RESUMO
BACKGROUND: Chronic myeloid leukemia (CML) is driven by the expression of the BCR-ABL oncoprotein. STAT5 is a BCR-ABL substrate and persistently activated by tyrosine phosphorylation in CML cells. Activated STAT5 (pSTAT5) drives proliferation and survival of leukemic cells and contributes to initial transformation and maintenance of the disease. In cytokine-induced STAT5 signaling, phosphorylation of STAT5A on Y694 leads to nuclear accumulation of the transcription factor, followed by DNA-binding and gene induction. However, Src-family kinases (SFK) mediate cytoplasmic retention of pSTAT5A leading to attenuated target gene expression and colony formation in CML cells. RESULTS: In this study we show that autophosphorylation of Y416 in the highly conserved activation loop of SFK generates a potent recruitment site for the SH2 domain of STAT5A. Binding of the SH2 domain to the activation loop is required for STAT5A(Y694) phosphorylation by SFK, but at the same time promotes the persistent cytoplasmic localization of the transcription factor as found in BCR-ABL(+) leukemia. As a consequence of the complex formation between tyrosine-phosphorylated SFK and the SH2 domain of STAT5A, the dimerization of STAT5A is impaired. We further demonstrate that constitutively active STAT5A(S710F) escapes from SFK-mediated cytoplasmic retention by enhancing STAT5A dimer stability. CONCLUSION: Our results reveal important structural aspects of cytoplasmic pSTAT5A found in myeloid leukemias and will contribute to the understanding of STAT5A mediated cytoplasmic signaling.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Multimerização Proteica , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Quinases da Família src/metabolismo , Animais , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Células HeLa , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Camundongos , Fosforilação/genética , Transporte Proteico/genética , Fator de Transcrição STAT5/genética , Proteínas Supressoras de Tumor/genética , Domínios de Homologia de src , Quinases da Família src/genéticaRESUMO
The inflammatory response involves a complex interplay of different cytokines which act in an auto- or paracrine manner to induce the so-called acute phase response. Cytokines are known to crosstalk on multiple levels, for instance by regulating the mRNA stability of targeted cytokines through activation of the p38-MAPK pathway. In our study we discovered a new mechanism that answers the long-standing question how pro-inflammatory cytokines and environmental stress restrict immediate signalling of interleukin (IL)-6-type cytokines. We show that p38, activated by IL-1beta, TNFalpha or environmental stress, impairs IL-6-induced JAK/STAT signalling through phosphorylation of the common cytokine receptor subunit gp130 and its subsequent internalisation and degradation. We identify MK2 as the kinase that phosphorylates serine 782 in the cytoplasmic part of gp130. Consequently, inhibition of p38 or MK2, deletion of MK2 or mutation of crucial amino acids within the MK2 target site or the di-leucine internalisation motif blocks receptor depletion and restores IL-6-dependent STAT activation as well as gene induction. Hence, a novel negative crosstalk mechanism for cytokine signalling is described, where cytokine receptor turnover is regulated in trans by pro-inflammatory cytokines and stress stimuli to coordinate the inflammatory response.
Assuntos
Receptor gp130 de Citocina/metabolismo , Endocitose , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Endocitose/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1beta/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The oncostatin M receptor (OSMR) is part of receptor complexes for oncostatin M and interleukin-31. Signaling events are triggered by Jaks (Janus kinases) that constitutively bind to membrane-proximal receptor regions. Besides their established role in signaling, Jaks are involved in the regulation of the surface expression of several cytokine receptors. Here, we analyzed the structural requirements within the human OSMR that underlie its limited surface expression in the absence of associated Jaks. We identified three dileucine-like motifs within the Jak-binding region of the OSMR that control receptor surface and overall expression. A receptor mutant in which all three motifs were mutated to alanine displayed markedly increased surface expression. Although the surface half-life of this mutant was increased compared with that of the wild-type receptor, no difference in the internalization rate was detectable, implying that these receptors differ in their post-endocytic fate. The protein stability of the wild-type receptor was markedly lower than that of mutant receptors, but could be strongly increased in the presence of the lysosomal inhibitor chloroquine. Our data are consistent with the dileucine motifs being involved in destabilization of receptors devoid of associated Jaks as part of a quality control ensuring signaling competence of OSMRs.