title: A shift in the salt bridge interaction of residues D620 and E621 mediates the constitutive activation of Jak2-H538Q/K539L.
abstract: Jak2 mutations in the exon 14 and exon 12 regions that cause constitutive activation have been associated with myeloproliferative neoplasms. We have previously shown that a pi stacking interaction between F617 and F595 is important for the constitutive activation of Jak2-V617F (Gnanasambandan et al., Biochemistry 49:9972-9984, 2010). Here, using a combination of molecular dynamics (MD) simulations and in vitro mutagenesis, we studied the molecular mechanism for the constitutive activation of the Jak2 exon 12 mutation, H538Q/K539L. The activation levels of Jak2-H538Q/K539L were found to be similar to that of Jak2-V617F, and Jak2-H538Q/K539L/V617F. Data from MD simulations indicated a shift in the salt bridge interactions of D620 and E621 with K539 in Jak2-WT to R541 in Jak2-H538Q/K539L. When compared to Jak2-WT, K539A mutation resulted in increased activation, while K539D or K539E mutations diminished Jak2 activation by 50 %. In the context of Jak2-H538Q/K539L, R541A mutation reduced its activation by 50 %, while R541D and R541E mutations returned its activation levels to that of Jak2-WT. Collectively, these results indicate that a shift in the salt bridge interaction of D620 and E621 with K539 in Jak2-WT to R541 in Jak2-H538Q/K539L is critical for constitutive activation of this Jak2 exon 12 mutant.
authors.affiliation: Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors.forename: Kavitha
authors.initials: K
authors.lastname: Gnanasambandan
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Magis
authors.forename: Peter P
authors.initials: PP
authors.lastname: Sayeski
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry*; D019556:COS Cells; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D018698:Glutamic Acid / Q000737:chemistry*; D006801:Humans; D053614:Janus Kinase 2 / Q000737:chemistry* / Q000235:genetics; D049409:Luciferases, Firefly / Q000096:biosynthesis / Q000235:genetics; D008239:Lysine / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D015398:Signal Transduction; D013816:Thermodynamics
chemical_list: D001224:Aspartic Acid; D018698:Glutamic Acid; D001120:Arginine; D049409:Luciferases, Firefly; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D008239:Lysine