Limits...
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.

Show MeSH

Related in: MedlinePlus

The highly conserved FIGQY amino acid stretch in the cytoplasmic domain of neurofascin is responsible for ankyrin binding.  (A) Cytoplasmic sequences of L1 family cell adhesion molecules and definition of the neurofascin cytoplasmic domain constructs. (B)  Epitope-tagged neurofascin was immunoprecipitated from 250 μg crude cell lysate, as previously described. 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 ankyrin-specific polyclonal antibody. After incubation with 125I-labeled protein A (2 h at 4°C),  immunoblots were visualized by autoradiography. (C) HA-epitope tagged neurofascin was expressed in B104 cells and subsequently immunoprecipitated from 250 μg crude cell lysate as described previously. Captured immune complexes (on protein A–labeled latex  beads) were then used for the in vitro ankyrin binding assays that have been described above.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139872&req=5

Figure 3: The highly conserved FIGQY amino acid stretch in the cytoplasmic domain of neurofascin is responsible for ankyrin binding. (A) Cytoplasmic sequences of L1 family cell adhesion molecules and definition of the neurofascin cytoplasmic domain constructs. (B) Epitope-tagged neurofascin was immunoprecipitated from 250 μg crude cell lysate, as previously described. 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 ankyrin-specific polyclonal antibody. After incubation with 125I-labeled protein A (2 h at 4°C), immunoblots were visualized by autoradiography. (C) HA-epitope tagged neurofascin was expressed in B104 cells and subsequently immunoprecipitated from 250 μg crude cell lysate as described previously. Captured immune complexes (on protein A–labeled latex beads) were then used for the in vitro ankyrin binding assays that have been described above.

Mentions: The finding that tyrosine phosphorylation abolished ankyrinbinding activity suggested the possibility that definition of the ankyrin-binding determinants of neurofascin would also lead to identification of the specific site of tyrosine phosphorylation. The ankyrin-binding site of neurofascin has been mapped previously to a 35–amino acid stretch in the COOH-terminal portion of the cytoplasmic domain encompassing two highly conserved tyrosine residues (Davis and Bennett, 1994). Deletion constructs targeted to this general location, as well as the full length and fully truncated constructs (Fig. 3 A), were evaluated for ankyrinbinding activity as determined by coimmunoprecipitation of endogenous ankyrin (Fig. 3 B) and by direct measurement of ankyrin-binding activity of isolated neurofascin (Fig. 3 C).


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)

The highly conserved FIGQY amino acid stretch in the cytoplasmic domain of neurofascin is responsible for ankyrin binding.  (A) Cytoplasmic sequences of L1 family cell adhesion molecules and definition of the neurofascin cytoplasmic domain constructs. (B)  Epitope-tagged neurofascin was immunoprecipitated from 250 μg crude cell lysate, as previously described. 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 ankyrin-specific polyclonal antibody. After incubation with 125I-labeled protein A (2 h at 4°C),  immunoblots were visualized by autoradiography. (C) HA-epitope tagged neurofascin was expressed in B104 cells and subsequently immunoprecipitated from 250 μg crude cell lysate as described previously. Captured immune complexes (on protein A–labeled latex  beads) were then used for the in vitro ankyrin binding assays that have been described above.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: The highly conserved FIGQY amino acid stretch in the cytoplasmic domain of neurofascin is responsible for ankyrin binding. (A) Cytoplasmic sequences of L1 family cell adhesion molecules and definition of the neurofascin cytoplasmic domain constructs. (B) Epitope-tagged neurofascin was immunoprecipitated from 250 μg crude cell lysate, as previously described. 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 ankyrin-specific polyclonal antibody. After incubation with 125I-labeled protein A (2 h at 4°C), immunoblots were visualized by autoradiography. (C) HA-epitope tagged neurofascin was expressed in B104 cells and subsequently immunoprecipitated from 250 μg crude cell lysate as described previously. Captured immune complexes (on protein A–labeled latex beads) were then used for the in vitro ankyrin binding assays that have been described above.
Mentions: The finding that tyrosine phosphorylation abolished ankyrinbinding activity suggested the possibility that definition of the ankyrin-binding determinants of neurofascin would also lead to identification of the specific site of tyrosine phosphorylation. The ankyrin-binding site of neurofascin has been mapped previously to a 35–amino acid stretch in the COOH-terminal portion of the cytoplasmic domain encompassing two highly conserved tyrosine residues (Davis and Bennett, 1994). Deletion constructs targeted to this general location, as well as the full length and fully truncated constructs (Fig. 3 A), were evaluated for ankyrinbinding activity as determined by coimmunoprecipitation of endogenous ankyrin (Fig. 3 B) and by direct measurement of ankyrin-binding activity of isolated neurofascin (Fig. 3 C).

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