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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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Neurofascin is phosphorylated in vivo by activation of tyrosine kinase signaling cascades or inactivation of tyrosine phosphatases. (A) HA epitope-tagged neurofascin was expressed in the rat neuroblastoma B104 cell line. 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). Neurofascin was subsequently immunoprecipitated from 500 μg crude cell extract using an HA-specific monoclonal antibody. Immune complexes were then electrophoresed on SDS-PAGE and transferred to nitrocellulose. Immunoblots were probed overnight at 4°C with a phosphotyrosine-specific  polyclonal antibody followed by incubation with 125I-radiolabeled protein A. Blots were visualized via autoradiography. (B) HA  epitope-tagged neurofascin was expressed in the rat neuroblastoma B104 cell line. Transfected cells were treated with one of the following agents (at the noted concentrations) for 30 min: vanadate, dephostatin, NGF, or bNGF. Neurofascin was immunoprecipitated from  500 μg crude cell lysate using the HA-specific monoclonal antibody. Immune complexes were electrophoresed on SDS-PAGE and  transferred to nitrocellulose. Blots were probed with the phosphotyrosine-specific polyclonal antibody followed by incubation with 125Iradiolabeled protein A. Subsequently, blots were subjected to Phosphorimage scanning to quantitate the extent of tyrosine phosphorylation of full length neurofascin under the given treatment conditions.
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Figure 1: Neurofascin is phosphorylated in vivo by activation of tyrosine kinase signaling cascades or inactivation of tyrosine phosphatases. (A) HA epitope-tagged neurofascin was expressed in the rat neuroblastoma B104 cell line. 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). Neurofascin was subsequently immunoprecipitated from 500 μg crude cell extract using an HA-specific monoclonal antibody. Immune complexes were then electrophoresed on SDS-PAGE and transferred to nitrocellulose. Immunoblots were probed overnight at 4°C with a phosphotyrosine-specific polyclonal antibody followed by incubation with 125I-radiolabeled protein A. Blots were visualized via autoradiography. (B) HA epitope-tagged neurofascin was expressed in the rat neuroblastoma B104 cell line. Transfected cells were treated with one of the following agents (at the noted concentrations) for 30 min: vanadate, dephostatin, NGF, or bNGF. Neurofascin was immunoprecipitated from 500 μg crude cell lysate using the HA-specific monoclonal antibody. Immune complexes were electrophoresed on SDS-PAGE and transferred to nitrocellulose. Blots were probed with the phosphotyrosine-specific polyclonal antibody followed by incubation with 125Iradiolabeled protein A. Subsequently, blots were subjected to Phosphorimage scanning to quantitate the extent of tyrosine phosphorylation of full length neurofascin under the given treatment conditions.

Mentions: Neurofascin and other members of the L1 family of nervous system cell adhesion molecules have four highly conserved tyrosine residues in their cytoplasmic domains. The possibility that one or more of these tyrosines are substrates for phosphorylation by protein kinases was evaluated using HA epitope-tagged neurofascin stably transfected into the B104 rat neuroblastoma cell line. Transfected cells were first treated with activators of receptor tyrosine kinases (NGF and bFGF) or inhibitors of protein tyrosine phosphatases (vanadate and dephostatin), after which neurofascin was immunoprecipitated using the HA-specific monoclonal antibody. Subsequent immunoblots were probed with an anti-phosphotyrosine antibody (Fig. 1 A). The protein tyrosine phosphatase inhibitor dephostatin (10 μM for 30 min) resulted in strong anti-phosphotyrosine immunoreactivity (Fig. 1 A, lane 2), as did sodium metavanadate (data not shown). Furthermore, incubation of the transfected cells with either nerve growth factor (100 ng/ml for 30 min; Fig. 1 A, lane 3) or basic fibroblast growth factor (50 ng/ml for 30 min; Fig. 1 A, lane 4) also generated a strong immunoreactive pool of tyrosine-phosphorylated neurofascin. Fig. 1 B quantitates the extent of tyrosine phosphorylation of neurofascin after treatment of transfected cells with either protein tyrosine phosphatase inhibitors (vanadate and dephostatin) or tyrosine kinase activators (NGF and bFGF) over an extended concentration range. These results demonstrate that neurofascin is a target for both protein tyrosine kinases and phosphatases in vivo when the relevant enzymes are pharmacologically perturbed in a dose-dependent manner.


