title: ATP binding to the pseudokinase domain of JAK2 is critical for pathogenic activation.
abstract: Pseudokinases lack conserved motifs typically required for kinase activity. Nearly half of pseudokinases bind ATP, but only few retain phosphotransfer activity, leaving the functional role of nucleotide binding in most cases unknown. Janus kinases (JAKs) are nonreceptor tyrosine kinases with a tandem pseudokinase-kinase domain configuration, where the pseudokinase domain (JAK homology 2, JH2) has important regulatory functions and harbors mutations underlying hematological and immunological diseases. JH2 of JAK1, JAK2, and TYK2 all bind ATP, but the significance of this is unclear. We characterize the role of nucleotide binding in normal and pathogenic JAK signaling using comprehensive structure-based mutagenesis. Disruption of JH2 ATP binding in wild-type JAK2 has only minor effects, and in the presence of type I cytokine receptors, the mutations do not affect JAK2 activation. However, JH2 mutants devoid of ATP binding ameliorate the hyperactivation of JAK2 V617F. Disrupting ATP binding in JH2 also inhibits the hyperactivity of other pathogenic JAK2 mutants, as well as of JAK1 V658F, and prevents induction of erythrocytosis in a JAK2 V617F myeloproliferative neoplasm mouse model. Molecular dynamic simulations and thermal-shift analysis indicate that ATP binding stabilizes JH2, with a pronounced effect on the C helix region, which plays a critical role in pathogenic activation of JAK2. Taken together, our results suggest that ATP binding to JH2 serves a structural role in JAKs, which is required for aberrant activity of pathogenic JAK mutants. The inhibitory effect of abrogating JH2 ATP binding in pathogenic JAK mutants may warrant novel therapeutic approaches.
authors.affiliation: School of Medicine, University of Tampere, FI-33014 Tampere, Finland;
authors.forename: Henrik M
authors.identifier: http://orcid.org/0000-0002-8534-2530
authors.initials: HM
authors.lastname: Hammarén
authors.affiliation: School of Medicine, University of Tampere, FI-33014 Tampere, Finland;
authors.forename: Daniela
authors.initials: D
authors.lastname: Ungureanu
authors.affiliation: Department of Biomedicine, Experimental Hematology, University Hospital Basel, CH-4031 Basel, Switzerland;
authors.forename: Jean
authors.initials: J
authors.lastname: Grisouard
authors.affiliation: Department of Biomedicine, Experimental Hematology, University Hospital Basel, CH-4031 Basel, Switzerland;
authors.forename: Radek C
authors.initials: RC
authors.lastname: Skoda
authors.affiliation: Kimmel Center for Biology and Medicine at the Skirball Institute and Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016; and.
authors.forename: Stevan R
authors.initials: SR
authors.lastname: Hubbard
authors.affiliation: School of Medicine, University of Tampere, FI-33014 Tampere, Finland; Clinical Hematology, Department of Internal Medicine, Tampere University Hospital, FI-33520 Tampere, Finland olli.silvennoinen@uta.fi.
