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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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Association of NCAM140 with lipid rafts is required for CaMKIIα activation. (A, left) Representative images of neurites of cultured hippocampal neurons treated with nonspecific rabbit IgG or NCAM polyclonal antibodies, extracted in cold 1% Triton X-100, and labeled with antibodies against activated Thr286-phosphorylated CaMKIIα and PI(4,5)P2 by indirect immunofluorescence. Note the higher levels of activated CaMKIIα and the higher degree of colocalization of activated CaMKIIα with PI(4,5)P2 along neurites of NCAM antibody-treated neurons. Arrows show clusters of activated CaMKIIα colocalizing with PI(4,5)P2 accumulations. Bar, 10 μm. (right) Graphs show mean levels ± SEM in arbitrary units (AU) of active Thr286-phosphorylated CaMKIIα (top) and coefficients of correlation between distributions of Thr286 phosphorylated CaMKIIα and PI(4,5)P2 (bottom) along neurites. n > 90 neurites from 45 neurons from 3 coverslips analyzed in each group. (B) Brain homogenates (BH), cytosolic fraction (CF), total membrane fraction (MF), and lipid raft fraction (RF) from NCAM+/+ and NCAM−/− brains were probed by Western blotting with antibodies against activated Thr286 phosphorylated CaMKIIα, total CaMKIIα, T- and L-type VDCC, lipid raft marker fyn, the soluble protein marker GAPDH, and the membrane protein marker CHL1. Note the accumulation of activated CaMKIIα in lipid rafts. (C) Lysates of CHO cells cotransfected with RPTPαWT and NCAM140, NCAM140Δcys, or GFP and treated with nonspecific rabbit IgG or NCAM polyclonal antibodies were probed with antibodies against NCAM, activated Thr286-phosphorylated CaMKIIα, and total CaMKIIα. Note the similar levels of expression of NCAM140 and NCAM140Δcys but the reduced activation of CaMKIIα in NCAM140Δcys- versus NCAM140-transfected cells. The graph shows quantitation of the blots with the levels of activated CaMKIIα in GFP-transfected cells treated with nonspecific IgG set to 100%. (D) RPTPα was immunoprecipitated from cell lysates in C, and serine phosphorylation of RPTPα was analyzed by alkaline hydrolysis. The amount of phosphate released by alkaline hydrolysis from RPTPα from cells transfected with GFP instead of NCAM and treated with nonspecific IgG was set to 100%. For C and D, mean values ± SEM are shown (n = 6). *, P < 0.05 (paired t test).
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fig6: Association of NCAM140 with lipid rafts is required for CaMKIIα activation. (A, left) Representative images of neurites of cultured hippocampal neurons treated with nonspecific rabbit IgG or NCAM polyclonal antibodies, extracted in cold 1% Triton X-100, and labeled with antibodies against activated Thr286-phosphorylated CaMKIIα and PI(4,5)P2 by indirect immunofluorescence. Note the higher levels of activated CaMKIIα and the higher degree of colocalization of activated CaMKIIα with PI(4,5)P2 along neurites of NCAM antibody-treated neurons. Arrows show clusters of activated CaMKIIα colocalizing with PI(4,5)P2 accumulations. Bar, 10 μm. (right) Graphs show mean levels ± SEM in arbitrary units (AU) of active Thr286-phosphorylated CaMKIIα (top) and coefficients of correlation between distributions of Thr286 phosphorylated CaMKIIα and PI(4,5)P2 (bottom) along neurites. n > 90 neurites from 45 neurons from 3 coverslips analyzed in each group. (B) Brain homogenates (BH), cytosolic fraction (CF), total membrane fraction (MF), and lipid raft fraction (RF) from NCAM+/+ and NCAM−/− brains were probed by Western blotting with antibodies against activated Thr286 phosphorylated CaMKIIα, total CaMKIIα, T- and L-type VDCC, lipid raft marker fyn, the soluble protein marker GAPDH, and the membrane protein marker CHL1. Note the accumulation of activated CaMKIIα in lipid rafts. (C) Lysates of CHO cells cotransfected with RPTPαWT and NCAM140, NCAM140Δcys, or GFP and treated with nonspecific rabbit IgG or NCAM polyclonal antibodies were probed with antibodies against NCAM, activated Thr286-phosphorylated CaMKIIα, and total CaMKIIα. Note the similar levels of expression of NCAM140 and NCAM140Δcys but the reduced activation of CaMKIIα in NCAM140Δcys- versus NCAM140-transfected cells. The graph shows quantitation of the blots with the levels of activated CaMKIIα in GFP-transfected cells treated with nonspecific IgG set to 100%. (D) RPTPα was immunoprecipitated from cell lysates in C, and serine phosphorylation of RPTPα was analyzed by alkaline hydrolysis. The amount of phosphate released by alkaline hydrolysis from RPTPα from cells transfected with GFP instead of NCAM and treated with nonspecific IgG was set to 100%. For C and D, mean values ± SEM are shown (n = 6). *, P < 0.05 (paired t test).

