Pseudokinase Domain Research Articles

Conformational control and regulation of the pseudokinase KSR via small molecule binding interactions.

Pseudokinases often operate through functionally related enzymes and receptors. A prime example is the pseudokinase KSR (Kinase Suppressor of RAS), which can act as both an amplifier and inhibitor of members in the RAS-MAPK (Mitogen Activated Protein Kinase) signaling pathway. KSR is structurally related to the active RAF kinases over multiple domains; moreover, the pseudokinase domain of KSR forms physical and regulatory complexes with both RAF and MEK through distinct interfaces. Characterization of small molecule interactions on KSR has been used to uncover novel chemical tools and understand the mechanism of action of clinical drugs. Here, we elaborate on assays and structural methods for measuring binding at orthosteric and interfacial binding sites on KSR. These distinct small molecule pockets provide therapeutic paths for targeting KSR1 and KSR2 pseudokinases in disease, including in RAS and RAF mutant cancers.

Analysis of human Tribbles 2 (TRIB2) pseudokinase.

Human Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a broad human protein interactome, including the well-studied AKT, C/EBPα and MAPK modules. Several lines of evidence indicate that human TRIB2 promotes cell survival and drug-resistance in solid tumors and blood cancers and is therefore of interest as a potential therapeutic target, although its physiological functions remain relatively poorly understood. The unique TRIB2 pseudokinase domain lacks the canonical 'DFG' motif, and subsequently possesses very low affinity for ATP in both the presence and absence of metal ions. However, TRIB2 also contains a unique cysteine-rich αC-helix, which interacts with a conserved peptide motif in its own carboxyl-terminal tail. This regulatory flanking region drives regulated interactions with distinct E3 ubiquitin ligases that serve to control the stability and turnover of TRIB2 client proteins. TRIB2 is also a low-affinity target of several known small-molecule protein kinase inhibitors, which were originally identified using purified recombinant TRIB2 proteins and a thermal shift assay. In this chapter, we discuss laboratory-based procedures for purification, stabilization and analysis of human TRIB2, including screening procedures that can be used for the identification of both reversible and covalent small molecule ligands.

Targeting the HER3 pseudokinase domain with small molecule inhibitors.

HER3 is a potent oncogenic growth factor receptor belonging to the human epidermal growth factor (HER/EGFR) family of receptor tyrosine kinases. In contrast to other EGFR family members, HER3 is a pseudokinase, lacking functional kinase activity. As such, efforts to develop small molecule tyrosine kinase inhibitors against this family member have been limited. In response to HER3-specific growth factors such as neuregulin (NRG, also known as heregulin or HRG), HER3 must couple with catalytically active family members, including its preferred partner HER2. Dimerization of the intracellular HER2:HER3 kinase domains is a critical part of the activation mechanism and HER3 plays a specialized role as an allosteric activator of the active HER2 kinase partner. Intriguingly, many pseudokinases retain functionally important nucleotide binding capacity, despite loss of kinase activity. We demonstrated that occupation of the nucleotide pocket of the pseudokinase HER3 retains functional importance for growth factor signaling through oncogenic HER2:HER3 heterodimers. Mutation of the HER3 nucleotide pocket both disrupts signaling and disrupts HER2:HER3 dimerization. Conversely, ATP competitive drugs which bind to HER3, but not HER2, can stabilize HER2:HER3 dimers, induce signaling and promote cell growth in breast cancer models. This indicates a nucleotide-dependent conformational role for the HER3 kinase domain. Critically, our recent proof-of-concept work demonstrated that HER3-directed small molecule inhibitors can also disrupt HER2:HER3 dimerization and signaling, supporting the prospect that HER3 can be a direct drug target despite its lack of intrinsic activity. In this chapter we will describe methods for identifying and validating small molecule inhibitors against the HER3 pseudokinase.

Identification of Small Molecule Inhibitors of RNase L by Fragment-Based Drug Discovery.

