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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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Effects of SSAT induction by BE-3-3-3 on adhesion and migration of α9-expressing CHO cells. (a) CHO cells were treated with BE-3-3-3. SSAT was detected by Western blotting with rabbit polyclonal anti-SSAT. Western blot for Crk was used as a control for equal protein loading. (b and c) α9-, α9α4-, and α9α5-expressing CHO cells were treated with a range of concentrations of BE-3-3-3 for 24 h and were suspended in serum-free medium. (b) Cells were seeded onto membranes coated with 5 μg/ml TNfn3RAA in the top well of 24-well plates in the presence or absence of the anti-α9β1 mAb Y9A2. After a 2-h incubation in the presence of 1% FCS in the bottom well, nonmigrated cells on the top side of the membrane were removed, and migrated cells on the bottom side were fixed, stained, and counted. (c) Cells were added to 96-well plates coated with 5 μg/ml TNfn3RAA in the presence or absence of the anti-α9β1 mAb Y9A2. They were allowed to attach for 60 min, and nonadherent cells were removed by centrifugation. Adherent cells were stained with crystal violet and quantified by measurement of absorbance at 595 nm. (d) Migration of α9-, α9α4-, and α9α5-expressing CHO cells was analyzed on plasma fibronectin (FN; 5 μg/ml). All data represent the means (±SEM) of triplicate experiments.
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fig3: Effects of SSAT induction by BE-3-3-3 on adhesion and migration of α9-expressing CHO cells. (a) CHO cells were treated with BE-3-3-3. SSAT was detected by Western blotting with rabbit polyclonal anti-SSAT. Western blot for Crk was used as a control for equal protein loading. (b and c) α9-, α9α4-, and α9α5-expressing CHO cells were treated with a range of concentrations of BE-3-3-3 for 24 h and were suspended in serum-free medium. (b) Cells were seeded onto membranes coated with 5 μg/ml TNfn3RAA in the top well of 24-well plates in the presence or absence of the anti-α9β1 mAb Y9A2. After a 2-h incubation in the presence of 1% FCS in the bottom well, nonmigrated cells on the top side of the membrane were removed, and migrated cells on the bottom side were fixed, stained, and counted. (c) Cells were added to 96-well plates coated with 5 μg/ml TNfn3RAA in the presence or absence of the anti-α9β1 mAb Y9A2. They were allowed to attach for 60 min, and nonadherent cells were removed by centrifugation. Adherent cells were stained with crystal violet and quantified by measurement of absorbance at 595 nm. (d) Migration of α9-, α9α4-, and α9α5-expressing CHO cells was analyzed on plasma fibronectin (FN; 5 μg/ml). All data represent the means (±SEM) of triplicate experiments.

Mentions: To determine whether the interaction between SSAT and the α9 cytoplasmic domain is critical for the enhancement of cell migration, we used CHO cells stably expressing wild-type or chimeric α9 subunits (Young et al., 2001) for cell adhesion and migration assays on an α9β1-specific substrate in the presence of a range of concentrations of BE-3-3-3 to increase endogenous levels of SSAT, and in the presence or absence of the α9β1-blocking antibody Y9A2. The α9β1-specific substrate we used (TNfn3RAA) was a recombinant form of the third fibronectin type III repeat in chicken tenascin C, in which the normal arginine-glycine-aspartic acid sequence (RGD) had been mutated to arginine-alanine-alanine (RAA) to prevent interaction with other integrins (Prieto et al., 1993; Yokosaki et al., 1994, 1998). The expression of SSAT was induced by BE-3-3-3 in a dose-dependent manner (Fig. 3 a). In the absence of BE-3-3-3, both the α9 and the α4 cytoplasmic domains caused similar enhancement of cell migration compared with the cytoplasmic domain of α5. However, BE-3-3-3 caused concentration-dependent enhancement of migration only in cells expressing wild-type α9 (Fig. 3 b). As we have previously reported (Young et al., 2001), all three versions of the α9 subunit supported equivalent adhesion to TNfn3RAA, and adhesion was unaffected by BE-3-3-3 treatment in all three cell lines (Fig. 3 c). As expected, adhesion and migration of all α9-expressing cells were inhibited by the anti-α9β1 antibody Y9A2. Migration on the irrelevant substrate, plasma fibronectin, was similar among all three cell lines and was unaffected by treatment with BE-3-3-3 (Fig. 3 d). Adhesion to fibronectin was also similar among all three cell lines and was unaffected by BE-3-3-3 (unpublished data). These results indicate that the effects of BE-3-3-3 are specific for α9β1-mediated cell migration and are dependent on the presence of the α9 cytoplasmic domain.


