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Open AccessArticle

Enzymatic Characterization of Wild-Type and Mutant Janus Kinase 1

Walter and Eliza Hall Institute, 1G Royal Parade, Parkville 3052, VIC, Australia
Department of Medical Biology, The University of Melbourne, Royal Parade, Parkville 3050, VIC, Australia
Authors to whom correspondence should be addressed.
Cancers 2019, 11(11), 1701;
Received: 4 October 2019 / Revised: 22 October 2019 / Accepted: 22 October 2019 / Published: 1 November 2019
(This article belongs to the Special Issue JAK-STAT Signalling Pathway in Cancer)
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 JAK2V617F 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 (JAK1V658F). 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. View Full-Text
Keywords: jak; jak/stat; myeloproliferative disease; v617f; jak; jak/stat; myeloproliferative disease; v617f jak; jak/stat; myeloproliferative disease; v617f; jak; jak/stat; myeloproliferative disease; v617f
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MDPI and ACS Style

Liau, N.P.D.; Laktyushin, A.; Morris, R.; Sandow, J.J.; Nicola, N.A.; Kershaw, N.J.; Babon, J.J. Enzymatic Characterization of Wild-Type and Mutant Janus Kinase 1. Cancers 2019, 11, 1701.

AMA Style

Liau NPD, Laktyushin A, Morris R, Sandow JJ, Nicola NA, Kershaw NJ, Babon JJ. Enzymatic Characterization of Wild-Type and Mutant Janus Kinase 1. Cancers. 2019; 11(11):1701.

Chicago/Turabian Style

Liau, Nicholas P.D.; Laktyushin, Artem; Morris, Rhiannon; Sandow, Jarrod J.; Nicola, Nicos A.; Kershaw, Nadia J.; Babon, Jeffrey J. 2019. "Enzymatic Characterization of Wild-Type and Mutant Janus Kinase 1" Cancers 11, no. 11: 1701.

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