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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 and Nr-CAM are tyrosine phosphorylated  in developing rat brain. Neurofascin (A) or Nr-CAM (B) were  immunoprecipitated, from each noted time point, with either the  mucin-specific neurofascin polyclonal antibody (A) or the NrCAM–specific polyclonal antibody (B) after normalization for  expressed neurofascin or Nr-CAM levels, respectively (see Materials and Methods). Resolved proteins were transferred to nitrocellulose, and blots were incubated overnight at 4°C with the antiphosphotyrosine antibody.
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Figure 7: Neurofascin and Nr-CAM are tyrosine phosphorylated in developing rat brain. Neurofascin (A) or Nr-CAM (B) were immunoprecipitated, from each noted time point, with either the mucin-specific neurofascin polyclonal antibody (A) or the NrCAM–specific polyclonal antibody (B) after normalization for expressed neurofascin or Nr-CAM levels, respectively (see Materials and Methods). Resolved proteins were transferred to nitrocellulose, and blots were incubated overnight at 4°C with the antiphosphotyrosine antibody.

Mentions: We wished to determine whether neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in vivo to establish a physiological correlation for what we have observed in vitro. Consequently, we looked at each molecule during neuronal development in rat to see if either or both of these molecules exhibit a period of phosphotyrosine immunoreactivity. Fig. 7, A (neurofascin) and B (Nr-CAM) demonstrate that both of these molecules are tyrosine phosphorylated in vivo in a time-dependent fashion, with the maximal level of phosphotyrosine immunoreactivity present during the embyronic period. Phosphorimage scanning (data not shown) indicates there is nearly a threefold reduction in Nr-CAM tyrosine phosphorylation from embryonic day 15 to adult brain. In comparison, neurofascin phosphotyrosine immunoreactivity is intense at embryonic day 15. However, this signal is dramatically reduced (an ∼20-fold decline from embryonic day 15 to adult) over the denoted developmental period. With respect to neurofascin, it is interesting to note that the 186-kD isoform is the preferential splice form to be tyrosine phosphorylated, even when minimally expressed, as in embryonic day 15. Immunoprecipitation first with the anti-phosphotyrosine antibody followed by immunoblotting with either neurofascin or Nr-CAM antibodies rendered qualitatively similar results (data not shown).


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 and Nr-CAM are tyrosine phosphorylated  in developing rat brain. Neurofascin (A) or Nr-CAM (B) were  immunoprecipitated, from each noted time point, with either the  mucin-specific neurofascin polyclonal antibody (A) or the NrCAM–specific polyclonal antibody (B) after normalization for  expressed neurofascin or Nr-CAM levels, respectively (see Materials and Methods). Resolved proteins were transferred to nitrocellulose, and blots were incubated overnight at 4°C with the antiphosphotyrosine antibody.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Neurofascin and Nr-CAM are tyrosine phosphorylated in developing rat brain. Neurofascin (A) or Nr-CAM (B) were immunoprecipitated, from each noted time point, with either the mucin-specific neurofascin polyclonal antibody (A) or the NrCAM–specific polyclonal antibody (B) after normalization for expressed neurofascin or Nr-CAM levels, respectively (see Materials and Methods). Resolved proteins were transferred to nitrocellulose, and blots were incubated overnight at 4°C with the antiphosphotyrosine antibody.
Mentions: We wished to determine whether neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in vivo to establish a physiological correlation for what we have observed in vitro. Consequently, we looked at each molecule during neuronal development in rat to see if either or both of these molecules exhibit a period of phosphotyrosine immunoreactivity. Fig. 7, A (neurofascin) and B (Nr-CAM) demonstrate that both of these molecules are tyrosine phosphorylated in vivo in a time-dependent fashion, with the maximal level of phosphotyrosine immunoreactivity present during the embyronic period. Phosphorimage scanning (data not shown) indicates there is nearly a threefold reduction in Nr-CAM tyrosine phosphorylation from embryonic day 15 to adult brain. In comparison, neurofascin phosphotyrosine immunoreactivity is intense at embryonic day 15. However, this signal is dramatically reduced (an ∼20-fold decline from embryonic day 15 to adult) over the denoted developmental period. With respect to neurofascin, it is interesting to note that the 186-kD isoform is the preferential splice form to be tyrosine phosphorylated, even when minimally expressed, as in embryonic day 15. Immunoprecipitation first with the anti-phosphotyrosine antibody followed by immunoblotting with either neurofascin or Nr-CAM antibodies rendered qualitatively similar results (data not shown).

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