Enzymatic Characterization of Wild-Type and Mutant Janus Kinase 1
Total Page：16
File Type：pdf, Size：1020Kb
cancers Article Enzymatic Characterization of Wild-Type and Mutant Janus Kinase 1 Nicholas P. D. Liau 1,2,* , Artem Laktyushin 1,2, Rhiannon Morris 1,2 , Jarrod J. Sandow 1,2, Nicos A. Nicola 1,2 , Nadia J. Kershaw 1,2 and Jeffrey J. Babon 1,2,* 1 Walter and Eliza Hall Institute, 1G Royal Parade, Parkville 3052, VIC, Australia; [email protected] (A.L.); [email protected] (R.M.); [email protected] (J.J.S.); [email protected] (N.A.N.); [email protected] (N.J.K.) 2 Department of Medical Biology, The University of Melbourne, Royal Parade, Parkville 3050, VIC, Australia * Correspondence: [email protected] (N.P.D.L.); [email protected] (J.J.B.); Tel.: +61-3-93452960 (N.P.D.L.); Fax: +61-3-93470852 (J.J.B.) Received: 4 October 2019; Accepted: 22 October 2019; Published: 1 November 2019 Abstract: Janus kinases (JAKs) are found constitutively associated with cytokine receptors and are present in an inactive state prior to cytokine exposure. Activating mutations of JAKs are causative for a number of leukemias, lymphomas, and myeloproliferative diseases. In particular, the JAK2V617F mutant is found in most human cases of polycythemia vera, a disease characterized by over-production of erythrocytes. The V617F mutation is found in the pseudokinase domain of JAK2 and it leads to cytokine-independent activation of the kinase, as does the orthologous mutation in other JAK-family members. The mechanism whereby this mutation hyperactivates these kinases is not well understood, primarily due to the fact that the full-length JAK proteins are difficult to produce for structural and kinetic studies. Here we have overcome this limitation to perform a series of enzymatic analyses on full-length JAK1 and its constitutively active mutant form (JAK1V658F). Consistent with previous studies, we show that the presence of the pseudokinase domain leads to a dramatic decrease in enzymatic activity with no further decrease from the presence of the FERM or SH2 domains. However, we find that the mutant kinase, in vitro, is indistinguishable from the wild-type enzyme in every measurable parameter tested: KM (ATP), KM (substrate), kcat, receptor binding, thermal stability, activation rate, dephosphorylation rate, and inhibitor affinity. These results show that the V658F mutation does not enhance the intrinsic enzymatic activity of JAK. Rather this data is more consistent with a model in which there are cellular processes and interactions that prevent JAK from being activated in the absence of cytokine and it is these constraints that are affected by disease-causing mutations. Keywords: jak; jak/stat; myeloproliferative disease; v617f; jak; jak/stat; myeloproliferative disease; v617f 1. Introduction Cytokines that signal through the JAK/STAT pathway are important modulators of hematopoiesis, inflammation, and the immune response [1,2]. Cytokines are small secreted proteins that bind to the extracellular domains of specific receptors on the surface of target cells to initiate a cytokine-specific transcriptional program. Cytokine receptors lack intrinsic kinase domains and so rely on the constitutively associated Janus kinases (JAKs) to phosphorylate downstream substrates and initiate the signaling cascade [3]. JAKs phosphorylate tyrosine residues on the cytoplasmic domains of the receptors to which they are attached, and these newly phosphorylated sites recruit downstream effector proteins and transcription factors, the most important of which are the signal transducers and Cancers 2019, 11, 1701; doi:10.3390/cancers11111701 www.mdpi.com/journal/cancers Cancers 2019, 11, x 2 of 15 Cancers 2019, 11, 1701 2 of 16 transducers and activators of transcription (STATs). STATs are in turn activated by JAK-mediated phosphorylation and then dissociate from the receptor and migrate into the nucleus to initiate the activators of transcription (STATs). STATs are in turn activated by JAK-mediated phosphorylation cytokine-induced transcriptional program [4]. and then dissociate from the receptor and migrate into the nucleus to initiate the cytokine-induced There are four JAK family members—JAK1, JAK2, JAK3, and TYK2, which all share the same transcriptional program [4]. basic domain architecture (see Figure 1A), in addition to 23% overall sequence identity and 48% There are four JAK family members—JAK1, JAK2, JAK3, and TYK2, which all share the same overall similarity [5]. JAK2 is known to be important for hematopoiesis, as JAK2 knockout mice die basic domain architecture (see Figure1A), in addition to 23% overall sequence identity and 48% overall before birth as a lack of erythrocytes [6,7]. JAK1, JAK3, and TYK2 are known to be important for similarity [5]. JAK2 is known to be important for hematopoiesis, as JAK2 