Protein Kinase Catalytic Domain Research Articles

Phylogenetic and transcriptomic characterization of insulin and growth factor receptor tyrosine kinases in crustaceans.

Receptor tyrosine kinases (RTKs) mediate the actions of growth factors in metazoans. In decapod crustaceans, RTKs are implicated in various physiological processes, such molting and growth, limb regeneration, reproduction and sexual differentiation, and innate immunity. RTKs are organized into two main types: insulin receptors (InsRs) and growth factor receptors, which include epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR). The identities of crustacean RTK genes are incomplete. A phylogenetic analysis of the CrusTome transcriptome database, which included all major crustacean taxa, showed that RTK sequences segregated into receptor clades representing InsR (72 sequences), EGFR (228 sequences), FGFR (129 sequences), and PDGFR/VEGFR (PVR; 235 sequences). These four receptor families were distinguished by the domain organization of the extracellular N-terminal region and motif sequences in the protein kinase catalytic domain in the C-terminus or the ligand-binding domain in the N-terminus. EGFR1 formed a single monophyletic group, while the other RTK sequences were divided into subclades, designated InsR1-3, FGFR1-3, and PVR1-2. In decapods, isoforms within the RTK subclades were common. InsRs were characterized by leucine-rich repeat, furin-like cysteine-rich, and fibronectin type 3 domains in the N-terminus. EGFRs had leucine-rich repeat, furin-like cysteine-rich, and growth factor IV domains. N-terminal regions of FGFR1 had one to three immunoglobulin-like domains, whereas FGFR2 had a cadherin tandem repeat domain. PVRs had between two and five immunoglobulin-like domains. A classification nomenclature of the four RTK classes, based on phylogenetic analysis and multiple sequence alignments, is proposed.

Interaction Networks Explain Holoenzyme Allostery in Protein Kinase A

Protein kinase A (PKA) signaling exemplifies phosphorylation-based signaling as we understand it today. Its catalytic-subunit structure and dynamics continue to advance our understanding of kinase mechanics as the first protein kinase catalytic domain to be identified, sequenced, cloned, and structurally detailed. The PKA holoenzyme elaborates on the role of its regulatory subunits and maintains our understanding of cAMP-dependent cellular signaling. The activation of PKA holoenzymes by cAMP is an example of specialized protein allostery, emphasizing the relevance of protein binding interfaces, unstructured regions, isoform diversity, and dynamics-based allostery. This review provides the most up-to-date overview of PKA structure and function, including a description of the catalytic and regulatory subunits’ structures. In addition, the structure, activation, and allostery of holoenzymes are covered.

Molecular and functional characterization of golden pompano (Trachinotus blochii) TBK1 on IFN regulation

The golden pompano (Trachinotus blochii), a pivotal commercial marine species in China, has gained significant popularity worldwide. However, accompanied with rapid growth and high density aquaculture, golden pompano has been seriously threatened by Nervous necrosis virus (NNV), while its molecular biology research regarding the innate immune system remains unexplored, which is crucial for understanding the activation of interferon (IFN) production and antiviral responses. In this study, we aimed to identify the characterization and function of golden pompano TANK-binding kinase 1 (gpTBK1), thereby providing evidence of the conservation of this classical factor in the RLR pathway among marine fish. Initially, we found the expression of gpTBK1 upregulation in diseased golden pompano with NNV infection and we successfully cloned the full-length open reading frame (ORF) of gpTBK1, consisting of 2172 nucleotides encoding 723 amino acids, from the head kidney. Subsequent analysis of the amino acid sequence revealed homology between gpTBK1 and other fish TBK1 proteins, with conserved N-terminal Serine/Threonine protein kinases catalytic domain (S_TKc) and C-terminal coiled coil domain (CCD). Moreover, the expression pattern showed that gpTBK1 exhibited ubiquitous expression across all evaluated tissues. Furthermore, functional identification experiments indicated that gpTBK1 activated interferon promoters’ activity in golden pompano and induced the expression of downstream IFN-stimulated genes (ISGs). Notably, gpTBK1 was found to co-localize and interact with gpIRF3 in the cytoplasm. Collectively, these data provide a comprehensive analysis of the characterization and functional role of gpTBK1 in promoting interferon production. This research may facilitate the further study of the innate antiviral response, particularly the anti-NNV mechanisms, in golden pompano.

