CRKL (CRK-Like) is an adapter protein predominantly phosphorylated in cells that

CRKL (CRK-Like) is an adapter protein predominantly phosphorylated in cells that express the tyrosine kinase p210BCR-ABL the fusion product of a (9;22) chromosomal translocation causative for chronic myeloid leukemia (CML). as an adapter protein is essential for p210BCR-ABL-induced transformation. fusion gene gives rise to a p210BCR-ABL oncoprotein (2 3 Forced expression of the p210BCR-ABL protein renders hematopoietic cells growth factor-independent (4 5 and induces proliferation of myeloid progenitors and bone marrow-derived B-lymphoid cells (6-9). Transplantation of p210BCR-ABL-transducedmurine bone tissue marrow cells into IPI-493 lethally irradiated syngeneic mice induces a CML-like myeloproliferative disorder demonstrating the leukemogenic potential ARHGDIG of p210BCR-ABL (10-13). The tyrosine kinase activity of p210BCR-ABL is vital because of its oncogenic potential both and (13 14 therefore the ABL inhibitor imatinib is a effective treatment for CML (15). CRKL (CRK-Like) an associate from the CRK category of adapter proteins includes an N-terminal SH2 site accompanied by two SH3 domains SH3n and SH3c (16). Although linked to is a distinct gene located on chromosome IPI-493 22q11.21 (16). Overexpression of CRKL enhances p190BCR-ABL-induced transformation and leukemogenesis in fibroblasts and in a transgenic mouse model (17 18 CRKL links tyrosine kinase substrates to downstream effectors containing SH3 binding motifs. CRKL is constitutively phosphorylated by BCR-ABL in neutrophils of CML patients and the degree of CRKL phosphorylation is a marker of BCR-ABL kinase activity (19 20 Direct binding of CRKL to p210BCR-ABL is mediated by association of the CRKL SH3n domain with a proline-rich motif in ABL IPI-493 (21). However a mutant BCR-ABL lacking the SH3 binding motif still transforms myeloid cells (21) suggesting that CRKL remains associated with the p210BCR-ABL complex by interactions with other proteins. Cell-penetrating peptides designed to bind with high affinity to the CRKL SH3n domain block the proliferation of primary CML blast cells (22). These SH3 binding peptides IPI-493 also bind to the gene products IPI-493 with high affinity (23) identifying a commonly-encountered difficulty in studies designed to provide definitive evidence for a role that CRKL may play in p210BCR-ABL induced transformation. We and others generated mouse mutants with targeted disruptions at the locus (24 25 “insensitive” background it was shown that To directly address the requirement for CRKL for p210BCR-ABL transformation we have therefore utilized hematopoietic progenitor cells from the fetal liver of sensitive genetic background. Our results are further supported by studies with a synthetic peptide that blocks association of CRKL with the p210BCR-ABL protein complex in mouse hematopoetic progenitor cells and in human K562 leukemia cells. The current study demonstrates an essential role for CRKL in p210BCR-ABL induced transformation of hematopoietic progenitor cells. Materials and Methods Mice The Institutional Animal Use and Care Committees (IAUCC) of the University of Chicago and the Oregon Health & Science University approved animal studies. and Crkl-deficient fetal liver cells were generated as described below. None of the cell lines used in this study were cultured for longer than 6-months from initial purchase or characterization. No further authentication of cell line characteristics was done. Retrovirus preparation Bosc23 cells (28) were maintained in Dulbecco’s Modified Eagles Medium (DMEM)supplemented with 10% fetal bovine serum (FBS) 1 U/mL penicillin and1 μg/mL streptomycin. Hematopoietic progenitor cells were infected with p210BCR-ABL retrovirus or control GFP retrovirus generated by transfecting Bosc23 cells with MSCV-p210-IRES-GFP (13) or empty MSCV-IRES GFP vector and the viral supernatant harvested at 48 hours post-transfection. Myeloid and lymphoid outgrowth assays Due to the lethal phenotype of or control retrovirus in lymphoid media (RPMI plus 10% FBS 1 U/mL penicillin 1 μg/mL streptomycin 1 L-glutamine and 0.05 mM 2-mercaptoethanol) containing 8 μg/mL polybrene by two rounds of spinoculation. Approximately 5 x 106 cells were plated per 60 mm dish in triplicate. Cultures were fed every 3 days by adding 2 mL lymphoid media. Viable non-adherent cells were counted at day 14 post-transduction and viability was determined by trypan blue dye exclusion. Analysis of cell surface markers.