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 gamma-activated 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-stimulated 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.
Department of Biochemistry and Molecular Biology and the Section of Hematology-Oncology, Department of Medicine, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
J
J
Du
Y M
YM
Alsayed
F
F
Xin
S J
SJ
Ackerman
L C
LC
Platanias
D048868:Adaptor Proteins, Signal Transducing; D002454:Cell Differentiation; D004804:Eosinophils / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D015848:Interleukin-5 / Q000494:pharmacology*; D015470:Leukemia, Myeloid, Acute; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D018123:Receptors, Interleukin / Q000502:physiology*; D053649:Receptors, Interleukin-5; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D015848:Interleukin-5; D009687:Nuclear Proteins; D010750:Phosphoproteins; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5