RESUMEN
The V617F mutation of the JAK2 tyrosine kinase is found in a majority of patients with myeloproliferative disorders. Flow cytometry assays for quantitation of phosphorylated and total protein for JAK2, STAT5, and heat shock proteins (HSPs) were developed to facilitate the study of the JAK/STAT pathway. A cell line homozygous for V617F (HEL) was treated with inhibitors of JAK2 tyrosine kinase activity and the HSP90 inhibitor 17-AAG. 17-AAG reduced HSP90 levels, but increased HSP70 levels. Phospho-STAT5, total STAT5, and total AKT levels were also reduced by 17-AAG treatment. Further, phospho-JAK2, total JAK2, and cell viability were reduced to a greater extent by 17-AAG than by the pan-JAK kinase family inhibitor JKII or the JAK2-specific inhibitor AG490, and these inhibitors failed to synergize with 17-AAG. Flow-cytometry-based assays for JAK/STAT signaling pathway and HSPs are likely to have broad clinical utility for monitoring patients with abnormalities in the JAK2 pathway.
Asunto(s)
Benzoquinonas/uso terapéutico , Citometría de Flujo/métodos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Janus Quinasa 2/metabolismo , Lactamas Macrocíclicas/uso terapéutico , Trastornos Mieloproliferativos/tratamiento farmacológico , Trastornos Mieloproliferativos/metabolismo , Benzoquinonas/farmacología , Evaluación Preclínica de Medicamentos , Proteínas HSP90 de Choque Térmico/análisis , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Janus Quinasa 2/análisis , Janus Quinasa 2/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacología , Proteínas Mutantes/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Factores de Transcripción STAT/metabolismo , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
OBJECTIVE: Identification of JAK2V617F in myeloproliferative disorders makes JAK2 an important marker for disease diagnosis and a highly attractive target for therapeutic drug development. This study is intended to identify a sensitive and specific substrate for assays of the JAK2 enzymatic activity. METHODS: We expressed a glutathione S-transferase (GST) fusion protein designated GST-JAKS, which carries a peptide sequence derived from the autophosphorylation sites of human JAK2. The protein was purified from Escherichia coli cells and was used to analyze to tyrosine kinase activities of purified enzymes and crude cell extracts from cells, including mononuclear cells of JAK2V617F -positive polycythemia vera blood. It was also used to perform JAK2 kinase assays to screen inhibitors of JAK2. RESULTS: GST-JAKS is strongly phosphorylated by activated forms of JAK2 including JAK2V617F and recombinant protein containing its catalytic domain alone. It showed minimal responses to wild-type JAK2 and was not phosphorylated by the epidermal growth receptor and the insulin receptor tyrosine kinases. Kinase assays with GST-JAKS provide a sharp contrast between wild-type and mutant JAK2,V617F and are sensitive enough to detect minute amounts of JAK2V617F found in crude cell extracts. Assays can be scaled up to screen for inhibitors of JAK2 in a dot blot format. CONCLUSION: GST-JAKS is sensitive and specific protein substrate for JAK2 assays. It may have clinical applications in diagnosis of diseases related to abnormal JAK2 activity. It is also an excellent substrate for development of large scale assays to screen JAK2 inhibitors.