Identification of FAM53C as a cytosolic-anchoring inhibitory binding protein of the kinase DYRK1A.

The protein kinase DYRK1A encoded in human chromosome 21 is the major contributor to the multiple symptoms observed in Down syndrome patients. In addition, DYRK1A malfunction is associated with various other neurodevelopmental disorders such as autism spectrum disorder. Here, we identified FAM53C with no hitherto known biological function as a novel suppressive binding partner of DYRK1A. FAM53C is bound to the catalytic protein kinase domain of DYRK1A, whereas DCAF7/WDR68, the major DYRK1A-binding protein, binds to the N-terminal domain of DYRK1A. The binding of FAM53C inhibited autophosphorylation activity of DYRK1A and its kinase activity to an exogenous substrate, MAPT/Tau. FAM53C did not bind directly to DCAF7/WDR68, whereas DYRK1A tethered FAM53C and DCAF7/WDR68 by binding concurrently to both of them, forming a tri-protein complex. DYRK1A possesses an NLS and accumulates in the nucleus when overexpressed in cells. Co-expression of FAM53C induced cytoplasmic re-localization of DYRK1A, revealing the cytoplasmic anchoring function of FAM53C to DYRK1A. Moreover, the binding of FAM53C to DYRK1A suppressed the DYRK1A-dependent nuclear localization of DCAF7/WDR68. All the results show that FAM53C binds to DYRK1A, suppresses its kinase activity, and anchors it in the cytoplasm. In addition, FAM53C is bound to the DYRK1A-related kinase DYRK1B with an Hsp90/Cdc37-independent manner. The results explain for the first time why endogenous DYRK1A is distributed in the cytoplasm in normal brain tissue. FAM53C-dependent regulation of the kinase activity and intracellular localization of DYRK1A may play a significant role in gene expression regulation caused by normal and aberrant levels of DYRK1A.

Exploration of chlorophyll fluorescence characteristics gene regulatory in rice (Oryza sativa L.): a genome-wide association study.

Chlorophyll content and fluorescence parameters are crucial indicators to evaluate the light use efficiency in rice; however, the correlations among these parameters and the underlying genetic mechanisms remain poorly understood. Here, to clarify these issues, we conducted a genome-wide association study (GWAS) on 225 rice accessions. In the phenotypic and Mendelian randomization (MR) analysis, a weak negative correlation was observed between the chlorophyll content and actual quantum yield of photosystem II (). The phenotypic diversity observed in SPAD, , , and among accessions was affected by genetic background. Furthermore, the GWAS identified 78 SNPs and 17 candidate genes significantly associated with SPAD, , , , and . Combining GWAS on 225 rice accessions with transcriptome analysis of two varieties exhibiting distinct fluorescence characteristics revealed two potential candidate genes (Os03g0583000 from & traits and Os06g0587200 from trait), which are respectively associated with peroxisomes, and protein kinase catalytic domains might involve in regulating the chlorophyll content and chlorophyll fluorescence. This study provides novel insights into the correlation among chlorophyll content and fluorescence parameters and the genetic mechanisms in rice, and offers valuable information for the breeding of rice with enhanced photosynthetic efficiency.

Molecular genetic characterization of sudden deaths due to thoracic aortic dissection or rupture

