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Tyrosine phosphorylation at a site highly conserved in the L1 family of cell adhesion molecules abolishes ankyrin binding and increases lateral mobility of neurofascin.

Garver TD, Ren Q, Tuvia S, Bennett V - J. Cell Biol. (1997)

Bottom Line: Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain.The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules.Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
This paper presents evidence that a member of the L1 family of ankyrin-binding cell adhesion molecules is a substrate for protein tyrosine kinase(s) and phosphatase(s), identifies the highly conserved FIGQY tyrosine in the cytoplasmic domain as the principal site of phosphorylation, and demonstrates that phosphorylation of the FIGQY tyrosine abolishes ankyrin-binding activity. Neurofascin expressed in neuroblastoma cells is subject to tyrosine phosphorylation after activation of tyrosine kinases by NGF or bFGF or inactivation of tyrosine phosphatases with vanadate or dephostatin. Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain. The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules. Phosphorylation of the FIGQY tyrosine abolishes ankyrin binding, as determined by coimmunoprecipitation of endogenous ankyrin and in vitro ankyrin-binding assays. Measurements of fluorescence recovery after photobleaching demonstrate that phosphorylation of the FIGQY tyrosine also increases lateral mobility of neurofascin expressed in neuroblastoma cells to the same extent as removal of the cytoplasmic domain. Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals. These findings suggest that tyrosine phosphorylation at the FIGQY site represents a highly conserved mechanism, used by the entire class of L1-related cell adhesion molecules, for regulation of ankyrin-dependent connections to the spectrin skeleton.

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In vivo activation of tyrosine phosphorylation of neurofascin decreases ankyrin binding capacity. (A) Neurofascin-transfected cells were treated with one of the following agents for 30  min: dephostatin (10 μM), NGF (100 ng/ml), or bFGF (50 ng/ml).  HA-labeled neurofascin (250 μg crude cell lysate) was subsequently immunoprecipitated as described above. Immune complexes were captured on protein A–Sepharose beads and electrophoresed on SDS-PAGE. Resolved proteins were transferred to  nitrocellulose and incubated overnight at 4°C with a brain ankyrinspecific polyclonal antibody. After incubation with 125I-labeled  protein A (2 h at 4°C), immunoblots were visualized with autoradiography. (B) Neurofascin-transfected cells were treated as described. HA-labeled neurofascin was immunoprecipitated, and  immune complexes were captured on protein A–labeled latex  beads, washed, and then incubated for 180 min at 4°C with 20 nM  125I-radiolabeled, bacterially expressed 82K membrane-binding  domain of the ankyrinB isoform plus a given amount of unlabeled  82K ankyrinB. After incubation the immune complexes were centrifuged over a 10% sucrose cushion to separate bound and free  radioactivity, and the beads were counted on a γ counter to assess  levels of ankyrin binding. Each point was obtained by averaging  duplicate assays from individual immunoprecipitations. (C) The  ankyrin binding assay data from above was converted into a form  suitable for Scatchard analysis. The tyrosine phosphorylation– induced changes in the Scatchard plots (downward shift with no  change in slope) demonstrate that the total ankyrin binding capacity is decreased with no change in ankyrin binding affinity.  This indicates that phosphorylation of a given neurofascin molecule abolishes its ability to bind ankyrin but does not alter the affinity of remaining neurofascin molecules that have not been  phosphorylated and, thus, maintain ankyrin-binding competence.
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Figure 2: In vivo activation of tyrosine phosphorylation of neurofascin decreases ankyrin binding capacity. (A) Neurofascin-transfected cells were treated with one of the following agents for 30 min: dephostatin (10 μM), NGF (100 ng/ml), or bFGF (50 ng/ml). HA-labeled neurofascin (250 μg crude cell lysate) was subsequently immunoprecipitated as described above. Immune complexes were captured on protein A–Sepharose beads and electrophoresed on SDS-PAGE. Resolved proteins were transferred to nitrocellulose and incubated overnight at 4°C with a brain ankyrinspecific polyclonal antibody. After incubation with 125I-labeled protein A (2 h at 4°C), immunoblots were visualized with autoradiography. (B) Neurofascin-transfected cells were treated as described. HA-labeled neurofascin was immunoprecipitated, and immune complexes were captured on protein A–labeled latex beads, washed, and then incubated for 180 min at 4°C with 20 nM 125I-radiolabeled, bacterially expressed 82K membrane-binding domain of the ankyrinB isoform plus a given amount of unlabeled 82K ankyrinB. After incubation the immune complexes were centrifuged over a 10% sucrose cushion to separate bound and free radioactivity, and the beads were counted on a γ counter to assess levels of ankyrin binding. Each point was obtained by averaging duplicate assays from individual immunoprecipitations. (C) The ankyrin binding assay data from above was converted into a form suitable for Scatchard analysis. The tyrosine phosphorylation– induced changes in the Scatchard plots (downward shift with no change in slope) demonstrate that the total ankyrin binding capacity is decreased with no change in ankyrin binding affinity. This indicates that phosphorylation of a given neurofascin molecule abolishes its ability to bind ankyrin but does not alter the affinity of remaining neurofascin molecules that have not been phosphorylated and, thus, maintain ankyrin-binding competence.

