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A more recent version of this article appeared on October 13, 2000
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M003655200v1
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Papers In Press, published online ahead of print August 3, 2000
J. Biol. Chem, 10.1074/jbc.M003655200
Submitted on April 28, 2000
Accepted on August 3, 2000

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

Jian Du, Yazan M. Alsayed, Feng Xin, Steven J. Ackerman, and Leonidas C. Platanias

Biochemistry and Molecular Biology, University of Illinois at Chicago, Chicago, IL 60612-7334

Corresponding Author: sackerma{at}uic.edu

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 AML14.3D10 eosinophil-differentiated cell line and purified peripheral blood eosinphils from normal donors. Stimulation of AML14.3D10 cells or blood eosinophils with IL-5 induces 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 translocate to the nucleus and bind 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 induces marked activity of an 8X GAS 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.


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