PrP). In parallel, NCAM140 clustering induces FGFR activation, promoting arachidonic acid production (not depicted), which results in arachidonic acid–dependent Ca2+ influx (Williams et al., 1994) via T- and L-type VDCC. An increase in Ca2+ concentration promotes binding of Ca2+/CaM to CaMKIIα, releasing the catalytic domain of CaMKIIα from inhibition by autoregulatory sequences proximal to the CaM binding site (not depicted). By associating with T- and L-type VDCC, NCAM anchors CaMKIIα, which is bound to NCAM140 via spectrin, near the Ca2+ influx sites. NCAM-induced aggregation of CaMKIIα in lipid rafts promotes transautophosphorylation of the CaMKIIα holoenzymes at Thr286, resulting in the constitutive activation of CaMKIIα. (C) In parallel to CaMKIIα activation, NCAM140 promotes redistribution of RPTPα to lipid rafts. In lipid rafts, activated CaMKIIα (only one CaMKIIα molecule is shown for simplicity) phosphorylates RPTPα at Ser180 and Ser204, which changes the conformation of RPTPα, resulting in enzyme activation. (D) Activated RPTPα binds and dephosphorylates fyn at Tyr531, activating the enzyme. Downstream targets of active fyn include the Ras–MAP (MAP) kinase pathway, the sustained activity of which is required for neuronal differentiation (not depicted).

Mentions: In this study, we expand our previous findings by showing that clustering of NCAM at the cell surface enhances serine phosphorylation and phosphatase activity of RPTPα, thus identifying NCAM as the first recognition molecule and surface receptor that not only associates with but also regulates the catalytic activity of RPTPα. The catalytic phosphatase activity of RPTPα can be enhanced by PKC-mediated Ser180 and/or Ser204 phosphorylation of the intracellular domain of RPTPα (den Hertog et al., 1995; Tracy et al., 1995; Stetak et al., 2001; Zheng et al., 2002), with PKCδ but not other PKC isoforms playing the major role in the phosphorylation of RPTPα on Ser180 and Ser204 (Brandt et al., 2003). However, we found that PKCδ is not involved in NCAM-induced activation. In agreement with this notion is the observation that 2.5–10 μM of the PKCδ inhibitor rottlerin did not inhibit RPTPα serine phosphorylation in nonstimulated NIH3T3 fibroblasts, and that very high concentrations of rottlerin (50 μM) only mildly affected RPTPα serine phosphorylation (unpublished data), which suggests that other enzymes are involved. Furthermore, we show that BisI, a PKCδ inhibitor, does not block RPTPα-mediated fyn activation in response to NCAM clustering. Accordingly, we identified CaMKIIα as a previously unrecognized enzyme that binds to and phosphorylates RPTPα at serine residues Ser180 and Ser204, increasing the phosphatase activity of RPTPα. We also show that CaMKIIα, which associates with NCAM via spectrin (Sytnyk et al., 2006), is activated in response to clustering of NCAM at the cell surface. Calmodulin, which associates with RPTPα in the presence of Ca2+, may thus play a role in CaMKIIα activation (Liang et al., 2000). NCAM-induced CaMKIIα activation then leads to phosphorylation of RPTPα at Ser180 and Ser204, which, in turn, leads to activation of lipid raft-enriched fyn (Fig. 10).


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)

A schematic model of molecular interactions induced by NCAM140 clustering and resulting in fyn activation. (A) Nonclustered NCAM140 resides outside of lipid rafts and does not associate with RPTPα, CaMKIIα (shown as a holoenzyme), and fyn, which are present in inactive forms in the plasma membrane outside of lipid rafts, in the cytosol and in lipid rafts, respectively. (B) NCAM140 clustering induces palmitoylation of the intracellular domain of NCAM140 that promotes NCAM140 redistribution to lipid rafts, where it binds to prion protein (PrP). In parallel, NCAM140 clustering induces FGFR activation, promoting arachidonic acid production (not depicted), which results in arachidonic acid–dependent Ca2+ influx (Williams et al., 1994) via T- and L-type VDCC. An increase in Ca2+ concentration promotes binding of Ca2+/CaM to CaMKIIα, releasing the catalytic domain of CaMKIIα from inhibition by autoregulatory sequences proximal to the CaM binding site (not depicted). By associating with T- and L-type VDCC, NCAM anchors CaMKIIα, which is bound to NCAM140 via spectrin, near the Ca2+ influx sites. NCAM-induced aggregation of CaMKIIα in lipid rafts promotes transautophosphorylation of the CaMKIIα holoenzymes at Thr286, resulting in the constitutive activation of CaMKIIα. (C) In parallel to CaMKIIα activation, NCAM140 promotes redistribution of RPTPα to lipid rafts. In lipid rafts, activated CaMKIIα (only one CaMKIIα molecule is shown for simplicity) phosphorylates RPTPα at Ser180 and Ser204, which changes the conformation of RPTPα, resulting in enzyme activation. (D) Activated RPTPα binds and dephosphorylates fyn at Tyr531, activating the enzyme. Downstream targets of active fyn include the Ras–MAP (MAP) kinase pathway, the sustained activity of which is required for neuronal differentiation (not depicted).
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Related In: Results  -  Collection

