CrkL functions as a nuclear adaptor and transcriptional activator in Bcr-Abl-expressing cells.
OBJECTIVE
To identify tyrosine phosphorylated proteins that interact with CrkL in Bcr-Abl-expressing cells and analyze the function of that association.


METHODS
Immunoprecipitation of CrkL was performed on lysates from parental cells (Rat-1, MO7e, or 32D) or Bcr-Abl-expressing cells (Rat-1p185, MO7p210, 32Dp210, K562) followed by immunoblotting for pTyr, Stat5, or CrkL. Interactions were confirmed in vitro using GST-CrkL fusion proteins. Electrophoretic mobility shift assays were performed on K562 nuclear extracts using a beta-casein promoter-derived probe. Supershift analysis was performed with CrkL, Stat5, Stat1, Grb2, and peptide-blocked CrkL and Stat5 antibodies. CrkL localization in Rat-1 and Rat-1p185 cells was detected with indirect immunofluorescence. Transcriptional activation was analyzed in COS7 cells transfected with a Stat-responsive luciferase reporter construct and Bcr-Abl, kinase-defective Bcr-Abl, CrkL, or Grb2.


RESULTS
We show that, in Bcr-Abl-expressing cells, CrkL+ interacts with tyrosine phosphorylated Stat5. Additionally, in the presence of Bcr-Abl, CrkL is found in the nucleus, can be detected in a Stat5/DNA complex, and increases transcriptional activation from a Stat-responsive reporter construct.


CONCLUSIONS
This suggests a novel role for CrkL, functioning as a nuclear adaptor protein that can associate with and activate Stat proteins in Bcr-Abl-expressing cells.
Rhodes
J
J
Oregon Health Sciences University, Portland, OR 97201, USA.
York
R D
RD
Tara
D
D
Tajinda
K
K
Druker
B J
BJ
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D016044:Fusion Proteins, bcr-abl / Q000096:biosynthesis / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008894:Milk Proteins; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D018909:src Homology Domains / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D004268:DNA-Binding Proteins; D008894:Milk Proteins; D009687:Nuclear Proteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D016044:Fusion Proteins, bcr-abl