The pseudokinase-endoribonuclease RNase L plays important roles in antiviral innate immunity and is also implicated in many other cellular activities. The inhibition of RNase L showed therapeutic potential for Aicardi-Goutières syndrome (AGS). Thus, RNase L is a promising drug target. In this study, using an enzyme assay and NMR screening, we discovered 13 inhibitory fragments against RNase L. Cocrystal structures of RNase L separately complexed with two different fragments were determined in which both fragments bound to the ATP-binding pocket of the pseudokinase domain. Myricetin, vitexin, and hyperoside, three natural products sharing similar scaffolds with the fragment AC40357, demonstrated a potent inhibitory activity in vitro. In addition, myricetin has a promising cellular inhibitory activity. A cocrystal structure of RNase L with myricetin provided a structural basis for inhibitor design by allosterically modulating the ribonuclease activity. Our findings demonstrate that fragment screening can lead to the discovery of natural product inhibitors of RNase L.

RFWD2 Knockdown as a Blocker to Reverse the Oncogenic Role of TRIB2 in Lung Adenocarcinoma.

RFWD2, an E3 ubiquitin ligase, is overexpressed in numerous human cancers, including leukemia, lung cancer, breast cancer, renal cell carcinoma, and colorectal cancer. The roles of RFWD2 in cancer are related to the targeting of its substrates for ubiquitination and degradation. This study aimed to investigate the role of TRIB2 in relation to the regulation of protein degradation through RFWD2. inBio Discover™ results demonstrated that TRIB2 can perform its functions by interacting with RFWD2 or other factors. TRIB2 can interact with and regulate RFWD2, which further attends the proteasome-mediated degradation of the RFWD2 substrate p-IκB-α. TRIB2 colocalizes with RFWD2-related IκB-α to form a ternary complex and further affects the IκB-α degradation by regulating its phosphorylation. Specific domain analysis showed that TRIB2 may bind to RFWD2 via its C-terminus, whereas it binds to IκB via its pseudokinase domain. TRIB2 acts as an oncogene and promotes cancer cell proliferation and migration, whereas RFWD2 knockdown reversed the role of TRIB2 in promoting cancer cell growth and colony formation in vitro and in vivo. In summary, this study reveals that TRIB2 promotes the progression of cancer by affecting the proteasome-mediated degradation of proteins through the interaction with RFWD2.

A novel activating JAK1 mutation in chronic eosinophilic leukemia

AbstractHypereosinophilia (HE) has been defined as persistent eosinophilia >1.5 × 109/L; it is broadly divided into primary HE (clonal or neoplastic; HEN), secondary/reactive HE (HER), or HE of undetermined significance (HEUS) when no cause is identified. The use of myeloid next-generation sequencing (NGS) panels has led to the detection of several mutations in patients previously diagnosed with HEUS, reassigning some patients to the category of HEN, specifically the World Health Organization category of chronic eosinophilic leukemia, not otherwise specified (CEL, NOS). Here, we describe a novel somatic JAK1 pseudokinase domain mutation (R629_S632delinsSA) in a patient with HE that had initially been characterized as a variant of uncertain significance. We performed functional studies that demonstrated that this mutation results in growth factor independence of Ba/F3 cells in vitro and activation of the JAK-STAT pathway. These effects were abrogated by the JAK1/JAK2 inhibitor ruxolitinib. R629_S632delinsSA is the first known somatic mutation in JAK1 linked to a clonal eosinophilic neoplasm, and highlights the importance of the JAK-STAT pathway in eosinophil survival.

The intracellular domains of the EphB6 and EphA10 receptor tyrosine pseudokinases function as dynamic signalling hubs.

