TyrosineKinaseJAK_2
Tyrosine Kinase Jak2 (4FVQ) from Homo Sapiens

Created By: Jacob Merlin

Jak 2 (PDB ID: 4FVQ) is a tyrosine kinase protein found in Homo Sapiens and is a member of the Janus family of protein tyrosine kinases (JAK1-JAK3, Tyk2).  The four kinases of the Janus family mediate essential functions in cytokine signaling (1).  Protein tyrosine kinases function by transferring a phosphate group from ATP to a tyrosine residue on a protein substrate affecting the substrate’s activation state in various signal transduction pathways (2).  Jak 2 associates with the cytoplasmic regions of cytokine receptors and is important for signaling for a variety of hormones, including growth hormone, leptin, IL-3 and IL-5 (3).    

Jak2 is 298 residues long.  The isoelectric point and molecular weight of Jak2 were calculated using the ExPASy Proteomics Server. The isoelectric point was found to occur at a pH of 6.77 and the molecular weight was calculated to be 33,070.94 daltons (4).  The ligands associated with Jak2 are ATP, a magnesium ion, and acetate ions.  The acetate ions were used to induce crystallization.  

Jak 2 possesses a pseudokinase domain (JH2) and a tyrosine kinase domain (JH1). Corresponding to its function in cytokine signaling, Jak2 contains an ATP binding site in the cleft between its two lobes (5).  Binding of Mg2+-ATP in the cleft displaces a water molecule that bridges an oxygen atom on Phe-594 and a nitrogen atom on Ala-598 and activates the protein (5).  Asn-678 coordinates the magnesium ion in the activation loop (5).  The structural stability of the JH2 domain is in part due to higher affinity binding cuaused by interactions with Mg-ATP (5,6). 

The apo structure of Jak2 (PDB ID: 4FVP) exhibits a closed lobe configuration.  Arg-715 mediates a C lobe- N lobe contact that partially accounts for the closed loop configuration in the apo and ATP-bound structure (5).  An intercalating water molecule disrupts alpha helix C in the apo form.   This intercalating water molecule causes the irregular backbone hydrogen bonding in alpha helix C (5).
     
The secondary structure of Jak2 is 40% helical with 17 helices and is 19% beta sheets with 12 strands (7).  The remaining 41% of the structure consists of other secondary structures including random coils and 3/10 helices.  The JH2 structure includes a short activation loop.   Gly-672 accommodates the shorter activation loop of JH2 because of its smaller size compared the arginine typically found there (5).  The presence of Gly-672 in the catalytic loop is partially responsible for JH2 being intrinsically flexible and more stable when compared to other protein kinases (5).
            
The pseudokinase domain JH2 was previously considered to be inactive, but recently, JH2 has been found to have catalytic activity and regulate the activity of JH1 (5).  Ser-523 and Tyr-570 are two negative regulatory sites phosphorylated by JH2 that help preserve the low basal activity of Jak 2 (6,8,9).  Other JAKs do not conserve these phosphorylation sites and may not posses these negative regulatory mechanisms.  JH2 lacks several residues that are important for catalysis.  An asparagine (Asn-673) ) replaces an aspartate in the catalytic loops and assists in a phosphoryl transfer reaction, while also contributing to activation of Jak2 (5).  The activation loop of JH2 begins with an Asp-Pro-Gly sequence, whereas the activation loop of canonical protein kinases begins with an Asp-Phe-Gly motif (5).
        
Mutations in human JAK genes cause myeloproliferative neoplasms (MPNs) (10).  Most often these mutations are in the JH2 region (10,11).  Mutations in the JH2 region have also been linked to leukemias (11).  The Jak2 pseudokinase domain mutant V617F (PDB ID: 4FVR) is the most commonly identified mutation in MPNs and has been associated with non-small cell lung cancer (12).   The mutation V617F stabilizes the JH2 stimulatory interaction required for Jak2 activation by rigidifying an alpha helix in the N lobe of JH2 (5).

In contrast to the wild type JH2, the V617F mutant exhibits structural deviations in the alpha helix C.  The V617F mutant is made larger by an additional turn and exhibits continuous backbone hydrogen bonding (5).  Phe-617 replaces valine in the mutant V617F and causes a rotation in the phenyl ring at Phe-595 and a shift in the side chain position of Phe-594 (5).  The deviation of Phe-594 in the catalytic site changes the side chain position of Lys-581.  This side chain deviation might explain why the catalytic activity is impaired in the mutant V617F (6).  

A Basic Local Alignment Search Tool (BLAST) database search was performed using the amino acid sequence of the JAK2 pseudokinase domain to find proteins with similar primary structure.  The BLAST search results in an E value which is determined by comparing the sequence of a subject protein to other proteins and assigning gaps where amino acids exist in the subject protein but not in the query proteins.  An E value below 0.5 indicates high similarity between proteins.  An E value of 2 x 10-96 indicates that proto-oncogene tyrosine-protein kinase Fes (PDB ID: 3CBL) has a similar primary structure to Jak2 (13).     

The Dali server was used to find proteins with comparable three-dimensional structure to the Jak2 pseudokinase domain.  The Dali server assigns a Z-score by comparing tertiary structures of proteins and calculating the differences in intramolecular distances.  A Z score of 2 or higher means the proteins have similar folds.  A Z score of 29.8 indicates that the ephrin type-B receptor 2 from mus musculus (PDB ID: 2HEN) has a similar three-dimensional structure to Jak2 (14).  Both molecules display increased inter-lobe flexibility and some structural plasticity that may account for their enhanced catalytic function (5,15).  Both molecules have a magnesium ion that binds centrally as a ligand and both have a form of intracellecular energy transfer.  ATP binds centrally on Jak2, whereas ADP binds centrally on the ephrin type-B receptor 2.  These ligand bindings are important for both molecules functions of mediating processes and signal transduction.  Ephrin type-B receptor 2 is differentiated because of a kink in the N-lobe alpha helix C not previously observed in active state protein kinase structures (15).  This kink is proposed to account for some of the repressed catalytic function of Eph receptors in their auto-inhibited states (15).