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AHNAK interaction with the annexin 2/S100A10 complex regulates cell membrane cytoarchitecture.

Benaud C, Gentil BJ, Assard N, Court M, Garin J, Delphin C, Baudier J - J. Cell Biol. (2003)

Bottom Line: Down-regulation of both annexin 2 and S100A10 using an annexin 2-specific small interfering RNA inhibits the association of AHNAK with plasma membrane.In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height.We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.

View Article: PubMed Central - PubMed

Affiliation: INSERM EMI-0104, DRDC-TS, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France.

ABSTRACT
Remodelling of the plasma membrane cytoarchitecture is crucial for the regulation of epithelial cell adhesion and permeability. In Madin-Darby canine kidney cells, the protein AHNAK relocates from the cytosol to the cytosolic surface of the plasma membrane during the formation of cell-cell contacts and the development of epithelial polarity. This targeting is reversible and regulated by Ca(2+)-dependent cell-cell adhesion. At the plasma membrane, AHNAK associates as a multimeric complex with actin and the annexin 2/S100A10 complex. The S100A10 subunit serves to mediate the interaction between annexin 2 and the COOH-terminal regulatory domain of AHNAK. Down-regulation of both annexin 2 and S100A10 using an annexin 2-specific small interfering RNA inhibits the association of AHNAK with plasma membrane. In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height. We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.

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Related in: MedlinePlus

Interaction of the annexin 2/S100A10 complex with AHNAK. (A). MCF-7 cell extracts were incubated with glutathione-Sepharose (lanes 2–4) or protein A–Sepharose (lanes 5–7) in the presence of recombinant GST alone (lane 2), recombinant GST fused to AHNAK central repeat domain M1 (lane 3), recombinant GST fused to AHNAK COOH-terminal domain (lane 4), anti-AHNAK-CQL antibody (lane 6), or anti-AHNAK-KIS antibody (lane 7). Bound proteins were analyzed by Western blotting with anti-annexin 2, -4, and -6 antibodies. Lane 1 is total cell extract. (B). MCF-7 or MDCK cell extracts were incubated with control protein A-Sepharose beads (lanes 1and 4), or with anti-AHNAK-CQL (lanes 2 and 6) or anti-AHNAK-KIS (lanes 3 and 5) antibodies. Coimmunoprecipitated proteins were analyzed by Western blotting with anti-AHNAK-KIS, anti-annexin 2, and anti-S100A10 antibodies. (C) S100A10 and annexin 2 are the major proteins that interact with the AHNAK Cter domain. [35S]Methionine/cysteine labeled MDCK whole-cell extracts (total; left) were incubated with GST (lane 1) and GST fusion AHNAK-Cter (lane 2) in EGTA/EDTA-containing buffer. Bound proteins were resolved on SDS-PAGE and were detected either by autoradiography (left) or blotted on nitrocellulose membrane and detected with mixed anti-annexin 2 and anti-S100A10 mAbs (right). (D) S100B antagonizes annexin 2/S100A10 binding to the AHNAK Cter domain. U87 cell extracts were incubated with GST fusion AHNAK-Cter in the absence (lane 1) or in the presence (lanes 2 and 3) of increasing S100B concentrations in Ca2+/Zn2+-containing buffer. Bound proteins were analyzed by Western blotting using anti-annexin 2 and anti-S100A10 antibodies (left), or with anti-annexin 2 and anti-S100B antibodies (right). Lane 4 is total cell extract. (E) [35S]Methionine/cysteine-labeled S100A10 was produced in rabbit reticulocyte (total), and the translation reaction was incubated with glutathione-Sepharose in the presence of recombinant GST (lane 1) or recombinant GST fused to the AHNAK COOH-terminal domain (lane 2) in Ca2+/Zn2+-containing buffer. Bound proteins were resolved on SDS-PAGE and detected by autoradiography. (F) S100A10 specifically interacts with AHNAK CterC and S100B in the yeast two-hybrid system. Top: diagram of the various AHNAK COOH-terminal fragments used. Yeast were cotransfected with S100A10 pACT2 vector and different baits corresponding to AHNAK Cter deletion mutants, S100B, and lamin cloned into the pLEX vector as indicated. The interactions were scored by the growth of transfected yeast on YC-UWLH medium lacking histidine (HIS) and confirmed by β-galactosidase activity assay (bgal).
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fig2: Interaction of the annexin 2/S100A10 complex with AHNAK. (A). MCF-7 cell extracts were incubated with glutathione-Sepharose (lanes 2–4) or protein A–Sepharose (lanes 5–7) in the presence of recombinant GST alone (lane 2), recombinant GST fused to AHNAK central repeat domain M1 (lane 3), recombinant GST fused to AHNAK COOH-terminal domain (lane 4), anti-AHNAK-CQL antibody (lane 6), or anti-AHNAK-KIS antibody (lane 7). Bound proteins were analyzed by Western blotting with anti-annexin 2, -4, and -6 antibodies. Lane 1 is total cell extract. (B). MCF-7 or MDCK cell extracts were incubated with control protein A-Sepharose beads (lanes 1and 4), or with anti-AHNAK-CQL (lanes 2 and 6) or anti-AHNAK-KIS (lanes 3 and 5) antibodies. Coimmunoprecipitated proteins were analyzed by Western blotting with anti-AHNAK-KIS, anti-annexin 2, and anti-S100A10 antibodies. (C) S100A10 and annexin 2 are the major proteins that interact with the AHNAK Cter domain. [35S]Methionine/cysteine labeled MDCK whole-cell extracts (total; left) were incubated with GST (lane 1) and GST fusion AHNAK-Cter (lane 2) in EGTA/EDTA-containing buffer. Bound proteins were resolved on SDS-PAGE and were detected either by autoradiography (left) or blotted on nitrocellulose membrane and detected with mixed anti-annexin 2 and anti-S100A10 mAbs (right). (D) S100B antagonizes annexin 2/S100A10 binding to the AHNAK Cter domain. U87 cell extracts were incubated with GST fusion AHNAK-Cter in the absence (lane 1) or in the presence (lanes 2 and 3) of increasing S100B concentrations in Ca2+/Zn2+-containing buffer. Bound proteins were analyzed by Western blotting using anti-annexin 2 and anti-S100A10 antibodies (left), or with anti-annexin 2 and anti-S100B antibodies (right). Lane 4 is total cell extract. (E) [35S]Methionine/cysteine-labeled S100A10 was produced in rabbit reticulocyte (total), and the translation reaction was incubated with glutathione-Sepharose in the presence of recombinant GST (lane 1) or recombinant GST fused to the AHNAK COOH-terminal domain (lane 2) in Ca2+/Zn2+-containing buffer. Bound proteins were resolved on SDS-PAGE and detected by autoradiography. (F) S100A10 specifically interacts with AHNAK CterC and S100B in the yeast two-hybrid system. Top: diagram of the various AHNAK COOH-terminal fragments used. Yeast were cotransfected with S100A10 pACT2 vector and different baits corresponding to AHNAK Cter deletion mutants, S100B, and lamin cloned into the pLEX vector as indicated. The interactions were scored by the growth of transfected yeast on YC-UWLH medium lacking histidine (HIS) and confirmed by β-galactosidase activity assay (bgal).

