CrkL functions as a nuclear adaptor and transcriptional activator in Bcr-Abl-expressing cells
| Autor: | Jennifer Rhodes, Randall D. York, Brian J. Druker, David Tara, Katsu Tajinda |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Transcriptional Activation
Cancer Research Immunoprecipitation Fusion Proteins bcr-abl src Homology Domains hemic and lymphatic diseases Genetics STAT5 Transcription Factor Tumor Cells Cultured Humans Molecular Biology STAT5 Adaptor Proteins Signal Transducing biology Chemistry Signal transducing adaptor protein Nuclear Proteins Cell Biology Hematology Transfection Milk Proteins Fusion protein Cell biology CRKL DNA-Binding Proteins Gene Expression Regulation Neoplastic Cancer research biology.protein Trans-Activators GRB2 K562 cells Signal Transduction |
| Zdroj: | Experimental hematology. 28(3) |
| ISSN: | 0301-472X |
| Popis: | Objective To identify tyrosine phosphorylated proteins that interact with CrkL in Bcr-Abl–expressing cells and analyze the function of that association. Materials and 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 β-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. Conclusion 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect