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T cell receptor-independent basal signaling via Erk and Abl kinases suppresses RAG gene expression.

Roose JP, Diehn M, Tomlinson MG, Lin J, Alizadeh AA, Botstein D, Brown PO, Weiss A - PLoS Biol. (2003)

Bottom Line: This TCR-like pathway results in constitutive low-level activity of Erk and Abl kinases.Inhibition of Abl by the drug STI-571 or inhibition of signaling events upstream of Erk increases RAG-1 expression.Our data suggest that physiologic gene expression programs depend upon tonic activity of signaling pathways independent of receptor ligation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Francisco, USA.

ABSTRACT
Signal transduction pathways guided by cellular receptors commonly exhibit low-level constitutive signaling in a continuous, ligand-independent manner. The dynamic equilibrium of positive and negative regulators establishes such a tonic signal. Ligand-independent signaling by the precursors of mature antigen receptors regulates development of B and T lymphocytes. Here we describe a basal signal that controls gene expression profiles in the Jurkat T cell line and mouse thymocytes. Using DNA microarrays and Northern blots to analyze unstimulated cells, we demonstrate that expression of a cluster of genes, including RAG-1 and RAG-2, is repressed by constitutive signals requiring the adapter molecules LAT and SLP-76. This TCR-like pathway results in constitutive low-level activity of Erk and Abl kinases. Inhibition of Abl by the drug STI-571 or inhibition of signaling events upstream of Erk increases RAG-1 expression. Our data suggest that physiologic gene expression programs depend upon tonic activity of signaling pathways independent of receptor ligation.

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Erk and Abl Synergize to Repress RAG-1 Expression and Require Tyrosine 132 in LAT(A) Jurkat T cells were stimulated with 5 ng/ml PMA for 24 h to mimic a low, constitutive MAPK signal. Intracellular FACS staining for phospho-Erk, phospho-Abl, and isotype control was performed on the indicated samples.(B) The combined effect of blocking both Abl and MEK-1 and MEK-2 on RAG-1 expression was determined by Northern blot analysis. Cells were treated for 24 h.(C) RAG-1 expression levels in LAT-deficient J.CaM2 cells treated with the designated inhibitors compared to vehicle-treated and Jurkat T cells as control.(D) Intracellular FACS analysis using phospho-Erk, phospho-Abl, and isotype control antibodies on Jurkat, J.CaM2, and J.CaM2 cells stably reconstituted with cDNA expression vectors carrying the indicated mutations in LAT.(E) Consequences of a signaling defective LAT molecule on RAG-1 mRNA levels in the specified resting cell lines. LATY→F6,7,8 reflects mutations in tyrosine residues 132, 171, and 191, whereas LATY→F6 harbors only a single mutation at tyrosine position 132.
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pbio.0000053-g007: Erk and Abl Synergize to Repress RAG-1 Expression and Require Tyrosine 132 in LAT(A) Jurkat T cells were stimulated with 5 ng/ml PMA for 24 h to mimic a low, constitutive MAPK signal. Intracellular FACS staining for phospho-Erk, phospho-Abl, and isotype control was performed on the indicated samples.(B) The combined effect of blocking both Abl and MEK-1 and MEK-2 on RAG-1 expression was determined by Northern blot analysis. Cells were treated for 24 h.(C) RAG-1 expression levels in LAT-deficient J.CaM2 cells treated with the designated inhibitors compared to vehicle-treated and Jurkat T cells as control.(D) Intracellular FACS analysis using phospho-Erk, phospho-Abl, and isotype control antibodies on Jurkat, J.CaM2, and J.CaM2 cells stably reconstituted with cDNA expression vectors carrying the indicated mutations in LAT.(E) Consequences of a signaling defective LAT molecule on RAG-1 mRNA levels in the specified resting cell lines. LATY→F6,7,8 reflects mutations in tyrosine residues 132, 171, and 191, whereas LATY→F6 harbors only a single mutation at tyrosine position 132.

Mentions: The unaltered phosphorylation status of Abl in U-0126-treated cells suggested that the MEK–Erk pathway does not contribute to basal Abl activity in our model. To further test this, we elevated the basal MAPK signal in Jurkat T cells by addition of 5 ng/ml PMA for 24 h. This treatment induced a higher level of phospho-Erk that is largely abrogated by U-0126, but not by PP2 (left panel of Figure 7A). By contrast, low amounts of PMA did not induce phosphorylation of Abl kinases (middle panel of Figure 7A). Therefore, whereas inhibition of Abl has modest effects on Erk phosphorylation, Erk activity does not enhance phosphorylation of tyrosine 245 in Abl. These observations further support the notion that Abl and Erk operate independently to regulate the expression of RAG-1. This notion is illustrated by the additive effects of STI-571 and U-0126 in inducing RAG-1 expression (Figure 7B). Moreover, it appears that the regulatory roles of both kinases in this pathway depend on LAT. Addition of STI-571, U-0126, or a combination of these inhibitors, or even targeting Src family kinases upstream of Abl and Erk, did not result in a hyperinduction of RAG-1 in the J.CaM2 line (Figure 7C).