Mentions: To gain further insight into AHNAK function at the plasma membrane, we sought to identify proteins interacting with AHNAK using coimmunoprecipitation coupled to mass spectrometry analysis. For convenient peptide sequence identification using human databases, we used human epithelial MCF-7 cells, which predominantly express AHNAK at the plasma membrane. Confluent MCF-7 cells were treated with the membrane-permeable cross-linker dithiobis succinimidyl propionate, and AHNAK was immunoprecipitated with anti-AHNAK-CQL antibodies. Proteins present within the AHNAK immunoprecipitate were reduced, separated by SDS-PAGE, and identified by mass spectrometry. One major protein recovered within AHNAK immunoprecipitate was identified as annexin 2 (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200307098/DC1). The interaction between AHNAK and annexin 2 was confirmed both by coimmunoprecipitation in absence of cross-linking with two independent antibodies directed against AHNAK and immunodetection, and by a pull-down approach using recombinant AHNAK fragments linked to the GST tag (Fig. 2 A). The COOH-terminal fragment of AHNAK (Cter; aa 4642–5643), but neither a central fragment of AHNAK (M1; aa 820–1330) nor the GST tag alone, interacted with annexin 2. The interaction of annexin 2 with AHNAK is specific because other annexin family members, annexin IV and annexin VI, are not recovered in the AHNAK immunoprecipitates or in the pull-down experiments.


