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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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Gene Expression Analysis of Resting Jurkat T Cells and Derived Signaling Mutants(A) Basal gene expression of wild-type Jurkat T cells compared to Lck-deficient J.CaM1, CD45 (PTPRC)-deficient J45, LAT-deficient J.CaM2, ZAP-70-deficient P116, and SLP-76 (LCP2)-deficient J14 signaling mutants. High-quality data were analyzed using the Statistical Analysis of Microarrays software package (Tusher et al. 2001). After selecting high-quality data, array elements that were 2.5-fold above or below the median on at least two microarrays were included (337 cDNA elements). These elements are displayed in hierarchical cluster format where rows represent genes and columns represent experimental samples. Colored pixels capture the magnitude of the response for any gene. Shades of red and green represent fold above and fold below the median, respectively. Black pixels reflect the median and gray pixels represent missing data. Data in 337 rows correspond to 321 unique cDNA clones, representing 269 unique genes. Jurkat T cells were analyzed in six individual samples and each mutant cell line in triplicate, and the sample dendrogram was generated by hierarchically clustering the arrays using the elements shown. To interactively explore the data, go to http://microarray-pubs.stanford.edu/cgi-bin/tonicsignal/fig1a/gx?n=fig1a. Abbreviations: FYB, Fyn-binding protein; SELL, CD62L. (B) Basal gene expression differences between unstimulated LAT deficient (J.CaM2) and wild-type Jurkat T cells. Data presented in the 273 rows correspond to 251 unique cDNA clones and represent the 193 unique genes most differentially expressed between the two lines that were selected (false discovery rate, <1%). Elements were further filtered for being at least 2-fold different between the lines and for technically adequate measurements in at least five of six samples analyzed. These elements are displayed as in (A). Abbreviations: MAGEA-8, melanoma antigen, family A, 8; MAGEA-11, melanoma antigen, family A, 11. To interactively explore the data, go to http://microarray-pubs.stanford.edu/cgi-bin/tonicsignal/fig1b/gx?n=fig1b.(C) Northern blot analysis of RAG-1 gene expression in Jurkat, J.CaM2, J14, and cDNA-reconstituted cell lines J.CaM2-LAT and J14-76-11. RNA levels are indicated by β-actin hybridization. All Northern blots presented in this study are representative examples of three independent experiments, unless mentioned otherwise.(D) Resting Jurkat, TCRα-deficient (J.RT-T3.1), TCRβ-deficient (J.RT3-T3.5), and LAT-deficient (J.CaM2) cell lines were analyzed for RAG-1 and β-actin mRNA expression by Northern blot analysis.
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pbio.0000053-g001: Gene Expression Analysis of Resting Jurkat T Cells and Derived Signaling Mutants(A) Basal gene expression of wild-type Jurkat T cells compared to Lck-deficient J.CaM1, CD45 (PTPRC)-deficient J45, LAT-deficient J.CaM2, ZAP-70-deficient P116, and SLP-76 (LCP2)-deficient J14 signaling mutants. High-quality data were analyzed using the Statistical Analysis of Microarrays software package (Tusher et al. 2001). After selecting high-quality data, array elements that were 2.5-fold above or below the median on at least two microarrays were included (337 cDNA elements). These elements are displayed in hierarchical cluster format where rows represent genes and columns represent experimental samples. Colored pixels capture the magnitude of the response for any gene. Shades of red and green represent fold above and fold below the median, respectively. Black pixels reflect the median and gray pixels represent missing data. Data in 337 rows correspond to 321 unique cDNA clones, representing 269 unique genes. Jurkat T cells were analyzed in six individual samples and each mutant cell line in triplicate, and the sample dendrogram was generated by hierarchically clustering the arrays using the elements shown. To interactively explore the data, go to http://microarray-pubs.stanford.edu/cgi-bin/tonicsignal/fig1a/gx?n=fig1a. Abbreviations: FYB, Fyn-binding protein; SELL, CD62L. (B) Basal gene expression differences between unstimulated LAT deficient (J.CaM2) and wild-type Jurkat T cells. Data presented in the 273 rows correspond to 251 unique cDNA clones and represent the 193 unique genes most differentially expressed between the two lines that were selected (false discovery rate, <1%). Elements were further filtered for being at least 2-fold different between the lines and for technically adequate measurements in at least five of six samples analyzed. These elements are displayed as in (A). Abbreviations: MAGEA-8, melanoma antigen, family A, 8; MAGEA-11, melanoma antigen, family A, 11. To interactively explore the data, go to http://microarray-pubs.stanford.edu/cgi-bin/tonicsignal/fig1b/gx?n=fig1b.(C) Northern blot analysis of RAG-1 gene expression in Jurkat, J.CaM2, J14, and cDNA-reconstituted cell lines J.CaM2-LAT and J14-76-11. RNA levels are indicated by β-actin hybridization. All Northern blots presented in this study are representative examples of three independent experiments, unless mentioned otherwise.(D) Resting Jurkat, TCRα-deficient (J.RT-T3.1), TCRβ-deficient (J.RT3-T3.5), and LAT-deficient (J.CaM2) cell lines were analyzed for RAG-1 and β-actin mRNA expression by Northern blot analysis.

