Regulation of IL-4-induced IRS-2 activation by serine phosphorylation. (CCR5P.248)

Abstract

Abstract Allergic asthma is characterized by acute inflammation of the airways with alternatively activated (M2) macrophages as key regulators of disease severity. Robust, yet transient tyrosine phosphorylation (pY) of IRS-2 in response to IL-4 leads to enhanced M2 gene activation. Mechanisms for downregulation of IL-4-induced IRS-2 signaling have not been determined although serine phosphorylation (pS) of the IRS proteins terminates insulin-activated IRS signaling. We hypothesize that serine phosphorylation of IRS-2 plays a role in inhibiting IL-4-induced responses. Human monocytic U937 cells were stimulated with IL-4 ± calyculin A, a Ser/Thr phosphatase inhibitor. In separate experiments, U937 cells and bone marrow derived macrophages (BMM) were stimulated with IL-4 and four kinase (JNK, GSK-3, TORC1, and TORC2) inhibitors. The amount of total IRS-2, pY-IRS-2 and pS-IRS-2 was determined. pY-IRS-2 inversely correlated with pS-IRS-2 following IL-4 stimulation. Calyculin A prevented pY-IRS-2, while increasing pS-IRS-2, thus diminishing the IL-4 signal. Rapamycin treatment prolonged pY-IRS-2 in U937 cells and BMM in a similar manner. More specifically, TORC2 inhibition did not prolong IRS-2 activation. These results suggest that inhibiting TORC1 activity prolonged pY-IRS-2 by diminishing pS-IRS-2. Understanding how IRS-2 signaling is regulated is crucial for finding novel targets to suppress the differentiation of M2 macrophages and lower severity of allergic lung inflammation.