AHNAK interaction with the annexin 2/S100A10 complex regulates cell membrane cytoarchitecture.

Benaud C, Gentil BJ, Assard N, Court M, Garin J, Delphin C, Baudier J - J. Cell Biol. (2003)

Interaction of the annexin 2/S100A10 complex with AHNAK. (A). MCF-7 cell extracts were incubated with glutathione-Sepharose (lanes 2–4) or protein A–Sepharose (lanes 5–7) in the presence of recombinant GST alone (lane 2), recombinant GST fused to AHNAK central repeat domain M1 (lane 3), recombinant GST fused to AHNAK COOH-terminal domain (lane 4), anti-AHNAK-CQL antibody (lane 6), or anti-AHNAK-KIS antibody (lane 7). Bound proteins were analyzed by Western blotting with anti-annexin 2, -4, and -6 antibodies. Lane 1 is total cell extract. (B). MCF-7 or MDCK cell extracts were incubated with control protein A-Sepharose beads (lanes 1and 4), or with anti-AHNAK-CQL (lanes 2 and 6) or anti-AHNAK-KIS (lanes 3 and 5) antibodies. Coimmunoprecipitated proteins were analyzed by Western blotting with anti-AHNAK-KIS, anti-annexin 2, and anti-S100A10 antibodies. (C) S100A10 and annexin 2 are the major proteins that interact with the AHNAK Cter domain. [35S]Methionine/cysteine labeled MDCK whole-cell extracts (total; left) were incubated with GST (lane 1) and GST fusion AHNAK-Cter (lane 2) in EGTA/EDTA-containing buffer. Bound proteins were resolved on SDS-PAGE and were detected either by autoradiography (left) or blotted on nitrocellulose membrane and detected with mixed anti-annexin 2 and anti-S100A10 mAbs (right). (D) S100B antagonizes annexin 2/S100A10 binding to the AHNAK Cter domain. U87 cell extracts were incubated with GST fusion AHNAK-Cter in the absence (lane 1) or in the presence (lanes 2 and 3) of increasing S100B concentrations in Ca2+/Zn2+-containing buffer. Bound proteins were analyzed by Western blotting using anti-annexin 2 and anti-S100A10 antibodies (left), or with anti-annexin 2 and anti-S100B antibodies (right). Lane 4 is total cell extract. (E) [35S]Methionine/cysteine-labeled S100A10 was produced in rabbit reticulocyte (total), and the translation reaction was incubated with glutathione-Sepharose in the presence of recombinant GST (lane 1) or recombinant GST fused to the AHNAK COOH-terminal domain (lane 2) in Ca2+/Zn2+-containing buffer. Bound proteins were resolved on SDS-PAGE and detected by autoradiography. (F) S100A10 specifically interacts with AHNAK CterC and S100B in the yeast two-hybrid system. Top: diagram of the various AHNAK COOH-terminal fragments used. Yeast were cotransfected with S100A10 pACT2 vector and different baits corresponding to AHNAK Cter deletion mutants, S100B, and lamin cloned into the pLEX vector as indicated. The interactions were scored by the growth of transfected yeast on YC-UWLH medium lacking histidine (HIS) and confirmed by β-galactosidase activity assay (bgal).
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Related In: Results  -  Collection