EphB6 and EphA10 are two poorly characterised pseudokinase members of the Eph receptor family, which collectively serves as mediators of contact-dependent cell–cell communication to transmit extracellular cues into intracellular signals. As per their active counterparts, EphB6 and EphA10 deregulation is strongly linked to proliferative diseases. However, unlike active Eph receptors, whose catalytic activities are thought to initiate an intracellular signalling cascade, EphB6 and EphA10 are classified as catalytically dead, raising the question of how non-catalytic functions contribute to Eph receptor signalling homeostasis. In this study, we have characterised the biochemical properties and topology of the EphB6 and EphA10 intracellular regions comprising the juxtamembrane (JM) region, pseudokinase and SAM domains. Using small-angle X-ray scattering and cross-linking-mass spectrometry, we observed high flexibility within their intracellular regions in solution and a propensity for interaction between the component domains. We identified tyrosine residues in the JM region of EphB6 as EphB4 substrates, which can bind the SH2 domains of signalling effectors, including Abl, Src and Vav3, consistent with cellular roles in recruiting these proteins for downstream signalling. Furthermore, our finding that EphB6 and EphA10 can bind ATP and ATP-competitive small molecules raises the prospect that these pseudokinase domains could be pharmacologically targeted to counter oncogenic signalling.

Binding of a pyrimidine RNA base-mimic to SARS-CoV-2 nonstructural protein 9

The coronaviral nonstructural protein 9 (Nsp9) is essential for viral replication; it is the primary substrate of Nsp12’s pseudokinase domain within the viral replication transcription complex, an association that also recruits other components during different stages of RNA reproduction. In the unmodified state, Nsp9 forms an obligate homodimer via an essential GxxxG protein-interaction motif, but its ssRNA-binding mechanism remains unknown. Using structural biological techniques, here we show that a base-mimicking compound identified from a small molecule fragment screen engages Nsp9 via a tetrameric Pi-Pi stacking interaction that induces the formation of a parallel trimer-of-dimers. This oligomerization mechanism allows an interchange of “latching” N-termini, the charges of which contribute to a series of electropositive channels that suggests a potential interface for viral RNA. The identified pyrrolo-pyrimidine compound may also serve as a potential starting point for the development of compounds seeking to probe Nsp9’s role within SARS-CoV-2 replication.

Indoloxytriazines as binding molecules for the JAK2 JH2 pseudokinase domain and its V617F variant

Small molecules that selectively bind to the pseudokinase JH2 domain over the JH1 kinase domain of JAK2 kinase are sought. Virtual screening led to the purchase of 17 compounds among which 9 were found to bind to V617F JAK2 JH2 with affinities of 40 – 300 μM in a fluorogenic assay. Ten analogues were then purchased yielding 9 additional active compounds. Aminoanilinyltriazine 22 was particularly notable as it shows no detectable binding to JAK2 JH1, and it has a 65-μM dissociation constant Kd with V617F JAK2 JH2. A crystal structure for 22 in complex with wild-type JAK2 JH2 was obtained to elucidate the binding mode. Additional de novo design led to the synthesis of 19 analogues of 22 with the most potent being 33n with Kd values of 2–3 μM for WT and V617F JAK2 JH2, and with 16-fold selectivity relative to binding with WT JAK2 JH1.

JAK3 Variants in Adults with Myeloid Malignancies and Potential for Response to JAK3 Inhibition