BackgroundSudden deaths due to thoracic aortic dissection or rupture (TADR) are often investigated by forensic pathologists in the United States. Up to a quarter of reported TADR result from a highly penetrant autosomal dominant single gene variant. Testing genes associated with familial TADR provides an underlying etiology for the cause of death and informs effective sudden death prevention for at-risk family members. At the New York City Office of Chief Medical Examiner (NYC-OCME), TADR cases are routinely tested by the in-house, CAP-accredited Molecular Genetics Laboratory. In this retrospective study, TADR and cardiovascular cases were reviewed to understand the burden of TADR in sudden deaths, value of molecular diagnostic testing in TADR, and genotype-phenotype correlations in a demographically diverse TADR cohort. MethodsBetween July 2019 and June 2022, cases with in-house cardiovascular genetic testing at NYC-OCME were retrospectively reviewed. Twenty genes associated with familial TADR were analyzed using high throughput massive parallel sequencing on postmortem tissues or bloodspot cards. Variant interpretation was conducted according to ACMG/AMP guidelines. ResultsA total of 1078 cases were tested for cardiovascular genetic conditions, of which 34 (3%) had TADR. Eight of those TADR cases had a pathogenic or likely pathogenic variant (P/LPV), 4 had a variant of uncertain significance (VUS), and 22 cases were negative for variants in TADR genes. The molecular diagnostic yield using the TADR subpanel was 23.5%. The genes with the greatest prevalence of P/LPV were FBN1 (6), followed by TGFBR2 (2), TGFBR1 (1), and MYLK (1). Highly penetrant P/LPV in TGFBR2, FBN1, and TGFBR1 were found in TADR individuals who died younger than 34 years old. Two P/LPV in FBN1 were secondary findings unrelated to cause of death. P/LPV in FBN1 included five truncating variants located in the N-terminal domains and one missense variant involved in the disulfide bonds of the EGF-like domain. All P/LPV in TGFBR1 and TGFBR2 were missense or in-frame deletion variants located in the protein kinase catalytic domain. Three variants were first reported in this study. ConclusionsMolecular testing of familial TADR-associated genes is a highly effective tool to identify the genetic cause of TADR sudden deaths and benefits surviving at-risk families.

Abstract 3679: DNAJB1-PRKACA fusion in fibrolamellar hepatocellular carcinoma induces glutamine addiction and an immunosuppressive tumor microenvironment

Abstract Fibrolamellar hepatocellular carcinoma (FLC) is a rare and often lethal form of liver cancer that primarily affects children and young adults. A fusion between DNAJB1, a heat shock chaperone protein, and PRKACA, the catalytic domain of protein kinase A (PKA) has been identified as a signature genomic event in FLC, but the effect of this fusion on the tumor immune microenvironment is not understood. We created an orthotopic, syngeneic model of FLC (TIBx-FLC) by inducing the DNAJB1-PRKACA fusion in a murine hepatoblastoma-derived cell line (TIBx). CD8 T cells isolated from TIBx-FLC tumors demonstrated markedly impaired activation as compared to CD8 T cells isolated from control TIBx tumors. We investigated metabolic programming as a potential mechanism for DNAJB1-PRKACA immunosuppression in FLC. Labeled glucose metabolomics performed on TIBx-FLC and TIBx tumor cells demonstrated a metabolic shift away from aerobic metabolism to an increased glucose contribution towards the hexosamine biosynthetic pathway and purine synthesis, which requires glutamine as a nitrogen source. As compared to the parental TIBx cell line, the TIBx-FLC cell line demonstrated high sensitivity to glutamine antagonism in vitro, consistent with glutamine addiction. Systemic treatment of BALB/c mice bearing TIBx-FLC tumors with JHU-083, a glutamine antagonist, in combination with immune checkpoint inhibitor therapy enhanced survival as compared to vehicle or monotherapy. These data identify altered glutamine metabolism as a target in FLC, and may provide an explanation for immune suppression seen in the FLC tumor microenvironment. Citation Format: Zeal Kamdar, Tamara Lopez-Vidal, Kathryn Howe, Kabeer Munjal, Ali Saeed, Daniel Zabransky, Daniel Shu, Gabriella Longway, Emma Kartalia, James Leatherman, Aditya Mohan, Pratik Khare, Cissy Zhang, Anne Le, Erika Pearce, Mark Furth, Marina Baretti, Robert Leone, Elizabeth Jaffee, Mark Yarchoan. DNAJB1-PRKACA fusion in fibrolamellar hepatocellular carcinoma induces glutamine addiction and an immunosuppressive tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3679.

Evolutionary divergence in the conformational landscapes of tyrosine vs serine/threonine kinases.

