Date Published: November 17, 2003
Publisher: Public Library of Science
Author(s): Jeroen P Roose, Maximilian Diehn, Michael G Tomlinson, Joseph Lin, Ash A Alizadeh, David Botstein, Patrick O Brown, Arthur Weiss
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.
Partial Text: Considerable evidence supports the notion that in most signal transduction systems regulated by cellular receptors some basal level of signaling occurs continuously in a ligand-independent manner, although the flux through such systems may vary considerably. The basal tone or the steady-state level of signaling in unstimulated cells is the result of an equilibrium of positive and negative regulators within a signaling pathway. This dynamic equilibrium is often revealed when the functions of negative regulators of signal transduction are impaired. For instance, inactivation of tyrosine phosphatase function by inhibitors (e.g., by pervanadate) frequently leads to an increased level of tyrosine phosphorylation of cellular proteins, in a ligand-independent manner. Recent studies in the yeast mating pathway have shown that inactivation of regulators of G-protein signaling (RGS proteins) can induce constitutive activation of downstream signaling pathways even in the absence of receptor expression (Siekhaus and Drubin 2003). Thus, the balanced actions of positive and negative regulators of signal transduction set the steady-state equilibrium. Receptor stimulation then perturbs the equilibrium state in various ways to initiate cellular responses. The steady-state level of signaling in the unstimulated state may itself have functional consequences, for instance, to maintain certain differentiated cellular properties or functions.
Here we have demonstrated a ligand-independent constitutive signaling pathway that is functional in Jurkat T cells, two murine thymomas, and in thymocytes. Our studies reveal that even without TCR engagement, the signaling pathways normally responsive to TCR stimulation are not inert. Instead the components of these pathways deliver unique constitutive instructions to the nucleus that maintains proper gene expression programs. RAG gene expression is tightly regulated during thymopoiesis and is expressed in at least two waves. The tonic basal signal we characterized in this study maintains repression of RAG gene expression and involves a TCR-like signal transduction pathway. One arm of the pathway requires Src family kinase activity, presence of LAT–SLP-76, and the enzyme activities of PLCγ1, PKC, PI3K, MEK-1, and calcineurin, culminating in basal kinase activity of Erk (Figure 8). We also identified a novel pathway that is responsible for constitutive phosphorylation of Abl. Proper signal transduction relies on Src family kinase activity and the presence of tyrosine 132 in LAT and can be blocked by STI-571 (Figure 8). We postulate that these constitutive signals may function to repress RAG gene expression during thymopoiesis and, as such, allow the locus to remain accessible for later usage without inappropriate expression.
Figure 1A and 1B can interactively explored at http://microarray-pubs.stanford.edu/tonicsignal/.