Related Experiment Video
Updated: Jul 2, 2026
A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors
Published on: December 7, 2014
Cellular LanthaScreen and beta-lactamase reporter assays for high-throughput screening of JAK2 inhibitors
Matthew B Robers1, Thomas Machleidt, Coby B Carlson
1Invitrogen Corporation, 501 Charmany Drive, Madison, WI 53719, USA.
Abstract:
The Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 5 pathway is responsible for regulation of cellular responses to a number of cytokines and growth factors. In hematopoietic cells, growth factors such as granulocyte macrophage-colony stimulating factor, interleukin-3, and erythropoietin induce the activation of JAK2, which leads to the phosphorylation, dimerization, and transactivation of STAT5 proteins. Dysregulation of JAK2 by activating mutations such as JAK2V617F results in constitutive phosphorylation of STAT5 and has been linked to numerous myeloproliferative disorders such as polycythemia vera. A cellular LanthaScreen (Invitrogen Corp., Carlsbad, CA) time-resolved Förster resonance energy transfer assay for wild-type JAK2 activity was developed. This assay utilized the growth factor-dependent human erythroleukemia TF1 cell line engineered to express a green fluorescent protein-STAT5 fusion protein. Furthermore, a complementary beta-lactamase reporter gene assay was developed to analyze the transcriptional activity of STAT5 downstream of JAK2 in TF1 cells. The same technologies were applied to the development of cellular assays for the interrogation of the disease-relevant JAK2V617F activating mutant. A small molecule inhibitor and Stealth (Invitrogen Corp.) RNA interference oligonucleotides were used to confirm the involvement of JAK2. Our results suggest that these cellular assays and validation tools represent powerful integrated methods for the analysis of physiological and disease-relevant JAK/STAT pathways within the physiological cellular context.
Insights
Researchers developed new cellular assays to study the Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 5 pathway. These methods analyze normal JAK/STAT signaling and its role in myeloproliferative disorders like polycythemia vera.
Area of Science:
- Cellular biology
- Molecular signaling
- Hematopoiesis
Background:
- The Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 5 pathway regulates cellular responses to cytokines and growth factors.
- Dysregulation of JAK2, particularly the JAK2V617F mutation, is implicated in myeloproliferative disorders, leading to constitutive STAT5 activation.
Purpose of the Study:
- To develop and validate cellular assays for analyzing Janus kinase (JAK) 2 activity and signal transducer and activator of transcription (STAT) 5 transcriptional activity.
- To investigate both physiological JAK/STAT signaling and disease-relevant mutant JAK2 activity within a cellular context.
Main Methods:
- Development of a LanthaScreen time-resolved Förster resonance energy transfer (TR-FRET) assay using TF1 cells expressing a green fluorescent protein-STAT5 fusion protein to measure JAK2 activity.
- Implementation of a beta-lactamase reporter gene assay to assess STAT5 transcriptional activity downstream of JAK2.
- Application of these assays to study wild-type JAK2 and the JAK2V617F mutant, with validation using small molecule inhibitors and RNA interference.
Main Results:
- Successful development of cellular assays capable of measuring JAK2 activity and STAT5 transcriptional activity.
- Demonstration of the utility of these assays for interrogating both normal JAK/STAT pathway function and the JAK2V617F mutant.
- Validation of JAK2 involvement in the observed signaling through inhibitor and RNA interference studies.
Conclusions:
- The developed cellular assays provide powerful, integrated tools for analyzing physiological and disease-relevant JAK/STAT pathways.
- These methods allow for the study of signaling within a relevant cellular context, aiding in understanding myeloproliferative disorders.