T cell receptor-independent basal signaling via Erk and Abl kinases suppresses RAG gene expression.

Roose JP, Diehn M, Tomlinson MG, Lin J, Alizadeh AA, Botstein D, Brown PO, Weiss A - PLoS Biol. (2003)

Erk and Abl Synergize to Repress RAG-1 Expression and Require Tyrosine 132 in LAT(A) Jurkat T cells were stimulated with 5 ng/ml PMA for 24 h to mimic a low, constitutive MAPK signal. Intracellular FACS staining for phospho-Erk, phospho-Abl, and isotype control was performed on the indicated samples.(B) The combined effect of blocking both Abl and MEK-1 and MEK-2 on RAG-1 expression was determined by Northern blot analysis. Cells were treated for 24 h.(C) RAG-1 expression levels in LAT-deficient J.CaM2 cells treated with the designated inhibitors compared to vehicle-treated and Jurkat T cells as control.(D) Intracellular FACS analysis using phospho-Erk, phospho-Abl, and isotype control antibodies on Jurkat, J.CaM2, and J.CaM2 cells stably reconstituted with cDNA expression vectors carrying the indicated mutations in LAT.(E) Consequences of a signaling defective LAT molecule on RAG-1 mRNA levels in the specified resting cell lines. LATY→F6,7,8 reflects mutations in tyrosine residues 132, 171, and 191, whereas LATY→F6 harbors only a single mutation at tyrosine position 132.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC261890&req=5

pbio.0000053-g007: Erk and Abl Synergize to Repress RAG-1 Expression and Require Tyrosine 132 in LAT(A) Jurkat T cells were stimulated with 5 ng/ml PMA for 24 h to mimic a low, constitutive MAPK signal. Intracellular FACS staining for phospho-Erk, phospho-Abl, and isotype control was performed on the indicated samples.(B) The combined effect of blocking both Abl and MEK-1 and MEK-2 on RAG-1 expression was determined by Northern blot analysis. Cells were treated for 24 h.(C) RAG-1 expression levels in LAT-deficient J.CaM2 cells treated with the designated inhibitors compared to vehicle-treated and Jurkat T cells as control.(D) Intracellular FACS analysis using phospho-Erk, phospho-Abl, and isotype control antibodies on Jurkat, J.CaM2, and J.CaM2 cells stably reconstituted with cDNA expression vectors carrying the indicated mutations in LAT.(E) Consequences of a signaling defective LAT molecule on RAG-1 mRNA levels in the specified resting cell lines. LATY→F6,7,8 reflects mutations in tyrosine residues 132, 171, and 191, whereas LATY→F6 harbors only a single mutation at tyrosine position 132.
Mentions: The unaltered phosphorylation status of Abl in U-0126-treated cells suggested that the MEK–Erk pathway does not contribute to basal Abl activity in our model. To further test this, we elevated the basal MAPK signal in Jurkat T cells by addition of 5 ng/ml PMA for 24 h. This treatment induced a higher level of phospho-Erk that is largely abrogated by U-0126, but not by PP2 (left panel of Figure 7A). By contrast, low amounts of PMA did not induce phosphorylation of Abl kinases (middle panel of Figure 7A). Therefore, whereas inhibition of Abl has modest effects on Erk phosphorylation, Erk activity does not enhance phosphorylation of tyrosine 245 in Abl. These observations further support the notion that Abl and Erk operate independently to regulate the expression of RAG-1. This notion is illustrated by the additive effects of STI-571 and U-0126 in inducing RAG-1 expression (Figure 7B). Moreover, it appears that the regulatory roles of both kinases in this pathway depend on LAT. Addition of STI-571, U-0126, or a combination of these inhibitors, or even targeting Src family kinases upstream of Abl and Erk, did not result in a hyperinduction of RAG-1 in the J.CaM2 line (Figure 7C).

Bottom Line: This TCR-like pathway results in constitutive low-level activity of Erk and Abl kinases.Inhibition of Abl by the drug STI-571 or inhibition of signaling events upstream of Erk increases RAG-1 expression.Our data suggest that physiologic gene expression programs depend upon tonic activity of signaling pathways independent of receptor ligation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Francisco, USA.

ABSTRACT
Signal transduction pathways guided by cellular receptors commonly exhibit low-level constitutive signaling in a continuous, ligand-independent manner. The dynamic equilibrium of positive and negative regulators establishes such a tonic signal. Ligand-independent signaling by the precursors of mature antigen receptors regulates development of B and T lymphocytes. Here we describe a basal signal that controls gene expression profiles in the Jurkat T cell line and mouse thymocytes. Using DNA microarrays and Northern blots to analyze unstimulated cells, we demonstrate that expression of a cluster of genes, including RAG-1 and RAG-2, is repressed by constitutive signals requiring the adapter molecules LAT and SLP-76. This TCR-like pathway results in constitutive low-level activity of Erk and Abl kinases. Inhibition of Abl by the drug STI-571 or inhibition of signaling events upstream of Erk increases RAG-1 expression. Our data suggest that physiologic gene expression programs depend upon tonic activity of signaling pathways independent of receptor ligation.

Show MeSH
Related in: MedlinePlus