Dimerization by a cytokine receptor is necessary for constitutive activation of JAK2V617F

The majority of the BCR-ABL-negative myeloproliferative disorders express the mutant JAK2, JAK2V617F. Previously we showed that constitutive activation of this oncogenic JAK2 mutant in Ba/F3 or 32D cells requires coexpression of a cognate homodimeric cytokine receptor, such as the EpoR. However, overexpression of JAK2V617F in Ba/F3 cells renders them cytokine-independent for growth in the absence of an exogenous cytokine receptor. Here, we demonstrated that JAK2V617F domains required for receptor association are essential for cytokine-independent growth by overexpressed JAK2V617F, suggesting JAK2V617F is binding to an unknown endogenous cytokine receptor(s) for its activation. We further showed that disruption of EpoR dimerization by coexpressing a truncated EpoR disrupted JAK2V617F-mediated transformation, indicating that EpoR dimerization plays an essential role in the activation of JAK2V617F. Interestingly, coexpression of JAK2V617F with EpoR mutants that retain JAK2 binding but are defective in mediating Epo-dependent JAK2 activation due to mutations in a conserved juxtamembrane motif does lead to cytokine-independent activation of JAK2V617F. Overall, these findings confirm that JAK2V617F requires binding to a dimerized cytokine receptor for its activation, and that the key EpoR juxtamembrane regulatory motif essential for Epo-dependent JAK2 activation is not essential for the activation of JAK2V617F. The structure of the activated JAK2V617F is thus likely to be different from that of the activated wild-type JAK2, raising the possibility of developing a specifically targeted therapy for myeloproliferative disorders.     

JAK1 and Tyk2 activation by the homologous polycythemia vera JAK2 V617F mutation: cross-talk with IGF1 receptor

The majority of polycythemia vera (PV) patients harbor a unique somatic mutation (V617F) in the pseudokinase domain of JAK2, which leads to constitutive signaling. Here we show that the homologous mutations in JAK1 (V658F) and in Tyk2 (V678F) lead to constitutive activation of these kinases. Their expression induces autonomous growth of cytokine-dependent cells and constitutive activation of STAT5, STAT3, mitogen-activated protein kinase, and Akt signaling in Ba/F3 cells. The mutant JAKs exhibit constitutive signaling also when expressed in fibrosarcoma cells deficient in JAK proteins. Expression of the JAK2 V617F mutant renders Ba/F3 cells hypersensitive to insulin-like growth factor 1 (IGF1), which is a hallmark of PV erythroid progenitors. Upon selection of Ba/F3 cells for autonomous growth induced by the JAK2 V617F mutant, cells respond to IGF1 by activating STAT5, STAT3, Erk1/2, and Akt on top of the constitutive activation characteristic of autonomous cells. The synergic effect on proliferation and STAT activation appears specific to the JAK2 V617F mutant. Our results show that the homologous V617F mutation induces activation of JAK1 and Tyk2, suggesting a common mechanism of activation for the JAK1, JAK2, and Tyk2 mutants. JAK3 is not activated by the homologous mutation M592F, despite the presence of the conserved GVC preceding sequence. We suggest that mutations in the JAK1 and Tyk2 genes may be identified as initial molecular defects in human cancers and autoimmune diseases.     

Complex effects of naturally occurring mutations in the JAK3 pseudokinase domain: evidence for interactions between the kinase and pseudokinase domains

The structure of Janus kinases (JAKs) is unique among protein tyrosine kinases in having tandem, nonidentical kinase and pseudokinase domains. Despite its conservation in evolution, however, the function of the pseudokinase domain remains poorly understood. Lack of JAK3 expression results in severe combined immunodeficiency (SCID). In this study, we analyze two SCID patients with mutations in the JAK3 pseudokinase domain, which allows for protein expression but disrupts the regulation of the kinase activity. Specifically, these mutant forms of JAK3 had undetectable kinase activity in vitro but were hyperphosphorylated both in patients' Epstein-Barr virus-transformed B cells and when overexpressed in COS7 cells. Moreover, reconstitution of cells with these mutants demonstrated that, although they were constitutively phosphorylated basally, they were unable to transmit cytokine-dependent signals. Further analysis showed that the isolated catalytic domain of JAK3 was functional whereas either the addition of the pseudokinase domain or its deletion from the full-length molecule reduced catalytic activity. Through coimmunoprecipitation of the isolated pseudokinase domain with the isolated catalytic domain, we provide the first evidence that these two domains interact. Furthermore, whereas the wild-type pseudokinase domain modestly inhibited kinase domain-mediated STAT5 phosphorylation, the patient-derived mutants markedly inhibited this phosphorylation. We thus conclude that the JAK3 pseudokinase domain is essential for JAK3 function by regulating its catalytic activity and autophosphorylation. We propose a model in which this occurs via intramolecular interaction with the kinase domain and that increased inhibition of kinase activity by the pseudokinase domain likely contributes to the disease pathogenesis in these two patients.     