Mentions: We employed a panel of Jurkat T cell mutants lacking specific TCR signaling proteins to study the transcriptional consequences of this constitutive signaling pathway, with the notion that the loss of key components, even in unstimulated cells, might have consequences on the expression of a set of genes. Using DNA microarrays, we compared gene expression profiles of Jurkat-derived T cell lines deficient for Lck (J.CaM1) (Straus and Weiss 1992), LAT (J.CaM2) (Finco et al. 1998), SLP-76 (J14) (Yablonski et al. 1998), ZAP-70 (P116) (Williams et al. 1998), and CD45 (J45) (Koretzky et al. 1991) to those of wild-type Jurkat T cells. We set out to analyze aberrant gene expression caused by the lack of constitutive signaling in the mutant lines. Confirming original reports, LAT and SLP-76 (LCP2) mRNA levels were reduced in J.CaM2 and J14 (Finco et al. 1998; Yablonski et al. 1998) (Figure 1A). In addition, all mutant lines demonstrated many unique alterations in expression levels of a large number of genes compared to wild-type Jurkat cells, even though they are all derived from the parental Jurkat T cell line. It is possible that altered gene expression resulting from random mutations caused by chemical or radiation mutagenesis, unrelated to the TCR signaling defect, complicated this analysis (Figure 1A).


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)

Gene Expression Analysis of Resting Jurkat T Cells and Derived Signaling Mutants(A) Basal gene expression of wild-type Jurkat T cells compared to Lck-deficient J.CaM1, CD45 (PTPRC)-deficient J45, LAT-deficient J.CaM2, ZAP-70-deficient P116, and SLP-76 (LCP2)-deficient J14 signaling mutants. High-quality data were analyzed using the Statistical Analysis of Microarrays software package (Tusher et al. 2001). After selecting high-quality data, array elements that were 2.5-fold above or below the median on at least two microarrays were included (337 cDNA elements). These elements are displayed in hierarchical cluster format where rows represent genes and columns represent experimental samples. Colored pixels capture the magnitude of the response for any gene. Shades of red and green represent fold above and fold below the median, respectively. Black pixels reflect the median and gray pixels represent missing data. Data in 337 rows correspond to 321 unique cDNA clones, representing 269 unique genes. Jurkat T cells were analyzed in six individual samples and each mutant cell line in triplicate, and the sample dendrogram was generated by hierarchically clustering the arrays using the elements shown. To interactively explore the data, go to http://microarray-pubs.stanford.edu/cgi-bin/tonicsignal/fig1a/gx?n=fig1a. Abbreviations: FYB, Fyn-binding protein; SELL, CD62L. (B) Basal gene expression differences between unstimulated LAT deficient (J.CaM2) and wild-type Jurkat T cells. Data presented in the 273 rows correspond to 251 unique cDNA clones and represent the 193 unique genes most differentially expressed between the two lines that were selected (false discovery rate, <1%). Elements were further filtered for being at least 2-fold different between the lines and for technically adequate measurements in at least five of six samples analyzed. These elements are displayed as in (A). Abbreviations: MAGEA-8, melanoma antigen, family A, 8; MAGEA-11, melanoma antigen, family A, 11. To interactively explore the data, go to http://microarray-pubs.stanford.edu/cgi-bin/tonicsignal/fig1b/gx?n=fig1b.(C) Northern blot analysis of RAG-1 gene expression in Jurkat, J.CaM2, J14, and cDNA-reconstituted cell lines J.CaM2-LAT and J14-76-11. RNA levels are indicated by β-actin hybridization. All Northern blots presented in this study are representative examples of three independent experiments, unless mentioned otherwise.(D) Resting Jurkat, TCRα-deficient (J.RT-T3.1), TCRβ-deficient (J.RT3-T3.5), and LAT-deficient (J.CaM2) cell lines were analyzed for RAG-1 and β-actin mRNA expression by Northern blot analysis.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC261890&req=5

