Interleukin-2 receptor β Thr-450 phosphorylation is a positive regulator for receptor complex stability and activation of signaling molecules

Abstract

T, B, and natural killer cells are required for normal immune response and are regulated by cytokines such as IL-2. These cell signals are propagated following receptor-ligand engagement, controlling recruitment and activation of effector proteins. The IL-2 receptor β subunit (IL-2Rβ) serves in this capacity and is known to be phosphorylated. Tyrosine phosphorylation of the β chain has been studied extensively. However, the identification and putative regulatory roles for serine and threonine phosphorylation sites have yet to be fully characterized. Using LC-MS/MS and phosphospecific antibodies, a novel IL-2/IL-15 inducible IL-2Rβ phosphorylation site (Thr-450) was identified. IL-2 phosphokinetic analysis revealed that phosphorylation of IL-2Rβ Thr-450 is rapid (2.5 min), transient (peaks at 15 min), and protracted compared with receptor tyrosine phosphorylation and occurs in multiple cell types, including primary human lymphocytes. Pharmacological and siRNA-mediated inhibition of various serine/threonine kinases revealed ERK1/2 as a positive regulator, whereas purified protein phosphatase 1 (PP1), dephosphorylated Thr-450 in vitro. Reconstitution assays demonstrated that Thr-450 is important for regulating IL-2R complex formation, recruitment of JAK3, and activation of AKT and ERK1/2 and a transcriptionally active STAT5. These results provide the first evidence of the identification and functional characterization for threonine phosphorylation of an interleukin receptor.