Activation of JAK2-V617F by Components of Heterodimeric Cytokine Receptors

The JAK2-V617F mutation is an important etiologic factor for the development of myeloproliferative neoplasms. The mechanism by which this mutated tyrosine kinase initiates deregulated signals in cells is not completely understood. It is believed that JAK2-V617F requires interactions with homodimeric cytokine receptors to elicit its transforming signal. In this study, we demonstrate that components of heterodimeric cytokine receptors can also activate JAK2-V617F. Expression of IL27Ra, a heterodimeric receptor component, enhanced the activation of JAK2-V617F and subsequent downstream signaling to activation of STAT5 and ERK. In addition, expression of components of the interleukin-3 receptor, IL3Ra and the common β chain, activated JAK2-V617F as well as STAT5 and ERK. Importantly, expression of IL27Ra functionally replaced the requirement of a homodimeric cytokine receptor to promote the activation and transforming activity of JAK2-V617F in BaF3 cells. Tyrosine phosphorylation of IL27Ra was not required to induce activation of JAK2-V617F or STAT5, or to enhance the transforming activity of JAK2-V617F. Expression of IL3Ra or the common β chain in BaF3 cells also enhanced the ability of JAK2-V617F to transform these hematopoietic cells. However, the heterodimeric receptor component IL12RB1 did not enhance the activation or transforming signals of JAK2-V617F in BaF3 cells. IL27Ra also activated the K539L and R683G JAK2 mutants. Together our data demonstrate that in addition to homodimeric receptors, some heterodimeric receptor components can support the activation and transforming signals of JAK2-V617F and other JAK2 mutants. Therefore, heterodimeric receptors may play unappreciated roles in JAK2 activation in the development of hematopoietic diseases including myeloproliferative neoplasms.     

Absence of JAK2 V617F mutation in thalassemia intermedia patients

JAK2 is a cytoplasmic tyrosine kinase that has a vital role in signal transduction from several hemopoietic growth factor receptors. The JAK2 V617F mutation has been implicated in a variety of diseases mainly related to myeloproliferative disorders including polycythemia Vera, essential thrombocythemia, and idiopathic Myelofibrosis but has not been previously described in Thalassemia patients. We studied 36 Lebanese patients diagnosed with thalassemia intermedia and assessed the presence or absence of the JAK2 V617F mutation using JAK2 activating mutation assay (In VivoScribe Technologies) and Polymerase Chain Reaction (PCR). None of the thalassemia intermedia patients were positive for this mutation. To our knowledge, this study is the first to determine the status of JAK2 V617F mutation in thalassemia intermedia patients and expands the international published literature on JAK2. The latter’s V617F mutation does not seem to play a role in this hematologically important clinical entity.     

Characteristic sequential residue environment of amino acids in proteins

The occurrence of all di- and tripeptide segments of proteins was counted in a large data base containing about 119 000 residues. It was found that the abundance of the amino acids does not determine the frequency of the various di- and tripeptide segments. In addition, the frequency of the various tripeptides cannot be predicted from the observed pair-frequency values. The pair-frequency distribution of amino acids is highly asymmetrical, pairs formed from identical residues are generally preferred and amino acids cannot be clustered on the basis of their first neighbour preferences. These data indicate the existence of general short range regularities in the primary structure of proteins. The consequences of these short range regularities were studied by comparing Chou-Fasman parameters with analogous parameters determined from the results of conformational energy calculations of single amino acids. This comparison shows that Chou-Fasman parameters carry significant information about the environment of each amino acid. The success of the Chou-Fasman's prediction and the properties of the pair and triplet distribution of the amino acid residues suggest that every amino acid has a characteristic sequential residue environment in proteins. The observed preferences could be invoked, for example, in protein design or in the study of the evolutionary relationship of proteins.     