Background: Janus kinases (JAK) family members (JAK1, JAK2, JAK3, TYK2) play an important role in tumorigenesis. JAK3 is involved in the signaling of a number of interleukins (IL) (including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21), as well as the phosphorylation of the signal transducers and activators of transcriptions (STAT) and PI3K/AKT. JAK3 activating variants, including V722I, demonstrably induce cellular transformation in vitro. The prevalence of JAK3 variants/mutations in myeloid neoplasms, particularly in acute myeloid leukemia (AML), and their role in leukemogenesis, remains unclear.Methods: We reviewed data for myeloid leukemia (AML, MDS and MPN) patients (pts) treated at MD Anderson Cancer Center between 2012 and 2017, whose marrow samples were tested for JAK3 variants/mutations by next generation sequencing. We evaluated the type of variants, the demographics, laboratory, treatment, and clinical information for pts with JAK3 variants. We performed an in vitro drug sensitivity assay using patient-derived xenograft leukemia cells from one patient with a JAK3 variant and using primary AML samples, to test for sensitivity to the JAK3 inhibitor tofacitinib.Results: A total of 35 leukemia pts with JAK3 variants were identified, and included: non-megakaryoblastic AML (n=31), MDS/MPN (n=2) and MDS (n=2). Four AML cases arose from prior MDS or MDS/MPN. All 4 non-AML pts had V722I variant, located in the pseudokinase domain. Of the 31 AML pts with JAK3 variants, 21 (68%) had V722I. Three (10%) other AML pts had P151R (FERM domain), 3 (10%) V718L (pseudokinase), 2 (6%) R925L (kinase), and 1 (3%) 940V (kinase); none of these variants has been previously described. A pt with T815M (between pseudokinase-kinase domains) was identified from outside genetic testing. The median allelic frequency for variants (where available) in AML pts was 53% (range, 41-88) and median marrow blasts was 45% (range 21-97). Overall, cytogenetics were complex in 19 (55%) pts and diploid in 11 (31%); 5 (14%) pts had miscellaneous clones. Concomitant somatic mutations most commonly occurred in IDH1/2 (n=12), TP53 (11), F LT3 (9), NPM1 (6), ASXL1 (5), RUNX1 (4), SRSF2 (4), and DNMT3A (3). Leukemia cells derived from a relapsed/refractory pt with JAK3 T815M variant had significant sensitivity to JAK3 inhibitor tofacitinib, with nearly 50% reduction in viability with 0.1uM drug concentration (Figure 1). Primary AML samples from 3 other pts showed no sensitivity to the drug.Conclusions: We report the largest survey of JAK3 variants among adult myeloid leukemia pts, including novel variants. Given allelic frequencies, some JAK3 variants may represent germline polymorphisms. The activating V722I variant may have increased prevalence among AML pts. The sensitivity of JAK3 T815M AML cells treated with the FDA-approved JAK3 inhibitor tofacitinib, as well as the prevalence and location of JAK3 variants in myeloid malignancies, provide a basis for further investigation. [Display omitted] DisclosuresDiNardo:Novartis: Honoraria, Research Funding; Agios: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding. Kantarjian:Bristol-Meyers Squibb: Research Funding; Novartis: Research Funding; Amgen: Research Funding; Pfizer: Research Funding; ARIAD: Research Funding; Delta-Fly Pharma: Research Funding. Verstovsek:Genentech: Research Funding; NS Pharma: Research Funding; Lilly Oncology: Research Funding; Galena BioPharma: Research Funding; Gilead: Research Funding; Promedior: Research Funding; Promedior: Research Funding; Gilead: Research Funding; CTI BioPharma Corp: Research Funding; Astrazeneca: Research Funding; Galena BioPharma: Research Funding; Genentech: Research Funding; Roche: Research Funding; Celgene: Research Funding; CTI BioPharma Corp: Research Funding; Blueprint Medicines Corp: Research Funding; Pfizer: Research Funding; Pfizer: Research Funding; NS Pharma: Research Funding; Blueprint Medicines Corp: Research Funding; Bristol Myers Squibb: Research Funding; Incyte: Research Funding; Seattle Genetics: Research Funding; Incyte: Research Funding; Seattle Genetics: Research Funding; Roche: Research Funding; Lilly Oncology: Research Funding; Astrazeneca: Research Funding; Bristol Myers Squibb: Research Funding; Celgene: Research Funding.

