RESUMO
Protein-tyrosine phosphatases (PTPs) are regulated by reversible inactivating oxidation of the catalytic-site cysteine. We have previously shown that reversible oxidation upon UVA irradiation is followed by calpain-mediated PTP degradation. Here, we address the mechanism of regulated cleavage and the physiological function of PTP degradation. Reversible oxidation of PTP1B in vitro strongly facilitated the association with calpain and led to greatly increased calpain-dependent inactivating cleavage. Both oxidation-induced association and cleavage depended exclusively on the presence of the catalytic (reversibly oxidized) cysteine residue 215. A major cleavage site was identified preceding amino acid position Ala77. In calpain-deficient cells, insulin signaling was apparently diminished, consistent with a possible role for calpain in removing a negative regulator of insulin signaling. Reversibly oxidized PTP1B may be a target of calpain in this context.
Assuntos
Calpaína/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Domínio Catalítico , Cisteína/química , Primers do DNA/genética , Técnicas In Vitro , Insulina/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de SinaisRESUMO
In an approach to gain insight into the sequence-dependent dephosphorylation of multiple phosphotyrosyl-containing peptides by the phosphatases SHP-1 and PTP1B, we applied a chromatographic technique for the analysis of the dephosphorylation products. Mono-, bi- and triphosphorylated reference peptides corresponding to positions 1999-2014 in the activation loop of the receptor tyrosine kinase Ros were first analyzed by reversed-phase HPLC and MALDI-TOF/TOF mass spectrometry. Then, the respective products from enzymatic treatment were investigated by HPLC and compared to the standard peptides. The results obtained in this study emphasize the advantage of monitoring phosphatase reactions for mono- and biphosphorylated peptides using the described procedure rather than spectrophotometric and fluorimetric methods that do not allow for a clear identification of the products formed.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fosfopeptídeos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosfopeptídeos/química , Fosforilação , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
FLT3 receptor tyrosine kinase is aberrantly active in many cases of acute myeloid leukemia (AML). Recently, bis(1H-indol-2-yl)methanones were found to inhibit FLT3 and PDGFR kinases. To optimize FLT3 activity and selectivity, 35 novel derivatives were synthesized and tested for inhibition of FLT3 and PDGFR autophosphorylation. The most potent FLT3 inhibitors 98 and 102 show IC50 values of 0.06 and 0.04 microM, respectively, and 1 order of magnitude lower PDGFR inhibiting activity. The derivatives 76 and 82 are 20- to 40-fold PDGFR selective. Docking at the recent FLT3 structure suggests a bidentate binding mode with the backbone of Cys-694. Activity and selectivity can be related to interactions of one indole moiety with a hydrophobic pocket including Phe-691, the only different binding site residue (PDGFR Thr-681). Compound 102 inhibited the proliferation of 32D cells expressing wildtype FLT3 or FLT3-ITD similarly as FLT3 autophosphorylation, and induced apoptosis in primary AML patient blasts.
