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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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LAT Is Required for Two Largely Separate Pathways Marked by Constitutive Phosphorylation of Erk and Abl Kinases(A) Intracellular FACS staining for phospho-Erk (I and IV), phospho-Abl (II and V), and isotype control (III and VI) in the indicated cell lines. Histograms are an example of a representative experiment. The accompanying bar graphs are depicted in the same color-coding and represent the mean levels of fluorescence and standard deviation of three independent assays for all experiments. Mean levels of phosphoproteins measured by fluorescence in a resting Jurkat population were arbitrarily set at 100, and the mean fluorescence of the isotype control samples is indicated by the dotted line as a point of reference. Specifics are mentioned in the text.(B) Analysis of tyrosine phosphorylation levels of c-Abl in the indicated cell samples. c-Abl or control immunopercipitations were immunoblotted for total tyrosine phosphorylation by 4G10. The same blot was stripped and reprobed for c-Abl. Doxycyclin was administered for 7 d to induce CD148 expression.(C) Resting Jurkat T cells were treated with the indicated inhibitors for 24 h, and intracellular FACS staining for phospho-Abl and isotype control was compared to that detected in vehicle-treated Jurkat cells and the J.CaM2 line. Bar graphs (representing three experiments) display the four conditions depicted in the histograms as well as three additional samples.
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pbio.0000053-g006: LAT Is Required for Two Largely Separate Pathways Marked by Constitutive Phosphorylation of Erk and Abl Kinases(A) Intracellular FACS staining for phospho-Erk (I and IV), phospho-Abl (II and V), and isotype control (III and VI) in the indicated cell lines. Histograms are an example of a representative experiment. The accompanying bar graphs are depicted in the same color-coding and represent the mean levels of fluorescence and standard deviation of three independent assays for all experiments. Mean levels of phosphoproteins measured by fluorescence in a resting Jurkat population were arbitrarily set at 100, and the mean fluorescence of the isotype control samples is indicated by the dotted line as a point of reference. Specifics are mentioned in the text.(B) Analysis of tyrosine phosphorylation levels of c-Abl in the indicated cell samples. c-Abl or control immunopercipitations were immunoblotted for total tyrosine phosphorylation by 4G10. The same blot was stripped and reprobed for c-Abl. Doxycyclin was administered for 7 d to induce CD148 expression.(C) Resting Jurkat T cells were treated with the indicated inhibitors for 24 h, and intracellular FACS staining for phospho-Abl and isotype control was compared to that detected in vehicle-treated Jurkat cells and the J.CaM2 line. Bar graphs (representing three experiments) display the four conditions depicted in the histograms as well as three additional samples.

Mentions: To resolve this question, we analyzed these low-level, constitutive phospho-c-Abl and phospho-Erk signals by intracellular FACS staining (we were unable to use the site-specific phospho-Y245 for c-Abl in Western blot analysis; data not shown). Panels I–III of Figure 6A demonstrate the feasibility of this approach. PMA stimulation of Jurkat T cells for 10 min resulted in 6-fold induction of phospho-Erk signal compared to unstimulated Jurkat (arbitrarily set at 100 in the accompanying bar graph of Figure 6A, panel I). As shown in the same panel, 24-h administration of a MEK-1 and MEK-2 inhibitor (U-0126) effectively blunted the phospho-Erk response, whereas the c-Abl inhibitor had a detectable but consistently smaller effect. The STI-571 inhibitor was, however, very potent in blocking the phospho-Abl signal in K562 cells, which reflects the kinase activity of BCR–Abl in this cell line (Figure 6A, panel II). Similar to the basal activity of the Erk kinases (Figure 6A, panel IV), phosphorylation of tyrosine 245 in Abl was consistently reduced in unstimulated J.CaM2 cells, resulting in a fluorescence signal not significantly above isotype control (Figure 6A, panels V and VI). The phospho-Abl signal observed in Jurkat T cells probably reflects both phosphorylated c-Abl and phosphorylated Arg since the two kinases are highly conserved in the tyrosine 245 region. Importantly, phospho-Abl levels were restored in the J.CaM2-LAT line, pointing to an essential role for LAT in Abl phosphorylation (Figure 6A, panel V; Figure 6B). The reduction of c-Abl-specific phosphorylation in J.CaM2 cells was confirmed by c-Abl immunoprecipitations followed by total phosphotyrosine immunoblotting and was restored in J.CaM2 cells complemented with LAT cDNA (Figure 6B). Induced expression of the phosphatase CD148 for 7 d also ablated this constitutive phospho-c-Abl signal (right panel in Figure 6B).


