Abstract
The gammac-family cytokine IL-2 activates signaling events that contribute to cell survival and proliferation, the best-studied of which are the STAT-5 and phosphatidylinositol 3-kinase (PI3K) pathways. The starting point of this study was to define genes regulated by the IL-2R-mediated PI3K pathway in T cells. Accordingly, we used an erythropoietin (EPO) receptor chimeric receptor system in which IL-2-dependent HT-2 T cells expressed a mutant EPO-IL-2Rbeta construct where Tyr-338 is mutated to Phe. Cells expressing this mutant IL-2Rbeta chain fail to induce phosphorylation of PI3K-p85alpha/beta or activate Akt, but mediate normal IL-2-dependent proliferation and activation of JAK1 and STAT-5A/B. Microarray analyses revealed differential regulation of numerous genes compared with cells expressing a wild-type IL-2Rbeta, including up-regulation of the IL-17 receptor subunit IL-17RA. Blockade of the PI3K pathway but not p70S6K led to up-regulation of IL-17RA, and constitutive Akt activation was associated with suppressed IL-17RA expression. Moreover, similar to the mutant EPO-IL-2Rbeta chimera, IL-15 and IL-21 induced IL-17RA preferentially compared with IL-2, and IL-2 but not IL-15 or IL-21 mediated prolonged activation of the PI3K p85 regulatory subunit. Thus, there are intrinsic signaling differences between IL-2 and IL-15 that can be attributed to differences in activation of the PI3K pathway.
Highlights
The ␥c-family cytokine IL-2 activates signaling events that contribute to cell survival and proliferation, the best-studied of which are the STAT-5 and phosphatidylinositol 3-kinase (PI3K) pathways
Genes Regulated by the IL-2-induced PI3K Pathway—IL-2 induces multiple signaling events in T cells, the best studied of which are the JAK1/3-STAT-5 and PI3K pathways (37)
We have extensively examined the mechanisms by which the IL-2R complex initiates these pathways using a chimeric receptor system, in which the extracellular domains of IL-2R and ␥c have been replaced with the erythropoietin (EPO) receptor and expressed stably in IL-2-dependent HT-2 T cells
Summary
Further filtering was performed to reduce variance between replicates of the same condition by first ranking those selected probe sets based on their intensity for each of the 4 GeneChips, followed by filtering out those probe sets with 10% rank order differences for replicated GeneChips in the same group. This led to another ϳ1% data reduction. Immunoprecipitations were performed as described (32) with ␣-JAK1 (Santa Cruz Biotechnology), ␣-p85 (Upstate Biotechnologies, Saranac, NY), and protein A- or protein G-agarose (Roche Applied Sciences). ELISA assays were performed with commercial kits from R&D and eBiosciences (San Diego, CA)
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