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Spermidine/spermine N1-acetyltransferase specifically binds to the integrin alpha9 subunit cytoplasmic domain and enhances cell migration.

Chen C, Young BA, Coleman CS, Pegg AE, Sheppard D - J. Cell Biol. (2004)

Bottom Line: Overexpression of SSAT increased alpha9beta1-mediated migration, and small interfering RNA knockdown of SSAT inhibited this migration without affecting cell adhesion or migration that was mediated by other integrin cytoplasmic domains.The enzyme activity of SSAT is critical for this effect, because a catalytically inactive version did not enhance migration.We conclude that SSAT directly binds to the alpha9 cytoplasmic domain and mediates alpha9-dependent enhancement of cell migration, presumably by localized effects on acetylation of polyamines or of unidentified substrates.

View Article: PubMed Central - PubMed

Affiliation: Lung Biology Center, Department of Medicine, University of California-San Francisco, San Francisco, CA 94110, USA.

ABSTRACT
The integrin alpha9beta1 is expressed on migrating cells, such as leukocytes, and binds to multiple ligands that are present at sites of tissue injury and inflammation. alpha9beta1, like the structurally related integrin alpha4beta1, mediates accelerated cell migration, an effect that depends on the alpha9 cytoplasmic domain. alpha4beta1 enhances migration through reversible binding to the adapter protein, paxillin, but alpha9beta1-dependent migration is paxillin independent. Using yeast two-hybrid screening, we identified the polyamine catabolizing enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) as a specific binding partner of the alpha9 cytoplasmic domain. Overexpression of SSAT increased alpha9beta1-mediated migration, and small interfering RNA knockdown of SSAT inhibited this migration without affecting cell adhesion or migration that was mediated by other integrin cytoplasmic domains. The enzyme activity of SSAT is critical for this effect, because a catalytically inactive version did not enhance migration. We conclude that SSAT directly binds to the alpha9 cytoplasmic domain and mediates alpha9-dependent enhancement of cell migration, presumably by localized effects on acetylation of polyamines or of unidentified substrates.

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Coimmunoprecipitation of α9β1 and SSAT. (a) Flow cytometric evaluation of cell surface expression of α9 integrin on α9-, α9α5-, and α9α4-expressing CHO cells. Open peaks represent fluorescence (FL) of unstained CHO cells, and shaded peaks represent fluorescence of CHO stained with the anti-α9β1 antibody Y9A2. (b) CHO cells transfected with Myc-tagged SSAT were treated with 50 μM BE-3-3-3 and immunoprecipitated by protein G–Sepharose coated with the anti-α9β1 antibody Y9A2. Precipitated proteins (or cell lysates) were separated by SDS-PAGE and detected with anti-Myc mAb or anti–integrin β1 cytoplasmic domain antiserum. (c) Cell surface proteins were labeled with thiol-cleavable biotin, captured with streptavidin agarose, and eluted by reduction. Eluted proteins were then immunoprecipitated and analyzed as in b.
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fig2: Coimmunoprecipitation of α9β1 and SSAT. (a) Flow cytometric evaluation of cell surface expression of α9 integrin on α9-, α9α5-, and α9α4-expressing CHO cells. Open peaks represent fluorescence (FL) of unstained CHO cells, and shaded peaks represent fluorescence of CHO stained with the anti-α9β1 antibody Y9A2. (b) CHO cells transfected with Myc-tagged SSAT were treated with 50 μM BE-3-3-3 and immunoprecipitated by protein G–Sepharose coated with the anti-α9β1 antibody Y9A2. Precipitated proteins (or cell lysates) were separated by SDS-PAGE and detected with anti-Myc mAb or anti–integrin β1 cytoplasmic domain antiserum. (c) Cell surface proteins were labeled with thiol-cleavable biotin, captured with streptavidin agarose, and eluted by reduction. Eluted proteins were then immunoprecipitated and analyzed as in b.

Mentions: To determine whether SSAT associates with α9 in cells, CHO cells stably expressing either wild-type α9 or chimeric α subunits containing the α9 extracellular and transmembrane domains, fused to the cytoplasmic domains of either α4 or α5 (Young et al., 2001), were transfected to stably express SSAT-Myc. Flow cytometry demonstrated that similar levels of α9, α9α4, and α9α5 were expressed on the cell surface (Fig. 2 a). Lysates from these cell lines were immunoprecipitated with the anti-α9β1 antibody Y9A2 (Wang et al., 1996) and immunoblotted with an anti-Myc antibody. Because endogenously and heterologously expressed SSAT have been shown to be rapidly degraded in CHO cells (McCloskey et al., 1999), the concentration of Myc-tagged SSAT was increased by treatment with the spermine analogue N1,N11-bis(ethyl)norspermine tetrahydrochloride (BE-3-3-3), which prevents proteosome-mediated SSAT degradation (Coleman and Pegg, 2001). Western blotting of immunoprecipitates with antiserum against the cytoplasmic domain of the integrin β1 subunit confirmed that similar amounts of α9β1 were precipitated from each lysate. SSAT was coimmunoprecipitated with full-length α9β1 in cells treated with BE-3-3-3, but no association with the α9α5 or α9α4 chimeras could be detected (Fig. 2 b).


