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Alpha-E-catenin binds to dynamitin and regulates dynactin-mediated intracellular traffic.

Lien WH, Gelfand VI, Vasioukhin V - J. Cell Biol. (2008)

Bottom Line: Dynactin-mediated organelle trafficking is increased in alpha-E-catenin(-/-) keratinocytes, an effect that is reversed by expression of exogenous alpha-E-catenin.Although neither the integrity of dynactin-dynein complexes nor their association with vesicles is affected by alpha-E-catenin, alpha-E-catenin is necessary for the attenuation of microtubule-dependent trafficking by the actin cytoskeleton.Because the actin-binding domain of alpha-E-catenin is necessary for this regulation, we hypothesize that alpha-E-catenin functions as a dynamic link between the dynactin complex and actin and, thus, integrates the microtubule and actin cytoskeleton during intracellular trafficking.

View Article: PubMed Central - PubMed

Affiliation: Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

ABSTRACT
Alpha-epithelial catenin (E-catenin) is an important cell-cell adhesion protein. In this study, we show that alpha-E-catenin also regulates intracellular traffic by binding to the dynactin complex component dynamitin. Dynactin-mediated organelle trafficking is increased in alpha-E-catenin(-/-) keratinocytes, an effect that is reversed by expression of exogenous alpha-E-catenin. Disruption of adherens junctions in low-calcium media does not affect dynactin-mediated traffic, indicating that alpha-E-catenin regulates traffic independently from its function in cell-cell adhesion. Although neither the integrity of dynactin-dynein complexes nor their association with vesicles is affected by alpha-E-catenin, alpha-E-catenin is necessary for the attenuation of microtubule-dependent trafficking by the actin cytoskeleton. Because the actin-binding domain of alpha-E-catenin is necessary for this regulation, we hypothesize that alpha-E-catenin functions as a dynamic link between the dynactin complex and actin and, thus, integrates the microtubule and actin cytoskeleton during intracellular trafficking.

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α–E-catenin binds to dynamitin and interacts with the dynactin protein complex. (A) Schematic of α–E-catenin containing three vinculin homology (VH) domains. Numbers indicate corresponding amino acids. (B) Interaction between dynamitin and α–E-catenin in a yeast two-hybrid assay. Cells were expressing plasmids containing Gal4 DNA–BD linked to various fragments of α–E-catenin and Gal4-AD linked to dynamitin (Dyn) or β-catenin (β-cat; positive control). (C) Interaction between dynamitin and α–E-catenin in mammalian cells. HEK 293FT cells were transfected with plasmids encoding GST and GST-linked α–E-catenin or vinculin and V5-tagged dynamitin, β-catenin (positive control), and Tbr1 (negative control). Protein extracts were pulled down (IP) with glutathione–Sepharose beads and analyzed by Western blotting (WB) with anti-V5 or anti-GST antibodies. (D) α–E-catenin associates not only with dynamitin but also with Arp1 and p150Glued proteins. GST-tagged full-length or VH2–VH3 fragments (α-cat 291–906) of α–E-catenin, V5-tagged dynamitin, and Arp1 were produced in HEK 293FT cells. GST-fused proteins were pulled down with glutathione–Sepharose beads, and protein complexes were analyzed by blotting with anti-V5, anti-p150Glued, and anti-GST antibodies. (E) α–E-catenin partially cofractionates with dynactin. Total keratinocyte extracts were sedimented on sucrose gradient, and fractions were analyzed by blotting with anti-p150Glued, anti–dynein intermediate chain (DIC), antidynamitin, anti-Arp1, and anti–α-catenin antibodies. (F) Interaction between endogenous α–E-catenin and dynactin. Dynactin-containing fractions after sucrose sedimentation of total extracts from wild-type (WT) and α–E-catenin−/− (KO) cells were immunoprecipitated with anti–α-catenin (C terminal) or β-galactosidase (β-gal; control) antibodies and analyzed by blotting with anti-p150Glued, antidynamitin (Dyn), anti-Arp1, and anti–α-catenin antibodies. M, position of a molecular weight standard band in the marker lane. (G) Interaction between endogenous α–E-catenin and dynamitin. Total proteins (input) from wild-type and α–E-catenin−/− keratinocytes were immunoprecipitated with anti–α-catenin (IP–α-cat; N terminal) antibodies and analyzed by blotting with antidynamitin and anti–α-catenin antibodies.
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fig1: α–E-catenin binds to dynamitin and interacts with the dynactin protein complex. (A) Schematic of α–E-catenin containing three vinculin homology (VH) domains. Numbers indicate corresponding amino acids. (B) Interaction between dynamitin and α–E-catenin in a yeast two-hybrid assay. Cells were expressing plasmids containing Gal4 DNA–BD linked to various fragments of α–E-catenin and Gal4-AD linked to dynamitin (Dyn) or β-catenin (β-cat; positive control). (C) Interaction between dynamitin and α–E-catenin in mammalian cells. HEK 293FT cells were transfected with plasmids encoding GST and GST-linked α–E-catenin or vinculin and V5-tagged dynamitin, β-catenin (positive control), and Tbr1 (negative control). Protein extracts were pulled down (IP) with glutathione–Sepharose beads and analyzed by Western blotting (WB) with anti-V5 or anti-GST antibodies. (D) α–E-catenin associates not only with dynamitin but also with Arp1 and p150Glued proteins. GST-tagged full-length or VH2–VH3 fragments (α-cat 291–906) of α–E-catenin, V5-tagged dynamitin, and Arp1 were produced in HEK 293FT cells. GST-fused proteins were pulled down with glutathione–Sepharose beads, and protein complexes were analyzed by blotting with anti-V5, anti-p150Glued, and anti-GST antibodies. (E) α–E-catenin partially cofractionates with dynactin. Total keratinocyte extracts were sedimented on sucrose gradient, and fractions were analyzed by blotting with anti-p150Glued, anti–dynein intermediate chain (DIC), antidynamitin, anti-Arp1, and anti–α-catenin antibodies. (F) Interaction between endogenous α–E-catenin and dynactin. Dynactin-containing fractions after sucrose sedimentation of total extracts from wild-type (WT) and α–E-catenin−/− (KO) cells were immunoprecipitated with anti–α-catenin (C terminal) or β-galactosidase (β-gal; control) antibodies and analyzed by blotting with anti-p150Glued, antidynamitin (Dyn), anti-Arp1, and anti–α-catenin antibodies. M, position of a molecular weight standard band in the marker lane. (G) Interaction between endogenous α–E-catenin and dynamitin. Total proteins (input) from wild-type and α–E-catenin−/− keratinocytes were immunoprecipitated with anti–α-catenin (IP–α-cat; N terminal) antibodies and analyzed by blotting with antidynamitin and anti–α-catenin antibodies.