Inactive conformations of protein kinase catalytic domains where the DFG motif has a "DFG-out" orientation and the activation loop is folded present a druggable binding pocket that is targeted by FDA-approved 'type-II inhibitors' in the treatment of cancers. Tyrosine kinases (TKs) typically show strong binding affinity with a wide spectrum of type-II inhibitors while serine/threonine kinases (STKs) usually bind more weakly which we suggest here is due to differences in the folded to extended conformational equilibrium of the activation loop between TKs vs. STKs. To investigate this, we use sequence covariation analysis with a Potts Hamiltonian statistical energy model to guide absolute binding free-energy molecular dynamics simulations of 74 protein-ligand complexes. Using the calculated binding free energies together with experimental values, we estimated free-energy costs for the large-scale (~17-20 Å) conformational change of the activation loop by an indirect approach, circumventing the very challenging problem of simulating the conformational change directly. We also used the Potts statistical potential to thread large sequence ensembles over active and inactive kinase states. The structure-based and sequence-based analyses are consistent; together they suggest TKs evolved to have free-energy penalties for the classical 'folded activation loop' DFG-out conformation relative to the active conformation, that is, on average, 4-6 kcal/mol smaller than the corresponding values for STKs. Potts statistical energy analysis suggests a molecular basis for this observation, wherein the activation loops of TKs are more weakly 'anchored' against the catalytic loop motif in the active conformation and form more stable substrate-mimicking interactions in the inactive conformation. These results provide insights into the molecular basis for the divergent functional properties of TKs and STKs, and have pharmacological implications for the target selectivity of type-II inhibitors.

Role of c-Jun NH2 -terminal kinase-mediated mitogen-activated protein kinase pathway in response to pesticides in Apis cerana cerana.

The mitogen-activated protein kinase (MAPK) cascade pathway plays an important role in regulating stress responses. The function of the c-Jun NH2 -terminal kinase (JNK), a component of the MAPK cascade pathway, in Apis cerana cerana (Acc) remains unclear. Here, JNK was isolated and identified from Acc. Bioinformatics analyses revealed there is a typical serine/threonine protein kinase catalytic domain in the AccJNK protein. An expression profile analysis showed that AccJNK was significantly induced by pesticide treatments. To further explore the functional mechanisms of AccJNK, a yeast 2-hybrid screen was performed, activator protein-1 (AP-1) was screened as the interaction partner of AccJNK, and the interaction relationship was further verified by pull-down assay. Quantitative real-time polymerase chain reaction showed the expression pattern of AccAP-1 was similar to that of AccJNK. After a knockdown of AccJNK or AccAP-1 by RNA interference, the survival rate of Acc after pesticide treatments increased. Additionally, the expression levels of antioxidant-related genes and the activities of antioxidant enzymes increased, suggesting that the knockdown of AccJNK or AccAP-1 increased the antioxidant capacity of bees. Our study revealed that the JNK-mediated MAPK pathway responds to pesticide stress by altering the antioxidant capacity of Acc.

Neurologic and neurodevelopmental complications in cardiofaciocutaneous syndrome are associated with genotype: A multinational cohort study

PurposeDysregulation of RAS or its major effector pathway is the molecular mechanism of RASopathies, a group of multisystemic congenital disorders. Neurologic complications are especially challenging in the management of the rare RASopathy cardiofaciocutaneous (CFC) syndrome. This study evaluated clinical neurologic and neurodevelopmental features and their associations with CFC syndrome gene variants. MethodsA multinational cohort of 138 individuals with CFC syndrome (BRAF = 90, MAP2K1 = 36, MAP2K2 = 10, KRAS = 2) was recruited. Neurologic presentation was captured via clinician review of medical records and caregiver-completed electronic surveys. Validated measures of seizure severity, adaptive function, and gross motor function were obtained. ResultsThe overall frequency of intellectual disability and seizures was 82% and 55%, respectively. The frequency and severity of seizures was higher among individuals with BRAF or MAP2K1 variants than in those with MAP2K2 variants. A disproportionate incidence of severe, treatment-resistant seizures was observed in patients with variants in the catalytic protein kinase domain of BRAF and at the common p.Y130 site of MAP2K1. Neurodevelopmental outcomes were associated with genotype as well as seizure severity. ConclusionMolecular genetic testing can aid in prediction of epilepsy and neurodevelopmental phenotypes in CFC syndrome. Study results identified potential CFC syndrome-associated variants in the development of relevant animal models for neurologic, neurocognitive, and motor function impairment.

