Engagement of the CrkL adapter in interleukin-5 signaling in eosinophils

Interleukin-5 (IL-5) drives the terminal differentiation of myeloid progenitors to the eosinophil lineage; blocks eosinophil apoptosis; and primes eosinophils for enhanced functional activities in allergic, parasitic, and other eosinophil-associated diseases. Here we describe a novel signaling pathway activated by the IL-5 receptor in eosinophils involving the CrkL adapter protein. We determined whether IL-5 induces activation of CrkL and STAT5 in eosinophils using both the human eosinophil-differentiated AML14.3D10 cell line and purified peripheral blood eosinophils from normal donors. Stimulation of AML14.3D10 cells or blood eosinophils with IL-5 induced rapid tyrosine phosphorylation of the CrkL adapter and STAT5 and the association of CrkL and STAT5 in vivo as evidenced by the detection of STAT5 in anti-CrkL immunoprecipitates. The resulting CrkL. STAT5 complexes translocated to the nucleus and bound STAT5 consensus DNA-binding sites present in the promoters of IL-5-regulated genes, as shown in gel mobility and antibody supershift assays. IL-5 also induced marked activity of an 8X-GAS (interferon γ-actirated site)-luciferase reporter construct in transient transfections of AML14.3D10 eosinophils, demonstrating that these complexes play a functional role in IL-5 signaling. CrkL was also found to interact, via its N-terminal SH3 domain, with C3G, a guanine exchange factor for the small G-protein Rap1, which was also rapidly activated in an IL-5-dependent manner in these cells, establishing that CrkL mediates downstream activation of at least two signaling cascades in IL-5-stimufated eosinophils. Thus, the CrkL adapter plays an important role in IL-5 signaling in the eosinophil, acting as a nuclear adapter for STAT5 and as an upstream regulator of the C3G-Rap1 signaling pathway.