The JH2 domain and SH2-JH2 linker regulate JAK2 activity: A detailed kinetic analysis of wild type and V617F mutant kinase domains.

JAK2 tyrosine kinase regulates many cellular functions. Its activity is controlled by the pseudokinase (JH2) domain by still poorly understood mechanisms. The V617F mutation in the pseudokinase domain activates JAK2 and causes myeloproliferative neoplasms. We conducted a detailed kinetic analysis of recombinant JAK2 tyrosine kinase domain (JH1) and wild-type and V617F tandem kinase (JH1JH2) domains using peptide microarrays to define the functions of the kinase domains. The results show that i) JAK2 follows a random Bi-Bi reaction mechanism ii) JH2 domain restrains the activity of the JH1 domain by reducing the affinity for ATP and ATP competitive inhibitors iii) V617F decreases affinity for ATP but increases catalytic activity compared to wild-type and iv) the SH2-JH2 linker region participates in controlling activity by reducing the affinity for ATP.

Analysis of steady-state Förster resonance energy transfer data by avoiding pitfalls: interaction of JAK2 tyrosine kinase with N-methylanthraniloyl nucleotides.

Förster resonance energy transfer (FRET) between the fluorescent ATP analogue 2'/3'-(N-methyl-anthraniloyl)-adenosine-5'-triphosphate (MANT-ATP) and enzymes is widely used to determine affinities for ATP-protein binding. However, in analysis of FRET fluorescence data, several important parameters are often ignored, resulting in poor accuracy of the calculated dissociation constant (Kd). In this study, we systematically analyze factors that interfere with Kd determination and describe methods for correction of primary and secondary inner filter effects that extend the use of the FRET method to higher MANT nucleotide concentrations. The interactions of the fluorescent nucleotide analogues MANT-ATP, MANT-ADP [2'/3'-O-(N-methylanthraniloyl) adenosine diphosphate], and MANT-AMP [2'/3'-O-(N-methylanthraniloyl) adenosine monophosphate] with the JAK2 tyrosine kinase domain are characterized. Taking all interfering factors into consideration, we found that JAK2 binds MANT-ATP tightly with a Kd of 15 to 25nM and excluded the presence of a second binding site. The affinity for MANT-ADP is also tight with a Kd of 50 to 80nM, whereas MANT-AMP does not bind. Titrations of JAK2 JH1 with nonhydrolyzable ATP analogue MANT-ATP-γ-S [2'/3'-O-(N-methylanthraniloyl) adenosine-5'-(thio)- triphosphate] yielded a Kd of 30 to 50nM. The methods demonstrated here are applicable to other enzyme-fluorophore combinations and are expected to help improve the analysis of steady-state FRET data in MANT nucleotide binding studies and to obtain more accurate results for the affinities of nucleotide binding proteins.