Limits...
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.

Show MeSH

Related in: MedlinePlus

NCAM promotes CaMKIIα activation and RPTPα–CaMKIIα complex formation. (A) RPTPα immunoprecipitates (IP) from NCAM+/+ and NCAM−/− brain lysates were probed by Western blotting with antibodies against CaMKIIα. Note that similar levels of RPTPα were immunoprecipitated. Mock immunoprecipitation with nonspecific IgG served as control. Coimmunoprecipitation of CaMKIIα with RPTPα is reduced in NCAM−/− brain lysates. (B) NCAM+/+ and NCAM−/− brain homogenates were probed by Western blotting with antibodies against total and active Thr286-phosphorylated CaMKIIα and GAPDH (loading control). The levels of active CaMKIIα were reduced in NCAM−/− brain homogenates. Graphs show quantitation of the blots (mean ± SEM, n = 6 for A and B) with optical density for NCAM+/+ probes set to 100%. *, P < 0.05, paired t test. (C) NCAM was clustered at the cell surface of cultured hippocampal neurons by NCAM antibodies (H28live). Neurons were then fixed and colabeled with antibodies against RPTPα and CaMKIIα. A high-magnification image of a neurite is shown. NCAM clusters overlap with accumulations of RPTPα and CaMKIIα (arrows). Bar, 5 μm. (D) CaMKIIα immunoprecipitates from NCAM+/+ and NCAM−/− brain lysates were probed by Western blotting with antibodies against NCAM. NCAM140 and NCAM180 coimmunoprecipitated with CaMKIIα. Mock immunoprecipitation with nonspecific IgG served as a control.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2542478&req=5

fig3: NCAM promotes CaMKIIα activation and RPTPα–CaMKIIα complex formation. (A) RPTPα immunoprecipitates (IP) from NCAM+/+ and NCAM−/− brain lysates were probed by Western blotting with antibodies against CaMKIIα. Note that similar levels of RPTPα were immunoprecipitated. Mock immunoprecipitation with nonspecific IgG served as control. Coimmunoprecipitation of CaMKIIα with RPTPα is reduced in NCAM−/− brain lysates. (B) NCAM+/+ and NCAM−/− brain homogenates were probed by Western blotting with antibodies against total and active Thr286-phosphorylated CaMKIIα and GAPDH (loading control). The levels of active CaMKIIα were reduced in NCAM−/− brain homogenates. Graphs show quantitation of the blots (mean ± SEM, n = 6 for A and B) with optical density for NCAM+/+ probes set to 100%. *, P < 0.05, paired t test. (C) NCAM was clustered at the cell surface of cultured hippocampal neurons by NCAM antibodies (H28live). Neurons were then fixed and colabeled with antibodies against RPTPα and CaMKIIα. A high-magnification image of a neurite is shown. NCAM clusters overlap with accumulations of RPTPα and CaMKIIα (arrows). Bar, 5 μm. (D) CaMKIIα immunoprecipitates from NCAM+/+ and NCAM−/− brain lysates were probed by Western blotting with antibodies against NCAM. NCAM140 and NCAM180 coimmunoprecipitated with CaMKIIα. Mock immunoprecipitation with nonspecific IgG served as a control.

Mentions: CaMKIIα is another serine/threonine protein kinase that associates with NCAM (Sytnyk et al., 2006). CaMKIIα coimmunoprecipitated with RPTPα from brain lysates (Fig. 3 A). Interestingly, binding of CaMKIIα to RPTPα was reduced in NCAM−/− brains (Fig. 3 A). Levels of activated Thr286-phosphorylated CaMKIIα were also reduced in NCAM−/− brains (Fig. 3 B). Hence, NCAM regulates CaMKIIα activation and RPTPα–CaMKIIα complex formation.


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 promotes CaMKIIα activation and RPTPα–CaMKIIα complex formation. (A) RPTPα immunoprecipitates (IP) from NCAM+/+ and NCAM−/− brain lysates were probed by Western blotting with antibodies against CaMKIIα. Note that similar levels of RPTPα were immunoprecipitated. Mock immunoprecipitation with nonspecific IgG served as control. Coimmunoprecipitation of CaMKIIα with RPTPα is reduced in NCAM−/− brain lysates. (B) NCAM+/+ and NCAM−/− brain homogenates were probed by Western blotting with antibodies against total and active Thr286-phosphorylated CaMKIIα and GAPDH (loading control). The levels of active CaMKIIα were reduced in NCAM−/− brain homogenates. Graphs show quantitation of the blots (mean ± SEM, n = 6 for A and B) with optical density for NCAM+/+ probes set to 100%. *, P < 0.05, paired t test. (C) NCAM was clustered at the cell surface of cultured hippocampal neurons by NCAM antibodies (H28live). Neurons were then fixed and colabeled with antibodies against RPTPα and CaMKIIα. A high-magnification image of a neurite is shown. NCAM clusters overlap with accumulations of RPTPα and CaMKIIα (arrows). Bar, 5 μm. (D) CaMKIIα immunoprecipitates from NCAM+/+ and NCAM−/− brain lysates were probed by Western blotting with antibodies against NCAM. NCAM140 and NCAM180 coimmunoprecipitated with CaMKIIα. Mock immunoprecipitation with nonspecific IgG served as a control.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2542478&req=5

fig3: NCAM promotes CaMKIIα activation and RPTPα–CaMKIIα complex formation. (A) RPTPα immunoprecipitates (IP) from NCAM+/+ and NCAM−/− brain lysates were probed by Western blotting with antibodies against CaMKIIα. Note that similar levels of RPTPα were immunoprecipitated. Mock immunoprecipitation with nonspecific IgG served as control. Coimmunoprecipitation of CaMKIIα with RPTPα is reduced in NCAM−/− brain lysates. (B) NCAM+/+ and NCAM−/− brain homogenates were probed by Western blotting with antibodies against total and active Thr286-phosphorylated CaMKIIα and GAPDH (loading control). The levels of active CaMKIIα were reduced in NCAM−/− brain homogenates. Graphs show quantitation of the blots (mean ± SEM, n = 6 for A and B) with optical density for NCAM+/+ probes set to 100%. *, P < 0.05, paired t test. (C) NCAM was clustered at the cell surface of cultured hippocampal neurons by NCAM antibodies (H28live). Neurons were then fixed and colabeled with antibodies against RPTPα and CaMKIIα. A high-magnification image of a neurite is shown. NCAM clusters overlap with accumulations of RPTPα and CaMKIIα (arrows). Bar, 5 μm. (D) CaMKIIα immunoprecipitates from NCAM+/+ and NCAM−/− brain lysates were probed by Western blotting with antibodies against NCAM. NCAM140 and NCAM180 coimmunoprecipitated with CaMKIIα. Mock immunoprecipitation with nonspecific IgG served as a control.
Mentions: CaMKIIα is another serine/threonine protein kinase that associates with NCAM (Sytnyk et al., 2006). CaMKIIα coimmunoprecipitated with RPTPα from brain lysates (Fig. 3 A). Interestingly, binding of CaMKIIα to RPTPα was reduced in NCAM−/− brains (Fig. 3 A). Levels of activated Thr286-phosphorylated CaMKIIα were also reduced in NCAM−/− brains (Fig. 3 B). Hence, NCAM regulates CaMKIIα activation and RPTPα–CaMKIIα complex formation.

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