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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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Proper Expression of RAG-1 and RAG-2 Requires a Signaling-Competent LAT Molecule(A) Graphic representation of human LAT. Conserved tyrosine residues in mammals are numbered 1 through 9, with the corresponding amino acid numbering below. Cysteine residues 26 and 29 are required for palmitoylation. Adapted from Lin and Weiss (2001).(B) RAG-1 expression by Northern blotting in Jurkat T cells induced to express the phosphatase CD148 for increasing periods of time.(C) RAG-1 and RAG-2 expression by DNA array analysis. Expression of the two genes was set at 1 in wild-type Jurkat cells and compared to expression in J.CaM2 cells, in J.CaM2 reconstituted with wild-type LAT (J.CaM2-LAT), or with signaling-mutant LAT molecules (J.CaM2-LATallF and J.CaM2-LATCtoS). In addition, expression profiles were compared to J.CaM2 treated with DMSO, J.CaM2 stimulated with PMA (25 ng/ml for 24 h), and Jurkat cells incubated with PP2 (20μM for 24 h).(D) Expression profiles of genes with increased expression in J.CaM2 and a similar expression behavior as RAG-1 and RAG-2. Abbreviations: ABCB10, ATP-binding cassette, subfamily B, member 10; DTR, diphteria toxin receptor; 1309069, similar to Rattus norvegicus nuclear-encoded mitochandrial elongation factor G; SCN2A2, sodium channel, voltage-gated, type II, α2 polypeptide; 2108230, unknown human expressed sequence tag.
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pbio.0000053-g003: Proper Expression of RAG-1 and RAG-2 Requires a Signaling-Competent LAT Molecule(A) Graphic representation of human LAT. Conserved tyrosine residues in mammals are numbered 1 through 9, with the corresponding amino acid numbering below. Cysteine residues 26 and 29 are required for palmitoylation. Adapted from Lin and Weiss (2001).(B) RAG-1 expression by Northern blotting in Jurkat T cells induced to express the phosphatase CD148 for increasing periods of time.(C) RAG-1 and RAG-2 expression by DNA array analysis. Expression of the two genes was set at 1 in wild-type Jurkat cells and compared to expression in J.CaM2 cells, in J.CaM2 reconstituted with wild-type LAT (J.CaM2-LAT), or with signaling-mutant LAT molecules (J.CaM2-LATallF and J.CaM2-LATCtoS). In addition, expression profiles were compared to J.CaM2 treated with DMSO, J.CaM2 stimulated with PMA (25 ng/ml for 24 h), and Jurkat cells incubated with PP2 (20μM for 24 h).(D) Expression profiles of genes with increased expression in J.CaM2 and a similar expression behavior as RAG-1 and RAG-2. Abbreviations: ABCB10, ATP-binding cassette, subfamily B, member 10; DTR, diphteria toxin receptor; 1309069, similar to Rattus norvegicus nuclear-encoded mitochandrial elongation factor G; SCN2A2, sodium channel, voltage-gated, type II, α2 polypeptide; 2108230, unknown human expressed sequence tag.

