JAK2

JAK2 associates with the cytoplasmic regions of numerous cytokine receptors including those for growth hormone, erythropoietin, leptin, interferon gamma, interleukin-3 and interleukin-5. JAKs contain tandem protein kinase domains: a pseudokinase domain (JH2) and a tyrosine kinase domain (JH1). In collaboration with Olli Silvennoinen's lab in Finland, we recently showed that the pseuodokinase domain of JAK2 is actually an active protein kinase, despite substitution of several residues that are conserved in canonical protein kinases. We determined a crystal structure of JAK2 JH2, which revealed that this domain adopts the eukaryotic protein kinase fold, but binds Mg-ATP more tightly than a canonical protein kinase (Fig. 1).  

Fig. 1. Crystal structure of JAK2 JH2. (a) Ribbon diagram of the structure of JH2. The N lobe of JH2 is colored light gray except for the nucleotide-binding loop (blue) and αC (yellow). The C lobe is colored dark gray except for the catalytic loop (orange) and the activation loop (green). The α-helices and β-strands are labeled (outlined letters), as are the N and C termini (N and C). ATP is shown in stick representation and colored cyan (carbon), red β7 (oxygen), blue (nitrogen) or black (phosphorus), and the Mg2+ ion is colored purple. The side chain of Val617, the site of the pathogenic mutation V617F (in the β4–β5 loop), is shown in stick representation. (b) Mode of ATP binding in JH2. The viewing angle is approximately the same as in a. Select side chains are shown, with carbon atoms colored according to the C residue’s location, as in a (for example, orange for catalytic loop). Superimposed is an electron- density map (Fo – Fc, pink mesh, contoured at 3σ) computed without Mg-ATP in the model (but present during refinement). Hydrogen bonds and salt bridges are represented by black dashed lines, and Mg2+ coordination (≤2.2 Å) is represented by green dashed lines. [Bandaranayake et al., Nat. Struct. Mol. Biol. 19, 754-759 (2012)]

 

Numerous mutations in the pseudokinase region of JAK2 result in constitutive tyrosine kinase activity and are causally linked to myeloproliferative neoplasms (MPNs) and leukemias in humans. These and other data indicate that the pseudokinase domain serves an autoinhibitory role in JAK2 regulation. Crystal structures of the individual pseudokinase (Fig. 1) and kinase domains of JAK2 have been determined previously, but the structure and organization of the tandem domains is unknown, and therefore the molecular bases for pseudokinase-mediated autoinhibition and pathogenic activation remain obscure. In collaboration with Yibing Shan at D.E. Shaw Research, we used long time-scale molecular dynamics simulations to generate a structural model for the autoinhibitory interaction between the pseudokinase and kinase domains of JAK2. A striking feature of our model, which is supported by charge-reversal mutagenesis experiments, is that nearly all of the activating disease mutations are present in the pseudokinase-kinase interface (Fig. 2). The simulations indicate that the kinase domain is stabilized in an inactive state by the pseudokinase domain, and they offer a molecular rationale for the hyperactivation of the predominant JAK2 MPN mutant, V617F.

Fig. 2. Model of JAK2 JH2-JH1 autoinhibitory interaction derived from MD simulations. Residues that cause JAK2 activation upon mutation (to the indicated residues)are shown in sphere representation (side chains) and colored pink (carbon atoms). Phosphorylated Ser523 and Tyr570 are shown in stick representation and colored according to their location. Oxygen atoms are colored red, nitrogen atoms blue, sulfur atoms yellow, and phosphorus atoms black.  [Shan et al., Nat. Struct. Mol. Biol. AOP June 11 (2014)]