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The Alzheimer amyloid precursor protein (APP) and FE65, an APP-binding protein, regulate cell movement.

Sabo SL, Ikin AF, Buxbaum JD, Greengard P - J. Cell Biol. (2001)

Bottom Line: APP and FE65 colocalize with actin and Mena, an Abl-associated signaling protein thought to regulate actin dynamics, in lamellipodia.Coexpression of APP and FE65 dramatically enhances the effect of APP on cell movement, probably by regulating the amount of APP at the cell surface.These data are consistent with a role for FE65 and APP, possibly in a Mena-containing macromolecular complex, in regulation of actin-based motility.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Cellular Neuroscience and the Zachary and Elizabeth M. Fisher Center, The Rockefeller University, New York, New York 10021, USA. slsabo@ucdavis.edu

ABSTRACT
FE65 binds to the Alzheimer amyloid precursor protein (APP), but the function of this interaction has not been identified. Here, we report that APP and FE65 are involved in regulation of cell movement. APP and FE65 colocalize with actin and Mena, an Abl-associated signaling protein thought to regulate actin dynamics, in lamellipodia. APP and FE65 specifically concentrate with beta 1-integrin in dynamic adhesion sites known as focal complexes, but not in more static adhesion sites known as focal adhesions. Overexpression of APP accelerates cell migration in an MDCK cell wound--healing assay. Coexpression of APP and FE65 dramatically enhances the effect of APP on cell movement, probably by regulating the amount of APP at the cell surface. These data are consistent with a role for FE65 and APP, possibly in a Mena-containing macromolecular complex, in regulation of actin-based motility.

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APP and Mena interact with FE65 simultaneously. (a and b) H4 cell lysates were incubated with GST fusion proteins corresponding to either the cytoplasmic domain of APP (APP C50-GST) or the WW domain of FE65 (FE65 WW-GST) for precipitation with glutathione-Sepaharose. Precipitated complexes were immunoblotted with FE65 (a) and Mena (b) antibodies, respectively. As a control for nonspecific coprecipitation, experiments were performed in parallel with GST alone. (c and d) Homogenates from MDCK cells that overexpressed both APP and FE65 (+FE65) and MDCK cells that overexpressed APP, but not FE65 (−FE65), were immunoprecipitated with Mena antibodies. The immunoprecipitates were then immunoblotted with APP (c) and FE65 (d) antibodies. APP coimmunoprecipitated with Mena only when FE65 was present.
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Figure 2: APP and Mena interact with FE65 simultaneously. (a and b) H4 cell lysates were incubated with GST fusion proteins corresponding to either the cytoplasmic domain of APP (APP C50-GST) or the WW domain of FE65 (FE65 WW-GST) for precipitation with glutathione-Sepaharose. Precipitated complexes were immunoblotted with FE65 (a) and Mena (b) antibodies, respectively. As a control for nonspecific coprecipitation, experiments were performed in parallel with GST alone. (c and d) Homogenates from MDCK cells that overexpressed both APP and FE65 (+FE65) and MDCK cells that overexpressed APP, but not FE65 (−FE65), were immunoprecipitated with Mena antibodies. The immunoprecipitates were then immunoblotted with APP (c) and FE65 (d) antibodies. APP coimmunoprecipitated with Mena only when FE65 was present.

Mentions: To determine if the APP–FE65–Mena tripartite complex exists, coprecipitations and immunoprecipitations were performed. When H4 cell lysates were incubated with a GST fusion protein containing the cytoplasmic domain of APP, FE65 was precipitated on glutathione-Sepharose beads through an interaction with the APP fusion protein (Fig. 2 a). Similarly, when the same lysates were incubated with a GST fusion protein containing the WW domain of FE65, Mena bound to the FE65 fusion protein was precipitated on glutathione-Sepharose beads (Fig. 2 b). Finally, when lysates from MDCK cells stably expressing both APP and FE65 were subjected to immunoprecipitation with antibodies raised against Mena, FE65 and APP were found in the immunoprecipitates by immunoblotting (Fig. 2c and Fig. d). When lysates from MDCK cells stably expressing only APP were used side-by-side in the same experiment, APP was not coimmunoprecipitated with Mena, indicating that the APP found in the immunoprecipitates associated with Mena indirectly through FE65. These data lend strong support to the idea that a macromolecular complex containing APP, FE65, and Mena exists in vivo. Since Mena is known to bind to profilin and is thought to regulate actin dynamics (Gertler et al. 1996; Lanier and Gertler 2000), the colocalization and interaction of APP and FE65 with Mena provides evidence for an indirect link between an APP–FE65 complex and the lamellipodial actin cytoskeleton.


