Janus kinase-2 (JAK2) mediates signaling by various cytokines, including erythropoietin and growth hormone. JAK2 possesses tandem pseudokinase and tyrosine kinase domains. Mutations in the pseudokinase domain are causally linked to myeloproliferative neoplasms (MPNs) in humans. The structure of the JAK2 tandem kinase domains is unknown, and therefore the molecular bases for pseudokinase-mediated autoinhibition and pathogenic activation remain obscure. Using unbiased molecular dynamics simulations of protein-protein docking, we produced a structural model for the autoinhibitory interaction between the JAK2 pseudokinase and kinase domains. A striking feature of our model, which is supported by mutagenesis experiments, is that nearly all of the disease mutations map to the domain interface. 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 hyperactivity of V617F, the predominant JAK2 MPN mutation.

Genetics play a significant role in venous thromboembolism (VTE), yet current clinical laboratory-based testing identifies a known heritable thrombophilia (factor V Leiden, prothrombin gene mutation G20210A, or a deficiency of protein C, protein S, or antithrombin) in only a minority of VTE patients. We hypothesized that a substantial number of VTE patients could have lesser-known thrombophilia mutations. To test this hypothesis, we performed whole-exome sequencing (WES) in 64 patients with VTE, focusing our analysis on a novel 55-gene extended thrombophilia panel that we compiled. Our extended thrombophilia panel identified a probable disease-causing genetic variant or variant of unknown significance in 39 of 64 study patients (60.9%), compared with 6 of 237 control patients without VTE (2.5%) ( < .0001). Clinical laboratory-based thrombophilia testing identified a heritable thrombophilia in only 14 of 54 study patients (25.9%). The majority of WES variants were either associated with thrombosis based on prior reports in the literature or predicted to affect protein structure based on protein modeling performed as part of this study. Variants were found in major thrombophilia genes, various genes, and highly conserved areas of other genes with established or potential roles in coagulation or fibrinolysis. Ten patients (15.6%) had>1 variant. Sanger sequencing performed in family members of 4 study patients with and without VTE showed generally concordant results with thrombotic history. WES and extended thrombophilia testing are promising tools for improving our understanding of VTE pathogenesis and identifying inherited thrombophilias.

MuSK (muscle-specific kinase) is a receptor tyrosine kinase that plays a central signaling role in formation of neuromuscular junctions (NMJs). MuSK is activated in a complex spatio-temporal manner to cluster acetylcholine receptors on the postsynaptic (muscle) side of the synapse and to induce differentiation of the nerve terminal on the presynaptic side. The ligand for MuSK is LRP4 (low-density lipoprotein receptor-related protein-4), a transmembrane protein in muscle, whose binding affinity for MuSK is potentiated by agrin, a neuronally derived heparan-sulfate proteoglycan. In addition, Dok7, a cytoplasmic adaptor protein, is also required for MuSK activation in vivo. This review focuses on the physical interplay between these proteins and MuSK for activation and downstream signaling, which culminates in NMJ formation.

Somatic mutations in the Jak2 allele that lead to constitutive kinase activation of the protein have been identified in human disease conditions such as the myeloproliferative neoplasms (MPNs). The most common mutation in these patients is a V617F substitution mutation, which is believed to play a causative role in the MPN pathogenesis. As such, identifying the molecular basis for the constitutive activation of Jak2-V617F is important for understanding its clinical implications and potential treatment. Here, we hypothesized that conversion of residue 617 from Val to Phe resulted in the formation of novel π stacking interactions with neighboring Phe residues. To test this, we first examined the Jak2 structure via molecular modeling and identified a potential π stacking interaction between F594, F595 and F617. Disruption of this interaction through site directed mutagenesis impaired Jak2 auto-phosphorylation, Jak2 dependent gene transcription and in vitro kinase activity of the Jak2-V617F protein. Further, substitution of F594 and F595 with Trp did not affect Jak2 function significantly, but replacement with charged residues did, showing the conservation of aromaticity and hydropathy index at these positions. Using molecular dynamics (MD) simulations, we found that the π stacking interaction between residues 595 and 617 in the Jak2-V617F protein was of much greater energy and conformed to the properties of π stacking, relative to the Jak2-WT or Jak2-V617F/F594A/F595A. In summary, we have identified a π stacking interaction between F595 and F617 that is specific to and is critical for the constitutive activation of Jak2-V617F. KeywordsJanus kinase 2 (Jak2); Pi stacking; Constitutive Activation Jak2 is a non-receptor tyrosine kinase belonging to the Janus (Jak) family of tyrosine kinases. Other family members include Jak1, Jak3 and Tyk2. Jak2 is essential for life as mice that are devoid of a functional Jak2 allele die during embryonic development due to a lack of definitive erythropoiesis (1,2). As such, it plays a critical role in a number of * To whom correspondence should be addressed: Dr. Peter P. Sayeski, Department of Physiology and Functional Genomics, P.O. Box 100274, University of Florida College of Medicine, Gainesville, FL 32610, Tel: 352-392-1816, Fax: 352-846-0270, psayeski@ufl.edu. † This work was supported by National Institutes of Health Award R01-HL67277, an American Heart Association Florida/Puerto Rico Affiliate Grant in Aid (#0855361E), and a University of Florida/Moffitt Cancer Center Cooperative Funding Initiative Award.Supporting Information Available: Movies corresponding to the quoted simulations are available as supporting information. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 November 23. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript cytokine-dependent signaling processes including those actions that are mediated by the er...