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Drosophila p120catenin plays a supporting role in cell adhesion but is not an essential adherens junction component.

Myster SH, Cavallo R, Anderson CT, Fox DT, Peifer M - J. Cell Biol. (2003)

Bottom Line: We generated alleles of p120 and found that mutants are viable and fertile and have no substantial changes in junction structure or function.However, p120 mutations strongly enhance mutations in the genes encoding DE-cadherin or Armadillo, the beta-catenin homologue.Finally, we examined the localization of p120 during embryogenesis. p120 localizes to adherens junctions, but its localization there is less universal than that of core adherens junction proteins.

View Article: PubMed Central - PubMed

Affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

ABSTRACT
Cadherin-catenin complexes, localized to adherens junctions, are essential for cell-cell adhesion. One means of regulating adhesion is through the juxtamembrane domain of the cadherin cytoplasmic tail. This region is the binding site for p120, leading to the hypothesis that p120 is a key regulator of cell adhesion. p120 has also been suggested to regulate the GTPase Rho and to regulate transcription via its binding partner Kaiso. To test these hypothesized functions, we turned to Drosophila, which has only a single p120 family member. It localizes to adherens junctions and binds the juxtamembrane region of DE-cadherin (DE-cad). We generated alleles of p120 and found that mutants are viable and fertile and have no substantial changes in junction structure or function. However, p120 mutations strongly enhance mutations in the genes encoding DE-cadherin or Armadillo, the beta-catenin homologue. Finally, we examined the localization of p120 during embryogenesis. p120 localizes to adherens junctions, but its localization there is less universal than that of core adherens junction proteins. Together, these data suggest that p120 is an important positive modulator of adhesion but that it is not an essential core component of adherens junctions.

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p120 is in AJ complexes and binds the JM region of DE-cad. (A) Cell extracts or IPs using anti-myc or anti-BicD (negative control) from wild-type or myc-p120 embryos were immunoblotted with antibodies against myc, p120, DE-cad, Arm, and BicD. Proteins identified are indicated to the right, and selected mol wt markers (kD) are on the left. Rat anti-p120 recognizes both endogenous and myc-p120. (B) Cell extracts or anti-Arm IPs from wild-type (wt), p120 mutant (mut), and myc-p120 expressing embryos were immunoblotted with antibodies against Arm, DE-cad, myc, p120, and Pnut (a negative control). (A and B) ∼1% of extract and ∼50% of each IP was loaded. (C–H) Yeast two-hybrid interactions assessed by β-galactosidase activity. (C) Interaction between p120 and the DE-cad cytoplasmic tail (DEC), DE-cad deletion constructs (DECXX; black bars) or vector control (white bars). (D) Schematic illustrating DEC deletion constructs in C. (E and F) Clustered point mutations in the JM region of the DE-cad cytoplasmic tail (DECM5 and DECM6, diagram in F; dE, DE-cad; mE, mouse E-cad) abolished binding of full-length p120 (E, black bar), but had no effect on interaction of the Arm repeats of Arm with DE-cad (E, white bars). (G and H) Arm repeats 1–10 of p120 are required to confer strong binding to DE-cad (black bars). Vector control (white bars). (H) Diagram of constructs used.
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fig3: p120 is in AJ complexes and binds the JM region of DE-cad. (A) Cell extracts or IPs using anti-myc or anti-BicD (negative control) from wild-type or myc-p120 embryos were immunoblotted with antibodies against myc, p120, DE-cad, Arm, and BicD. Proteins identified are indicated to the right, and selected mol wt markers (kD) are on the left. Rat anti-p120 recognizes both endogenous and myc-p120. (B) Cell extracts or anti-Arm IPs from wild-type (wt), p120 mutant (mut), and myc-p120 expressing embryos were immunoblotted with antibodies against Arm, DE-cad, myc, p120, and Pnut (a negative control). (A and B) ∼1% of extract and ∼50% of each IP was loaded. (C–H) Yeast two-hybrid interactions assessed by β-galactosidase activity. (C) Interaction between p120 and the DE-cad cytoplasmic tail (DEC), DE-cad deletion constructs (DECXX; black bars) or vector control (white bars). (D) Schematic illustrating DEC deletion constructs in C. (E and F) Clustered point mutations in the JM region of the DE-cad cytoplasmic tail (DECM5 and DECM6, diagram in F; dE, DE-cad; mE, mouse E-cad) abolished binding of full-length p120 (E, black bar), but had no effect on interaction of the Arm repeats of Arm with DE-cad (E, white bars). (G and H) Arm repeats 1–10 of p120 are required to confer strong binding to DE-cad (black bars). Vector control (white bars). (H) Diagram of constructs used.

