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 associati on. 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 protei ns. Electrophoretic mobility shift assays were performed on K562 nuclear ex tracts 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 wi th 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 tyr osine phosphorylated Stat5. Additionally, in the presence of Bcr-Abl, CrkL is found in the nucleus, can be detected in a Stat5/DNA complex, and increa ses transcriptional activation from a Stat-responsive reporter construct. Conclusion. This suggests a novel role for CrkL, functioning as a nuclear a daptor protein that can associate with and activate Stat proteins in Bcr-Ab l-expressing cells. (C) 2000 International Society for Experimental Hematol ogy. Published by Elsevier Science Inc.