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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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Phosphorylation  of the FIGQY tyrosine increases detergent extractability of neurofascin. (A) Control cells were first fixed with  2% paraformaldehyde for 20  min at 4°C and then treated  with a PBS blocking buffer  (150 mM NaCl, 30 mM sodium phosphate, pH 7.4, 1%  BSA, and 10% normal goat  serum) for 30 min. Cells were  subsequently treated with  0.2% Triton X-100 in the  PBS blocking solution for 10  min at room temperature  and washed with a 0.2%  Tween-20 solution. (B) Cells  were first treated with 0.2%  Triton X-100 in DMEM cell  culture media (GIBCO  BRL) for 10 min at room  temperature and then subsequently fixed with 2%  paraformaldehyde for 20 min  at 4°C. Cells were then incubated with PBS blocking  buffer for 30 min and washed with a 0.2% Tween-20 solution. (C)  Cells were incubated with 100 ng/ml NGF for 90 min before the  treatment described in (B). After fixation, all cells were incubated overnight at 4°C with the HA-specific monoclonal antibody  (1:1,000). Cells were washed in 0.2% Tween-20 buffer and subsequently incubated with anti–rabbit FITC-conjugated secondary  antibody (1:2,000) for 2 h at 4°C. Finally, cells were washed with  the 0.2% Tween-20 solution. Confocal images and fluorescence  intensities (intensity/μm2) were obtained with an LSM Zeiss confocal microscope using a Zeiss data analysis software package.  (D) Quantitative analysis of fluorescence intensities. Cells  treated with 0.2% Triton X-100 before paraformaldehyde fixation are presented relative to fluorescence intensities for cells  that were paraformaldehyde fixed before Triton X-100 extraction.
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Figure 6: Phosphorylation of the FIGQY tyrosine increases detergent extractability of neurofascin. (A) Control cells were first fixed with 2% paraformaldehyde for 20 min at 4°C and then treated with a PBS blocking buffer (150 mM NaCl, 30 mM sodium phosphate, pH 7.4, 1% BSA, and 10% normal goat serum) for 30 min. Cells were subsequently treated with 0.2% Triton X-100 in the PBS blocking solution for 10 min at room temperature and washed with a 0.2% Tween-20 solution. (B) Cells were first treated with 0.2% Triton X-100 in DMEM cell culture media (GIBCO BRL) for 10 min at room temperature and then subsequently fixed with 2% paraformaldehyde for 20 min at 4°C. Cells were then incubated with PBS blocking buffer for 30 min and washed with a 0.2% Tween-20 solution. (C) Cells were incubated with 100 ng/ml NGF for 90 min before the treatment described in (B). After fixation, all cells were incubated overnight at 4°C with the HA-specific monoclonal antibody (1:1,000). Cells were washed in 0.2% Tween-20 buffer and subsequently incubated with anti–rabbit FITC-conjugated secondary antibody (1:2,000) for 2 h at 4°C. Finally, cells were washed with the 0.2% Tween-20 solution. Confocal images and fluorescence intensities (intensity/μm2) were obtained with an LSM Zeiss confocal microscope using a Zeiss data analysis software package. (D) Quantitative analysis of fluorescence intensities. Cells treated with 0.2% Triton X-100 before paraformaldehyde fixation are presented relative to fluorescence intensities for cells that were paraformaldehyde fixed before Triton X-100 extraction.

Mentions: Effects of tyrosine phosphorylation on the static in vivo interaction between forms of expressed neurofascin with detergent-insoluble components of the spectrin-based cytoskeleton were evaluated by analyzing the changes in rhodamine-labeled, HA epitope-tagged neurofascin intensity after Triton X-100 extraction. Detergent-extraction experiments allow one to examine the steady state interaction between neurofascin and ankyrin and the factors that may modulate this interaction. B104 cells expressing either native neurofascin, cytoplasmic domain-deleted neurofascin, or the FIGQY to F tyrosine mutant form of neurofascin were either untreated or treated with 100 ng/ml NGF for 90 min (Fig. 6, A–D). Cells (extracted with 0.2% Triton X-100 before fixation) expressing full length neurofascin exhibited a 65% retention of fluorescence, as compared to cells fixed before detergent treatment. The fluorescence was reduced to 28% after treatment with NGF. Neurofascin lacking the cytoplasmic domain was retained at only 10% in the presence of detergent, indicating that NGF abolished most, but not all, of the interactions of the cytoplasmic domain with detergent-insoluble proteins. Interestingly, the reduction in fluorescence due to NGF-induced tyrosine phosphorylation of native neurofascin is quantitatively similar to the reduction in the in vitro binding capacity of neurofascin after tyrosine phosphorylation (Fig. 2 B). In contrast to native neurofascin, cells expressing the FIGQY to F tyrosine mutant neurofascin were unaffected by NGF treatment and retained ∼65% of the label in the presence and absence of NGF, once again indicating that this site is the primary one responsible for phosphotyrosine-dependent regulation of neurofascin–ankyrin interactions.