Mentions: The question of whether tyrosine phosphorylation modulates ankyrin-binding activity of neurofascin was evaluated by determining the effects of phosphorylation on the ability of neurofascin to coimmunoprecipitate endogenous ankyrin expressed in B104 cells (Fig. 2 A). Neurofascin immunoprecipitates were simultaneously enriched for polypeptides of 210 and 190 kD as well as a doublet at 102 and 104 kD that crossreact with a brain ankyrin-specific polyclonal antibody (ankyrinB; Fig. 2 A, lane 2). The 210- and 190-kD crossreacting polypeptides are of the expected size for ankyrin with membrane-binding, spectrin-binding, and COOH-terminal domains. The smaller polypeptides presumably represent either alternatively spliced variants missing one or more of these domains or proteolytically cleaved forms of the 210- and 190-kD polypeptides. Activation of tyrosine kinases (Fig. 2 A, NGF or bFGF; lanes 4 and 5) or inactivation of tyrosine phosphatases (Fig. 2 A, dephostatin; lane 3) completely eliminate the ability of neurofascin to coimmunoprecipitate 210- and 190-kD ankyrin as well as the 102 and 104-kD crossreacting polypeptides.


Tyrosine phosphorylation at a site highly conserved in the L1 family of cell adhesion molecules abolishes ankyrin binding and increases lateral mobility of neurofascin.

Garver TD, Ren Q, Tuvia S, Bennett V - J. Cell Biol. (1997)

In vivo activation of tyrosine phosphorylation of neurofascin decreases ankyrin binding capacity. (A) Neurofascin-transfected cells were treated with one of the following agents for 30  min: dephostatin (10 μM), NGF (100 ng/ml), or bFGF (50 ng/ml).  HA-labeled neurofascin (250 μg crude cell lysate) was subsequently immunoprecipitated as described above. Immune complexes were captured on protein A–Sepharose beads and electrophoresed on SDS-PAGE. Resolved proteins were transferred to  nitrocellulose and incubated overnight at 4°C with a brain ankyrinspecific polyclonal antibody. After incubation with 125I-labeled  protein A (2 h at 4°C), immunoblots were visualized with autoradiography. (B) Neurofascin-transfected cells were treated as described. HA-labeled neurofascin was immunoprecipitated, and  immune complexes were captured on protein A–labeled latex  beads, washed, and then incubated for 180 min at 4°C with 20 nM  125I-radiolabeled, bacterially expressed 82K membrane-binding  domain of the ankyrinB isoform plus a given amount of unlabeled  82K ankyrinB. After incubation the immune complexes were centrifuged over a 10% sucrose cushion to separate bound and free  radioactivity, and the beads were counted on a γ counter to assess  levels of ankyrin binding. Each point was obtained by averaging  duplicate assays from individual immunoprecipitations. (C) The  ankyrin binding assay data from above was converted into a form  suitable for Scatchard analysis. The tyrosine phosphorylation– induced changes in the Scatchard plots (downward shift with no  change in slope) demonstrate that the total ankyrin binding capacity is decreased with no change in ankyrin binding affinity.  This indicates that phosphorylation of a given neurofascin molecule abolishes its ability to bind ankyrin but does not alter the affinity of remaining neurofascin molecules that have not been  phosphorylated and, thus, maintain ankyrin-binding competence.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139872&req=5