Mentions: Mammalian p120 subfamily members interact with cadherins via conserved sequences in the JM region (for review see Anastasiadis and Reynolds, 2000). We thus examined whether Drosophila p120 interacts with DE-cad and Arm in AJ complexes and whether p120 binds the DE-cad JM region. We immunoprecipitated myc-p120 with anti-myc antibodies and looked for coimmunoprecipitation of DE-cad (Fig. 3 A). DE-cad was specifically detected in myc-immunoprecipitates (IPs) from embryos expressing myc-p120 and not from wild-type embryos. Interestingly, Arm was also detected in the IPs, although endogenous p120 was not. The absence of endogenous p120 suggests we did not immunoprecipitate large oligimeric cadherin complexes; the presence of Arm thus supports the idea that Arm and p120 may bind the same cadherin cis-dimer. IPs with a control antibody, anti-BicD, confirmed the specificity of this coimmunoprecipitation (Fig. 3 A). Next, we immunoprecipitated endogenous cadherin–catenin complexes using anti-Arm (Fig. 3 B). As expected, Arm antibodies coimmunoprecipitated DE-cad. They also coimmunoprecipitated both endogenous p120 and myc-p120 (Fig. 3 B), suggesting that both are part of cadherin–catenin complexes. Controls with p120 mutants or embryos not expressing myc-p120 confirmed the specificity of this interaction (Fig. 3 B).


Drosophila p120catenin plays a supporting role in cell adhesion but is not an essential adherens junction component.

Myster SH, Cavallo R, Anderson CT, Fox DT, Peifer M - J. Cell Biol. (2003)

p120 is in AJ complexes and binds the JM region of DE-cad. (A) Cell extracts or IPs using anti-myc or anti-BicD (negative control) from wild-type or myc-p120 embryos were immunoblotted with antibodies against myc, p120, DE-cad, Arm, and BicD. Proteins identified are indicated to the right, and selected mol wt markers (kD) are on the left. Rat anti-p120 recognizes both endogenous and myc-p120. (B) Cell extracts or anti-Arm IPs from wild-type (wt), p120 mutant (mut), and myc-p120 expressing embryos were immunoblotted with antibodies against Arm, DE-cad, myc, p120, and Pnut (a negative control). (A and B) ∼1% of extract and ∼50% of each IP was loaded. (C–H) Yeast two-hybrid interactions assessed by β-galactosidase activity. (C) Interaction between p120 and the DE-cad cytoplasmic tail (DEC), DE-cad deletion constructs (DECXX; black bars) or vector control (white bars). (D) Schematic illustrating DEC deletion constructs in C. (E and F) Clustered point mutations in the JM region of the DE-cad cytoplasmic tail (DECM5 and DECM6, diagram in F; dE, DE-cad; mE, mouse E-cad) abolished binding of full-length p120 (E, black bar), but had no effect on interaction of the Arm repeats of Arm with DE-cad (E, white bars). (G and H) Arm repeats 1–10 of p120 are required to confer strong binding to DE-cad (black bars). Vector control (white bars). (H) Diagram of constructs used.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172674&req=5

