title: CrkL functions as a nuclear adaptor and transcriptional activator in Bcr-Abl-expressing cells.
abstract: 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.
authors.affiliation: Oregon Health Sciences University, Portland, OR 97201, USA.
authors.forename: J
authors.initials: J
authors.lastname: Rhodes
authors.forename: R D
authors.initials: RD
authors.lastname: York
authors.forename: D
authors.initials: D
authors.lastname: Tara
authors.forename: K
authors.initials: K
authors.lastname: Tajinda
authors.forename: B J
authors.initials: BJ
authors.lastname: Druker
mesh_terms: 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
chemical_list: 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