Mentions: To gain new insights into the mechanism of α–E-catenin function, we performed a yeast two-hybrid screen to identify novel α–E-catenin–interacting proteins. α–E-catenin contains three vinculin homology domains: VH1, VH2, and VH3 (Fig. 1 A). Although the VH1 domain is responsible for binding to β-catenin, the functional significance of the VH2 and VH3 domains is not as well understood. We used ΔVH1 α–E-catenin (291–906 aa) as a bait to screen an embryonic mouse brain cDNA library. We identified 84 individual clones, and subsequent analysis revealed that 74 of them contained cDNA encoding dynamitin (Dctn2), which is a central component of the dynactin protein complex.


Alpha-E-catenin binds to dynamitin and regulates dynactin-mediated intracellular traffic.

Lien WH, Gelfand VI, Vasioukhin V - J. Cell Biol. (2008)

α–E-catenin binds to dynamitin and interacts with the dynactin protein complex. (A) Schematic of α–E-catenin containing three vinculin homology (VH) domains. Numbers indicate corresponding amino acids. (B) Interaction between dynamitin and α–E-catenin in a yeast two-hybrid assay. Cells were expressing plasmids containing Gal4 DNA–BD linked to various fragments of α–E-catenin and Gal4-AD linked to dynamitin (Dyn) or β-catenin (β-cat; positive control). (C) Interaction between dynamitin and α–E-catenin in mammalian cells. HEK 293FT cells were transfected with plasmids encoding GST and GST-linked α–E-catenin or vinculin and V5-tagged dynamitin, β-catenin (positive control), and Tbr1 (negative control). Protein extracts were pulled down (IP) with glutathione–Sepharose beads and analyzed by Western blotting (WB) with anti-V5 or anti-GST antibodies. (D) α–E-catenin associates not only with dynamitin but also with Arp1 and p150Glued proteins. GST-tagged full-length or VH2–VH3 fragments (α-cat 291–906) of α–E-catenin, V5-tagged dynamitin, and Arp1 were produced in HEK 293FT cells. GST-fused proteins were pulled down with glutathione–Sepharose beads, and protein complexes were analyzed by blotting with anti-V5, anti-p150Glued, and anti-GST antibodies. (E) α–E-catenin partially cofractionates with dynactin. Total keratinocyte extracts were sedimented on sucrose gradient, and fractions were analyzed by blotting with anti-p150Glued, anti–dynein intermediate chain (DIC), antidynamitin, anti-Arp1, and anti–α-catenin antibodies. (F) Interaction between endogenous α–E-catenin and dynactin. Dynactin-containing fractions after sucrose sedimentation of total extracts from wild-type (WT) and α–E-catenin−/− (KO) cells were immunoprecipitated with anti–α-catenin (C terminal) or β-galactosidase (β-gal; control) antibodies and analyzed by blotting with anti-p150Glued, antidynamitin (Dyn), anti-Arp1, and anti–α-catenin antibodies. M, position of a molecular weight standard band in the marker lane. (G) Interaction between endogenous α–E-catenin and dynamitin. Total proteins (input) from wild-type and α–E-catenin−/− keratinocytes were immunoprecipitated with anti–α-catenin (IP–α-cat; N terminal) antibodies and analyzed by blotting with antidynamitin and anti–α-catenin antibodies.
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fig1: α–E-catenin binds to dynamitin and interacts with the dynactin protein complex. (A) Schematic of α–E-catenin containing three vinculin homology (VH) domains. Numbers indicate corresponding amino acids. (B) Interaction between dynamitin and α–E-catenin in a yeast two-hybrid assay. Cells were expressing plasmids containing Gal4 DNA–BD linked to various fragments of α–E-catenin and Gal4-AD linked to dynamitin (Dyn) or β-catenin (β-cat; positive control). (C) Interaction between dynamitin and α–E-catenin in mammalian cells. HEK 293FT cells were transfected with plasmids encoding GST and GST-linked α–E-catenin or vinculin and V5-tagged dynamitin, β-catenin (positive control), and Tbr1 (negative control). Protein extracts were pulled down (IP) with glutathione–Sepharose beads and analyzed by Western blotting (WB) with anti-V5 or anti-GST antibodies. (D) α–E-catenin