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Molecular Regulation of Janus Kinases (Jaks) Focus on the Pseudokinase Domain

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Molecular Regulation of Janus Kinases (Jaks) Focus on the Pseudokinase Domain JUULI RAIVOLA Molecular Regulation of Janus Kinases (JAKs) Focus on the Pseudokinase Domain Tampere University Dissertations 366 7DPSHUH8QLYHUVLW\'LVVHUWDWLRQV -88/,5$,92/$ 0ROHFXODU5HJXODWLRQRI-DQXV.LQDVHV -$.V )RFXVRQWKH3VHXGRNLQDVH'RPDLQ $&$'(0,&',66(57$7,21 7REHSUHVHQWHGZLWKWKHSHUPLVVLRQRI WKH)DFXOW\RI0HGLFLQHDQG+HDOWK7HFKQRORJ\ RI7DPSHUH8QLYHUVLW\ IRUSXEOLFGLVFXVVLRQLQ$UYRYlpön NDWX7DPSHUH RQ-DQXDU\DWR¶FORFN $&$'(0,&',66(57$7,21 7DPSHUH8QLYHUVLW\)DFXOW\RI0HGLFLQHDQG+HDOWK7HFKQRORJ\ )LQODQG Responsible 3URIHVVRU2OOL6LOYHQQRLQHQ supervisor 7DPSHUH8QLYHUVLW\ and Custos )LQODQG Pre-examiners 3URIHVVRU-DUL<OlQQH 'RFHQW9LYHN6KDUPD 8QLYHUVLW\RI-\YlVN\Ol 8QLYHUVLW\RI+HOVLQNL )LQODQG )LQODQG Opponent 3URIHVVRU6WHIDQ1&RQVWDQWLQHVFX 8QLYHUVLWpFDWKROLTXHGH/RXYDLQ %HOJLXP 7KHRULJLQDOLW\RIWKLVWKHVLVKDVEHHQFKHFNHGXVLQJWKH7XUQLWLQ2ULJLQDOLW\&KHFN VHUYLFH &RS\ULJKWDXWKRU &RYHUGHVLJQ5RLKX,QF ,6%1 SULQW ,6%1 SGI ,661 SULQW ,661 SGI KWWSXUQIL851,6%1 3XQD0XVWD2\±<OLRSLVWRSDLQR -RHQVXX To my Family i ii TIIVISTELMÄ JAK-STAT-(vapaasti suomennettuna Janus-kinaasi - signaalinvälittäjä ja transkriptioaktivaattori) reitti välittää yli 50 sytokiinin signaaleja, jotka säätelevät solun selviytymistä, jakaantumista, migraatiota, geeniekspressiota, sekä muita elintärkeitä prosesseja kuten immuunivastetta. Siksi myös virheellisesti toimiva JAK- STAT signalointi aikaansaa vakavia seurauksia. Aktivoivat JAK-mutaatiot aiheuttavat hematologisia syöpiä sekä myeloproliferatiivisia tauteja, kun taas vajaatoimintainen JAK-signalointi voi johtaa muun muassa vakavaan immuunivajaukseen sekä autoimmuunisairauksiin. Tämä tutkimus keskittyi JAKeissa (JAK1-3 ja tyrosiinikinaasi 2, TYK2) olevaan pseudokinaasiosaan (JH2) joka ei ole kinaasiaktiivinen, kuten sitä muistuttava kinaasiosa (JH1). Biokemialliset tutkimuksemme osoittavat, että JAK pseudokinaasiosat eroavat muun muassa nukleotidin (ATP:n) sitoutumisominaisuuksien osalta. Solupohjaisten kokeiden avulla näytimme, että mutatoimalla kohdennetusti tiettyjä JH2 alueita, pystymme vaikuttamaan JAK- aktiivisuuteen. Vertailimme myös näiden mutanttien vaikutusta signalointiin, riippuen siitä mihin JAK-perheen jäseneen mutaatio kohdentuu. Havaitsimme, että yksittäisen JAKin ja sen pseudokinaasiosan rooli on keskeinen toiminnallisessa signaloinnissa, mutta se voi vaihdella riippuen reseptorikompleksista, jossa JAK kulloinkin toimii. Koska hyvälaatuista täyspitkää JAK rakennetta ei ole saatavilla, D.E. Shaw research (N.Y.) toteutti laskennallisen mallin JAK2-erytropoientin -reseptori kompleksista, jonka me yhteistyössä vahvistimme rakenneperustaisen mutaatioanalyysin avulla. Mallimme kattaa sekä aktiivisen dimeerin, että inaktiivisen monomeerisen JAK2 rakenteen. Inaktiivisessa konformaatiossa JAKin sisäisen JH2-JH1 interaktio sulkee rakenteen ja estää aktiivisten osien transfosforylaation. Aktiivisessa, aukinaisessa rakenteessa JH2-JH2 interaktio kahden JAK2 proteiinin välillä vahvistaa aktiivisen iii reseptorikompleksin muodostumista. Myös viimeaikaset tutkimukset tukevat pseudokinaasiosan osallistumista dimerisaatioon, ja sitä kautta aktivaatioon. Mallimme antaa myös teoreettista taustaa huomiollemme, jonka mukaan ATP:n sitoutuminen pseudokinaasiosaan mahdollistaa patogeenisten mutaatioiden aktivoitumisen: ATP-sitoutumiskohta on rakenteellisesti tärkeä osa pseudokinaasia, ja vaikuttaa suoraan sen rakenteeseen, sekä dynaamiseen vaihteluun aktiivisen ja inaktiivisen konformaation välillä. Edellä kuvatut tulokset lisäävät ymmärrystä niistä ominaisuuksista, jotka määrittävät moninaisten JAK-signalointireittien spesifisyyden. Lisäksi työmme valottaa mekanismeja, joilla patogeeniset JAK-mutaatiot aiheuttavat pysyvän signaloinnin aktivoitumisen, sekä tukee uusien, entistä vaikuttavampien JAK-inhibiittoreiden kehitystä. iv ABSTRACT The Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway mediates the transduction of over 50 cytokines that regulate cell survival, proliferation, migration, gene expression and other vital processes such as immune response. On the other hand, defects in the JAK-STAT signalling have severe impacts. Activating JAK (JAK1-3 and tyrosine kinase 2, TYK2) mutations cause haematological cancers and myeloproliferative disorders while impaired JAK- signalling leads to severe combined immunodeficiency (SCID) and autoimmune diseases. The results presented in this thesis focus on the JAK pseudokinase domain (JH2) that is inactive but has a crucial role in regulating the JAK activity. Our biochemical and cell-based studies show that all JAK JH2s bind ATP, but that the binding properties vary among the JAK-family. Clinical and structure-based mutation studies show that modulation