TmIKKε Is Required to Confer Protection Against Gram-Negative Bacteria, E. coli by the Regulation of Antimicrobial Peptide Production in the Tenebrio molitor Fat Body.

The inhibitor of nuclear factor-kappa B (NF-κB) kinase (IKK) is the core regulator of the NF-κB pathway against pathogenic invasion in vertebrates or invertebrates. IKKβ, -ε and -γ have pivotal roles in the Toll and immune deficiency (IMD) pathways. In this study, a homolog of IKKε (TmIKKε) was identified from Tenebrio molitor RNA sequence database and functionally characterized for its role in regulating immune signaling pathways in insects. The TmIKKε gene is characterized by two exons and one intron comprising an open reading frame (ORF) of 2,196 bp that putatively encodes a polypeptide of 731 amino acid residues. TmIKKε contains a serine/threonine protein kinases catalytic domain. Phylogenetic analysis established the close homology of TmIKKε to Tribolium castaneum IKKε (TcIKKε) and its proximity with other IKK-related kinases. The expression of TmIKKε mRNA was elevated in the gut, integument, and hemocytes of the last-instar larva and the fat body, Malpighian tubules, and testis of 5-day-old adults. TmIKKε expression was significantly induced by Escherichia coli, Staphylococcus aureus, and Candida albicans challenge in whole larvae and tissues, such as hemocytes, gut, and fat body. The knockdown of the TmIKKε messenger RNA (mRNA) expression significantly reduced the survival of the larvae against microbial challenges. Further, we investigated the induction patterns of 14 T. molitor antimicrobial peptides (AMPs) genes in TmIKKε gene-silencing model after microbial challenges. While in hemocytes, the transcriptional regulation of most AMPs was negatively regulated in the gut and fat body tissue of T. molitor, AMPs, such as TmTenecin 1, TmTenecin 4, TmDefensin, TmColeoptericin A, TmColeoptericin B, TmAttacin 1a, and TmAttacin 2, were positively regulated in TmIKKε-silenced individuals after microbial challenge. Collectively, the results implicate TmIKKε as an important factor in antimicrobial innate immune responses in T. molitor.

The Eriocheir sinensis calcium/calmodulin-dependent protein kinase II activates apoptosis to resist Spiroplasma eriocheiris infection

Calcium/calmodulin-dependent protein kinase II is a downstream mediator of calcium signalling and participates in the regulation of various cellular physiological functions. In previous studies, the expression of Eriocheir sinensis CaMKII (EsCaMKII) was significantly decreased in the thoracic ganglion after Spiroplasma eriocheiris infection, as shown using TMT-based quantitative proteomic analysis; however, the specific functions of EsCaMKII are still unclear. In this study, the full-length cDNA of EsCaMKII was 3314 bp long, consisting of a 1605 bp open reading frame encoding a protein of 535 amino acids, including a 258 aa serine/threonine protein kinase catalytic domain (EsCaMKII-CD). EsCaMKII is highly transcribed in haemocytes, nerves (thoracic ganglion), gills, and muscles, but lowly transcribed in the hepatopancreas, heart, and intestines. The transcription levels of EsCaMKII were altered in E. sinensis haemocytes after S. eriocheiris infection. After the over-expression of EsCaMKII-CD in RAW264.7 cells, the apoptosis rate of RAW264.7 cells was significantly increased. After the over-expression of EsCaMKII-CD, the morphology of RAW264.7 cells became worse after being infected with S. eriocheiris. Meanwhile, the copy number of S. eriocheiris in RAW264.7 cells was significantly decreased. From 48 h to 96 h after EsCaMKII RNA interference, the transcription levels of EsCaMKII decreased significantly. The transcription of apoptosis genes and cell apoptosis were also inhibited in haemocytes after EsCaMKII RNAi. The knockdown of EsCaMKII by RNAi resulted in significant increases in the copy number of S. eriocheiris and in the mortality of crabs during S. eriocheiris infection. These results indicate that EsCaMKII could promote the apoptosis of E. sinensis and enhance its ability to resist S. eriocheiris infection.

