Interactions of JAK2 pseudokinase and kinase domains in health and disease
Karjalainen, Anzhelika (2017)
2017
Bioengineering
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Hyväksymispäivämäärä
2017-06-07
Julkaisun pysyvä osoite on
http://urn.fi/URN:NBN:fi:tty-201705241468
http://urn.fi/URN:NBN:fi:tty-201705241468
Tiivistelmä
Homeostasis of the human body depends on the immune system regulated via specific signaling pathways mediated by cytokines. Many cytokines exert their unique effect by the means of JAK/STAT signaling pathway. Janus Kinase (JAK) family of enzymes are tyrosine kinase proteins which include 4 members: JAK1, JAK2, JAK3 and TYK2. JAKs consist of four domains: FERM domain and SH2 domain, which are engaged in cytokine-receptor association, and two protein-kinase domains: a pseudokinase domain (JH2) and a canonical tyrosine kinase domain (JH1). Tyrosine kinase domain is responsible for the catalytic activity of the protein whereas the pseudokinase domain was found to have an important role of negatively regulating the tyrosine kinase domain function. JAK kinases are recognized as pharmaceutical targets for treatment of numerous myeloproliferative diseases as well as several types of blood cancer. However, the structure and mechanism of JAK activation and inhibition are still elusive. To design effective pharmaceutical agents, it is of paramount importance to determine the differences in interdomain interactions between pseudokinase and catalytic domains of JAKS in health and disease.
In this study, we apply a novel approach to studying the conformational dynamics between JH2 and JH1. Fluorescence resonance energy transfer (FRET) based on cyan fluorescent protein and new Tetra Cysteine labelling technology that relies on small biarsenical FlAsH-EDT2 dye was used to study the orientational change in JH2-JH1 domains in wild type and under function disruptive mutation, V617F. The FRET measurements were performed in gamma2a cells by acceptor photobleaching. The results demonstrate an altered FRET efficiency in pathogenic constructs which suggests that V617F induces a conformational shift between JH2 and JH1 domains by increasing the distance between the C-terminus of JH1 and the 726-728 residues in the C-lobe of JH2 which brings closer the insertion loop of JH1 domain towards the N-terminus of JH2. Such change might underlie the hyperactivatory effect of V617F mutation and disruption of inhibitory regulation by JH2. Translating our results into precise structural mechanism will help provide better understanding of JAK-specific architectural traits in intramolecular interactions and contribute to design of more effective and selective drugs.
In this study, we apply a novel approach to studying the conformational dynamics between JH2 and JH1. Fluorescence resonance energy transfer (FRET) based on cyan fluorescent protein and new Tetra Cysteine labelling technology that relies on small biarsenical FlAsH-EDT2 dye was used to study the orientational change in JH2-JH1 domains in wild type and under function disruptive mutation, V617F. The FRET measurements were performed in gamma2a cells by acceptor photobleaching. The results demonstrate an altered FRET efficiency in pathogenic constructs which suggests that V617F induces a conformational shift between JH2 and JH1 domains by increasing the distance between the C-terminus of JH1 and the 726-728 residues in the C-lobe of JH2 which brings closer the insertion loop of JH1 domain towards the N-terminus of JH2. Such change might underlie the hyperactivatory effect of V617F mutation and disruption of inhibitory regulation by JH2. Translating our results into precise structural mechanism will help provide better understanding of JAK-specific architectural traits in intramolecular interactions and contribute to design of more effective and selective drugs.