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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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Rescue of siRNA knockdown of hamster SSAT by overexpression of human wild-type SSAT cDNA in CHO cells. (a and b) α9-expressing CHO cells stably expressing either empty vector or wild-type human SSAT (SSAT(WT)) were transfected with siRNA-1 (nonfunctional), siRNA-3, and siRNA-4 in 6-well plates. (a) 24 h later, hamster SSAT mRNA was assayed by quantitative PCR and expressed as percent basal level (normalized to siRNA-1). (b) Human SSAT mRNA was assayed by quantitative PCR and expressed as percent basal level (normalized to siRNA-1). (c) BE-3-3-3 was used to stabilize SSAT 24 h after transfection with nonfunctional siRNA (1) or two functional siRNAs (3 and 4) in CHO cells stably transfected with empty plasmid (CHO-α9-Mock) or wild-type human SSAT (CHO-α9-SSAT(WT)). SSAT protein was detected by Western blot. Western blot for Crk was used as a control for equal protein loading. (d) α9- or α9α5-expressing CHO cells with or without human SSAT expression were transfected with siRNA-1, siRNA-3, and siRNA-4; 24 h later, cells were suspended in serum-free medium and used for migration assays without BE-3-3-3 (0 μM) or with BE-3-3-3 (50 μM), as described in the Fig. 3 legend.
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fig7: Rescue of siRNA knockdown of hamster SSAT by overexpression of human wild-type SSAT cDNA in CHO cells. (a and b) α9-expressing CHO cells stably expressing either empty vector or wild-type human SSAT (SSAT(WT)) were transfected with siRNA-1 (nonfunctional), siRNA-3, and siRNA-4 in 6-well plates. (a) 24 h later, hamster SSAT mRNA was assayed by quantitative PCR and expressed as percent basal level (normalized to siRNA-1). (b) Human SSAT mRNA was assayed by quantitative PCR and expressed as percent basal level (normalized to siRNA-1). (c) BE-3-3-3 was used to stabilize SSAT 24 h after transfection with nonfunctional siRNA (1) or two functional siRNAs (3 and 4) in CHO cells stably transfected with empty plasmid (CHO-α9-Mock) or wild-type human SSAT (CHO-α9-SSAT(WT)). SSAT protein was detected by Western blot. Western blot for Crk was used as a control for equal protein loading. (d) α9- or α9α5-expressing CHO cells with or without human SSAT expression were transfected with siRNA-1, siRNA-3, and siRNA-4; 24 h later, cells were suspended in serum-free medium and used for migration assays without BE-3-3-3 (0 μM) or with BE-3-3-3 (50 μM), as described in the Fig. 3 legend.

Mentions: The target sequences of the functional siRNA-3 and siRNA-4 are the same in mouse and hamster SSAT cDNA. Therefore, we used these two siRNAs to inhibit the expression of endogenous SSAT in CHO cells. Both mRNA and protein concentrations of hamster SSAT were substantially reduced by each siRNA (Fig. 7, a and c). However, the sequence of human SSAT is different from the mouse and hamster sequences in the regions targeted by each of these siRNAs. Thus, neither siRNA-3 nor siRNA-4 affects the mRNA or protein concentration in cells transfected to express human SSAT (Fig. 7, b and c). In CHO cells that only express endogenous hamster SSAT, as in MEFs, the enhancement of α9-dependent cell migration by BE-3-3-3 was decreased by siRNA transfection (Fig. 7 d). However, α9-dependent enhancement of cell migration was not inhibited by siRNA-targeting mouse (and hamster) SSAT in CHO cells overexpressing human SSAT (Fig. 7 d). This reconstitution of α9-dependent enhancement of cell migration in siRNA-treated CHO cells with human SSAT that is resistant to these siRNAs suggests that the effects of the siRNAs tested are likely specifically due to the knockdown 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)

Rescue of siRNA knockdown of hamster SSAT by overexpression of human wild-type SSAT cDNA in CHO cells. (a and b) α9-expressing CHO cells stably expressing either empty vector or wild-type human SSAT (SSAT(WT)) were transfected with siRNA-1 (nonfunctional), siRNA-3, and siRNA-4 in 6-well plates. (a) 24 h later, hamster SSAT mRNA was assayed by quantitative PCR and expressed as percent basal level (normalized to siRNA-1). (b) Human SSAT mRNA was assayed by quantitative PCR and expressed as percent basal level (normalized to siRNA-1). (c) BE-3-3-3 was used to stabilize SSAT 24 h after transfection with nonfunctional siRNA (1) or two functional siRNAs (3 and 4) in CHO cells stably transfected with empty plasmid (CHO-α9-Mock) or wild-type human SSAT (CHO-α9-SSAT(WT)). SSAT protein was detected by Western blot. Western blot for Crk was used as a control for equal protein loading. (d) α9- or α9α5-expressing CHO cells with or without human SSAT expression were transfected with siRNA-1, siRNA-3, and siRNA-4; 24 h later, cells were suspended in serum-free medium and used for migration assays without BE-3-3-3 (0 μM) or with BE-3-3-3 (50 μM), as described in the Fig. 3 legend.
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Related In: Results  -  Collection

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fig7: Rescue of siRNA knockdown of hamster SSAT by overexpression of human wild-type SSAT cDNA in CHO cells. (a and b) α9-expressing CHO cells stably expressing either empty vector or wild-type human SSAT (SSAT(WT)) were transfected with siRNA-1 (nonfunctional), siRNA-3, and siRNA-4 in 6-well plates. (a) 24 h later, hamster SSAT mRNA was assayed by quantitative PCR and expressed as percent basal level (normalized to siRNA-1). (b) Human SSAT mRNA was assayed by quantitative PCR and expressed as percent basal level (normalized to siRNA-1). (c) BE-3-3-3 was used to stabilize SSAT 24 h after transfection with nonfunctional siRNA (1) or two functional siRNAs (3 and 4) in CHO cells stably transfected with empty plasmid (CHO-α9-Mock) or wild-type human SSAT (CHO-α9-SSAT(WT)). SSAT protein was detected by Western blot. Western blot for Crk was used as a control for equal protein loading. (d) α9- or α9α5-expressing CHO cells with or without human SSAT expression were transfected with siRNA-1, siRNA-3, and siRNA-4; 24 h later, cells were suspended in serum-free medium and used for migration assays without BE-3-3-3 (0 μM) or with BE-3-3-3 (50 μM), as described in the Fig. 3 legend.
Mentions: The target sequences of the functional siRNA-3 and siRNA-4 are the same in mouse and hamster SSAT cDNA. Therefore, we used these two siRNAs to inhibit the expression of endogenous SSAT in CHO cells. Both mRNA and protein concentrations of hamster SSAT were substantially reduced by each siRNA (Fig. 7, a and c). However, the sequence of human SSAT is different from the mouse and hamster sequences in the regions targeted by each of these siRNAs. Thus, neither siRNA-3 nor siRNA-4 affects the mRNA or protein concentration in cells transfected to express human SSAT (Fig. 7, b and c). In CHO cells that only express endogenous hamster SSAT, as in MEFs, the enhancement of α9-dependent cell migration by BE-3-3-3 was decreased by siRNA transfection (Fig. 7 d). However, α9-dependent enhancement of cell migration was not inhibited by siRNA-targeting mouse (and hamster) SSAT in CHO cells overexpressing human SSAT (Fig. 7 d). This reconstitution of α9-dependent enhancement of cell migration in siRNA-treated CHO cells with human SSAT that is resistant to these siRNAs suggests that the effects of the siRNAs tested are likely specifically due to the knockdown 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