Substitution of asparagine 76 by a tyrosine residue induces domain swapping in Helicobacter pylori phosphopantetheine adenylyltransferase

Phosphopantetheine adenylyltransferase (PPAT) catalyses the penultimate step in coenzyme A biosynthesis in bacteria and is therefore a candidate target for antibacterial drug development. We randomly mutated the residues in the Helicobacter pylori PPAT sequence to identify those that govern protein folding and ligand binding, and we describe the crystal structure of one of these mutants (I4V/N76Y) that contains the mutations I4?→?V and N76?→?Y. Unlike other PPATs, which are homohexamers, I4V/N76Y is a domain-swapped homotetramer. The protomer structure of this mutant is an open conformation in which the 65 C-terminal residues are intertwined with those of a neighbouring protomer. Despite structural differences between wild-type PPAT and IV4/N76Y, they had similar ligand-binding properties. ATP binding to these two proteins was enthalpically driven, whereas that for Escherichia coli PPAT is entropically driven. The structural packing of the subunits may affect the thermal denaturation of wild-type PPAT and I4V/N76Y. Mutations in hinge regions often induce domain swapping, i.e. the spatial exchange of portions of adjacent protomers, but residues 4 and 76 of H. pylori PPAT are not located in or near to the hinge region. However, one or both of these residues is responsible for the large conformational change in the C-terminal region of each protomer. To identify the residue(s) responsible, we constructed the single-site mutant, N76Y, and found a large displacement of α-helix 4, which indicated that its flexibility allowed the domain swap to occur.     

Substitution of asparagine 76 by a tyrosine residue induces domain swapping in Helicobacter pylori phosphopantetheine adenylyltransferase

Phosphopantetheine adenylyltransferase (PPAT) catalyses the penultimate step in coenzyme A biosynthesis in bacteria and is therefore a candidate target for antibacterial drug development. We randomly mutated the residues in the Helicobacter pylori PPAT sequence to identify those that govern protein folding and ligand binding, and we describe the crystal structure of one of these mutants (I4V/N76Y) that contains the mutations I4?→?V and N76?→?Y. Unlike other PPATs, which are homohexamers, I4V/N76Y is a domain-swapped homotetramer. The protomer structure of this mutant is an open conformation in which the 65 C-terminal residues are intertwined with those of a neighbouring protomer. Despite structural differences between wild-type PPAT and IV4/N76Y, they had similar ligand-binding properties. ATP binding to these two proteins was enthalpically driven, whereas that for Escherichia coli PPAT is entropically driven. The structural packing of the subunits may affect the thermal denaturation of wild-type PPAT and I4V/N76Y. Mutations in hinge regions often induce domain swapping, i.e. the spatial exchange of portions of adjacent protomers, but residues 4 and 76 of H. pylori PPAT are not located in or near to the hinge region. However, one or both of these residues is responsible for the large conformational change in the C-terminal region of each protomer. To identify the residue(s) responsible, we constructed the single-site mutant, N76Y, and found a large displacement of α-helix 4, which indicated that its flexibility allowed the domain swap to occur.     

NEMO binding domain of IKK-2 encompasses amino acids 735-745

NF-kappaB activation is mediated by the IKK signalsome. Though this signalsome is comprised of IKK-1, IKK-2, and NEMO/IKKgamma, it is the interaction between IKK-2 and NEMO that is critical to formation of a functional signalsome. More specifically, previous reports have indicated that this interaction involves the C-terminal LDWSWL residues of IKK-2 (called the Nemo Binding Domain (NBD)) and the N-terminus of NEMO. In an effort to characterize the IKK-2:NEMO interaction, we have investigated several NBD-containing peptides for their ability to bind NEMO and inhibit the critical IKK-2:NEMO interaction. The six residue NBD peptide, LDWSWL, showed modest binding to NEMO and little inhibition of the IKK-2:NEMO interaction, whereas peptides containing the NBD plus additional flanking amino acids (NBD-containing peptides) more effectively bound NEMO and inhibited the interaction. These longer NBD-containing peptides may be required to give the NBD an appropriate conformation for recognition by NEMO and/or to provide for additional interactions with NEMO.     

Nature disfavors sequences of alternating polar and non-polar amino acids: implications for amyloidogenesis

Recent experiments with combinatorial libraries of de novo proteins have demonstrated that sequences designed to contain polar and non-polar amino acid residues arranged in an alternating pattern form fibrillar structures resembling beta-amyloid. This finding prompted us to probe the distribution of alternating patterns in the sequences of natural proteins. Analysis of a database of 250,514 protein sequences (79,708,024 residues) for all possible binary patterns of polar and non-polar amino acid residues revealed that alternating patterns occur significantly less often than other patterns with similar compositions. The under-representation of alternating binary patterns in natural protein sequences, coupled with the observation that such patterns promote amyloid-like structures in de novo proteins, suggests that sequences of alternating polar and non-polar amino acids are inherently amyloidogenic and consequently have been disfavored by evolutionary selection.     

