Enzymatic characterization of wild-type and mutant janus kinase 1
Details
Publication Year 2019-11-01, Volume 11, Issue #11, Page pii: E1701
Journal Title
Cancers (Basel)
Publication Type
Journal Article
Abstract
Janus kinases (JAKs) are found constitutively associated with cytokine receptors and are present in an inactive state prior to cytokine exposure. Activating mutations of JAKs are causative for a number of leukemias, lymphomas, and myeloproliferative diseases. In particular, the JAK2(V617F) mutant is found in most human cases of polycythemia vera, a disease characterized by over-production of erythrocytes. The V617F mutation is found in the pseudokinase domain of JAK2 and it leads to cytokine-independent activation of the kinase, as does the orthologous mutation in other JAK-family members. The mechanism whereby this mutation hyperactivates these kinases is not well understood, primarily due to the fact that the full-length JAK proteins are difficult to produce for structural and kinetic studies. Here we have overcome this limitation to perform a series of enzymatic analyses on full-length JAK1 and its constitutively active mutant form (JAK1(V658F)). Consistent with previous studies, we show that the presence of the pseudokinase domain leads to a dramatic decrease in enzymatic activity with no further decrease from the presence of the FERM or SH2 domains. However, we find that the mutant kinase, in vitro, is indistinguishable from the wild-type enzyme in every measurable parameter tested: KM (ATP), KM (substrate), kcat, receptor binding, thermal stability, activation rate, dephosphorylation rate, and inhibitor affinity. These results show that the V658F mutation does not enhance the intrinsic enzymatic activity of JAK. Rather this data is more consistent with a model in which there are cellular processes and interactions that prevent JAK from being activated in the absence of cytokine and it is these constraints that are affected by disease-causing mutations.
Publisher
MDPI
WEHI Research Division(s)
Blood Cells And Blood Cancer; Advanced Technology And Biology; Structural Biology
PubMed ID
31683831
Open Access at Publisher's Site
https://doi.org/10.3390/cancers11111701
NHMRC Grants
NHMRC/1113577 NHMRC/1121755 NHMRC/1122999
Rights Notice
Refer to copyright notice on published article.


Creation Date: 2019-11-20 02:45:22
Last Modified: 2019-11-21 10:01:28
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