A Novel JAK2 Mutation Confers Cytokine-Independence in the Absence of a Type I Dimeric Cytokine Receptor

JAK2 fusions reported in hematologic malignancies are often associated with eosinophilia. In addition to JAK2 fusions such as PCM1-JAK2, BCR-JAK2 and ETV6-JAK2, the JAK2V617Fpoint mutant has been recurrently observed in patients with hypereosinophilic syndrome (HES), occurring in ~4% of patients with hypereosinophilia of unknown significance (Schwaab, Am J Hematol, 2015).Here, we describe a novel JAK2 mutation in a 69 year-old woman with a history of immune thrombocytopenia referred for steroid-refractory HES. Peripheral blood eosinophilia was present for at least five years, with absolute eosinophil counts ranging up to 17,500/μL. Bone marrow biopsy revealed hypercellular marrow with trilineage hematopoiesis, increased atypical megakaryocytes, and markedly increased eosinophils with abnormal morphology, consistent with myeloproliferative neoplasm. Results from an eosinophilia FISH panel for FIP1L1/CHIC2/PDGFRα, FGFR, PDGFRβ, and CBFβ fusions were negative, as were T-cell clonality studies by next-generation sequencing (NGS). An NGS-based myeloid malignancies panel revealed a novel mutation in JAK2 consisting of a ten base deletion and insertion of a single thymine (JAK2 c.1747_1756delinsT, p.Leu583_Ala586delinsSer;JAK2indel). Treatment with ruxolitinib improved the peripheral blood eosinophilia, but dose intensity was limited by thrombocytopenia (Table 1).For transformation of cells to cytokine-independence, JAK2V617F requires co-expression of a type I dimeric cytokine receptor, such as Epo-R, to enable downstream signaling in the absence of growth factors (Lu et al, Proc Natl Acad Sci, 2005). To characterize the signaling properties of the new JAK2indel mutant, we cloned JAK2WT, JAK2V617F, and JAK2indel into the MSCV-IRES-GFP (MIG) retroviral vector. IL-3-dependent Ba/F3 cells lacking type I cytokine receptors were infected with retrovirus to assess cytokine independence.Parental Ba/F3 cells and those transduced with JAK2WT, JAK2V617F, or JAK2indel proliferated at similar rates when grown in WEHI conditioned medium (CM). However, upon IL-3 (WEHI) withdrawal, only GFP-positive JAK2indel Ba/F3 cells demonstrated growth factor independence (n=3). Furthermore, parental and GFP-sorted JAK2WT- and JAK2V617F-expressingBa/F3 cells display acceleration of growth when cultured in graded dilutions of WEHI CM, while growth of JAK2indel Ba/F3 cells is not further enhanced by WEHI CM. JAK2indel-expressing Ba/F3 cells also demonstrated cytokine-independent growth in colony assays, whereas parental, JAK2WT-, and JAK2V617F-expressing cells displayed little to no colony formation in the absence of cytokines. Immunoblot analyses revealed that after 4 hours of culture ± IL-3, all cells grown in IL-3 demonstrated activation of STAT5 and ERK, but only JAK2indel Ba/F3 cells demonstrated cytokine-independent signaling activation.JAK2indel activates STAT5 and ERK without co-expression of a type I cytokine receptor, implicating a mechanism of JAK2 kinase activation that is distinct from JAK2V617F. Unexpectedly, preliminary structural analysis suggests that the conformational change induced by JAK2indel resembles that of JAK2V617F. Mutation of glutamic acid 596 to arginine (E596R) has been shown to abolish the constitutive activity of JAK2V617F via modulation of the C-terminal residues of the SH2-JH2 linker, thereby restoring pseudokinase domain autoinhibitory function (Leroy et al, Biochem J, 2016). To test whether this regulatory mechanism is preserved in JAK2indel, we introduced E596R into JAK2indel and are currently characterizing JAK2indel/E596R in cellular and biochemical assays. Additional modeling and biochemical work is underway to clarify whether JAK2indel confers a new functionality that supports signaling activation in the absence of a type I cytokine receptor.In summary we report a novel JAK2 mutation in a patient with myeloproliferative-variant HES that results in cytokine-independent growth, is associated with STAT5 and ERK phosphorylation and may be employing an alternative mechanism to achieve constitutive activation of signaling. The fact that the patient's eosinophilia improved with ruxolitinib treatment illustrates the importance of performing NGS in patients with HES to identify potentially targetable genetic lesions. [Display omitted] DisclosuresDeininger:Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Incyte: Consultancy; Novartis: Consultancy, Research Funding; Celgene: Research Funding; Pfizer: Consultancy; BMS: Consultancy, Research Funding; ARIAD: Consultancy.

