Association of CrkL with STAT5 in hematopoietic cells stimulated by granulocyte-macrophage colony-stimulating factor or erythropoietin

Citation
J. Ota et al., Association of CrkL with STAT5 in hematopoietic cells stimulated by granulocyte-macrophage colony-stimulating factor or erythropoietin, BIOC BIOP R, 252(3), 1998, pp. 779-786
Citations number
46
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ISSN journal
0006291X → ACNP
Volume
252
Issue
3
Year of publication
1998
Pages
779 - 786
Database
ISI
SICI code
0006-291X(19981127)252:3<779:AOCWSI>2.0.ZU;2-I
Abstract
CrkL is an adapter protein comprising Src homology (SH) 2 and SH3 domains. We investigated the molecule(s) associated with CrkL in factor-dependent ce ll lines. In the granulocyte-macrophage colony-stimulating factor (GM-CSF)- dependent cell lines TF-1 and UT-7, an approximately 95-kDa tyrosine-phosph orylated protein was precipitated along with CrkL after GM-CSF stimulation. The same protein was also observed when we used the erythropoietin (EPO)de pendent cell line UT-7/EPO, in an EPO stimulation-dependent manner. We iden tified it as STAT5 (signal transducer and activator of transcription 5, 96 kDa) by STAT5-specific antibodies. The direct binding of the SH2 domain of CrkL to STAT5 was demonstrated in far Western blotting and pull-down experi ments using the glutathione S-transferase (GST) fusion construct CrkL-SH2. The addition of the oligopeptide containing phosphotyrosine 694 in STAT5A i mpaired the association between GST-CrkL-SH2 and STAT5. Furthermore, in a g el shift assay using prolactin-inducible element (PIE) as the probe, the DN A binding activity of STAT5 was inhibited by the interaction with GST-CrkL- SH2 in vitro. Finally, we found that STAT5 associated with CrkL did not bin d to PIE sequence. These results suggest that CrkL participates in the Janu s kinase (JAK)STAT pathway by direct association with STAT5. (C) 1998 Acade mic Press.