Mechanistic insights into activation and SOCS3-mediated inhibition of myeloproliferative neoplasm-associated JAK2 mutants from biochemical and structural analyses

JAK2 (Janus kinase 2) initiates the intracellular signalling cascade downstream of cell surface receptor activation by cognate haematopoietic cytokines, including erythropoietin and thrombopoietin. The pseudokinase (JH2) domain of JAK2 negatively regulates the catalytic activity of the adjacent tyrosine kinase (JH1) domain and mutations within the pseudokinase domain underlie human myeloproliferative neoplasms, including polycythaemia vera and essential thrombocytosis. To date, the mechanism of JH2-mediated inhibition of JH1 kinase activation as well as the susceptibility of pathological mutant JAK2 to inhibition by the physiological negative regulator, SOCS3, have remained unclear. Here, using recombinant, purified JAK2JH1-JH2 proteins, we demonstrate that, when activated, wild-type and myeloproliferative neoplasm-associated mutants of JAK2 exhibit comparable enzymatic activity and inhibition by SOCS3 in in vitro kinase assays. Small angle X-ray scattering (SAXS) showed that JAK2JH1-JH2 exists in an elongated configuration in solution with no evidence for interaction between JH1 and JH2 domains in cis. Collectively, these data are consistent with a model in which JAK2’s pseudokinase domain binds and inhibits the activation of the tyrosine kinase domain of a neighbouring JAK2 molecule within a cytokine receptor complex, but does not influence the activity of JAK2 once it has been activated. Our data indicate that, in the absence of the N-terminal FERM domain and thus c...

Source: BJ Signal - December 19, 2013 Category: Biochemistry Authors: L N Varghese, D Ungureanu, N P.D. Liau, S N Young, A Laktyushin, H Hammarén, I S. Lucet, N A Nicola, O Silvennoinen, J J Babon, J M Murphy Tags: BJ Signal Source Type: research