Selective Tyrosine Kinase 2 Inhibition for Treatment of Inflammatory Bowel Disease: New Hope on the Rise.

Conventional systemic and biologic agents are the mainstay of inflammatory bowel disease (IBD) management; however, many of these agents are associated with loss of clinical response, highlighting the need for effective, novel targeted therapies. Janus kinase (JAK) 1-3 and tyrosine kinase 2 (TYK2) mediate signal transduction events downstream of multiple cytokine receptors that regulate targeted gene transcription, including the interleukin-12, interleukin-23, and type I interferon receptors for TYK2. This review summarizes the role of TYK2 signaling in IBD pathogenesis, the differential selectivity of TYK2 inhibitors, and the potential clinical implications of TYK2 inhibition in IBD. A PubMed literature review was conducted to identify studies of JAK1-3 and TYK2 inhibitors in IBD and other immune-mediated inflammatory diseases. Key efficacy and safety information was extracted and summarized. Pan-JAK inhibitors provide inconsistent efficacy in patients with IBD and are associated with toxicities resulting from a lack of selectivity at therapeutic dosages. Selective inhibition of TYK2 signaling via an allosteric mechanism, with an agent that binds to the regulatory (pseudokinase) domain, may reduce potential toxicities typically associated with JAK1-3 inhibitors. Deucravacitinib, a novel, oral, selective TYK2 inhibitor, and brepocitinib and PF-06826647, TYK2 inhibitors that bind to the active site in the catalytic domain, are in development for IBD and other immune-mediated inflammatory diseases. Allosteric TYK2 inhibition is more selective than JAK1-3 inhibition and has the potential to limit toxicities typically associated with JAK1-3 inhibitors. Future studies will be important in establishing the role of selective, allosteric TYK2 inhibition in the management of IBD.

The Critical Role of TRIB2 in Cancer and Therapy Resistance.

Simple SummaryThe Tribbles proteins are members of CAMK Ser/Thr protein kinase family. They are evolutionary conserved pseudokinases found in most tissues of eukaryotic organisms. This ubiquitously expressed protein family is characterized by containing a catalytically deficient kinase domain which lacks amino acid residues required for the productive interaction with ATP and metal ions. Tribbles proteins exert their biological functions mainly through direct interaction with MAPKK and AKT proteins, therefore regulating important pathways involved in cell proliferation, apoptosis and differentiation. Due to the role of MAPKK and AKT signalling in the context of cancer development, Tribbles proteins have been recently considered as biomarkers of cancer progression. Furthermore, as the atypical pseudokinase domain retains a binding platform for substrates, Tribbles targeting provides an attractive opportunity for drug development.The Tribbles pseudokinases family consists of TRIB1, TRIB2, TRIB3 and STK40 and, although evolutionarily conserved, they have distinctive characteristics. Tribbles members are expressed in a context and cell compartment-dependent manner. For example, TRIB1 and TRIB2 have potent oncogenic activities in vertebrate cells. Since the identification of Tribbles proteins as modulators of multiple signalling pathways, recent studies have linked their expression with several pathologies, including cancer. Tribbles proteins act as protein adaptors involved in the ubiquitin-proteasome degradation system, as they bridge the gap between substrates and E3 ligases. Between TRIB family members, TRIB2 is the most ancestral member of the family. TRIB2 is involved in protein homeostasis regulation of C/EBPα, β-catenin and TCF4. On the other hand, TRIB2 interacts with MAPKK, AKT and NFkB proteins, involved in cell survival, proliferation and immune response. Here, we review the characteristic features of TRIB2 structure and signalling and its role in many cancer subtypes with an emphasis on TRIB2 function in therapy resistance in melanoma, leukemia and glioblastoma. The strong evidence between TRIB2 expression and chemoresistance provides an attractive opportunity for targeting TRIB2.

