title: Detection in primary chronic myeloid leukaemia cells of p210BCR-ABL1 in complexes with adaptor proteins CBL, CRKL, and GRB2.
abstract: Chronic myeloid leukemia (CML) arises as a consequence of the expression of a chimeric fusion protein, p210BCR-ABL1, which is localized to the cytoplasm and has constitutive protein tyrosine kinase activity. Extensive publications report that p210BCR-ABL1 complexed with multiple cytoplasmic proteins can modulate several cell signaling pathways. However, while altered signaling states can be demonstrated in primary CML material, most of the reported analytical work on complexed proteins has been done in cell lines expressing p210BCR-ABL1. This has been necessary because primary hemopoietic cell lysates contain a degradative activity which rapidly and permanently destroys p210BCR-ABL1, precluding accurate p210BCR-ABL1 quantification by Western blotting or investigation of coimmunoprecipitating proteins in primary cells. This degradative activity has proven intractable to inhibition by conventional protease inhibitors. We show here that the degradative activity in primary cells is associated with cell lysosomes and is most likely to be an acid-dependent hydrolase. By lysing primary hemopoietic cells at high pH, we have demonstrated substantial inhibition of the p210BCR-ABL1-degradative activity and now report, to the best of our knowledge, the first published demonstration by coimmunoprecipitation of the association between p210BCR-ABL1 and cytoplasmic effector proteins in primary CML material.
authors.affiliation: Department of Haematology, Faculty of Medicine, Imperial College, Hammersmith Campus, London, UK.
authors.forename: Hetal
authors.initials: H
authors.lastname: Patel
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Marley
authors.forename: Myrtle Y
authors.initials: MY
authors.lastname: Gordon
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002738:Chloroquine / Q000494:pharmacology; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism*; D051380:GRB2 Adaptor Protein / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D047468:Immunoprecipitation / Q000379:methods; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D009687:Nuclear Proteins; D002738:Chloroquine; D050721:Proto-Oncogene Proteins c-cbl; D016044:Fusion Proteins, bcr-abl; D020169:Caspases; C067419:CBL protein, human