RESUMO
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.
Assuntos
Domínio Catalítico , Proteínas Tirosina Quinases/química , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Glutationa Transferase/metabolismo , Immunoblotting , Fosforilação , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas c-cbl , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Tirosina/química , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/isolamento & purificação , Domínios de Homologia de srcRESUMO
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.
Assuntos
Antineoplásicos/toxicidade , Proteínas de Fusão bcr-abl/genética , Piperazinas/toxicidade , Pirimidinas/toxicidade , Benzamidas , Crise Blástica , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citarabina/toxicidade , Daunorrubicina/toxicidade , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Hidroxiureia/toxicidade , Mesilato de Imatinib , Interleucina-3/farmacologia , Leucemia Megacarioblástica Aguda , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcr , Fator de Células-Tronco/farmacologia , Transfecção , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
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.