authors.forename: Olli
authors.initials: O
authors.lastname: Silvennoinen
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000235:genetics; D005260:Female; D006801:Humans; D015151:Immunoblotting; D053614:Janus Kinase 2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D056004:Molecular Dynamics Simulation; D020125:Mutation, Missense; D009196:Myeloproliferative Disorders / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017467:Receptors, Erythropoietin / Q000378:metabolism
chemical_list: D017467:Receptors, Erythropoietin; D000255:Adenosine Triphosphate; C507924:JAK2 protein, human; D053614:Janus Kinase 2
keywords: JAK; cytokine; myeloid neoplasia; nucleotide binding; pseudokinase domain
references.citation: Anal Biochem. 2013 Nov 15;442(2):213-22
references.pmid: 23891636
references.citation: Curr Opin Pharmacol. 2014 Aug;17:58-63
references.pmid: 25113945
references.citation: J Mol Biol. 2005 Sep 2;351(5):1110-22
references.pmid: 16054648
references.citation: Mol Biol Cell. 2003 Apr;14(4):1448-59
references.pmid: 12686600
references.citation: Cancer Cell. 2005 Apr;7(4):387-97
references.pmid: 15837627
references.citation: J Cell Mol Med. 2010 Mar;14(3):504-27
references.pmid: 20132407
references.citation: Proc Natl Acad Sci U S A. 2014 Jun 3;111(22):8025-30
references.pmid: 24843152
references.citation: Science. 2009 Dec 18;326(5960):1707-11
references.pmid: 19892943
references.citation: Nat Struct Mol Biol. 2014 Jul;21(7):579-84
references.pmid: 24918548
references.citation: Proc Natl Acad Sci U S A. 2005 Dec 27;102(52):18962-7
references.pmid: 16365288
references.citation: Science. 2002 Dec 6;298(5600):1912-34
references.pmid: 12471243
references.citation: Nat Struct Mol Biol. 2012 Aug;19(8):754-9
references.pmid: 22820988
references.citation: Biochemistry. 2010 Nov 23;49(46):9972-84
references.pmid: 20958061
references.citation: Cancer Discov. 2012 Jun;2(6):512-23
references.pmid: 22684457
references.citation: Nature. 2009 Oct 15;461(7266):1002-6
references.pmid: 19829382
references.citation: Mol Cell Biol. 2000 May;20(10):3387-95
references.pmid: 10779328
references.citation: Blood. 2008 Apr 15;111(8):3931-40
references.pmid: 18160670
references.citation: PLoS Biol. 2009 Jun 9;7(6):e1000126
references.pmid: 19513107
references.citation: J Mol Evol. 2002 Jun;54(6):725-33
references.pmid: 12029354
references.citation: J Biol Chem. 2008 Feb 29;283(9):5258-66
references.pmid: 18158285
references.citation: Proc Natl Acad Sci U S A. 2000 Aug 1;97(16):8991-6
references.pmid: 10908660
references.citation: Nat Struct Mol Biol. 2009 Jun;16(6):624-30
references.pmid: 19465915
references.citation: N Engl J Med. 2005 Apr 28;352(17):1779-90
references.pmid: 15858187
references.citation: Chem Biol. 2014 Apr 24;21(4):453-8
references.pmid: 24656791
references.citation: J Mol Graph. 1996 Feb;14(1):33-8, 27-8
references.pmid: 8744570
references.citation: Lancet. 2005 Mar 19-25;365(9464):1054-61
references.pmid: 15781101
references.citation: J Biol Chem. 2002 Dec 6;277(49):47954-63
references.pmid: 12351625
references.citation: Biochim Biophys Acta. 2014 Oct;1844(10):1835-41
references.pmid: 25107665
references.citation: Ther Adv Hematol. 2013 Feb;4(1):15-35
references.pmid: 23610611
references.citation: PLoS One. 2010;5(6):e11157
references.pmid: 20585391
references.citation: Nat Med. 2012 Mar;18(3):382-4
references.pmid: 22327622
references.citation: Oncogene. 2013 May 23;32(21):2601-13
references.pmid: 22869151
references.citation: Biochem J. 2014 Jan 15;457(2):323-34
references.pmid: 24107129
references.citation: Nature. 2005 Apr 28;434(7037):1144-8
references.pmid: 15793561
references.citation: Structure. 2009 Jan 14;17(1):128-38
references.pmid: 19141289
references.citation: Blood. 1997 Nov 15;90(10):3996-4003
references.pmid: 9354668
references.citation: Nat Struct Mol Biol. 2011 Sep;18(9):971-6
references.pmid: 21841788
references.citation: J Biol Chem. 2011 Jun 17;286(24):21886-95
references.pmid: 21524996
references.citation: Mol Cell Biol. 2000 Feb;20(3):947-56
references.pmid: 10629052
references.citation: Nat Struct Mol Biol. 2013 Oct;20(10):1221-3
references.pmid: 24013208