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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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FIGQY is the primary site of tyrosine phosphorylation on neurofascin and decreases ankyrin-binding capacity in a site-specific manner. (A) Neurofascin was immunoprecipitated after a 30-min treatment of the LVDY to F and the FIGQY to F neurofascin  transfected cells with one of the following agents: dephostatin (10 μM), NGF (100 ng/ml), or bFGF (50 ng/ml). Subsequent immunoblots  were probed with the anti-phosphotyrosine antibody overnight at 4°C followed by 125I-labeled protein A. (B) The immunoblot visualized above was subjected to Phosphorimage scanning to quantitate the extent of tyrosine phosphorylation of full length neurofascin under the given treatment conditions. The phosphotyrosine signals for both tyrosine mutants are expressed relative to the signals obtained  from treatment of B104 cells expressing native, full length neurofascin (Fig. 1 A). All cells were treated during the same experiment. (C)  Neurofascin was immunoprecipitated from each treatment group for each denoted cell line. Immune complexes were electrophoresed  on SDS-PAGE, and resolved proteins were transferred to nitrocellulose. Blots were incubated with the brain ankyrin-specific polyclonal antibody followed by 125I-labeled protein A. (D) Neurofascin was immunoprecipitated following noted treatments from each cell  line. Immune complexes, captured via protein A-labeled latex beads, were then used in the ankyrin binding assay that has been described above. The LVDY to F tyrosine mutant, with an intact FIGQY tyrosine residue, is subject to ankyrin binding regulation via tyrosine phosphorylation, whereas the FIGQY to F tyrosine mutant is resistant to phosphorylation-induced decreases in the in vitro  ankyrin binding assays.
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Figure 4: FIGQY is the primary site of tyrosine phosphorylation on neurofascin and decreases ankyrin-binding capacity in a site-specific manner. (A) Neurofascin was immunoprecipitated after a 30-min treatment of the LVDY to F and the FIGQY to F neurofascin transfected cells with one of the following agents: dephostatin (10 μM), NGF (100 ng/ml), or bFGF (50 ng/ml). Subsequent immunoblots were probed with the anti-phosphotyrosine antibody overnight at 4°C followed by 125I-labeled protein A. (B) The immunoblot visualized above was subjected to Phosphorimage scanning to quantitate the extent of tyrosine phosphorylation of full length neurofascin under the given treatment conditions. The phosphotyrosine signals for both tyrosine mutants are expressed relative to the signals obtained from treatment of B104 cells expressing native, full length neurofascin (Fig. 1 A). All cells were treated during the same experiment. (C) Neurofascin was immunoprecipitated from each treatment group for each denoted cell line. Immune complexes were electrophoresed on SDS-PAGE, and resolved proteins were transferred to nitrocellulose. Blots were incubated with the brain ankyrin-specific polyclonal antibody followed by 125I-labeled protein A. (D) Neurofascin was immunoprecipitated following noted treatments from each cell line. Immune complexes, captured via protein A-labeled latex beads, were then used in the ankyrin binding assay that has been described above. The LVDY to F tyrosine mutant, with an intact FIGQY tyrosine residue, is subject to ankyrin binding regulation via tyrosine phosphorylation, whereas the FIGQY to F tyrosine mutant is resistant to phosphorylation-induced decreases in the in vitro ankyrin binding assays.

Mentions: The results with deletion mutants implicated the five residues, FIGQY, as essential for full ankyrin binding, with the NH2-terminal residues SLVDY potentially playing a secondary role, and suggested that phosphorylation of one or both of these tyrosines could be the basis for regulation of ankyrin-binding activity. The role of these tyrosines in regulation was evaluated by site-directed mutagenesis of each to phenylalanine (Fig. 3 A) followed by evaluation of tyrosine phosphorylation and ankyrin-binding activity. These constructs were stably transfected in B104 cells that were subsequently treated with NGF, bFGF, or dephostatin. The LVDY to F tyrosine mutant has a tyrosine phosphorylation pattern (Fig. 4 A) similar to that seen for the full length, native neurofascin (Fig. 1 A). However, the FIGQY to F tyrosine mutant is significantly less phosphorylated under the same pharmacological paradigm. Fig. 4 B quantitatively depicts the effects of the treatments on tyrosine phosphorylation. These results identify the FIGQY tyrosine residue as the primary site of tyrosine phosphorylation under the given treatment conditions.