Structural Insights into Pseudokinase Domains of Receptor Tyrosine Kinases

Receptor tyrosine kinases (RTKs) orchestrate embryonic development and tissue homeostasis by controlling key cellular processes like growth and proliferation. Consequently, the vast majority of the 58 RTKs in humans are linked to cancer or metabolic disorders. For instance, RTKs represent known oncogenic drivers (such as EGFR in non-small cell lung cancer), contribute to cancer hallmarks (such as VEGFR in pro-tumor angiogenesis), and mediate resistance to therapeutics or hormones (such as the Insulin Receptor in diabetes). Classically, RTKs transduce signals through various modes of ligand-induced receptor dimerization and subsequent tyrosine kinase-mediated signaling; however, ~10% of all metazoan RTKs are pseudokinases—that is, they lack the capability to catalyze phosphotransfer. Our objective is to ascertain how RTK pseudokinases relay extracellular cues without kinase activity and whether they can be targeted therapeutically. Using structural, biophysical, and cell signaling approaches, we investigated this class of RTKs to shed light on potential non-catalytic signaling mechanisms. We identified structural elements conserved between the pseudokinases and their kinase relatives, found their pseudokinase domains to be conformationally dynamic, and uncovered a putative route to drugging these receptors via “conformational disrupting” small molecules. Our findings suggest that RTK pseudokinases could serve as viable drug targets and set the scene for future dissection of signaling pathways that have long been considered enigmatic.

Conformational interconversion of MLKL and disengagement from RIPK3 precede cell death by necroptosis

Phosphorylation of the MLKL pseudokinase by the RIPK3 kinase leads to MLKL oligomerization, translocation to, and permeabilization of, the plasma membrane to induce necroptotic cell death. The precise choreography of MLKL activation remains incompletely understood. Here, we report Monobodies, synthetic binding proteins, that bind the pseudokinase domain of MLKL within human cells and their crystal structures in complex with the human MLKL pseudokinase domain. While Monobody-32 constitutively binds the MLKL hinge region, Monobody-27 binds MLKL via an epitope that overlaps the RIPK3 binding site and is only exposed after phosphorylated MLKL disengages from RIPK3 following necroptotic stimulation. The crystal structures identified two distinct conformations of the MLKL pseudokinase domain, supporting the idea that a conformational transition accompanies MLKL disengagement from RIPK3. These studies provide further evidence that MLKL undergoes a large conformational change upon activation, and identify MLKL disengagement from RIPK3 as a key regulatory step in the necroptosis pathway.

For Whom the Bell Tolls: The Structure of the Dead Kinase, IRAK3

In this issue of Structure, Lange etal. (2020) report the structure of the pseudokinase domain of IRAK3, a negative regulator of Myddosome inflammatory signaling. The IRAK3 pseudokinase domain forms a head-to-head dimer, suggesting a new mode of kinase/pseudokinase allostery by which IRAK3 could attenuate the activity of IRAK4 in cells.

Disruption of the integrin-linked kinase (ILK) pseudokinase domain affects kidney development in mice

Disruption of the integrin-linked kinase (ILK) pseudokinase domain affects kidney development in mice

Embryonic lethality and defective mammary gland development of activator-function impaired conditional knock-in Erbb3 V943R mice.

