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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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Chemical Inhibitors Map the Constitutive Signal to a TCR-Like Pathway(A) Northern blot analysis for RAG-1 expression in Jurkat T cells treated with the indicated inhibitors. Cells were incubated for 24 h prior to RNA isolation. The relative expression level of RAG-1 was calculated from three independent experiments, and the mean expression and standard deviation (SD) are indicated.(B) Northern blot analysis for RAG-1 mRNA in Jurkat expressing the phosphatase PTEN treated with indicated inhibitors. (B) and (D) are representative of two independent experiments.(C) Basal signaling was blocked in wild-type thymocytes by treating with the indicated inhibitors for 20 h. CD4 and CD8 FACS profiles of thymocytes were determined and expression of Rag-1 and β-actin was analyzed by Northern blotting.(D) Northern blot analysis for Rag-1 expression in β2M−/−/MHCII−/− thymocytes treated for 20 h with the enzyme inhibitors.
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pbio.0000053-g004: Chemical Inhibitors Map the Constitutive Signal to a TCR-Like Pathway(A) Northern blot analysis for RAG-1 expression in Jurkat T cells treated with the indicated inhibitors. Cells were incubated for 24 h prior to RNA isolation. The relative expression level of RAG-1 was calculated from three independent experiments, and the mean expression and standard deviation (SD) are indicated.(B) Northern blot analysis for RAG-1 mRNA in Jurkat expressing the phosphatase PTEN treated with indicated inhibitors. (B) and (D) are representative of two independent experiments.(C) Basal signaling was blocked in wild-type thymocytes by treating with the indicated inhibitors for 20 h. CD4 and CD8 FACS profiles of thymocytes were determined and expression of Rag-1 and β-actin was analyzed by Northern blotting.(D) Northern blot analysis for Rag-1 expression in β2M−/−/MHCII−/− thymocytes treated for 20 h with the enzyme inhibitors.

Mentions: To further trace specific molecular components of this tonic signaling pathway, we treated Jurkat cells for 24 h with chemical inhibitors to various TCR-regulatable signaling molecules. Resting Jurkat T cells were incubated with the following inhibitors (molecular target indicated in parenthesis): PP2 (Src kinases), D609 (PLCγ?), Ro-318220 (novel PKC family members), LY294002 (PI3K), PD98059 (MEK-1), U-0126 (MEK-1 and MEK-2), FK506 (calcineurin), and DMSO (vehicle control) (see Figure 4A). Addition of DMSO did not alter the RAG-1 expression level. Inhibition of many of these signaling molecules in wild-type Jurkat T cells induced marked increases in RAG-1 gene expression (Figure 4A). All these inhibitors except LY294002 yielded an approximately 4-fold induction of expression. The effects of the inhibitors shown were specific since no change in RAG-1 expression was observed when Jurkat cells were treated with inhibitors specific for protein kinase A (PKA) (H89) and the mammalian target of rapamycin (mTOR) (rapamycin) (data not shown).


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)

Chemical Inhibitors Map the Constitutive Signal to a TCR-Like Pathway(A) Northern blot analysis for RAG-1 expression in Jurkat T cells treated with the indicated inhibitors. Cells were incubated for 24 h prior to RNA isolation. The relative expression level of RAG-1 was calculated from three independent experiments, and the mean expression and standard deviation (SD) are indicated.(B) Northern blot analysis for RAG-1 mRNA in Jurkat expressing the phosphatase PTEN treated with indicated inhibitors. (B) and (D) are representative of two independent experiments.(C) Basal signaling was blocked in wild-type thymocytes by treating with the indicated inhibitors for 20 h. CD4 and CD8 FACS profiles of thymocytes were determined and expression of Rag-1 and β-actin was analyzed by Northern blotting.(D) Northern blot analysis for Rag-1 expression in β2M−/−/MHCII−/− thymocytes treated for 20 h with the enzyme inhibitors.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC261890&req=5

pbio.0000053-g004: Chemical Inhibitors Map the Constitutive Signal to a TCR-Like Pathway(A) Northern blot analysis for RAG-1 expression in Jurkat T cells treated with the indicated inhibitors. Cells were incubated for 24 h prior to RNA isolation. The relative expression level of RAG-1 was calculated from three independent experiments, and the mean expression and standard deviation (SD) are indicated.(B) Northern blot analysis for RAG-1 mRNA in Jurkat expressing the phosphatase PTEN treated with indicated inhibitors. (B) and (D) are representative of two independent experiments.(C) Basal signaling was blocked in wild-type thymocytes by treating with the indicated inhibitors for 20 h. CD4 and CD8 FACS profiles of thymocytes were determined and expression of Rag-1 and β-actin was analyzed by Northern blotting.(D) Northern blot analysis for Rag-1 expression in β2M−/−/MHCII−/− thymocytes treated for 20 h with the enzyme inhibitors.
Mentions: To further trace specific molecular components of this tonic signaling pathway, we treated Jurkat cells for 24 h with chemical inhibitors to various TCR-regulatable signaling molecules. Resting Jurkat T cells were incubated with the following inhibitors (molecular target indicated in parenthesis): PP2 (Src kinases), D609 (PLCγ?), Ro-318220 (novel PKC family members), LY294002 (PI3K), PD98059 (MEK-1), U-0126 (MEK-1 and MEK-2), FK506 (calcineurin), and DMSO (vehicle control) (see Figure 4A). Addition of DMSO did not alter the RAG-1 expression level. Inhibition of many of these signaling molecules in wild-type Jurkat T cells induced marked increases in RAG-1 gene expression (Figure 4A). All these inhibitors except LY294002 yielded an approximately 4-fold induction of expression. The effects of the inhibitors shown were specific since no change in RAG-1 expression was observed when Jurkat cells were treated with inhibitors specific for protein kinase A (PKA) (H89) and the mammalian target of rapamycin (mTOR) (rapamycin) (data not shown).

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