Figure 2: In vivo activation of tyrosine phosphorylation of neurofascin decreases ankyrin binding capacity. (A) Neurofascin-transfected cells were treated with one of the following agents for 30 min: dephostatin (10 μM), NGF (100 ng/ml), or bFGF (50 ng/ml). HA-labeled neurofascin (250 μg crude cell lysate) was subsequently immunoprecipitated as described above. Immune complexes were captured on protein A–Sepharose beads and electrophoresed on SDS-PAGE. Resolved proteins were transferred to nitrocellulose and incubated overnight at 4°C with a brain ankyrinspecific polyclonal antibody. After incubation with 125I-labeled protein A (2 h at 4°C), immunoblots were visualized with autoradiography. (B) Neurofascin-transfected cells were treated as described. HA-labeled neurofascin was immunoprecipitated, and immune complexes were captured on protein A–labeled latex beads, washed, and then incubated for 180 min at 4°C with 20 nM 125I-radiolabeled, bacterially expressed 82K membrane-binding domain of the ankyrinB isoform plus a given amount of unlabeled 82K ankyrinB. After incubation the immune complexes were centrifuged over a 10% sucrose cushion to separate bound and free radioactivity, and the beads were counted on a γ counter to assess levels of ankyrin binding. Each point was obtained by averaging duplicate assays from individual immunoprecipitations. (C) The ankyrin binding assay data from above was converted into a form suitable for Scatchard analysis. The tyrosine phosphorylation– induced changes in the Scatchard plots (downward shift with no change in slope) demonstrate that the total ankyrin binding capacity is decreased with no change in ankyrin binding affinity. This indicates that phosphorylation of a given neurofascin molecule abolishes its ability to bind ankyrin but does not alter the affinity of remaining neurofascin molecules that have not been phosphorylated and, thus, maintain ankyrin-binding competence.
Mentions: The question of whether tyrosine phosphorylation modulates ankyrin-binding activity of neurofascin was evaluated by determining the effects of phosphorylation on the ability of neurofascin to coimmunoprecipitate endogenous ankyrin expressed in B104 cells (Fig. 2 A). Neurofascin immunoprecipitates were simultaneously enriched for polypeptides of 210 and 190 kD as well as a doublet at 102 and 104 kD that crossreact with a brain ankyrin-specific polyclonal antibody (ankyrinB; Fig. 2 A, lane 2). The 210- and 190-kD crossreacting polypeptides are of the expected size for ankyrin with membrane-binding, spectrin-binding, and COOH-terminal domains. The smaller polypeptides presumably represent either alternatively spliced variants missing one or more of these domains or proteolytically cleaved forms of the 210- and 190-kD polypeptides. Activation of tyrosine kinases (Fig. 2 A, NGF or bFGF; lanes 4 and 5) or inactivation of tyrosine phosphatases (Fig. 2 A, dephostatin; lane 3) completely eliminate the ability of neurofascin to coimmunoprecipitate 210- and 190-kD ankyrin as well as the 102 and 104-kD crossreacting polypeptides.

Bottom Line: Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain.The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules.Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.

ABSTRACT
This paper presents evidence that a member of the L1 family of ankyrin-binding cell adhesion molecules is a substrate for protein tyrosine kinase(s) and phosphatase(s), identifies the highly conserved FIGQY tyrosine in the cytoplasmic domain as the principal site of phosphorylation, and demonstrates that phosphorylation of the FIGQY tyrosine abolishes ankyrin-binding activity. Neurofascin expressed in neuroblastoma cells is subject to tyrosine phosphorylation after activation of tyrosine kinases by NGF or bFGF or inactivation of tyrosine phosphatases with vanadate or dephostatin. Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain. The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules. Phosphorylation of the FIGQY tyrosine abolishes ankyrin binding, as determined by coimmunoprecipitation of endogenous ankyrin and in vitro ankyrin-binding assays. Measurements of fluorescence recovery after photobleaching demonstrate that phosphorylation of the FIGQY tyrosine also increases lateral mobility of neurofascin expressed in neuroblastoma cells to the same extent as removal of the cytoplasmic domain. Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals. These findings suggest that tyrosine phosphorylation at the FIGQY site represents a highly conserved mechanism, used by the entire class of L1-related cell adhesion molecules, for regulation of ankyrin-dependent connections to the spectrin skeleton.

Show MeSH
Related in: MedlinePlus