CrkL functions as a nuclear adaptor and transcriptional activator in Bcr-Abl-expressing cells

Jennifer Rhodes, Randall D. York, David Tara, Katsu Tajinda, Brian Druker

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

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. Copyright (C) 2000 International Society for Experimental Hematology.

Original languageEnglish (US)
Pages (from-to)305-310
Number of pages6
JournalExperimental Hematology
Volume28
Issue number3
DOIs
StatePublished - Mar 2000

Fingerprint

Transcriptional Activation
Tyrosine
Proteins
Electrophoretic Mobility Shift Assay
Indirect Fluorescent Antibody Technique
Nuclear Proteins
Caseins
Luciferases
Immunoprecipitation
Immunoblotting
Phosphotransferases
Peptides
Antibodies
DNA
In Vitro Techniques

Keywords

  • Bcr-Abl
  • CrkL
  • Stat5

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Hematology
  • Oncology
  • Transplantation

Cite this

CrkL functions as a nuclear adaptor and transcriptional activator in Bcr-Abl-expressing cells. / Rhodes, Jennifer; York, Randall D.; Tara, David; Tajinda, Katsu; Druker, Brian.

In: Experimental Hematology, Vol. 28, No. 3, 03.2000, p. 305-310.

Research output: Contribution to journalArticle

Rhodes, Jennifer ; York, Randall D. ; Tara, David ; Tajinda, Katsu ; Druker, Brian. / CrkL functions as a nuclear adaptor and transcriptional activator in Bcr-Abl-expressing cells. In: Experimental Hematology. 2000 ; Vol. 28, No. 3. pp. 305-310.
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abstract = "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. Copyright (C) 2000 International Society for Experimental Hematology.",
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AB - 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. Copyright (C) 2000 International Society for Experimental Hematology.

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