Spermidine/spermine N1-acetyltransferase specifically binds to the integrin alpha9 subunit cytoplasmic domain and enhances cell migration.

Chen C, Young BA, Coleman CS, Pegg AE, Sheppard D - J. Cell Biol. (2004)

Coimmunoprecipitation of α9β1 and SSAT. (a) Flow cytometric evaluation of cell surface expression of α9 integrin on α9-, α9α5-, and α9α4-expressing CHO cells. Open peaks represent fluorescence (FL) of unstained CHO cells, and shaded peaks represent fluorescence of CHO stained with the anti-α9β1 antibody Y9A2. (b) CHO cells transfected with Myc-tagged SSAT were treated with 50 μM BE-3-3-3 and immunoprecipitated by protein G–Sepharose coated with the anti-α9β1 antibody Y9A2. Precipitated proteins (or cell lysates) were separated by SDS-PAGE and detected with anti-Myc mAb or anti–integrin β1 cytoplasmic domain antiserum. (c) Cell surface proteins were labeled with thiol-cleavable biotin, captured with streptavidin agarose, and eluted by reduction. Eluted proteins were then immunoprecipitated and analyzed as in b.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172529&req=5

fig2: Coimmunoprecipitation of α9β1 and SSAT. (a) Flow cytometric evaluation of cell surface expression of α9 integrin on α9-, α9α5-, and α9α4-expressing CHO cells. Open peaks represent fluorescence (FL) of unstained CHO cells, and shaded peaks represent fluorescence of CHO stained with the anti-α9β1 antibody Y9A2. (b) CHO cells transfected with Myc-tagged SSAT were treated with 50 μM BE-3-3-3 and immunoprecipitated by protein G–Sepharose coated with the anti-α9β1 antibody Y9A2. Precipitated proteins (or cell lysates) were separated by SDS-PAGE and detected with anti-Myc mAb or anti–integrin β1 cytoplasmic domain antiserum. (c) Cell surface proteins were labeled with thiol-cleavable biotin, captured with streptavidin agarose, and eluted by reduction. Eluted proteins were then immunoprecipitated and analyzed as in b.
Mentions: To determine whether SSAT associates with α9 in cells, CHO cells stably expressing either wild-type α9 or chimeric α subunits containing the α9 extracellular and transmembrane domains, fused to the cytoplasmic domains of either α4 or α5 (Young et al., 2001), were transfected to stably express SSAT-Myc. Flow cytometry demonstrated that similar levels of α9, α9α4, and α9α5 were expressed on the cell surface (Fig. 2 a). Lysates from these cell lines were immunoprecipitated with the anti-α9β1 antibody Y9A2 (Wang et al., 1996) and immunoblotted with an anti-Myc antibody. Because endogenously and heterologously expressed SSAT have been shown to be rapidly degraded in CHO cells (McCloskey et al., 1999), the concentration of Myc-tagged SSAT was increased by treatment with the spermine analogue N1,N11-bis(ethyl)norspermine tetrahydrochloride (BE-3-3-3), which prevents proteosome-mediated SSAT degradation (Coleman and Pegg, 2001). Western blotting of immunoprecipitates with antiserum against the cytoplasmic domain of the integrin β1 subunit confirmed that similar amounts of α9β1 were precipitated from each lysate. SSAT was coimmunoprecipitated with full-length α9β1 in cells treated with BE-3-3-3, but no association with the α9α5 or α9α4 chimeras could be detected (Fig. 2 b).

Bottom Line: Overexpression of SSAT increased alpha9beta1-mediated migration, and small interfering RNA knockdown of SSAT inhibited this migration without affecting cell adhesion or migration that was mediated by other integrin cytoplasmic domains.The enzyme activity of SSAT is critical for this effect, because a catalytically inactive version did not enhance migration.We conclude that SSAT directly binds to the alpha9 cytoplasmic domain and mediates alpha9-dependent enhancement of cell migration, presumably by localized effects on acetylation of polyamines or of unidentified substrates.

View Article: PubMed Central - PubMed

Affiliation: Lung Biology Center, Department of Medicine, University of California-San Francisco, San Francisco, CA 94110, USA.

ABSTRACT
The integrin alpha9beta1 is expressed on migrating cells, such as leukocytes, and binds to multiple ligands that are present at sites of tissue injury and inflammation. alpha9beta1, like the structurally related integrin alpha4beta1, mediates accelerated cell migration, an effect that depends on the alpha9 cytoplasmic domain. alpha4beta1 enhances migration through reversible binding to the adapter protein, paxillin, but alpha9beta1-dependent migration is paxillin independent. Using yeast two-hybrid screening, we identified the polyamine catabolizing enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) as a specific binding partner of the alpha9 cytoplasmic domain. Overexpression of SSAT increased alpha9beta1-mediated migration, and small interfering RNA knockdown of SSAT inhibited this migration without affecting cell adhesion or migration that was mediated by other integrin cytoplasmic domains. The enzyme activity of SSAT is critical for this effect, because a catalytically inactive version did not enhance migration. We conclude that SSAT directly binds to the alpha9 cytoplasmic domain and mediates alpha9-dependent enhancement of cell migration, presumably by localized effects on acetylation of polyamines or of unidentified substrates.

Show MeSH
Related in: MedlinePlus