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fig2: Interaction of the annexin 2/S100A10 complex with AHNAK. (A). MCF-7 cell extracts were incubated with glutathione-Sepharose (lanes 2–4) or protein A–Sepharose (lanes 5–7) in the presence of recombinant GST alone (lane 2), recombinant GST fused to AHNAK central repeat domain M1 (lane 3), recombinant GST fused to AHNAK COOH-terminal domain (lane 4), anti-AHNAK-CQL antibody (lane 6), or anti-AHNAK-KIS antibody (lane 7). Bound proteins were analyzed by Western blotting with anti-annexin 2, -4, and -6 antibodies. Lane 1 is total cell extract. (B). MCF-7 or MDCK cell extracts were incubated with control protein A-Sepharose beads (lanes 1and 4), or with anti-AHNAK-CQL (lanes 2 and 6) or anti-AHNAK-KIS (lanes 3 and 5) antibodies. Coimmunoprecipitated proteins were analyzed by Western blotting with anti-AHNAK-KIS, anti-annexin 2, and anti-S100A10 antibodies. (C) S100A10 and annexin 2 are the major proteins that interact with the AHNAK Cter domain. [35S]Methionine/cysteine labeled MDCK whole-cell extracts (total; left) were incubated with GST (lane 1) and GST fusion AHNAK-Cter (lane 2) in EGTA/EDTA-containing buffer. Bound proteins were resolved on SDS-PAGE and were detected either by autoradiography (left) or blotted on nitrocellulose membrane and detected with mixed anti-annexin 2 and anti-S100A10 mAbs (right). (D) S100B antagonizes annexin 2/S100A10 binding to the AHNAK Cter domain. U87 cell extracts were incubated with GST fusion AHNAK-Cter in the absence (lane 1) or in the presence (lanes 2 and 3) of increasing S100B concentrations in Ca2+/Zn2+-containing buffer. Bound proteins were analyzed by Western blotting using anti-annexin 2 and anti-S100A10 antibodies (left), or with anti-annexin 2 and anti-S100B antibodies (right). Lane 4 is total cell extract. (E) [35S]Methionine/cysteine-labeled S100A10 was produced in rabbit reticulocyte (total), and the translation reaction was incubated with glutathione-Sepharose in the presence of recombinant GST (lane 1) or recombinant GST fused to the AHNAK COOH-terminal domain (lane 2) in Ca2+/Zn2+-containing buffer. Bound proteins were resolved on SDS-PAGE and detected by autoradiography. (F) S100A10 specifically interacts with AHNAK CterC and S100B in the yeast two-hybrid system. Top: diagram of the various AHNAK COOH-terminal fragments used. Yeast were cotransfected with S100A10 pACT2 vector and different baits corresponding to AHNAK Cter deletion mutants, S100B, and lamin cloned into the pLEX vector as indicated. The interactions were scored by the growth of transfected yeast on YC-UWLH medium lacking histidine (HIS) and confirmed by β-galactosidase activity assay (bgal).
Mentions: To gain further insight into AHNAK function at the plasma membrane, we sought to identify proteins interacting with AHNAK using coimmunoprecipitation coupled to mass spectrometry analysis. For convenient peptide sequence identification using human databases, we used human epithelial MCF-7 cells, which predominantly express AHNAK at the plasma membrane. Confluent MCF-7 cells were treated with the membrane-permeable cross-linker dithiobis succinimidyl propionate, and AHNAK was immunoprecipitated with anti-AHNAK-CQL antibodies. Proteins present within the AHNAK immunoprecipitate were reduced, separated by SDS-PAGE, and identified by mass spectrometry. One major protein recovered within AHNAK immunoprecipitate was identified as annexin 2 (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200307098/DC1). The interaction between AHNAK and annexin 2 was confirmed both by coimmunoprecipitation in absence of cross-linking with two independent antibodies directed against AHNAK and immunodetection, and by a pull-down approach using recombinant AHNAK fragments linked to the GST tag (Fig. 2 A). The COOH-terminal fragment of AHNAK (Cter; aa 4642–5643), but neither a central fragment of AHNAK (M1; aa 820–1330) nor the GST tag alone, interacted with annexin 2. The interaction of annexin 2 with AHNAK is specific because other annexin family members, annexin IV and annexin VI, are not recovered in the AHNAK immunoprecipitates or in the pull-down experiments.

Bottom Line: Down-regulation of both annexin 2 and S100A10 using an annexin 2-specific small interfering RNA inhibits the association of AHNAK with plasma membrane.In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height.We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.

View Article: PubMed Central - PubMed

Affiliation: INSERM EMI-0104, DRDC-TS, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France.

ABSTRACT
Remodelling of the plasma membrane cytoarchitecture is crucial for the regulation of epithelial cell adhesion and permeability. In Madin-Darby canine kidney cells, the protein AHNAK relocates from the cytosol to the cytosolic surface of the plasma membrane during the formation of cell-cell contacts and the development of epithelial polarity. This targeting is reversible and regulated by Ca(2+)-dependent cell-cell adhesion. At the plasma membrane, AHNAK associates as a multimeric complex with actin and the annexin 2/S100A10 complex. The S100A10 subunit serves to mediate the interaction between annexin 2 and the COOH-terminal regulatory domain of AHNAK. Down-regulation of both annexin 2 and S100A10 using an annexin 2-specific small interfering RNA inhibits the association of AHNAK with plasma membrane. In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height. We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.

Show MeSH
Related in: MedlinePlus