knockout mice die before development of the immune system, though a JAK1 knockout results in a more severe phenotype birth as a lack of erythrocytes [6,7]. JAK1, JAK3, and TYK2 are known to be important for development than JAK3 or TYK2, suggesting that JAKs are at least partially redundant for some functions [8–11]. of the immune system, though a JAK1 knockout results in a more severe phenotype than JAK3 or The N-terminal half of the protein contains a FERM-SH2 module which anchors the protein to the TYK2, suggesting that JAKs are at least partially redundant for some functions [8–11]. The N-terminal intracellular Box1 and Box2 motifs of a cytokine receptor [12,13]. The C-terminal half consists of two half of the protein contains a FERM-SH2 module which anchors the protein to the intracellular Box1 kinase domains, the first of which is catalytically inactive (termed the JH2 or pseudokinase domain) and Box2 motifs of a cytokine receptor [12,13]. The C-terminal half consists of two kinase domains, the whilst the second is catalytically active (the JH1 or kinase domain) and phosphorylates downstream first of which is catalytically inactive (termed the JH2 or pseudokinase domain) whilst the second is substrates. catalytically active (the JH1 or kinase domain) and phosphorylates downstream substrates. Figure 1. Purification of JAK1 constructs. (A), Schematic representation of the JAK1 domains used Figure 1. Purification of JAK1 constructs. (A), Schematic representation of the JAK1 domains used for for expression. The internal domain boundaries are numbered on the full-length construct, whilst expression. The internal domain boundaries are numbered on the full-length construct, whilst N and N and C terminal boundaries are numbered at each end. (B), Purified JAK1 constructs shown on C terminal boundaries are numbered at each end. (B), Purified JAK1 constructs shown on SDS-PAGE SDS-PAGE gel stained with Coomassie blue. KD = kinase domain, FL WT = full length wild type JAK1, gel stained with Coomassie blue. KD = kinase domain, FL WT = full length wild type JAK1, FL V658F FL V658F = full length JAK1V658F, PK-KD WT = pseudokinase-kinase domain wild type, PK-KD V658F = full length JAK1V658F, PK-KD WT = pseudokinase-kinase domain wild type, PK-KD V658F = = pseudokinase-kinase domain V658F, FERM-SH2 = FERM-SH2 domain. pseudokinase-kinase domain V658F, FERM-SH2 = FERM-SH2 domain. JAKs are found to be associated with receptors but in an inactive state prior to cytokine exposure. TheirJAKs kinase are domainsfound to be contain associated a structural with receptors feature knownbut in an as inactive the activation state prior loop, to whichcytokine contains exposure. two Theirconsecutive kinase domains tyrosine contain residues. a structural When the feature first of known these tyrosines as the activation is unphosphorylated loop, which contains the kinase two is consecutiveinactive. Phosphorylation tyrosine residues. of this When tyrosine the leadsfirst of to th re-orientationese tyrosines of is the unphosphorylated activation loop and the activates kinase theis inactive.domain. Phosphorylation Phosphorylation of of this tyrosinetyrosine isleads catalyzed to re-orientation in trans by theof the kinase activation domain loop of the and neighboring activates theJAK domain. molecule Phosphorylation attached to the otherof this chain tyrosine of the is cytokine catalyzed receptor in trans [3]. by This the auto-activation kinase domain occurs of the in neighboringresponse to cytokineJAK molecule engaging attached the extracellular to the other domain chain of the of receptorthe cytokine and inducing receptor either [3]. dimerizationThis auto- activation(or multimerization) occurs in response [14,15] or reorientationto cytokine engagi [16]. Theng pseudokinasethe extracellular domain domain plays of an the important receptor part and in inducingregulating either the activation dimerization process (or sincemultimerization) its removal results [14,15] in or constitutive reorientation partial [16]. activation The pseudokinase of the kinase domainas well asplays a failure an important to fully-activate part in in regulating the presence the of activation cytokine [process17]. The since importance its removal of this results domain in is constitutivehighlighted partial by the activation fact that the of majoritythe kinase of as activating well as a (and failure therefore to fully-activate disease-causing) in the mutationspresence of of cytokineJAKs are [17]. found The in importance this domain. of this domain is highlighted by the fact that the majority of activating (and thereforeThe most disease-causing) common disease-causing mutations JAK of mutationJAKs are isfound the V617F in this point domain. mutation in the pseudokinase domainThe ofmost JAK2. common This mutation disease-causing