Mentions: We next examined whether signaling via LAT is essential for the tonic suppression of RAG-1 expression. Phosphorylation of conserved tyrosines in LAT (Figure 3A) is required for the recruitment of downstream signaling effectors and adaptors. It is technically very difficult to detect basally phosphorylated LAT in resting cells since the antibody that immunoprecipitates LAT recognizes phosphorylated LAT very poorly (data not shown). However, we have previously demonstrated that these phosphorylated tyrosine residues in LAT can be substrates for the phosphatase CD148. Doxycyclin-inducible expression of CD148 in Jurkat T cells rather selectively inhibits TCR-stimulated phosphorylation of LAT and PLCγ1; the latter effect may be secondary to the effect on LAT (Baker et al. 2001). Induction of CD148 expression for 1, 3, or 7 d resulted in increasingly high RAG-1 levels, suggesting that basal phosphorylation of LAT in unstimulated cells is crucial for proper RAG-1 expression (Figure 3B). To further address the importance of signaling via LAT, we generated two new LAT mutant J.CaM2 cell lines. The first (J.CaM2-LATallF) stably expresses normal levels of a LAT molecule with all tyrosine residues replaced by phenylalanine residues. The second (J.CaM2-LATCtoS) stably expresses a LAT molecule in which sites for palmitoylation, cysteines 26 and 29, were both replaced by serine residues, displacing LAT from localization to the GEMs (data not shown). Both cell lines demonstrated the same impaired TCR-induced phosphorylation of Erk as we had previously observed in transient assays (Lin et al. 1999; Lin and Weiss 2001). We used DNA microarrays to compare global gene expression patterns in Jurkat, J.CaM2, J.CaM2 reconstituted with wild-type LAT (J.CaM2-LAT), J.CaM2-LATallF, and J.CaM2-LATCtoS cells. These samples were also compared to J.CaM2 treated with dimethyl sulphoxide (DMSO) as a control, J.CaM2 stimulated with PMA that bypasses the requirement for LAT, and Jurkat treated with the Src kinase inhibitor PP2. Figure 3C shows the relative expression level of RAG-1 and RAG-2 in the different samples compared to expression in Jurkat control cells (expression = 1). Expression of both RAG genes was elevated in the absence of the adapter LAT and reduced in J.CaM2 reconstituted with wild-type LAT cDNA (Figure 3Ca–c). By contrast, reconstitution with a signaling-defective LAT molecule without tyrosine residues, and thus without docking sites for other signaling proteins, did not result in a reduction of RAG gene expression (Figure 3Cd). Reconstitution with a LAT molecule that does not get palmitoylated resulted only in partial reduction, suggesting that there is a partial, constitutive signal generated by LAT molecules independent of GEM localization (Figure 3Ce). These results clearly indicate that proper localization and signaling through the adapter LAT are required for normal suppression of RAG gene expression in resting cells. Src and Syk family kinases initiate signaling events upstream of LAT, leading to its tyrosine phosphorylation. Since a potent and selective Syk kinase inhibitor is not available, we used the Src kinase selective inhibitor PP2 to inhibit Lck and Fyn function in Jurkat cells. Addition of a related inhibitor, PP1, has been reported to displace ZAP-70 from the cortical membrane of Jurkat T cells (Huby et al. 1998), presumably resulting in reduced phosphorylation of LAT. Treatment of unstimulated Jurkat cells with PP2 increased expression of RAG-1 and RAG-2 to a level similar to that in J.CaM2 cells, suggesting that basal Lck and/or Fyn kinase activity is required for constitutive repression of these genes (Figure 3Ch).


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)

Proper Expression of RAG-1 and RAG-2 Requires a Signaling-Competent LAT Molecule(A) Graphic representation of human LAT. Conserved tyrosine residues in mammals are numbered 1 through 9, with the corresponding amino acid numbering below. Cysteine residues 26 and 29 are required for palmitoylation. Adapted from Lin and Weiss (2001).(B) RAG-1 expression by Northern blotting in Jurkat T cells induced to express the phosphatase CD148 for increasing periods of time.(C) RAG-1 and RAG-2 expression by DNA array analysis. Expression of the two genes was set at 1 in wild-type Jurkat cells and compared to expression in J.CaM2 cells, in J.CaM2 reconstituted with wild-type LAT (J.CaM2-LAT), or with signaling-mutant LAT molecules (J.CaM2-LATallF and J.CaM2-LATCtoS). In addition, expression profiles were compared to J.CaM2 treated with DMSO, J.CaM2 stimulated with PMA (25 ng/ml for 24 h), and Jurkat cells incubated with PP2 (20μM for 24 h).(D) Expression profiles of genes with increased expression in J.CaM2 and a similar expression behavior as RAG-1 and RAG-2. Abbreviations: ABCB10, ATP-binding cassette, subfamily B, member 10; DTR, diphteria toxin receptor; 1309069, similar to Rattus norvegicus nuclear-encoded mitochandrial elongation factor G; SCN2A2, sodium channel, voltage-gated, type II, α2 polypeptide; 2108230, unknown human expressed sequence tag.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC261890&req=5