Mentions: Clustering of NCAM140 and NCAM180 induces their redistribution to lipid rafts (Niethammer et al., 2002; Leshchyns'ka et al., 2003; Bodrikov et al., 2005; Santuccione et al., 2005). Therefore, we analyzed whether lipid rafts play a role in NCAM-induced CaMKIIα activation. In cultured hippocampal neurons extracted with cold 1% Triton X-100 and colabeled with antibodies against Thr286-phosphorylated CaMKIIα and the lipid raft marker PI(4,5)P2 (Niethammer et al., 2002), accumulations of activated CaMKIIα partially overlapped with the clusters of PI(4,5)P2 (Fig. 6 A), which indicates that cytoskeleton- and/or lipid raft–associated detergent-insoluble pools of CaMKIIα were present in neurites. Clustering of NCAM at the cell surface with NCAM antibodies resulted in an approximately threefold increase in the levels of detergent-insoluble active CaMKIIα and a higher overlap in distribution of CaMKIIα and PI(4,5)P2 along neurites when compared with neurons treated with nonspecific immunoglobulins (Fig. 6 A). This observation suggests that activation of CaMKIIα in response to NCAM clustering occurs in lipid rafts.


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)

Association of NCAM140 with lipid rafts is required for CaMKIIα activation. (A, left) Representative images of neurites of cultured hippocampal neurons treated with nonspecific rabbit IgG or NCAM polyclonal antibodies, extracted in cold 1% Triton X-100, and labeled with antibodies against activated Thr286-phosphorylated CaMKIIα and PI(4,5)P2 by indirect immunofluorescence. Note the higher levels of activated CaMKIIα and the higher degree of colocalization of activated CaMKIIα with PI(4,5)P2 along neurites of NCAM antibody-treated neurons. Arrows show clusters of activated CaMKIIα colocalizing with PI(4,5)P2 accumulations. Bar, 10 μm. (right) Graphs show mean levels ± SEM in arbitrary units (AU) of active Thr286-phosphorylated CaMKIIα (top) and coefficients of correlation between distributions of Thr286 phosphorylated CaMKIIα and PI(4,5)P2 (bottom) along neurites. n > 90 neurites from 45 neurons from 3 coverslips analyzed in each group. (B) Brain homogenates (BH), cytosolic fraction (CF), total membrane fraction (MF), and lipid raft fraction (RF) from NCAM+/+ and NCAM−/− brains were probed by Western blotting with antibodies against activated Thr286 phosphorylated CaMKIIα, total CaMKIIα, T- and L-type VDCC, lipid raft marker fyn, the soluble protein marker GAPDH, and the membrane protein marker CHL1. Note the accumulation of activated CaMKIIα in lipid rafts. (C) Lysates of CHO cells cotransfected with RPTPαWT and NCAM140, NCAM140Δcys, or GFP and treated with nonspecific rabbit IgG or NCAM polyclonal antibodies were probed with antibodies against NCAM, activated Thr286-phosphorylated CaMKIIα, and total CaMKIIα. Note the similar levels of expression of NCAM140 and NCAM140Δcys but the reduced activation of CaMKIIα in NCAM140Δcys- versus NCAM140-transfected cells. The graph shows quantitation of the blots with the levels of activated CaMKIIα in GFP-transfected cells treated with nonspecific IgG set to 100%. (D) RPTPα was immunoprecipitated from cell lysates in C, and serine phosphorylation of RPTPα was analyzed by alkaline hydrolysis. The amount of phosphate released by alkaline hydrolysis from RPTPα from cells transfected with GFP instead of NCAM and treated with nonspecific IgG was set to 100%. For C and D, mean values ± SEM are shown (n = 6). *, P < 0.05 (paired t test).