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fig10: A schematic model of molecular interactions induced by NCAM140 clustering and resulting in fyn activation. (A) Nonclustered NCAM140 resides outside of lipid rafts and does not associate with RPTPα, CaMKIIα (shown as a holoenzyme), and fyn, which are present in inactive forms in the plasma membrane outside of lipid rafts, in the cytosol and in lipid rafts, respectively. (B) NCAM140 clustering induces palmitoylation of the intracellular domain of NCAM140 that promotes NCAM140 redistribution to lipid rafts, where it binds to prion protein (PrP). In parallel, NCAM140 clustering induces FGFR activation, promoting arachidonic acid production (not depicted), which results in arachidonic acid–dependent Ca2+ influx (Williams et al., 1994) via T- and L-type VDCC. An increase in Ca2+ concentration promotes binding of Ca2+/CaM to CaMKIIα, releasing the catalytic domain of CaMKIIα from inhibition by autoregulatory sequences proximal to the CaM binding site (not depicted). By associating with T- and L-type VDCC, NCAM anchors CaMKIIα, which is bound to NCAM140 via spectrin, near the Ca2+ influx sites. NCAM-induced aggregation of CaMKIIα in lipid rafts promotes transautophosphorylation of the CaMKIIα holoenzymes at Thr286, resulting in the constitutive activation of CaMKIIα. (C) In parallel to CaMKIIα activation, NCAM140 promotes redistribution of RPTPα to lipid rafts. In lipid rafts, activated CaMKIIα (only one CaMKIIα molecule is shown for simplicity) phosphorylates RPTPα at Ser180 and Ser204, which changes the conformation of RPTPα, resulting in enzyme activation. (D) Activated RPTPα binds and dephosphorylates fyn at Tyr531, activating the enzyme. Downstream targets of active fyn include the Ras–MAP (MAP) kinase pathway, the sustained activity of which is required for neuronal differentiation (not depicted).
Mentions: In this study, we expand our previous findings by showing that clustering of NCAM at the cell surface enhances serine phosphorylation and phosphatase activity of RPTPα, thus identifying NCAM as the first recognition molecule and surface receptor that not only associates with but also regulates the catalytic activity of RPTPα. The catalytic phosphatase activity of RPTPα can be enhanced by PKC-mediated Ser180 and/or Ser204 phosphorylation of the intracellular domain of RPTPα (den Hertog et al., 1995; Tracy et al., 1995; Stetak et al., 2001; Zheng et al., 2002), with PKCδ but not other PKC isoforms playing the major role in the phosphorylation of RPTPα on Ser180 and Ser204 (Brandt et al., 2003). However, we found that PKCδ is not involved in NCAM-induced activation. In agreement with this notion is the observation that 2.5–10 μM of the PKCδ inhibitor rottlerin did not inhibit RPTPα serine phosphorylation in nonstimulated NIH3T3 fibroblasts, and that very high concentrations of rottlerin (50 μM) only mildly affected RPTPα serine phosphorylation (unpublished data), which suggests that other enzymes are involved. Furthermore, we show that BisI, a PKCδ inhibitor, does not block RPTPα-mediated fyn activation in response to NCAM clustering. Accordingly, we identified CaMKIIα as a previously unrecognized enzyme that binds to and phosphorylates RPTPα at serine residues Ser180 and Ser204, increasing the phosphatase activity of RPTPα. We also show that CaMKIIα, which associates with NCAM via spectrin (Sytnyk et al., 2006), is activated in response to clustering of NCAM at the cell surface. Calmodulin, which associates with RPTPα in the presence of Ca2+, may thus play a role in CaMKIIα activation (Liang et al., 2000). NCAM-induced CaMKIIα activation then leads to phosphorylation of RPTPα at Ser180 and Ser204, which, in turn, leads to activation of lipid raft-enriched fyn (Fig. 10).

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