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)

LAT Is Required for Two Largely Separate Pathways Marked by Constitutive Phosphorylation of Erk and Abl Kinases(A) Intracellular FACS staining for phospho-Erk (I and IV), phospho-Abl (II and V), and isotype control (III and VI) in the indicated cell lines. Histograms are an example of a representative experiment. The accompanying bar graphs are depicted in the same color-coding and represent the mean levels of fluorescence and standard deviation of three independent assays for all experiments. Mean levels of phosphoproteins measured by fluorescence in a resting Jurkat population were arbitrarily set at 100, and the mean fluorescence of the isotype control samples is indicated by the dotted line as a point of reference. Specifics are mentioned in the text.(B) Analysis of tyrosine phosphorylation levels of c-Abl in the indicated cell samples. c-Abl or control immunopercipitations were immunoblotted for total tyrosine phosphorylation by 4G10. The same blot was stripped and reprobed for c-Abl. Doxycyclin was administered for 7 d to induce CD148 expression.(C) Resting Jurkat T cells were treated with the indicated inhibitors for 24 h, and intracellular FACS staining for phospho-Abl and isotype control was compared to that detected in vehicle-treated Jurkat cells and the J.CaM2 line. Bar graphs (representing three experiments) display the four conditions depicted in the histograms as well as three additional samples.
© Copyright Policy
Related In: Results  -  Collection

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

pbio.0000053-g006: LAT Is Required for Two Largely Separate Pathways Marked by Constitutive Phosphorylation of Erk and Abl Kinases(A) Intracellular FACS staining for phospho-Erk (I and IV), phospho-Abl (II and V), and isotype control (III and VI) in the indicated cell lines. Histograms are an example of a representative experiment. The accompanying bar graphs are depicted in the same color-coding and represent the mean levels of fluorescence and standard deviation of three independent assays for all experiments. Mean levels of phosphoproteins measured by fluorescence in a resting Jurkat population were arbitrarily set at 100, and the mean fluorescence of the isotype control samples is indicated by the dotted line as a point of reference. Specifics are mentioned in the text.(B) Analysis of tyrosine phosphorylation levels of c-Abl in the indicated cell samples. c-Abl or control immunopercipitations were immunoblotted for total tyrosine phosphorylation by 4G10. The same blot was stripped and reprobed for c-Abl. Doxycyclin was administered for 7 d to induce CD148 expression.(C) Resting Jurkat T cells were treated with the indicated inhibitors for 24 h, and intracellular FACS staining for phospho-Abl and isotype control was compared to that detected in vehicle-treated Jurkat cells and the J.CaM2 line. Bar graphs (representing three experiments) display the four conditions depicted in the histograms as well as three additional samples.
Mentions: To resolve this question, we analyzed these low-level, constitutive phospho-c-Abl and phospho-Erk signals by intracellular FACS staining (we were unable to use the site-specific phospho-Y245 for c-Abl in Western blot analysis; data not shown). Panels I–III of Figure 6A demonstrate the feasibility of this approach. PMA stimulation of Jurkat T cells for 10 min resulted in 6-fold induction of phospho-Erk signal compared to unstimulated Jurkat (arbitrarily set at 100 in the accompanying bar graph of Figure 6A, panel I). As shown in the same panel, 24-h administration of a MEK-1 and MEK-2 inhibitor (U-0126) effectively blunted the phospho-Erk response, whereas the c-Abl inhibitor had a detectable but consistently smaller effect. The STI-571 inhibitor was, however, very potent in blocking the phospho-Abl signal in K562 cells, which reflects the kinase activity of BCR–Abl in this cell line (Figure 6A, panel II). Similar to the basal activity of the Erk kinases (Figure 6A, panel IV), phosphorylation of tyrosine 245 in Abl was consistently reduced in unstimulated J.CaM2 cells, resulting in a fluorescence signal not significantly above isotype control (Figure 6A, panels V and VI). The phospho-Abl signal observed in Jurkat T cells probably reflects both phosphorylated c-Abl and phosphorylated Arg since the two kinases are highly conserved in the tyrosine 245 region. Importantly, phospho-Abl levels were restored in the J.CaM2-LAT line, pointing to an essential role for LAT in Abl phosphorylation (Figure 6A, panel V; Figure 6B). The reduction of c-Abl-specific phosphorylation in J.CaM2 cells was confirmed by c-Abl immunoprecipitations followed by total phosphotyrosine immunoblotting and was restored in J.CaM2 cells complemented with LAT cDNA (Figure 6B). Induced expression of the phosphatase CD148 for 7 d also ablated this constitutive phospho-c-Abl signal (right panel in Figure 6B).

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