The Alzheimer amyloid precursor protein (APP) and FE65, an APP-binding protein, regulate cell movement.

Sabo SL, Ikin AF, Buxbaum JD, Greengard P - J. Cell Biol. (2001)

APP and Mena interact with FE65 simultaneously. (a and b) H4 cell lysates were incubated with GST fusion proteins corresponding to either the cytoplasmic domain of APP (APP C50-GST) or the WW domain of FE65 (FE65 WW-GST) for precipitation with glutathione-Sepaharose. Precipitated complexes were immunoblotted with FE65 (a) and Mena (b) antibodies, respectively. As a control for nonspecific coprecipitation, experiments were performed in parallel with GST alone. (c and d) Homogenates from MDCK cells that overexpressed both APP and FE65 (+FE65) and MDCK cells that overexpressed APP, but not FE65 (−FE65), were immunoprecipitated with Mena antibodies. The immunoprecipitates were then immunoblotted with APP (c) and FE65 (d) antibodies. APP coimmunoprecipitated with Mena only when FE65 was present.
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Related In: Results  -  Collection

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Figure 2: APP and Mena interact with FE65 simultaneously. (a and b) H4 cell lysates were incubated with GST fusion proteins corresponding to either the cytoplasmic domain of APP (APP C50-GST) or the WW domain of FE65 (FE65 WW-GST) for precipitation with glutathione-Sepaharose. Precipitated complexes were immunoblotted with FE65 (a) and Mena (b) antibodies, respectively. As a control for nonspecific coprecipitation, experiments were performed in parallel with GST alone. (c and d) Homogenates from MDCK cells that overexpressed both APP and FE65 (+FE65) and MDCK cells that overexpressed APP, but not FE65 (−FE65), were immunoprecipitated with Mena antibodies. The immunoprecipitates were then immunoblotted with APP (c) and FE65 (d) antibodies. APP coimmunoprecipitated with Mena only when FE65 was present.
Mentions: To determine if the APP–FE65–Mena tripartite complex exists, coprecipitations and immunoprecipitations were performed. When H4 cell lysates were incubated with a GST fusion protein containing the cytoplasmic domain of APP, FE65 was precipitated on glutathione-Sepharose beads through an interaction with the APP fusion protein (Fig. 2 a). Similarly, when the same lysates were incubated with a GST fusion protein containing the WW domain of FE65, Mena bound to the FE65 fusion protein was precipitated on glutathione-Sepharose beads (Fig. 2 b). Finally, when lysates from MDCK cells stably expressing both APP and FE65 were subjected to immunoprecipitation with antibodies raised against Mena, FE65 and APP were found in the immunoprecipitates by immunoblotting (Fig. 2c and Fig. d). When lysates from MDCK cells stably expressing only APP were used side-by-side in the same experiment, APP was not coimmunoprecipitated with Mena, indicating that the APP found in the immunoprecipitates associated with Mena indirectly through FE65. These data lend strong support to the idea that a macromolecular complex containing APP, FE65, and Mena exists in vivo. Since Mena is known to bind to profilin and is thought to regulate actin dynamics (Gertler et al. 1996; Lanier and Gertler 2000), the colocalization and interaction of APP and FE65 with Mena provides evidence for an indirect link between an APP–FE65 complex and the lamellipodial actin cytoskeleton.

Bottom Line: APP and FE65 colocalize with actin and Mena, an Abl-associated signaling protein thought to regulate actin dynamics, in lamellipodia.Coexpression of APP and FE65 dramatically enhances the effect of APP on cell movement, probably by regulating the amount of APP at the cell surface.These data are consistent with a role for FE65 and APP, possibly in a Mena-containing macromolecular complex, in regulation of actin-based motility.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Cellular Neuroscience and the Zachary and Elizabeth M. Fisher Center, The Rockefeller University, New York, New York 10021, USA. slsabo@ucdavis.edu

ABSTRACT
FE65 binds to the Alzheimer amyloid precursor protein (APP), but the function of this interaction has not been identified. Here, we report that APP and FE65 are involved in regulation of cell movement. APP and FE65 colocalize with actin and Mena, an Abl-associated signaling protein thought to regulate actin dynamics, in lamellipodia. APP and FE65 specifically concentrate with beta 1-integrin in dynamic adhesion sites known as focal complexes, but not in more static adhesion sites known as focal adhesions. Overexpression of APP accelerates cell migration in an MDCK cell wound--healing assay. Coexpression of APP and FE65 dramatically enhances the effect of APP on cell movement, probably by regulating the amount of APP at the cell surface. These data are consistent with a role for FE65 and APP, possibly in a Mena-containing macromolecular complex, in regulation of actin-based motility.

Show MeSH
Related in: MedlinePlus