Tyrosine 813 is a site of JAK2 autophosphorylation critical for activation of JAK2 by SH2-B beta

The tyrosine kinase Janus kinase 2 (JAK2) binds to the majority of the known members of the cytokine family of receptors. Ligand-receptor binding leads to activation of the associated JAK2 molecules, resulting in rapid autophosphorylation of multiple tyrosines within JAK2. Phosphotyrosines can then serve as docking sites for downstream JAK2 signaling molecules. Despite the importance of these phosphotyrosines in JAK2 function, only a few sites and binding partners have been identified. Using two-dimensional phosphopeptide mapping and a phosphospecific antibody, we identified tyrosine 813 as a site of JAK2 autophosphorylation of overexpressed JAK2 and endogenous JAK2 activated by growth hormone. Tyrosine 813 is contained within a YXXL sequence motif associated with several other identified JAK2 phosphorylation sites. We show that phosphorylation of tyrosine 813 is required for the SH2 domain-containing adapter protein SH2-B beta to bind JAK2 and to enhance the activity of JAK2 and STAT5B. The homologous tyrosine in JAK3, tyrosine 785, is autophosphorylated in response to interleukin-2 stimulation and is required for SH2-B beta to bind JAK3. Taken together these data strongly suggest that tyrosine 813 is a site of autophosphorylation in JAK2 and is the SH2-B beta-binding site within JAK2 that is required for SH2-B beta to enhance activation of JAK2.     

Generation and Characterization of a JAK2V617F-Containing Erythroleukemia Cell Line

The JAK2V617F mutation is found in the majority of patients with myeloproliferative neoplasms (MPNs). Transgenic expression of the mutant gene causes MPN-like phenotypes in mice. We have produced JAK2V617F mice with p53 null background. Some of these mice developed acute erythroleukemia. From one of these mice, we derived a cell line designated J53Z1. J53Z1 cells were stained positive for surface markers CD71 and CD117 but negative for Sca-1, TER-119, CD11b, Gr-1, F4/80, CD11c, CD317, CD4, CD8a, CD3e, B220, CD19, CD41, CD42d, NK-1.1, and FceR1. Real time PCR analyses demonstrated expressions of erythropoietin receptor EpoR, GATA1, and GATA2 in these cells. J53Z1 cells grew rapidly in suspension culture containing fetal bovine serum with a doubling time of ∼18 hours. When transplanted into C57Bl/6 mice, J53Z1 cells induced acute erythroleukemia with massive infiltration of tumor cells in the spleen and liver. J53Z1 cells were responsive to stimulation with erythropoietin and stem cell factor and were selectively inhibited by JAK2 inhibitors which induced apoptosis of the cells. Together, J53Z1 cells belong to the erythroid lineage, and they may be useful for studying the role of JAK2V617F in proliferation and differentiation of erythroid cells and for identifying potential therapeutic drugs targeting JAK2.     

Identification of amino acids in the factor XI apple 3 domain required for activation of factor IX

Activated coagulation factor XI (factor XIa) proteolytically cleaves its substrate, factor IX, in an interaction requiring the factor XI A3 domain (Sun, Y., and Gailani, D. (1996) J. Biol. Chem. 271, 29023-29028). To identify key amino acids involved in factor IX activation, recombinant factor XIa proteins containing alanine substitutions for wild-type sequence were expressed in 293 fibroblasts and tested in a plasma clotting assay. Substitutions for Ile(183)-Val(191) and Ser(195)-Ile(197) at the N terminus and for Ser(258)-Ser(264) at the C terminus of the A3 domain markedly decreased factor XI coagulant activity. The plasma protease prekallikrein is structurally homologous to factor XI, but activated factor IX poorly. A chimeric factor XIa molecule with the A3 domain replaced with A3 from prekallikrein (FXI/PKA3) activated factor IX with a K(m) 35-fold greater than that of wild-type factor XI. FXI/PKA3 was used as a template for a series of proteins in which prekallikrein A3 sequence was replaced with factor XI sequence to restore factor IX activation. Clotting and kinetics studies using these chimeras confirmed the results obtained with alanine mutants. Amino acids between Ile(183) and Val(191) are necessary for proper factor IX activation, but additional sequence between Ser(195) and Ile(197) or between Phe(260) and Ser(265) is required for complete restoration of activation.