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)

Effects of SSAT induction by BE-3-3-3 on adhesion and migration of α9-expressing CHO cells. (a) CHO cells were treated with BE-3-3-3. SSAT was detected by Western blotting with rabbit polyclonal anti-SSAT. Western blot for Crk was used as a control for equal protein loading. (b and c) α9-, α9α4-, and α9α5-expressing CHO cells were treated with a range of concentrations of BE-3-3-3 for 24 h and were suspended in serum-free medium. (b) Cells were seeded onto membranes coated with 5 μg/ml TNfn3RAA in the top well of 24-well plates in the presence or absence of the anti-α9β1 mAb Y9A2. After a 2-h incubation in the presence of 1% FCS in the bottom well, nonmigrated cells on the top side of the membrane were removed, and migrated cells on the bottom side were fixed, stained, and counted. (c) Cells were added to 96-well plates coated with 5 μg/ml TNfn3RAA in the presence or absence of the anti-α9β1 mAb Y9A2. They were allowed to attach for 60 min, and nonadherent cells were removed by centrifugation. Adherent cells were stained with crystal violet and quantified by measurement of absorbance at 595 nm. (d) Migration of α9-, α9α4-, and α9α5-expressing CHO cells was analyzed on plasma fibronectin (FN; 5 μg/ml). All data represent the means (±SEM) of triplicate experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172529&req=5

fig3: Effects of SSAT induction by BE-3-3-3 on adhesion and migration of α9-expressing CHO cells. (a) CHO cells were treated with BE-3-3-3. SSAT was detected by Western blotting with rabbit polyclonal anti-SSAT. Western blot for Crk was used as a control for equal protein loading. (b and c) α9-, α9α4-, and α9α5-expressing CHO cells were treated with a range of concentrations of BE-3-3-3 for 24 h and were suspended in serum-free medium. (b) Cells were seeded onto membranes coated with 5 μg/ml TNfn3RAA in the top well of 24-well plates in the presence or absence of the anti-α9β1 mAb Y9A2. After a 2-h incubation in the presence of 1% FCS in the bottom well, nonmigrated cells on the top side of the membrane were removed, and migrated cells on the bottom side were fixed, stained, and counted. (c) Cells were added to 96-well plates coated with 5 μg/ml TNfn3RAA in the presence or absence of the anti-α9β1 mAb Y9A2. They were allowed to attach for 60 min, and nonadherent cells were removed by centrifugation. Adherent cells were stained with crystal violet and quantified by measurement of absorbance at 595 nm. (d) Migration of α9-, α9α4-, and α9α5-expressing CHO cells was analyzed on plasma fibronectin (FN; 5 μg/ml). All data represent the means (±SEM) of triplicate experiments.
Mentions: To determine whether the interaction between SSAT and the α9 cytoplasmic domain is critical for the enhancement of cell migration, we used CHO cells stably expressing wild-type or chimeric α9 subunits (Young et al., 2001) for cell adhesion and migration assays on an α9β1-specific substrate in the presence of a range of concentrations of BE-3-3-3 to increase endogenous levels of SSAT, and in the presence or absence of the α9β1-blocking antibody Y9A2. The α9β1-specific substrate we used (TNfn3RAA) was a recombinant form of the third fibronectin type III repeat in chicken tenascin C, in which the normal arginine-glycine-aspartic acid sequence (RGD) had been mutated to arginine-alanine-alanine (RAA) to prevent interaction with other integrins (Prieto et al., 1993; Yokosaki et al., 1994, 1998). The expression of SSAT was induced by BE-3-3-3 in a dose-dependent manner (Fig. 3 a). In the absence of BE-3-3-3, both the α9 and the α4 cytoplasmic domains caused similar enhancement of cell migration compared with the cytoplasmic domain of α5. However, BE-3-3-3 caused concentration-dependent enhancement of migration only in cells expressing wild-type α9 (Fig. 3 b). As we have previously reported (Young et al., 2001), all three versions of the α9 subunit supported equivalent adhesion to TNfn3RAA, and adhesion was unaffected by BE-3-3-3 treatment in all three cell lines (Fig. 3 c). As expected, adhesion and migration of all α9-expressing cells were inhibited by the anti-α9β1 antibody Y9A2. Migration on the irrelevant substrate, plasma fibronectin, was similar among all three cell lines and was unaffected by treatment with BE-3-3-3 (Fig. 3 d). Adhesion to fibronectin was also similar among all three cell lines and was unaffected by BE-3-3-3 (unpublished data). These results indicate that the effects of BE-3-3-3 are specific for α9β1-mediated cell migration and are dependent on the presence of the α9 cytoplasmic domain.

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