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fig6: Association of NCAM140 with lipid rafts is required for CaMKIIα activation. (A, left) Representative images of neurites of cultured hippocampal neurons treated with nonspecific rabbit IgG or NCAM polyclonal antibodies, extracted in cold 1% Triton X-100, and labeled with antibodies against activated Thr286-phosphorylated CaMKIIα and PI(4,5)P2 by indirect immunofluorescence. Note the higher levels of activated CaMKIIα and the higher degree of colocalization of activated CaMKIIα with PI(4,5)P2 along neurites of NCAM antibody-treated neurons. Arrows show clusters of activated CaMKIIα colocalizing with PI(4,5)P2 accumulations. Bar, 10 μm. (right) Graphs show mean levels ± SEM in arbitrary units (AU) of active Thr286-phosphorylated CaMKIIα (top) and coefficients of correlation between distributions of Thr286 phosphorylated CaMKIIα and PI(4,5)P2 (bottom) along neurites. n > 90 neurites from 45 neurons from 3 coverslips analyzed in each group. (B) Brain homogenates (BH), cytosolic fraction (CF), total membrane fraction (MF), and lipid raft fraction (RF) from NCAM+/+ and NCAM−/− brains were probed by Western blotting with antibodies against activated Thr286 phosphorylated CaMKIIα, total CaMKIIα, T- and L-type VDCC, lipid raft marker fyn, the soluble protein marker GAPDH, and the membrane protein marker CHL1. Note the accumulation of activated CaMKIIα in lipid rafts. (C) Lysates of CHO cells cotransfected with RPTPαWT and NCAM140, NCAM140Δcys, or GFP and treated with nonspecific rabbit IgG or NCAM polyclonal antibodies were probed with antibodies against NCAM, activated Thr286-phosphorylated CaMKIIα, and total CaMKIIα. Note the similar levels of expression of NCAM140 and NCAM140Δcys but the reduced activation of CaMKIIα in NCAM140Δcys- versus NCAM140-transfected cells. The graph shows quantitation of the blots with the levels of activated CaMKIIα in GFP-transfected cells treated with nonspecific IgG set to 100%. (D) RPTPα was immunoprecipitated from cell lysates in C, and serine phosphorylation of RPTPα was analyzed by alkaline hydrolysis. The amount of phosphate released by alkaline hydrolysis from RPTPα from cells transfected with GFP instead of NCAM and treated with nonspecific IgG was set to 100%. For C and D, mean values ± SEM are shown (n = 6). *, P < 0.05 (paired t test).
Mentions: Clustering of NCAM140 and NCAM180 induces their redistribution to lipid rafts (Niethammer et al., 2002; Leshchyns'ka et al., 2003; Bodrikov et al., 2005; Santuccione et al., 2005). Therefore, we analyzed whether lipid rafts play a role in NCAM-induced CaMKIIα activation. In cultured hippocampal neurons extracted with cold 1% Triton X-100 and colabeled with antibodies against Thr286-phosphorylated CaMKIIα and the lipid raft marker PI(4,5)P2 (Niethammer et al., 2002), accumulations of activated CaMKIIα partially overlapped with the clusters of PI(4,5)P2 (Fig. 6 A), which indicates that cytoskeleton- and/or lipid raft–associated detergent-insoluble pools of CaMKIIα were present in neurites. Clustering of NCAM at the cell surface with NCAM antibodies resulted in an approximately threefold increase in the levels of detergent-insoluble active CaMKIIα and a higher overlap in distribution of CaMKIIα and PI(4,5)P2 along neurites when compared with neurons treated with nonspecific immunoglobulins (Fig. 6 A). This observation suggests that activation of CaMKIIα in response to NCAM clustering occurs in lipid rafts.

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