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 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. 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.
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 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. 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.
Authors: Ji-Heui Seo; Lisa J Wood; Anupriya Agarwal; Thomas O'Hare; Collin R Elsea; Ian J Griswold; Michael W N Deininger; Akira Imamoto; Brian J Druker Journal: Cancer Res Date: 2010-08-31 Impact factor: 12.701
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