pbio.0000053-g001: Gene Expression Analysis of Resting Jurkat T Cells and Derived Signaling Mutants(A) Basal gene expression of wild-type Jurkat T cells compared to Lck-deficient J.CaM1, CD45 (PTPRC)-deficient J45, LAT-deficient J.CaM2, ZAP-70-deficient P116, and SLP-76 (LCP2)-deficient J14 signaling mutants. High-quality data were analyzed using the Statistical Analysis of Microarrays software package (Tusher et al. 2001). After selecting high-quality data, array elements that were 2.5-fold above or below the median on at least two microarrays were included (337 cDNA elements). These elements are displayed in hierarchical cluster format where rows represent genes and columns represent experimental samples. Colored pixels capture the magnitude of the response for any gene. Shades of red and green represent fold above and fold below the median, respectively. Black pixels reflect the median and gray pixels represent missing data. Data in 337 rows correspond to 321 unique cDNA clones, representing 269 unique genes. Jurkat T cells were analyzed in six individual samples and each mutant cell line in triplicate, and the sample dendrogram was generated by hierarchically clustering the arrays using the elements shown. To interactively explore the data, go to http://microarray-pubs.stanford.edu/cgi-bin/tonicsignal/fig1a/gx?n=fig1a. Abbreviations: FYB, Fyn-binding protein; SELL, CD62L. (B) Basal gene expression differences between unstimulated LAT deficient (J.CaM2) and wild-type Jurkat T cells. Data presented in the 273 rows correspond to 251 unique cDNA clones and represent the 193 unique genes most differentially expressed between the two lines that were selected (false discovery rate, <1%). Elements were further filtered for being at least 2-fold different between the lines and for technically adequate measurements in at least five of six samples analyzed. These elements are displayed as in (A). Abbreviations: MAGEA-8, melanoma antigen, family A, 8; MAGEA-11, melanoma antigen, family A, 11. To interactively explore the data, go to http://microarray-pubs.stanford.edu/cgi-bin/tonicsignal/fig1b/gx?n=fig1b.(C) Northern blot analysis of RAG-1 gene expression in Jurkat, J.CaM2, J14, and cDNA-reconstituted cell lines J.CaM2-LAT and J14-76-11. RNA levels are indicated by β-actin hybridization. All Northern blots presented in this study are representative examples of three independent experiments, unless mentioned otherwise.(D) Resting Jurkat, TCRα-deficient (J.RT-T3.1), TCRβ-deficient (J.RT3-T3.5), and LAT-deficient (J.CaM2) cell lines were analyzed for RAG-1 and β-actin mRNA expression by Northern blot analysis.
Mentions: We employed a panel of Jurkat T cell mutants lacking specific TCR signaling proteins to study the transcriptional consequences of this constitutive signaling pathway, with the notion that the loss of key components, even in unstimulated cells, might have consequences on the expression of a set of genes. Using DNA microarrays, we compared gene expression profiles of Jurkat-derived T cell lines deficient for Lck (J.CaM1) (Straus and Weiss 1992), LAT (J.CaM2) (Finco et al. 1998), SLP-76 (J14) (Yablonski et al. 1998), ZAP-70 (P116) (Williams et al. 1998), and CD45 (J45) (Koretzky et al. 1991) to those of wild-type Jurkat T cells. We set out to analyze aberrant gene expression caused by the lack of constitutive signaling in the mutant lines. Confirming original reports, LAT and SLP-76 (LCP2) mRNA levels were reduced in J.CaM2 and J14 (Finco et al. 1998; Yablonski et al. 1998) (Figure 1A). In addition, all mutant lines demonstrated many unique alterations in expression levels of a large number of genes compared to wild-type Jurkat cells, even though they are all derived from the parental Jurkat T cell line. It is possible that altered gene expression resulting from random mutations caused by chemical or radiation mutagenesis, unrelated to the TCR signaling defect, complicated this analysis (Figure 1A).

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