of JH2 can be used to effectively alter the activity. In addition, by introducing homologous mutations into all JAK members, we observed that an individual JAK can have varying functions depending of the signaling systems it is attached to. The results highlight that each JAK within the signaling complex, and specifically JH2, is important for the signaling. As no well-defined structures exist of the full-length JAKs, a molecular dynamic (MD) simulation model of the full-length JAK2 with erythropoietin receptor was constructed in collaboration with D.E. Shaw research (N.Y.). The model depicts JAK2 in different states of activation and is supported by structure-based mutation analysis. In the inactive, monomeric conformation the interaction between JH2 and the kinase domain (JH1) in part closes the JAK2 structure and hinders the transphosphorylation of the active kinase domains. The active conformation is a more open, dimeric structure formed partly via JH2-JH2 interactions between the opposing JAK2 proteins. The model also gives theoretical background to our observation that ATP-binding to JH2 is crucial for the pathogenic JAK activation. It implies that the ATP-binding site is structurally important region within the pseudokinase that directly affects to the dynamic shifting between the active and inactive JH2 conformations. v The results summarized above allow us to better comprehend the characteristics that dictate the specificity among JAK-signaling pathways. Moreover, they bring insight into the mechanism of pathologic JAK activation and support the development of novel, more potent JAK inhibitors. vi CONTENTS 1 Introduction .................................................................................................................... 13 2 Review of the Literature ................................................................................................ 15 2.1 Eukaryotic Protein kinases and cytokine signalling ......................................... 15 2.1.1 The eukaryotic protein kinase family .............................................. 15 2.1.2 Structure of a canonical kinase ........................................................ 16 2.1.3 Pseudokinases ................................................................................... 18 2.2 JAK-STAT signalling ......................................................................................... 21 2.2.1 Cytokines and their receptors .......................................................... 21 2.2.2 The JAK-STAT signalling pathway ................................................ 27 2.2.3 Structure and function of the JAK domains .................................. 28 2.2.3.1 FERM-SH2 ..................................................................... 29 2.2.3.2 JH1-JH2 ........................................................................... 30 2.2.3.3 ATP binding and activity of JAK JH2 .......................... 31 2.2.4 STATs ................................................................................................ 32 2.2.5 Regulation of the JAK-STAT signalling ......................................... 33 2.2.5.1 Protein phosphatases ...................................................... 33 2.2.5.2 SOCS................................................................................ 34 2.2.5.3 Other regulatory proteins ............................................... 35 2.3 Disease-driving mutations in the JAK-STAT pathway ................................... 35 2.3.1 JAK loss of function (LOF) mutations .......................................... 36 2.3.2 JAK gain of function mutations (GOFs) ....................................... 39 2.4 Inhibitors for JAK-signalling............................................................................. 41 3 Aims of the study ........................................................................................................... 46 4 Materials and methods ................................................................................................... 47 4.1 Plasmid constructs, cloning, and site-directed mutagenesis ........................... 47 4.1.1 Mammalian expression constructs (I–IV) ...................................... 47 4.1.2 Expression constructs for recombinant protein production in insect cells ................................................................. 47 4.1.3 Homology modelling of JAK3 JH2 ................................................ 48 4.1.4 Site-directed mutagenesis ................................................................. 48 4.2 Mammalian cell culture, transfection, and cytokine stimulation .................... 50 4.3 Luciferase reporter assay to decipher JAK downstream signaling ................. 51 4.4 SDS-PAGE and immunoblotting ..................................................................... 51 4.5 Protein expression and
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