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)

Neurofascin is phosphorylated in vivo by activation of tyrosine kinase signaling cascades or inactivation of tyrosine phosphatases. (A) HA epitope-tagged neurofascin was expressed in the rat neuroblastoma B104 cell line. 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). Neurofascin was subsequently immunoprecipitated from 500 μg crude cell extract using an HA-specific monoclonal antibody. Immune complexes were then electrophoresed on SDS-PAGE and transferred to nitrocellulose. Immunoblots were probed overnight at 4°C with a phosphotyrosine-specific  polyclonal antibody followed by incubation with 125I-radiolabeled protein A. Blots were visualized via autoradiography. (B) HA  epitope-tagged neurofascin was expressed in the rat neuroblastoma B104 cell line. Transfected cells were treated with one of the following agents (at the noted concentrations) for 30 min: vanadate, dephostatin, NGF, or bNGF. Neurofascin was immunoprecipitated from  500 μg crude cell lysate using the HA-specific monoclonal antibody. Immune complexes were electrophoresed on SDS-PAGE and  transferred to nitrocellulose. Blots were probed with the phosphotyrosine-specific polyclonal antibody followed by incubation with 125Iradiolabeled protein A. Subsequently, blots were subjected to Phosphorimage scanning to quantitate the extent of tyrosine phosphorylation of full length neurofascin under the given treatment conditions.
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Related In: Results  -  Collection

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Figure 1: Neurofascin is phosphorylated in vivo by activation of tyrosine kinase signaling cascades or inactivation of tyrosine phosphatases. (A) HA epitope-tagged neurofascin was expressed in the rat neuroblastoma B104 cell line. 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). Neurofascin was subsequently immunoprecipitated from 500 μg crude cell extract using an HA-specific monoclonal antibody. Immune complexes were then electrophoresed on SDS-PAGE and transferred to nitrocellulose. Immunoblots were probed overnight at 4°C with a phosphotyrosine-specific polyclonal antibody followed by incubation with 125I-radiolabeled protein A. Blots were visualized via autoradiography. (B) HA epitope-tagged neurofascin was expressed in the rat neuroblastoma B104 cell line. Transfected cells were treated with one of the following agents (at the noted concentrations) for 30 min: vanadate, dephostatin, NGF, or bNGF. Neurofascin was immunoprecipitated from 500 μg crude cell lysate using the HA-specific monoclonal antibody. Immune complexes were electrophoresed on SDS-PAGE and transferred to nitrocellulose. Blots were probed with the phosphotyrosine-specific polyclonal antibody followed by incubation with 125Iradiolabeled protein A. Subsequently, blots were subjected to Phosphorimage scanning to quantitate the extent of tyrosine phosphorylation of full length neurofascin under the given treatment conditions.
Mentions: Neurofascin and other members of the L1 family of nervous system cell adhesion molecules have four highly conserved tyrosine residues in their cytoplasmic domains. The possibility that one or more of these tyrosines are substrates for phosphorylation by protein kinases was evaluated using HA epitope-tagged neurofascin stably transfected into the B104 rat neuroblastoma cell line. Transfected cells were first treated with activators of receptor tyrosine kinases (NGF and bFGF) or inhibitors of protein tyrosine phosphatases (vanadate and dephostatin), after which neurofascin was immunoprecipitated using the HA-specific monoclonal antibody. Subsequent immunoblots were probed with an anti-phosphotyrosine antibody (Fig. 1 A). The protein tyrosine phosphatase inhibitor dephostatin (10 μM for 30 min) resulted in strong anti-phosphotyrosine immunoreactivity (Fig. 1 A, lane 2), as did sodium metavanadate (data not shown). Furthermore, incubation of the transfected cells with either nerve growth factor (100 ng/ml for 30 min; Fig. 1 A, lane 3) or basic fibroblast growth factor (50 ng/ml for 30 min; Fig. 1 A, lane 4) also generated a strong immunoreactive pool of tyrosine-phosphorylated neurofascin. Fig. 1 B quantitates the extent of tyrosine phosphorylation of neurofascin after treatment of transfected cells with either protein tyrosine phosphatase inhibitors (vanadate and dephostatin) or tyrosine kinase activators (NGF and bFGF) over an extended concentration range. These results demonstrate that neurofascin is a target for both protein tyrosine kinases and phosphatases in vivo when the relevant enzymes are pharmacologically perturbed in a dose-dependent manner.

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