ABSTRACT
Catalytic (SH1) domains of protein tyrosine kinases (PTKs) demonstrate specificity for peptide substrates. Whether SH1 domains differentiate between tyrosines in a physiological substrate has not been confirmed. Using purified proteins, we studied the ability of Syk, Fyn, and Abl to differentiate between tyrosines in a common PTK substrate, c-Cbl. We found that each kinase produced a distinct pattern of c-Cbl phosphorylation, which altered the phosphotyrosine-dependent interactions between c-Cbl and CrkL or phosphatidylinositol 3'-kinase (PI3-K). Our data support the concept that SH1 domains determine the final sites of phosphorylation once PTKs reach their target proteins.
Subject(s)
Catalytic Domain , Protein-Tyrosine Kinases/chemistry , Proto-Oncogene Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Amino Acid Sequence , Binding Sites , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Glutathione Transferase/metabolism , Immunoblotting , Phosphorylation , Plasmids/metabolism , Protein Structure, Tertiary , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/isolation & purification , Proto-Oncogene Proteins c-cbl , Recombinant Fusion Proteins/metabolism , Substrate Specificity , Tyrosine/chemistry , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/isolation & purification , src Homology DomainsABSTRACT
Chronic myelogenous leukemia (CML), a malignancy of a hematopoietic stem cell, is caused by the Bcr-Abl tyrosine kinase. STI571(formerly CGP 57148B), an Abl tyrosine kinase inhibitor, has specific in vitro antileukemic activity against Bcr-Abl-positive cells and is currently in Phase II clinical trials. As it is likely that resistance to a single agent would be observed, combinations of STI571 with other antileukemic agents have been evaluated for activity against Bcr-Abl-positive cell lines and in colony-forming assays in vitro. The specific antileukemic agents tested included several agents currently used for the treatment of CML: interferon-alpha (IFN), hydroxyurea (HU), daunorubicin (DNR), and cytosine arabinoside (Ara-C). In proliferation assays that use Bcr-Abl-expressing cells lines, the combination of STI571 with IFN, DNR, and Ara-C showed additive or synergistic effects, whereas the combination of STI571 and HU demonstrated antagonistic effects. However, in colony-forming assays that use CML patient samples, all combinations showed increased antiproliferative effects as compared with STI571 alone. These data indicate that combinations of STI571 with IFN, DNR, or Ara-C may be more useful than STI571 alone in the treatment of CML and suggest consideration of clinical trials of these combinations.
Subject(s)
Antineoplastic Agents/toxicity , Fusion Proteins, bcr-abl/genetics , Piperazines/toxicity , Pyrimidines/toxicity , Benzamides , Blast Crisis , Cell Division/drug effects , Cell Survival/drug effects , Cytarabine/toxicity , Daunorubicin/toxicity , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Hydroxyurea/toxicity , Imatinib Mesylate , Interleukin-3/pharmacology , Leukemia, Megakaryoblastic, Acute , Protein-Tyrosine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-bcr , Stem Cell Factor/pharmacology , Transfection , Tumor Cells, Cultured , Tumor Stem Cell AssayABSTRACT
The SH2-SH3 domain-containing adaptor protein CRKL is the predominant tyrosine phosphorylated protein in chronic myelogenous leukemia (CML) neutrophils and BCR-ABL-expressing cell lines. The amino terminal CRKL SH3 domain binds directly to a proline-rich region in the C-terminus of BCR-ABL. BCR-ABL mutants with deletions of this region were constructed to assess biologic effects of eliminating the CRKL binding site. Yeast two-hybrid analysis and gel overlay assays show eradication of the direct interaction of CRKL with BCR-ABL in the proline deletion mutants. However, these BCR-ABL mutants transform myeloid cells to growth factor independence, and in these cells CRKL is tyrosine phosphorylated and associates with BCR-ABL. These findings suggest both direct and indirect interactions of CRKL with BCR-ABL. Thus, disruption of the direct interaction with BCR-ABL has not excluded a role for CRKL in BCR-ABL-mediated transformation.
