Janus Kinase 1 (PDB ID: 3EHY)
created by Christina Choi
The Janus kinases (JAK1, JAK2, JAK3, and TYK2), a family of nonreceptor protein kinases, catalyze the ATP-dependent phosphorylation of tyrosine residues on its substrates and participate in various cytokine signaling and growth factor signaling pathways, regulating the immune system and oncogenesis. The JAKs share seven homologous domains, JH7 to JH1 from N terminus to C terminus respectively. The JH1 domain contains the protein tyrosine kinase (PTK) domain, which is catalytic and crucial to protein function (1). While structurally similar to JH1, the JH2 domain, known as the pseudokinase domain, lacks catalytic activity and is involved in regulating JH1. The JH3 and JH4 domains stabilize protein conformation (2) while JH5, JH6, and JH7 compose the FERM domain, which participates in receptor protein binding and seemingly in JAK regulation (Figure 1).
The JAK proteins participate in the JAK-STAT (Signal Transducer and Activator of Transcription) pathway, in which a cytokine from outside the cell binds to its JAK-bound receptors (1); the receptors dimerize, and the JAK proteins are phosphorylated at its serine residue in its active site by the binding of ATP, becoming catalytically active. The JAK proteins then transfer the phosphate groups to tyrosine residues of STAT proteins, which dimerize and bind to DNA, activating gene transcription in the nucleus (1, Figure 2)
JAK1 (PDB ID= 3EHY) noncovalently associates with cytokine receptors; according to Williams et al (2), it “plays an essential role in types I and II interferon signaling and elicits signals from the interleukin-2, interleukin-4, gp130 and class II receptor families”, controlling lymphoid cell proliferation. The PTK domain of JAK1 has an amino acid sequence of 290 amino acids partitioned into two lobes, an N-terminal and a larger C-terminal lobe, with 15 turns, 11 beta strands, 7 3/10-helices, 2 beta bridges, and 9 alpha helices. The substrate-biding cleft, the active site in which ATP associates, is between the N-terminal and C-terminal lobes and is largely hydrophobic to facilitate adenine binding. Behind the cleft resides a polar pocket that coordinates magnesium ions and accommodates ribose and the triphosphates of ATP (2). The functional domain consists of a glycine loop, hinge region, catalytic loop, and activation loop. In its active state, the activation loop folds forward and is expelled from the active site, allowing ATP to enter the cleft and phosphorylate tyrosine residues 1034 and 1035(2).
The PTK domain of JAK1 is similar to that of JAK2 and JAK3, and the substrate-binding site is highly conserved. JAK1 and JAK2 have a sequence similarity of 53% while JAK1 and JAK3 have a similarity of 50% (2). The N-terminal and C-terminal lobes of JAK1 were found to be further apart than in JAK2 by measuring the angle between the two domains, which creates a more open conformation; in order to completely superimpose the structures, the C-terminal of JAK1 must undergo a 3.3o rotation. The substrate-binding site of JAK2 is more positively charged with residues Lys-1053, Lys-857, Ser-1054, Glu-1015, and Gln-854, and whereas JAK1 residues Asp-1042, Asp-880, Asp-1081, Ser-1080, and Glu-883, contribute to its greater electronegativity (2).
Inhibitors 2-tbutyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-one and (3R,4R)-3-[4-methyl-3-[N-methyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-1-yl]-3-oxopropionitrile, in short CMP6 and CP-690,550 respectively, bind competitively in the substrate-binding cleft by structural complementarity with JAK1 PTK as well as JAK2 PTK. Structurally, CMP6 and CP-690,550 are similar with an r.m.s.d. of 0.18 Å for 271 carbon atoms. CMP6, when bound to the cleft of JAK1, orients its pyridone moiety into the polar pocket, which interacts via Van Der Waals with “Leu-881, Val-889, Ala-906 and Val-938 of the N-terminal lobe, Met-956 and Phe-958 of the hinge and Gly-962 and Leu-1010 of the C-terminal lobe” according to Williams et al (2), while its imidazole ring forms hydrogen bonds with water. CMP6 does not associate with the glycine loop, which instead coordinates with the substrate-binding cleft. The IC50 values for JAK1-CMP6 is 20nM and 5nM for JAK2-CMP6; CMP6 has a higher affinity for JAK2 and creates a more “closed” structure in comparison to JAK1 (2).
CP-690,550, when bound, faces its pyrimidine ring in the direction of the hinge region and the piperidine ring is placed in the polar pocket, its methyl moiety on the side of the C-terminal lobe. It has a high structural complementarity with JAK1, and two hydrogen bonds are formed between the hinge region and the pyrrolopyrimidine rings of CP-690,550. As opposed to CMP6, CP-690,550 associates with the glycine loop, and its nitrileN24 hydrogen bonds with Gly-882, Gly-884, Gly-887 in the loop. Van der Waals interactions between JAK1 PTK and CP-690,550 impart stability in the inhibitory complex, and its pyrrolopyrimidine and piperidine rings associate with the hydrophobic Leu-881, Val-889, Ala-906, Val-938 residues of the N-terminal lobe , Leu-1010 of the C-terminal lobe, and Met-956, Phe-958, Leu-959 of the hinge region. Cells with the JAK2 V617F mutation are inhibited by CP-690,550, which binds in a similar fashion as JAK1. (2).
Point mutations of Phe-958 and Pro-960 in the hinge region of the JAK1 PTK domain results in resistance to inhibitor complexes, resulting in constitutive kinase activity and impairing the production of B-Cells and T-Cells. Patients with these point mutations were diagnosed with acute myeloid leukemia and T-cell acute lymphoblastic leukemia (3). As for JAK2, point mutation V617K results in resistance to inhibitors, hypersensitivity to cytokines, and a variety of myoproliferative diseases, such as chronic myelofibrosis and thrombocythemia. (4).