fig3: p120 is in AJ complexes and binds the JM region of DE-cad. (A) Cell extracts or IPs using anti-myc or anti-BicD (negative control) from wild-type or myc-p120 embryos were immunoblotted with antibodies against myc, p120, DE-cad, Arm, and BicD. Proteins identified are indicated to the right, and selected mol wt markers (kD) are on the left. Rat anti-p120 recognizes both endogenous and myc-p120. (B) Cell extracts or anti-Arm IPs from wild-type (wt), p120 mutant (mut), and myc-p120 expressing embryos were immunoblotted with antibodies against Arm, DE-cad, myc, p120, and Pnut (a negative control). (A and B) ∼1% of extract and ∼50% of each IP was loaded. (C–H) Yeast two-hybrid interactions assessed by β-galactosidase activity. (C) Interaction between p120 and the DE-cad cytoplasmic tail (DEC), DE-cad deletion constructs (DECXX; black bars) or vector control (white bars). (D) Schematic illustrating DEC deletion constructs in C. (E and F) Clustered point mutations in the JM region of the DE-cad cytoplasmic tail (DECM5 and DECM6, diagram in F; dE, DE-cad; mE, mouse E-cad) abolished binding of full-length p120 (E, black bar), but had no effect on interaction of the Arm repeats of Arm with DE-cad (E, white bars). (G and H) Arm repeats 1–10 of p120 are required to confer strong binding to DE-cad (black bars). Vector control (white bars). (H) Diagram of constructs used.
Mentions: Mammalian p120 subfamily members interact with cadherins via conserved sequences in the JM region (for review see Anastasiadis and Reynolds, 2000). We thus examined whether Drosophila p120 interacts with DE-cad and Arm in AJ complexes and whether p120 binds the DE-cad JM region. We immunoprecipitated myc-p120 with anti-myc antibodies and looked for coimmunoprecipitation of DE-cad (Fig. 3 A). DE-cad was specifically detected in myc-immunoprecipitates (IPs) from embryos expressing myc-p120 and not from wild-type embryos. Interestingly, Arm was also detected in the IPs, although endogenous p120 was not. The absence of endogenous p120 suggests we did not immunoprecipitate large oligimeric cadherin complexes; the presence of Arm thus supports the idea that Arm and p120 may bind the same cadherin cis-dimer. IPs with a control antibody, anti-BicD, confirmed the specificity of this coimmunoprecipitation (Fig. 3 A). Next, we immunoprecipitated endogenous cadherin–catenin complexes using anti-Arm (Fig. 3 B). As expected, Arm antibodies coimmunoprecipitated DE-cad. They also coimmunoprecipitated both endogenous p120 and myc-p120 (Fig. 3 B), suggesting that both are part of cadherin–catenin complexes. Controls with p120 mutants or embryos not expressing myc-p120 confirmed the specificity of this interaction (Fig. 3 B).

Bottom Line: We generated alleles of p120 and found that mutants are viable and fertile and have no substantial changes in junction structure or function.However, p120 mutations strongly enhance mutations in the genes encoding DE-cadherin or Armadillo, the beta-catenin homologue.Finally, we examined the localization of p120 during embryogenesis. p120 localizes to adherens junctions, but its localization there is less universal than that of core adherens junction proteins.

View Article: PubMed Central - PubMed

Affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

ABSTRACT
Cadherin-catenin complexes, localized to adherens junctions, are essential for cell-cell adhesion. One means of regulating adhesion is through the juxtamembrane domain of the cadherin cytoplasmic tail. This region is the binding site for p120, leading to the hypothesis that p120 is a key regulator of cell adhesion. p120 has also been suggested to regulate the GTPase Rho and to regulate transcription via its binding partner Kaiso. To test these hypothesized functions, we turned to Drosophila, which has only a single p120 family member. It localizes to adherens junctions and binds the juxtamembrane region of DE-cadherin (DE-cad). We generated alleles of p120 and found that mutants are viable and fertile and have no substantial changes in junction structure or function. However, p120 mutations strongly enhance mutations in the genes encoding DE-cadherin or Armadillo, the beta-catenin homologue. Finally, we examined the localization of p120 during embryogenesis. p120 localizes to adherens junctions, but its localization there is less universal than that of core adherens junction proteins. Together, these data suggest that p120 is an important positive modulator of adhesion but that it is not an essential core component of adherens junctions.

Show MeSH
Related in: MedlinePlus