Association of CrkL with STAT5 in hematopoietic cells stimulated by granulocyte-macrophage colony-stimulating factor or erythropoietin.
CrkL is an adapter protein comprising Src homology (SH) 2 and SH3 domains. We investigated the molecule(s) associated with CrkL in factor-dependent cell lines. In the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent cell lines TF-1 and UT-7, an approximately 95-kDa tyrosine-phosphorylated protein was precipitated along with CrkL after GM-CSF stimulation. The same protein was also observed when we used the erythropoietin (EPO)-dependent cell line UT-7/EPO, in an EPO stimulation-dependent manner. We identified 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 experiments using the glutathione S-transferase (GST) fusion construct CrkL-SH2. The addition of the oligopeptide containing phosphotyrosine 694 in STAT5A impaired the association between GST-CrkL-SH2 and STAT5. Furthermore, in a gel shift assay using prolactin-inducible element (PIE) as the probe, the DNA 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 bind to PIE sequence. These results suggest that CrkL participates in the Janus kinase (JAK)-STAT pathway by direct association with STAT5.
Ota
J
J
Biochemical Research Laboratory, Morinaga Milk Industry.
Kimura
F
F
Sato
K
K
Wakimoto
N
N
Nakamura
Y
Y
Nagata
N
N
Suzu
S
S
Yamada
M
M
Shimamura
S
S
Motoyoshi
K
K
D048868:Adaptor Proteins, Signal Transducing; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000494:pharmacology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D008894:Milk Proteins; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D050799:STAT5 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism*; D025521:Tumor Suppressor Proteins; D014443:Tyrosine / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D004268:DNA-Binding Proteins; D008894:Milk Proteins; D009687:Nuclear Proteins; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; D004921:Erythropoietin; D014443:Tyrosine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D004247:DNA