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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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Lateral mobility of neurofascin is increased by site-specific phosphorylation of FIGQY tyrosine. B104 cells expressing  either native neurofascin or the cytoplasmic domain deleted neurofascin (A) or the FIGQY to F tyrosine mutant form of neurofascin (B) were either untreated or treated with 100 ng/ml NGF  for 30 min and then immunolabeled with the HA-specific monoclonal antibody and an FITC-tagged secondary antibody. Cells  were subsequently monitored using an Odyssey confocal imaging  system (Noran Inc.) mounted on an inverted Nikon Diaphot microscope in combination with an imaging board (Bitflow) and eye  imager calculator software (IO Industries), a system that captures  images every 16.7 ms. FITC was excited at 488 nm and was monitored above 515 nm. FITC was photobleached using a focused  UV laser at 365 nm. Fluorescence measurements were determined by the National Institutes of Health Image method of analysis.
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Figure 5: Lateral mobility of neurofascin is increased by site-specific phosphorylation of FIGQY tyrosine. B104 cells expressing either native neurofascin or the cytoplasmic domain deleted neurofascin (A) or the FIGQY to F tyrosine mutant form of neurofascin (B) were either untreated or treated with 100 ng/ml NGF for 30 min and then immunolabeled with the HA-specific monoclonal antibody and an FITC-tagged secondary antibody. Cells were subsequently monitored using an Odyssey confocal imaging system (Noran Inc.) mounted on an inverted Nikon Diaphot microscope in combination with an imaging board (Bitflow) and eye imager calculator software (IO Industries), a system that captures images every 16.7 ms. FITC was excited at 488 nm and was monitored above 515 nm. FITC was photobleached using a focused UV laser at 365 nm. Fluorescence measurements were determined by the National Institutes of Health Image method of analysis.

Mentions: Native neurofascin exhibits essentially no recovery of fluorescence after photobleaching, implying its involvement in highly constraining intermolecular interactions (Fig. 5 A). In contrast, neurofascin with a fully truncated cytoplasmic domain exhibits a 10–15% recovery after photobleaching (Fig. 5 A), indicating that the cytoplasmic domain of neurofascin makes a small but significant contribution to the restriction in lateral mobility. The effect of the cytoplasmic domain presumably results from connections to the spectrin skeleton through ankyrin binding. Interactions involving the ectodomain and/or membrane-spanning domain of neurofascin, however, probably participate in the majority of restriction in the lateral mobility of neurofascin.


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)

Lateral mobility of neurofascin is increased by site-specific phosphorylation of FIGQY tyrosine. B104 cells expressing  either native neurofascin or the cytoplasmic domain deleted neurofascin (A) or the FIGQY to F tyrosine mutant form of neurofascin (B) were either untreated or treated with 100 ng/ml NGF  for 30 min and then immunolabeled with the HA-specific monoclonal antibody and an FITC-tagged secondary antibody. Cells  were subsequently monitored using an Odyssey confocal imaging  system (Noran Inc.) mounted on an inverted Nikon Diaphot microscope in combination with an imaging board (Bitflow) and eye  imager calculator software (IO Industries), a system that captures  images every 16.7 ms. FITC was excited at 488 nm and was monitored above 515 nm. FITC was photobleached using a focused  UV laser at 365 nm. Fluorescence measurements were determined by the National Institutes of Health Image method of analysis.
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Related In: Results  -  Collection

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Figure 5: Lateral mobility of neurofascin is increased by site-specific phosphorylation of FIGQY tyrosine. B104 cells expressing either native neurofascin or the cytoplasmic domain deleted neurofascin (A) or the FIGQY to F tyrosine mutant form of neurofascin (B) were either untreated or treated with 100 ng/ml NGF for 30 min and then immunolabeled with the HA-specific monoclonal antibody and an FITC-tagged secondary antibody. Cells were subsequently monitored using an Odyssey confocal imaging system (Noran Inc.) mounted on an inverted Nikon Diaphot microscope in combination with an imaging board (Bitflow) and eye imager calculator software (IO Industries), a system that captures images every 16.7 ms. FITC was excited at 488 nm and was monitored above 515 nm. FITC was photobleached using a focused UV laser at 365 nm. Fluorescence measurements were determined by the National Institutes of Health Image method of analysis.
Mentions: Native neurofascin exhibits essentially no recovery of fluorescence after photobleaching, implying its involvement in highly constraining intermolecular interactions (Fig. 5 A). In contrast, neurofascin with a fully truncated cytoplasmic domain exhibits a 10–15% recovery after photobleaching (Fig. 5 A), indicating that the cytoplasmic domain of neurofascin makes a small but significant contribution to the restriction in lateral mobility. The effect of the cytoplasmic domain presumably results from connections to the spectrin skeleton through ankyrin binding. Interactions involving the ectodomain and/or membrane-spanning domain of neurofascin, however, probably participate in the majority of restriction in the lateral mobility of neurofascin.

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