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The junction-associated protein AF-6 interacts and clusters with specific Eph receptor tyrosine kinases at specialized sites of cell-cell contact in the brain.

Buchert M, Schneider S, Meskenaite V, Adams MT, Canaani E, Baechi T, Moelling K, Hovens CM - J. Cell Biol. (1999)

Bottom Line: Furthermore, AF-6 is a substrate for a subgroup of Eph receptors and phosphorylation of AF-6 is dependent on a functional kinase domain of the receptor.The physical interaction of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipitation from whole rat brain lysates.AF-6 is a candidate for mediating the clustering of Eph receptors at postsynaptic specializations in the adult rat brain.

View Article: PubMed Central - PubMed

Affiliation: Institut für Medizinische Virologie, Universität Zürich, Switzerland.

ABSTRACT
The AF-6/afadin protein, which contains a single PDZ domain, forms a peripheral component of cell membranes at specialized sites of cell-cell junctions. To identify potential receptor-binding targets of AF-6 we screened the PDZ domain of AF-6 against a range of COOH-terminal peptides selected from receptors having potential PDZ domain-binding termini. The PDZ domain of AF-6 interacts with a subset of members of the Eph subfamily of RTKs via its COOH terminus both in vitro and in vivo. Cotransfection of a green fluorescent protein-tagged AF-6 fusion protein with full-length Eph receptors into heterologous cells induces a clustering of the Eph receptors and AF-6 at sites of cell-cell contact. Immunohistochemical analysis in the adult rat brain reveals coclustering of AF-6 with Eph receptors at postsynaptic membrane sites of excitatory synapses in the hippocampus. Furthermore, AF-6 is a substrate for a subgroup of Eph receptors and phosphorylation of AF-6 is dependent on a functional kinase domain of the receptor. The physical interaction of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipitation from whole rat brain lysates. AF-6 is a candidate for mediating the clustering of Eph receptors at postsynaptic specializations in the adult rat brain.

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Phosphorylation of  full-length AF-6 by the overexpressed EphB2 and EphB3  receptors but not by a kinase-deficient mutant. 293T  cells were transiently transfected with either a FLAG-tagged AF-6 (AF-6) alone or  together with the wild-type  EphB3 receptor (EphB3),  a kinase-deficient mutant  (EphB3K665R) or with the  EphB2 receptor (EphB2).  Transfected cells were lysed  and equal amounts of each  lysate were probed on a  Western blot (WB) either  with (a) a monoclonal anti-pTyr antibody, (b) with a  monoclonal anti-FLAG M2  antibody, (c) with polyclonal  antisera against EphB2, or (d)  against EphB3. (e) 293T cells  were transiently transfected  with either FLAG-tagged AF-6  (AF-6) alone or together with  wild-type EphB3 (EphB3), a  kinase-deficient mutant (Eph  B3K665R), EphB2 (EphB2).  Cell lysates were immunoprecipitated (IP) with anti-FLAG M2 antibodies. Immunoprecipitates were probed  on a Western blot (WB) either with a monoclonal anti-pTyr antibody (top) or with  anti-FLAG M2 antibodies  (bottom). Y is Tyr.
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Figure 7: Phosphorylation of full-length AF-6 by the overexpressed EphB2 and EphB3 receptors but not by a kinase-deficient mutant. 293T cells were transiently transfected with either a FLAG-tagged AF-6 (AF-6) alone or together with the wild-type EphB3 receptor (EphB3), a kinase-deficient mutant (EphB3K665R) or with the EphB2 receptor (EphB2). Transfected cells were lysed and equal amounts of each lysate were probed on a Western blot (WB) either with (a) a monoclonal anti-pTyr antibody, (b) with a monoclonal anti-FLAG M2 antibody, (c) with polyclonal antisera against EphB2, or (d) against EphB3. (e) 293T cells were transiently transfected with either FLAG-tagged AF-6 (AF-6) alone or together with wild-type EphB3 (EphB3), a kinase-deficient mutant (Eph B3K665R), EphB2 (EphB2). Cell lysates were immunoprecipitated (IP) with anti-FLAG M2 antibodies. Immunoprecipitates were probed on a Western blot (WB) either with a monoclonal anti-pTyr antibody (top) or with anti-FLAG M2 antibodies (bottom). Y is Tyr.

Mentions: To obtain some information on the biological significance of the interaction between AF-6 and Eph receptors, especially whether AF-6 is a substrate of the Eph receptor tyrosine kinases, we transiently transfected 293T cells with AF-6 along with either wild-type EphB3, EphB2, or a kinase-deficient mutant EphB3 receptor (EphB3K665R). The result shown in Fig. 7 a demonstrates that AF-6 is tyrosine phosphorylated only when coexpressed with wild-type EphB2 or EphB3 receptors. No phosphorylation occurred with a kinase-negative mutant receptor. Phosphorylation of AF-6 is dependent on previous activation of the receptor which is achieved by overexpression in 293T cells (Fig. 7 a and data not shown). When transfected AF-6 is immunoprecipitated from 293T cells, strong phosphorylation of the immunoprecipitated AF-6 is detected when it is cotransfected with the wild-type EphB2 and EphB3 receptors, whereas AF-6 is not phosphorylated when transfected alone or with the kinase-negative mutant EphB3K665R (Fig. 7 e, top). Taken together, these results demonstrate that AF-6 is phosphorylated by the EphB2 and EphB3 receptors in a kinase-dependent fashion and demonstrates that AF-6 is a substrate for a subgroup of Eph receptors.