ERBB3 is a pseudokinase domain-containing member of the ERBB family of receptor tyrosine kinases (RTKs). Following ligand binding, ERBB receptors homo- or hetero-dimerize, leading to a head-to-tail arrangement of the intracellular kinase domains, where the "receiver" kinase domain of one ERBB is activated by the "activator" domain of the other ERBB in the dimer. In ERBB3, a conserved valine at codon 943 (V943) in the kinase C-terminal domain has been shown to be important for its function as an "activator" kinase in vitro. Here we report a knock-in mouse model where we have modified the endogenous Erbb3 allele to allow for tissue-specific conditional expression of Erbb3 V943R (Erbb3 CKI-V943R ). Additionally, we generated an Erbb3 D850N (Erbb3 CKI-D850N ) conditional knock-in mouse model where the conserved aspartate in the DFG motif of the pseudokinase domain was mutated to abolish any potential residual kinase activity. While Erbb3 D850N/D850N animals developed normally, homozygous Erbb3 V943R/V943R expression during development resulted in embryonic lethality. Further, tissue specific expression of Erbb3 V943R/V943R in the mammary gland epithelium following its activation using MMTV-Cre resulted in delayed elongation of the ductal network during puberty. Single-cell RNA-seq analysis of Erbb3 V943R/V943R mammary glands showed a reduction in a specific subset of fibrinogen-producing luminal epithelial cells.

Dimeric Structure of the Pseudokinase IRAK3 Suggests an Allosteric Mechanism for Negative Regulation

SummaryInterleukin-1 receptor associated kinases (IRAKs) are key players in innate immune signaling that mediate the host response to pathogens. In contrast to the active kinases IRAK1 and IRAK4, IRAK2 and IRAK3 are pseudokinases lacking catalytic activity and their functions are poorly understood. IRAK3 is thought to be a negative regulator of innate immune signaling and mutations in IRAK3 are associated with asthma and cancer. Here, we report the crystal structure of the human IRAK3 pseudokinase domain in a closed, pseudoactive conformation. IRAK3 dimerizes in a unique way through a head-to-head arrangement not observed in any other kinases. Multiple conserved cysteine residues imply a potential redox control of IRAK3 conformation and dimerization. By analyzing asthma-associated mutations, we identify an evolutionarily conserved surface on IRAK3 that could form an interaction interface with IRAK4, suggesting a model for the negative regulation of IRAK4 by IRAK3.

Prevalence of JAK2 V617F Mutation in Iranian Patients with Myeloproliferative Neoplasms

Background and Aim: Multiple lines of evidence have been suggested that JAK2 is likely the main candidate gene responsible for the pathogenesis of myeloproliferative neoplasms. The V617F mutation in the pseudokinase domain of JAK2 protein has been detected in a majority of patients. We aimed to evaluate the frequency of this somatic missense substitution among Iranian patients with myeloproliferative neoplasms. Methods Peripheral blood samples were collected from patients with myeloproliferative neoplasms across different regions of Iran. The JAK2 V617F mutation was identified by allele-specific PCR. To confirm the PCR results, randomly selected positive and negative samples were sequenced. Results: Among 72 identified patients, 45 (62.5%) were found to harbor JAK2 V617F. The frequencies of the mutation ranged 100% for primary myelofibrosis, 75% for chronic myelogenous leukemia, 67% for polycythemia vera, 62.5% for myelodysplastic/myeloproliferative neoplasms, and 52% for essential thrombocythemia. Our findings revealed that the mutation was more common among men in comparison with women and the correlation between the mutation and gender was statistically significant (p-value<0.01). Additionally, the presence of JAK2 V617F was associated with older ages (p-value =0.009). Conclusion: The JAK2 V617F mutation was detected in 62.5% of patients with myeloproliferative neoplasms. We have shown that this single acquired point mutation was presented in at least half of the patients. Hence, it seems that the identification of JAK2 V617F mutation in myeloproliferative neoplasms can be very effective in disease diagnosing and management. *Corresponding Author: Mohammad Hamid; Email: hamidi@pasteur.ac.ir Please cite this article as: Hamid M, Shahbaz Z. Prevalence of JAK2 V617F Mutation in Iranian Patients with Myeloproliferative Neoplasms. Arch Med Lab Sci. 2020;6:1-7 (e5). https://doi.org/10.22037/amls.v6.32758