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)

FIGQY is the primary site of tyrosine phosphorylation on neurofascin and decreases ankyrin-binding capacity in a site-specific manner. (A) Neurofascin was immunoprecipitated after a 30-min treatment of the LVDY to F and the FIGQY to F neurofascin  transfected cells with one of the following agents: dephostatin (10 μM), NGF (100 ng/ml), or bFGF (50 ng/ml). Subsequent immunoblots  were probed with the anti-phosphotyrosine antibody overnight at 4°C followed by 125I-labeled protein A. (B) The immunoblot visualized above was subjected to Phosphorimage scanning to quantitate the extent of tyrosine phosphorylation of full length neurofascin under the given treatment conditions. The phosphotyrosine signals for both tyrosine mutants are expressed relative to the signals obtained  from treatment of B104 cells expressing native, full length neurofascin (Fig. 1 A). All cells were treated during the same experiment. (C)  Neurofascin was immunoprecipitated from each treatment group for each denoted cell line. Immune complexes were electrophoresed  on SDS-PAGE, and resolved proteins were transferred to nitrocellulose. Blots were incubated with the brain ankyrin-specific polyclonal antibody followed by 125I-labeled protein A. (D) Neurofascin was immunoprecipitated following noted treatments from each cell  line. Immune complexes, captured via protein A-labeled latex beads, were then used in the ankyrin binding assay that has been described above. The LVDY to F tyrosine mutant, with an intact FIGQY tyrosine residue, is subject to ankyrin binding regulation via tyrosine phosphorylation, whereas the FIGQY to F tyrosine mutant is resistant to phosphorylation-induced decreases in the in vitro  ankyrin binding assays.
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Figure 4: FIGQY is the primary site of tyrosine phosphorylation on neurofascin and decreases ankyrin-binding capacity in a site-specific manner. (A) Neurofascin was immunoprecipitated after a 30-min treatment of the LVDY to F and the FIGQY to F neurofascin transfected cells with one of the following agents: dephostatin (10 μM), NGF (100 ng/ml), or bFGF (50 ng/ml). Subsequent immunoblots were probed with the anti-phosphotyrosine antibody overnight at 4°C followed by 125I-labeled protein A. (B) The immunoblot visualized above was subjected to Phosphorimage scanning to quantitate the extent of tyrosine phosphorylation of full length neurofascin under the given treatment conditions. The phosphotyrosine signals for both tyrosine mutants are expressed relative to the signals obtained from treatment of B104 cells expressing native, full length neurofascin (Fig. 1 A). All cells were treated during the same experiment. (C) Neurofascin was immunoprecipitated from each treatment group for each denoted cell line. Immune complexes were electrophoresed on SDS-PAGE, and resolved proteins were transferred to nitrocellulose. Blots were incubated with the brain ankyrin-specific polyclonal antibody followed by 125I-labeled protein A. (D) Neurofascin was immunoprecipitated following noted treatments from each cell line. Immune complexes, captured via protein A-labeled latex beads, were then used in the ankyrin binding assay that has been described above. The LVDY to F tyrosine mutant, with an intact FIGQY tyrosine residue, is subject to ankyrin binding regulation via tyrosine phosphorylation, whereas the FIGQY to F tyrosine mutant is resistant to phosphorylation-induced decreases in the in vitro ankyrin binding assays.
Mentions: The results with deletion mutants implicated the five residues, FIGQY, as essential for full ankyrin binding, with the NH2-terminal residues SLVDY potentially playing a secondary role, and suggested that phosphorylation of one or both of these tyrosines could be the basis for regulation of ankyrin-binding activity. The role of these tyrosines in regulation was evaluated by site-directed mutagenesis of each to phenylalanine (Fig. 3 A) followed by evaluation of tyrosine phosphorylation and ankyrin-binding activity. These constructs were stably transfected in B104 cells that were subsequently treated with NGF, bFGF, or dephostatin. The LVDY to F tyrosine mutant has a tyrosine phosphorylation pattern (Fig. 4 A) similar to that seen for the full length, native neurofascin (Fig. 1 A). However, the FIGQY to F tyrosine mutant is significantly less phosphorylated under the same pharmacological paradigm. Fig. 4 B quantitatively depicts the effects of the treatments on tyrosine phosphorylation. These results identify the FIGQY tyrosine residue as the primary site of tyrosine phosphorylation under the given treatment conditions.

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