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)

Phosphorylation  of the FIGQY tyrosine increases detergent extractability of neurofascin. (A) Control cells were first fixed with  2% paraformaldehyde for 20  min at 4°C and then treated  with a PBS blocking buffer  (150 mM NaCl, 30 mM sodium phosphate, pH 7.4, 1%  BSA, and 10% normal goat  serum) for 30 min. Cells were  subsequently treated with  0.2% Triton X-100 in the  PBS blocking solution for 10  min at room temperature  and washed with a 0.2%  Tween-20 solution. (B) Cells  were first treated with 0.2%  Triton X-100 in DMEM cell  culture media (GIBCO  BRL) for 10 min at room  temperature and then subsequently fixed with 2%  paraformaldehyde for 20 min  at 4°C. Cells were then incubated with PBS blocking  buffer for 30 min and washed with a 0.2% Tween-20 solution. (C)  Cells were incubated with 100 ng/ml NGF for 90 min before the  treatment described in (B). After fixation, all cells were incubated overnight at 4°C with the HA-specific monoclonal antibody  (1:1,000). Cells were washed in 0.2% Tween-20 buffer and subsequently incubated with anti–rabbit FITC-conjugated secondary  antibody (1:2,000) for 2 h at 4°C. Finally, cells were washed with  the 0.2% Tween-20 solution. Confocal images and fluorescence  intensities (intensity/μm2) were obtained with an LSM Zeiss confocal microscope using a Zeiss data analysis software package.  (D) Quantitative analysis of fluorescence intensities. Cells  treated with 0.2% Triton X-100 before paraformaldehyde fixation are presented relative to fluorescence intensities for cells  that were paraformaldehyde fixed before Triton X-100 extraction.
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Figure 6: Phosphorylation of the FIGQY tyrosine increases detergent extractability of neurofascin. (A) Control cells were first fixed with 2% paraformaldehyde for 20 min at 4°C and then treated with a PBS blocking buffer (150 mM NaCl, 30 mM sodium phosphate, pH 7.4, 1% BSA, and 10% normal goat serum) for 30 min. Cells were subsequently treated with 0.2% Triton X-100 in the PBS blocking solution for 10 min at room temperature and washed with a 0.2% Tween-20 solution. (B) Cells were first treated with 0.2% Triton X-100 in DMEM cell culture media (GIBCO BRL) for 10 min at room temperature and then subsequently fixed with 2% paraformaldehyde for 20 min at 4°C. Cells were then incubated with PBS blocking buffer for 30 min and washed with a 0.2% Tween-20 solution. (C) Cells were incubated with 100 ng/ml NGF for 90 min before the treatment described in (B). After fixation, all cells were incubated overnight at 4°C with the HA-specific monoclonal antibody (1:1,000). Cells were washed in 0.2% Tween-20 buffer and subsequently incubated with anti–rabbit FITC-conjugated secondary antibody (1:2,000) for 2 h at 4°C. Finally, cells were washed with the 0.2% Tween-20 solution. Confocal images and fluorescence intensities (intensity/μm2) were obtained with an LSM Zeiss confocal microscope using a Zeiss data analysis software package. (D) Quantitative analysis of fluorescence intensities. Cells treated with 0.2% Triton X-100 before paraformaldehyde fixation are presented relative to fluorescence intensities for cells that were paraformaldehyde fixed before Triton X-100 extraction.
Mentions: Effects of tyrosine phosphorylation on the static in vivo interaction between forms of expressed neurofascin with detergent-insoluble components of the spectrin-based cytoskeleton were evaluated by analyzing the changes in rhodamine-labeled, HA epitope-tagged neurofascin intensity after Triton X-100 extraction. Detergent-extraction experiments allow one to examine the steady state interaction between neurofascin and ankyrin and the factors that may modulate this interaction. B104 cells expressing either native neurofascin, cytoplasmic domain-deleted neurofascin, or the FIGQY to F tyrosine mutant form of neurofascin were either untreated or treated with 100 ng/ml NGF for 90 min (Fig. 6, A–D). Cells (extracted with 0.2% Triton X-100 before fixation) expressing full length neurofascin exhibited a 65% retention of fluorescence, as compared to cells fixed before detergent treatment. The fluorescence was reduced to 28% after treatment with NGF. Neurofascin lacking the cytoplasmic domain was retained at only 10% in the presence of detergent, indicating that NGF abolished most, but not all, of the interactions of the cytoplasmic domain with detergent-insoluble proteins. Interestingly, the reduction in fluorescence due to NGF-induced tyrosine phosphorylation of native neurofascin is quantitatively similar to the reduction in the in vitro binding capacity of neurofascin after tyrosine phosphorylation (Fig. 2 B). In contrast to native neurofascin, cells expressing the FIGQY to F tyrosine mutant neurofascin were unaffected by NGF treatment and retained ∼65% of the label in the presence and absence of NGF, once again indicating that this site is the primary one responsible for phosphotyrosine-dependent regulation of neurofascin–ankyrin interactions.

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