pbio.0000053-g003: Proper Expression of RAG-1 and RAG-2 Requires a Signaling-Competent LAT Molecule(A) Graphic representation of human LAT. Conserved tyrosine residues in mammals are numbered 1 through 9, with the corresponding amino acid numbering below. Cysteine residues 26 and 29 are required for palmitoylation. Adapted from Lin and Weiss (2001).(B) RAG-1 expression by Northern blotting in Jurkat T cells induced to express the phosphatase CD148 for increasing periods of time.(C) RAG-1 and RAG-2 expression by DNA array analysis. Expression of the two genes was set at 1 in wild-type Jurkat cells and compared to expression in J.CaM2 cells, in J.CaM2 reconstituted with wild-type LAT (J.CaM2-LAT), or with signaling-mutant LAT molecules (J.CaM2-LATallF and J.CaM2-LATCtoS). In addition, expression profiles were compared to J.CaM2 treated with DMSO, J.CaM2 stimulated with PMA (25 ng/ml for 24 h), and Jurkat cells incubated with PP2 (20μM for 24 h).(D) Expression profiles of genes with increased expression in J.CaM2 and a similar expression behavior as RAG-1 and RAG-2. Abbreviations: ABCB10, ATP-binding cassette, subfamily B, member 10; DTR, diphteria toxin receptor; 1309069, similar to Rattus norvegicus nuclear-encoded mitochandrial elongation factor G; SCN2A2, sodium channel, voltage-gated, type II, α2 polypeptide; 2108230, unknown human expressed sequence tag.
Mentions: We next examined whether signaling via LAT is essential for the tonic suppression of RAG-1 expression. Phosphorylation of conserved tyrosines in LAT (Figure 3A) is required for the recruitment of downstream signaling effectors and adaptors. It is technically very difficult to detect basally phosphorylated LAT in resting cells since the antibody that immunoprecipitates LAT recognizes phosphorylated LAT very poorly (data not shown). However, we have previously demonstrated that these phosphorylated tyrosine residues in LAT can be substrates for the phosphatase CD148. Doxycyclin-inducible expression of CD148 in Jurkat T cells rather selectively inhibits TCR-stimulated phosphorylation of LAT and PLCγ1; the latter effect may be secondary to the effect on LAT (Baker et al. 2001). Induction of CD148 expression for 1, 3, or 7 d resulted in increasingly high RAG-1 levels, suggesting that basal phosphorylation of LAT in unstimulated cells is crucial for proper RAG-1 expression (Figure 3B). To further address the importance of signaling via LAT, we generated two new LAT mutant J.CaM2 cell lines. The first (J.CaM2-LATallF) stably expresses normal levels of a LAT molecule with all tyrosine residues replaced by phenylalanine residues. The second (J.CaM2-LATCtoS) stably expresses a LAT molecule in which sites for palmitoylation, cysteines 26 and 29, were both replaced by serine residues, displacing LAT from localization to the GEMs (data not shown). Both cell lines demonstrated the same impaired TCR-induced phosphorylation of Erk as we had previously observed in transient assays (Lin et al. 1999; Lin and Weiss 2001). We used DNA microarrays to compare global gene expression patterns in Jurkat, J.CaM2, J.CaM2 reconstituted with wild-type LAT (J.CaM2-LAT), J.CaM2-LATallF, and J.CaM2-LATCtoS cells. These samples were also compared to J.CaM2 treated with dimethyl sulphoxide (DMSO) as a control, J.CaM2 stimulated with PMA that bypasses the requirement for LAT, and Jurkat treated with the Src kinase inhibitor PP2. Figure 3C shows the relative expression level of RAG-1 and RAG-2 in the different samples compared to expression in Jurkat control cells (expression = 1). Expression of both RAG genes was elevated in the absence of the adapter LAT and reduced in J.CaM2 reconstituted with wild-type LAT cDNA (Figure 3Ca–c). By contrast, reconstitution with a signaling-defective LAT molecule without tyrosine residues, and thus without docking sites for other signaling proteins, did not result in a reduction of RAG gene expression (Figure 3Cd). Reconstitution with a LAT molecule that does not get palmitoylated resulted only in partial reduction, suggesting that there is a partial, constitutive signal generated by LAT molecules independent of GEM localization (Figure 3Ce). These results clearly indicate that proper localization and signaling through the adapter LAT are required for normal suppression of RAG gene expression in resting cells. Src and Syk family kinases initiate signaling events upstream of LAT, leading to its tyrosine phosphorylation. Since a potent and selective Syk kinase inhibitor is not available, we used the Src kinase selective inhibitor PP2 to inhibit Lck and Fyn function in Jurkat cells. Addition of a related inhibitor, PP1, has been reported to displace ZAP-70 from the cortical membrane of Jurkat T cells (Huby et al. 1998), presumably resulting in reduced phosphorylation of LAT. Treatment of unstimulated Jurkat cells with PP2 increased expression of RAG-1 and RAG-2 to a level similar to that in J.CaM2 cells, suggesting that basal Lck and/or Fyn kinase activity is required for constitutive repression of these genes (Figure 3Ch).

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