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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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Adhesion and migration of α9-expressing CHO cells transfected with SSAT. α9-expressing CHO cells were transfected with empty plasmid (Mock), wild-type SSAT (WT), or enzymatically inactive mutant SSAT (dominant negative [DN]). Cells were treated with or without 50 μM BE-3-3-3 for 24 h. (a) Cell migration on 5 μg/ml TNfn3RAA, (b) cell adhesion on 5 μg/ml TNfn3RAA, and (c) cell migration on 5 μg/ml fibronectin were assayed and analyzed as described in the Fig. 3 legend. All data represent the means (±SEM) of triplicate experiments. (d) Western blot showing level of SSAT protein detection in mock- and SSAT-transfected α9-expressing CHO cells in the presence or absence of treatment with BE-3-3-3. Western blot for Crk was used as a control for equal protein loading.
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fig5: Adhesion and migration of α9-expressing CHO cells transfected with SSAT. α9-expressing CHO cells were transfected with empty plasmid (Mock), wild-type SSAT (WT), or enzymatically inactive mutant SSAT (dominant negative [DN]). Cells were treated with or without 50 μM BE-3-3-3 for 24 h. (a) Cell migration on 5 μg/ml TNfn3RAA, (b) cell adhesion on 5 μg/ml TNfn3RAA, and (c) cell migration on 5 μg/ml fibronectin were assayed and analyzed as described in the Fig. 3 legend. All data represent the means (±SEM) of triplicate experiments. (d) Western blot showing level of SSAT protein detection in mock- and SSAT-transfected α9-expressing CHO cells in the presence or absence of treatment with BE-3-3-3. Western blot for Crk was used as a control for equal protein loading.

Mentions: Endogenous levels of SSAT are below the level of detection by existing anti-SSAT antibodies. To confirm the relationship between the SSAT expression level and α9β1-dependent cell migration, and to determine whether the effects of SSAT depend on catalytic activity, we also performed adhesion and migration assays in CHO cells stably transfected to express either wild-type or catalytically inactive (R101A/E152K mutant) c-Myc–tagged SSAT. In the absence of BE-3-3-3, cells coexpressing wild-type α9 and exogenous wild-type SSAT demonstrated enhancement of cell migration on TNfn3RAA, compared with cells expressing wild-type α9 alone (Fig. 5 a); cell migration was further enhanced by treatment with BE-3-3-3. In contrast, neither treatment with BE-3-3-3 nor overexpression of wild-type SSAT had any effect on migration in cells expressing the chimeric subunits composed of the extracellular and transmembrane domains of α9 fused to the cytoplasmic domains of α4 (α9α4) or α5 (α9α5). The relative SSAT protein concentrations in mock- and SSAT-transfected cells in the presence and absence of treatment with BE-3-3-3–treated cells are shown in Fig. 5 d. Expression of catalytically inactive SSAT (Coleman et al., 1996) did not affect the baseline enhancement of cell migration in cells expressing wild-type α9, perhaps because the level of expression was not adequate to effectively compete with that of endogenous SSAT. However, catalytically inactive SSAT did not enhance cell migration, and it prevented even the increase in migration caused by BE-3-3-3 in cells expressing wild-type α9 alone, a result consistent with previous reports that this construct can function as a dominant negative inhibitor of wild-type SSAT (Coleman et al., 1996). Similar to the previous experimental results, overexpression of either form of SSAT had no effect on cell adhesion on TNfn3RAA or on migration on plasma fibronectin in the presence or absence of BE-3-3-3 (Fig. 5, b and c). These results confirm the specificity of SSAT for enhancement of α9-dependent cell migration and suggest that this effect depends on the catalytic activity of SSAT.


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)

Adhesion and migration of α9-expressing CHO cells transfected with SSAT. α9-expressing CHO cells were transfected with empty plasmid (Mock), wild-type SSAT (WT), or enzymatically inactive mutant SSAT (dominant negative [DN]). Cells were treated with or without 50 μM BE-3-3-3 for 24 h. (a) Cell migration on 5 μg/ml TNfn3RAA, (b) cell adhesion on 5 μg/ml TNfn3RAA, and (c) cell migration on 5 μg/ml fibronectin were assayed and analyzed as described in the Fig. 3 legend. All data represent the means (±SEM) of triplicate experiments. (d) Western blot showing level of SSAT protein detection in mock- and SSAT-transfected α9-expressing CHO cells in the presence or absence of treatment with BE-3-3-3. Western blot for Crk was used as a control for equal protein loading.
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fig5: Adhesion and migration of α9-expressing CHO cells transfected with SSAT. α9-expressing CHO cells were transfected with empty plasmid (Mock), wild-type SSAT (WT), or enzymatically inactive mutant SSAT (dominant negative [DN]). Cells were treated with or without 50 μM BE-3-3-3 for 24 h. (a) Cell migration on 5 μg/ml TNfn3RAA, (b) cell adhesion on 5 μg/ml TNfn3RAA, and (c) cell migration on 5 μg/ml fibronectin were assayed and analyzed as described in the Fig. 3 legend. All data represent the means (±SEM) of triplicate experiments. (d) Western blot showing level of SSAT protein detection in mock- and SSAT-transfected α9-expressing CHO cells in the presence or absence of treatment with BE-3-3-3. Western blot for Crk was used as a control for equal protein loading.
Mentions: Endogenous levels of SSAT are below the level of detection by existing anti-SSAT antibodies. To confirm the relationship between the SSAT expression level and α9β1-dependent cell migration, and to determine whether the effects of SSAT depend on catalytic activity, we also performed adhesion and migration assays in CHO cells stably transfected to express either wild-type or catalytically inactive (R101A/E152K mutant) c-Myc–tagged SSAT. In the absence of BE-3-3-3, cells coexpressing wild-type α9 and exogenous wild-type SSAT demonstrated enhancement of cell migration on TNfn3RAA, compared with cells expressing wild-type α9 alone (Fig. 5 a); cell migration was further enhanced by treatment with BE-3-3-3. In contrast, neither treatment with BE-3-3-3 nor overexpression of wild-type SSAT had any effect on migration in cells expressing the chimeric subunits composed of the extracellular and transmembrane domains of α9 fused to the cytoplasmic domains of α4 (α9α4) or α5 (α9α5). The relative SSAT protein concentrations in mock- and SSAT-transfected cells in the presence and absence of treatment with BE-3-3-3–treated cells are shown in Fig. 5 d. Expression of catalytically inactive SSAT (Coleman et al., 1996) did not affect the baseline enhancement of cell migration in cells expressing wild-type α9, perhaps because the level of expression was not adequate to effectively compete with that of endogenous SSAT. However, catalytically inactive SSAT did not enhance cell migration, and it prevented even the increase in migration caused by BE-3-3-3 in cells expressing wild-type α9 alone, a result consistent with previous reports that this construct can function as a dominant negative inhibitor of wild-type SSAT (Coleman et al., 1996). Similar to the previous experimental results, overexpression of either form of SSAT had no effect on cell adhesion on TNfn3RAA or on migration on plasma fibronectin in the presence or absence of BE-3-3-3 (Fig. 5, b and c). These results confirm the specificity of SSAT for enhancement of α9-dependent cell migration and suggest that this effect depends on the catalytic activity of SSAT.

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