Cloning and functional analysis of the novel rice blast resistance gene Pi65 in japonica rice.

Pi65, a leucine-rich repeat receptor-like kinase (LRR-RLK) domain cloned from Oryza sativa japonica, is a novel rice blast disease resistance gene. Rice blast seriously threatens rice production worldwide. Utilizing the rice blast resistance gene to breed rice blast-resistant varieties is one of the best ways to control rice blast disease. Using a map-based cloning strategy, we cloned a novel rice blast resistance gene, Pi65, from the resistant variety GangYu129 (abbreviated GY129, Oryza sativa japonica). Overexpression of Pi65 in the susceptible variety LiaoXing1 (abbreviated LX1, Oryza sativa japonica) enhanced rice blast resistance, while knockout of Pi65 in GY129 resulted in susceptibility to rice blast disease. Pi65 encodes two transmembrane domains, with 15 LRR domains and one serine/threonine protein kinase catalytic domain, conferring resistance to isolates of Magnaporthe oryzae (abbreviated M. oryzae) collected from Northeast China. There were sixteen amino acid differences between the Pi65 resistance and susceptible alleles. Compared with the Pi65-resistant allele, the susceptible allele exhibited one LRR domain deletion. Pi65 was constitutively expressed in whole plants, and it could be induced in the early stage of M. oryzae infection. Transcriptome analysis revealed that numerous genes associated with disease resistance were specifically upregulated in GY129 24h post inoculation (HPI); in contrast, photosynthesis and carbohydrate metabolism-related genes were particularly downregulated at 24 HPI, demonstrating that disease resistance-associated genes were activated in GY129 (carrying Pi65) after rice blast fungal infection and that cellular basal metabolism and energy metabolism were inhibited simultaneously. Our study provides genetic resources for improving rice blast resistance and enriches the study of rice blast resistance mechanisms.

Glycogen Synthase Kinase 3 Gene Is Important in Growth and Molting of the Pacific White Shrimp Litopenaeus vannamei

Glycogen synthase kinase 3 (GSK3) is a vital multifunctional molecule that is widely distributed in invertebrates and vertebrates. GSK3 is a highly conserved serine/threonine (Ser/Thr) protein kinase, which plays an important role in insulin, Wnt, and various signaling pathways. In this study, a GSK3 gene were identified in the genome of the Pacific white shrimp, Litopenaeus vannamei, and analyzed its gene structure, phylogeny, and expression profiles. The deduced LvGSK3 protein contains a highly conserved Ser/Thr protein kinase catalytic (S_TKc) domain, the LvGSK3 gene exhibited high expression in different early developmental stages, most adult tissues, and premolting stages. RNA interference of LvGSK3 significantly retarded the increment of body weight and affected the expressions of molting-related genes compared with control groups. These results will improve our understanding of the conserved structure and functions of the LvGSK3 gene and show potential applications of shrimp growth.

A newly identified Hippo homologue from the oriental river prawn Macrobrachium nipponense is involved in the antimicrobial immune response