associates not only with dynamitin but also with Arp1 and p150Glued proteins. GST-tagged full-length or VH2–VH3 fragments (α-cat 291–906) of α–E-catenin, V5-tagged dynamitin, and Arp1 were produced in HEK 293FT cells. GST-fused proteins were pulled down with glutathione–Sepharose beads, and protein complexes were analyzed by blotting with anti-V5, anti-p150Glued, and anti-GST antibodies. (E) α–E-catenin partially cofractionates with dynactin. Total keratinocyte extracts were sedimented on sucrose gradient, and fractions were analyzed by blotting with anti-p150Glued, anti–dynein intermediate chain (DIC), antidynamitin, anti-Arp1, and anti–α-catenin antibodies. (F) Interaction between endogenous α–E-catenin and dynactin. Dynactin-containing fractions after sucrose sedimentation of total extracts from wild-type (WT) and α–E-catenin−/− (KO) cells were immunoprecipitated with anti–α-catenin (C terminal) or β-galactosidase (β-gal; control) antibodies and analyzed by blotting with anti-p150Glued, antidynamitin (Dyn), anti-Arp1, and anti–α-catenin antibodies. M, position of a molecular weight standard band in the marker lane. (G) Interaction between endogenous α–E-catenin and dynamitin. Total proteins (input) from wild-type and α–E-catenin−/− keratinocytes were immunoprecipitated with anti–α-catenin (IP–α-cat; N terminal) antibodies and analyzed by blotting with antidynamitin and anti–α-catenin antibodies.
Mentions: To gain new insights into the mechanism of α–E-catenin function, we performed a yeast two-hybrid screen to identify novel α–E-catenin–interacting proteins. α–E-catenin contains three vinculin homology domains: VH1, VH2, and VH3 (Fig. 1 A). Although the VH1 domain is responsible for binding to β-catenin, the functional significance of the VH2 and VH3 domains is not as well understood. We used ΔVH1 α–E-catenin (291–906 aa) as a bait to screen an embryonic mouse brain cDNA library. We identified 84 individual clones, and subsequent analysis revealed that 74 of them contained cDNA encoding dynamitin (Dctn2), which is a central component of the dynactin protein complex.

Bottom Line: Dynactin-mediated organelle trafficking is increased in alpha-E-catenin(-/-) keratinocytes, an effect that is reversed by expression of exogenous alpha-E-catenin.Although neither the integrity of dynactin-dynein complexes nor their association with vesicles is affected by alpha-E-catenin, alpha-E-catenin is necessary for the attenuation of microtubule-dependent trafficking by the actin cytoskeleton.Because the actin-binding domain of alpha-E-catenin is necessary for this regulation, we hypothesize that alpha-E-catenin functions as a dynamic link between the dynactin complex and actin and, thus, integrates the microtubule and actin cytoskeleton during intracellular trafficking.

View Article: PubMed Central - PubMed

Affiliation: Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

ABSTRACT
Alpha-epithelial catenin (E-catenin) is an important cell-cell adhesion protein. In this study, we show that alpha-E-catenin also regulates intracellular traffic by binding to the dynactin complex component dynamitin. Dynactin-mediated organelle trafficking is increased in alpha-E-catenin(-/-) keratinocytes, an effect that is reversed by expression of exogenous alpha-E-catenin. Disruption of adherens junctions in low-calcium media does not affect dynactin-mediated traffic, indicating that alpha-E-catenin regulates traffic independently from its function in cell-cell adhesion. Although neither the integrity of dynactin-dynein complexes nor their association with vesicles is affected by alpha-E-catenin, alpha-E-catenin is necessary for the attenuation of microtubule-dependent trafficking by the actin cytoskeleton. Because the actin-binding domain of alpha-E-catenin is necessary for this regulation, we hypothesize that alpha-E-catenin functions as a dynamic link between the dynactin complex and actin and, thus, integrates the microtubule and actin cytoskeleton during intracellular trafficking.

Show MeSH
Related in: MedlinePlus