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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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SSAT specifically binds to the α9 cytoplasmic domain. (a) Plasmid DNA isolated from positive colonies from α9 yeast two-hybrid library screening were cotransformed into yeast with pAS2-1–containing integrin α2, α4, α5, α9, or β1 cytoplasmic domain cDNA. 72 h after the transformation, yeast growth on YPD (-Ade/-His/-Leu/-Trp/α-X-gal) plate was shown. (b) GST pull-down assays with the α9, α2, α4, and α5 cytoplasmic domains fused to GST and immobilized on Glutathione Sepharose 4B. Wild-type SSAT protein was produced by in vitro transcription and translation (IVTT) in the presence of [35S]methionine. Bound proteins were separated by 15% SDS-PAGE and analyzed by autoradiography. 25% of IVTT products were also analyzed and showed the same amount of input. (c) Wild-type and full-length SSAT, NH2-terminal deletion SSAT (21–171), COOH-terminal deletion mutants (1–161) and (1–141), and enzymatically inactive (dominant negative [DN]) mutant SSAT (R101A/E152K) proteins were produced by IVTT, pulled down by GST-α9, and analyzed as in b.
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fig1: SSAT specifically binds to the α9 cytoplasmic domain. (a) Plasmid DNA isolated from positive colonies from α9 yeast two-hybrid library screening were cotransformed into yeast with pAS2-1–containing integrin α2, α4, α5, α9, or β1 cytoplasmic domain cDNA. 72 h after the transformation, yeast growth on YPD (-Ade/-His/-Leu/-Trp/α-X-gal) plate was shown. (b) GST pull-down assays with the α9, α2, α4, and α5 cytoplasmic domains fused to GST and immobilized on Glutathione Sepharose 4B. Wild-type SSAT protein was produced by in vitro transcription and translation (IVTT) in the presence of [35S]methionine. Bound proteins were separated by 15% SDS-PAGE and analyzed by autoradiography. 25% of IVTT products were also analyzed and showed the same amount of input. (c) Wild-type and full-length SSAT, NH2-terminal deletion SSAT (21–171), COOH-terminal deletion mutants (1–161) and (1–141), and enzymatically inactive (dominant negative [DN]) mutant SSAT (R101A/E152K) proteins were produced by IVTT, pulled down by GST-α9, and analyzed as in b.

Mentions: Proteins that interact with the α9 cytoplasmic domain were identified by yeast two-hybrid screening of a human leukocyte cDNA library, using the entire cytoplasmic domain of human α9 (residues 974–1006) as bait. Several candidate cDNAs were isolated and sequenced. These candidates were rescreened by cotransformation with bait vectors containing the α9, α5, α4, α2, or β1 cytoplasmic domains. Only a single candidate, human spermidine/spermine N1-acetyltransferase (SSAT), was found to specifically interact with α9 and not with α5, α4, α2, or β1 (Fig. 1 a).


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)

SSAT specifically binds to the α9 cytoplasmic domain. (a) Plasmid DNA isolated from positive colonies from α9 yeast two-hybrid library screening were cotransformed into yeast with pAS2-1–containing integrin α2, α4, α5, α9, or β1 cytoplasmic domain cDNA. 72 h after the transformation, yeast growth on YPD (-Ade/-His/-Leu/-Trp/α-X-gal) plate was shown. (b) GST pull-down assays with the α9, α2, α4, and α5 cytoplasmic domains fused to GST and immobilized on Glutathione Sepharose 4B. Wild-type SSAT protein was produced by in vitro transcription and translation (IVTT) in the presence of [35S]methionine. Bound proteins were separated by 15% SDS-PAGE and analyzed by autoradiography. 25% of IVTT products were also analyzed and showed the same amount of input. (c) Wild-type and full-length SSAT, NH2-terminal deletion SSAT (21–171), COOH-terminal deletion mutants (1–161) and (1–141), and enzymatically inactive (dominant negative [DN]) mutant SSAT (R101A/E152K) proteins were produced by IVTT, pulled down by GST-α9, and analyzed as in b.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: SSAT specifically binds to the α9 cytoplasmic domain. (a) Plasmid DNA isolated from positive colonies from α9 yeast two-hybrid library screening were cotransformed into yeast with pAS2-1–containing integrin α2, α4, α5, α9, or β1 cytoplasmic domain cDNA. 72 h after the transformation, yeast growth on YPD (-Ade/-His/-Leu/-Trp/α-X-gal) plate was shown. (b) GST pull-down assays with the α9, α2, α4, and α5 cytoplasmic domains fused to GST and immobilized on Glutathione Sepharose 4B. Wild-type SSAT protein was produced by in vitro transcription and translation (IVTT) in the presence of [35S]methionine. Bound proteins were separated by 15% SDS-PAGE and analyzed by autoradiography. 25% of IVTT products were also analyzed and showed the same amount of input. (c) Wild-type and full-length SSAT, NH2-terminal deletion SSAT (21–171), COOH-terminal deletion mutants (1–161) and (1–141), and enzymatically inactive (dominant negative [DN]) mutant SSAT (R101A/E152K) proteins were produced by IVTT, pulled down by GST-α9, and analyzed as in b.
Mentions: Proteins that interact with the α9 cytoplasmic domain were identified by yeast two-hybrid screening of a human leukocyte cDNA library, using the entire cytoplasmic domain of human α9 (residues 974–1006) as bait. Several candidate cDNAs were isolated and sequenced. These candidates were rescreened by cotransformation with bait vectors containing the α9, α5, α4, α2, or β1 cytoplasmic domains. Only a single candidate, human spermidine/spermine N1-acetyltransferase (SSAT), was found to specifically interact with α9 and not with α5, α4, α2, or β1 (Fig. 1 a).

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