Enzymatic Characterization of Wild-Type and Mutant Janus Kinase 1.

Nicholas P D Liau,Nicos A Nicola,Rhiannon Morris,Jeffrey J Babon,Jarrod J Sandow,Artem Laktyushin,Nadia J Kershaw

doi:10.3390/cancers11111701

Nicholas P D Liau, Nicos A Nicola + Show 5 more

Open Access

https://doi.org/10.3390/cancers11111701

Copy DOI

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 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.

Highlights

Cytokines that signal through the Janus kinases (JAKs)/STAT pathway are important modulators of hematopoiesis, inflammation, and the immune response [1,2]
STATs are in turn activated by JAK-mediated phosphorylation and dissociate from the receptor and migrate into the nucleus to initiate the activators of transcription (STATs)
After an extensive screening of different constructs, expression conditions, and purification protocols, we developed a protocol capable of producing JAK1 at the quantities and purity required for enzymatic analysis

Summary

Introduction

Cytokines that signal through the JAK/STAT pathway are important modulators of hematopoiesis, inflammation, and the immune response [1,2]. Cytokines are small secreted proteins that bind to the extracellular domains of specific receptors on the surface of target cells to initiate a cytokine-specific transcriptional program. Cytokine receptors lack intrinsic kinase domains and so rely on the constitutively associated Janus kinases (JAKs) to phosphorylate downstream substrates and initiate the signaling cascade [3]. JAKs phosphorylate tyrosine residues on the cytoplasmic domains of the receptors to which they are attached, and these newly phosphorylated sites recruit downstream effector proteins and transcription factors, the most important of which are the signal transducers and Cancers 2019, 11, 1701; doi:10.3390/cancers11111701 www.mdpi.com/journal/cancers. STATs are in turn activated by JAK-mediated phosphorylation and dissociate from the receptor and migrate into the nucleus to initiate the activators of transcription (STATs). STATs are in turn activated by JAK-mediated phosphorylation cytokine-induced transcriptional program [4]

Methods

Results

Discussion

Conclusion