The junction-associated protein AF-6 interacts and clusters with specific Eph receptor tyrosine kinases at specialized sites of cell-cell contact in the brain.

Buchert M, Schneider S, Meskenaite V, Adams MT, Canaani E, Baechi T, Moelling K, Hovens CM - J. Cell Biol. (1999)

Phosphorylation of  full-length AF-6 by the overexpressed EphB2 and EphB3  receptors but not by a kinase-deficient mutant. 293T  cells were transiently transfected with either a FLAG-tagged AF-6 (AF-6) alone or  together with the wild-type  EphB3 receptor (EphB3),  a kinase-deficient mutant  (EphB3K665R) or with the  EphB2 receptor (EphB2).  Transfected cells were lysed  and equal amounts of each  lysate were probed on a  Western blot (WB) either  with (a) a monoclonal anti-pTyr antibody, (b) with a  monoclonal anti-FLAG M2  antibody, (c) with polyclonal  antisera against EphB2, or (d)  against EphB3. (e) 293T cells  were transiently transfected  with either FLAG-tagged AF-6  (AF-6) alone or together with  wild-type EphB3 (EphB3), a  kinase-deficient mutant (Eph  B3K665R), EphB2 (EphB2).  Cell lysates were immunoprecipitated (IP) with anti-FLAG M2 antibodies. Immunoprecipitates were probed  on a Western blot (WB) either with a monoclonal anti-pTyr antibody (top) or with  anti-FLAG M2 antibodies  (bottom). Y is Tyr.
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Related In: Results  -  Collection

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Figure 7: Phosphorylation of full-length AF-6 by the overexpressed EphB2 and EphB3 receptors but not by a kinase-deficient mutant. 293T cells were transiently transfected with either a FLAG-tagged AF-6 (AF-6) alone or together with the wild-type EphB3 receptor (EphB3), a kinase-deficient mutant (EphB3K665R) or with the EphB2 receptor (EphB2). Transfected cells were lysed and equal amounts of each lysate were probed on a Western blot (WB) either with (a) a monoclonal anti-pTyr antibody, (b) with a monoclonal anti-FLAG M2 antibody, (c) with polyclonal antisera against EphB2, or (d) against EphB3. (e) 293T cells were transiently transfected with either FLAG-tagged AF-6 (AF-6) alone or together with wild-type EphB3 (EphB3), a kinase-deficient mutant (Eph B3K665R), EphB2 (EphB2). Cell lysates were immunoprecipitated (IP) with anti-FLAG M2 antibodies. Immunoprecipitates were probed on a Western blot (WB) either with a monoclonal anti-pTyr antibody (top) or with anti-FLAG M2 antibodies (bottom). Y is Tyr.
Mentions: To obtain some information on the biological significance of the interaction between AF-6 and Eph receptors, especially whether AF-6 is a substrate of the Eph receptor tyrosine kinases, we transiently transfected 293T cells with AF-6 along with either wild-type EphB3, EphB2, or a kinase-deficient mutant EphB3 receptor (EphB3K665R). The result shown in Fig. 7 a demonstrates that AF-6 is tyrosine phosphorylated only when coexpressed with wild-type EphB2 or EphB3 receptors. No phosphorylation occurred with a kinase-negative mutant receptor. Phosphorylation of AF-6 is dependent on previous activation of the receptor which is achieved by overexpression in 293T cells (Fig. 7 a and data not shown). When transfected AF-6 is immunoprecipitated from 293T cells, strong phosphorylation of the immunoprecipitated AF-6 is detected when it is cotransfected with the wild-type EphB2 and EphB3 receptors, whereas AF-6 is not phosphorylated when transfected alone or with the kinase-negative mutant EphB3K665R (Fig. 7 e, top). Taken together, these results demonstrate that AF-6 is phosphorylated by the EphB2 and EphB3 receptors in a kinase-dependent fashion and demonstrates that AF-6 is a substrate for a subgroup of Eph receptors.

Bottom Line: Furthermore, AF-6 is a substrate for a subgroup of Eph receptors and phosphorylation of AF-6 is dependent on a functional kinase domain of the receptor.The physical interaction of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipitation from whole rat brain lysates.AF-6 is a candidate for mediating the clustering of Eph receptors at postsynaptic specializations in the adult rat brain.

View Article: PubMed Central - PubMed

Affiliation: Institut für Medizinische Virologie, Universität Zürich, Switzerland.

ABSTRACT
The AF-6/afadin protein, which contains a single PDZ domain, forms a peripheral component of cell membranes at specialized sites of cell-cell junctions. To identify potential receptor-binding targets of AF-6 we screened the PDZ domain of AF-6 against a range of COOH-terminal peptides selected from receptors having potential PDZ domain-binding termini. The PDZ domain of AF-6 interacts with a subset of members of the Eph subfamily of RTKs via its COOH terminus both in vitro and in vivo. Cotransfection of a green fluorescent protein-tagged AF-6 fusion protein with full-length Eph receptors into heterologous cells induces a clustering of the Eph receptors and AF-6 at sites of cell-cell contact. Immunohistochemical analysis in the adult rat brain reveals coclustering of AF-6 with Eph receptors at postsynaptic membrane sites of excitatory synapses in the hippocampus. Furthermore, AF-6 is a substrate for a subgroup of Eph receptors and phosphorylation of AF-6 is dependent on a functional kinase domain of the receptor. The physical interaction of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipitation from whole rat brain lysates. AF-6 is a candidate for mediating the clustering of Eph receptors at postsynaptic specializations in the adult rat brain.

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