The Hippo signalling pathway plays a vital role in organ size control, cell proliferation, apoptosis, and immune regulation. In this study, a Hippo homologue with three isoforms (named MnHippo-a, MnHippo-b, and MnHippo-c) was isolated and characterized for the first time from the freshwater prawn Macrobrachium nipponense. The deduced amino acid sequences of MnHippo-a (698 aa), MnHippo-b (688 aa), and MnHippo-c (656 aa) were highly similar, and they all contained an N-terminal S_TKc (serine/threonine protein kinase catalytic) domain and a C-terminal Mst1_SARAH (Sav/Rassf/Hpo) domain. MnHippo-a and MnHippo-c were derived from alternative splicing. Phylogenetic analysis was performed, and the results revealed that MnHippo was a member of the clade containing STPK4 and Hippo of Penaeus vannamei. The expression distribution showed that MnHippo was constitutively expressed in various tissues of uninfected prawns and highly expressed in the hepatopancreas and intestine. In prawns challenged with Vibrio parahaemolyticus and Staphylococcus aureus, the expression of MnHippo in haemocytes was significantly upregulated. Furthermore, in MnHippo-knockdown prawns injected with V. parahaemolyticus or S. aureus, the transcription levels of five antimicrobial peptides were downregulated. MnHippo silencing weakened the clearance of V. parahaemolyticus and S. aureus in prawns. The survival rate of the MnHippo-dsRNA group was obviously decreased from 2 to 6 days post-injection with V. parahaemolyticus or S. aureus. Hence, MnHippo might be involved in the antibacterial immune defence of M. nipponense.

NLK suppresses MAVS-mediated signaling in black carp antiviral innate immunity

Mammalian Nemo-like kinase (NLK) plays important roles in multiple biological processes including immune response; however, the roles of teleost NLK remain largely unknown. In the present study, the NLK homolog (bcNLK) of black carp (Mylopharyngodon piceus) has been cloned and characterized. The coding region of bcNLK consists of 1427 nucleotides and encodes 476 amino acid, including two low complexity region (LCR) domains at the N-terminus and a serine/threonine protein kinase catalytic (S-TKc) domain in the middle region. The transcription of bcNLK are promoted after spring viremia of carp virus (SVCV) infection and poly (I:C) stimulation in host cells, but not post LPS treatment. bcNLK exhibits weak impact on the transcription of interferon (IFN) promoter in the reporter assay, however, black carp MAVS (bcMAVS)-mediated IFN promoter transcription is remarkably dampened by bcNLK. The interaction between bcNLK and bcMAVS is detected through the co-immunoprecipitation assay. Accordingly, the plaque assay results show that bcMAVS-mediated antiviral ability is impaired by bcNLK. Moreover, knockdown of bcNLK in host cells leads to the enhanced antiviral ability against SVCV. All these data support the conclusion that black carp NLK associates with MAVS and inhibited MAVS-mediated antiviral signaling.

Expression of the Testis-Specific Serine/Threonine Kinases Suggests Their Role in Spermiogenesis of Bay Scallop Argopecten irradians.

Members of the testis-specific serine/threonine kinases (Tssk) family play critical roles in spermatogenesis in vertebrates. But in mollusks, research on Tssk family is still lagging. In this study, we systematically identified Tssk family based on the genomic and transcriptomic data from a commercially important scallop Argopecten irradians and detected the spatiotemporal expression in adult gonads. Five members were identified, with the gene length varying from 1,068 to 10,729 bp and the protein length ranging from 294 to 731 aa. All the Tssks possess a serine/threonine protein kinase catalytic (S_TKc) domain. Phylogenetic analysis revealed existence of four homologs of vertebrate Tssk1/2, Tssk3, Tssk4, Tssk5, and absence of Tssk6 in the scallop. The remaining gene (Tssk7) formed an independent clade with Tssks of other mollusks and arthropods, indicating that it may be a new member of Tssk family unique to protostomes. By investigating the expression of Tssks in four developmental stages of testes and ovaries, we found all five Tssks were primarily expressed in mature testis. In situ hybridization experiment revealed the five Tssks were localized in the spermatids and spermatozoa. The testis-predominant expression of Tssk family suggests Tssks may play pivotal roles in spermiogenesis in the scallop. Our study provides basic information on the characteristics and expression profiles of Tssk family of A. irradians. To our knowledge, it represents the first comprehensive analysis of Tssk family in mollusks.

Structural and Biochemical Studies Confirming the Mechanism of Action of Asciminib, an Agent Specifically Targeting the ABL Myristoyl Pocket (STAMP)

Tyrosine kinase inhibitors (TKIs) that inhibit the transphosphorylation activity of the BCR-ABL1 oncoprotein by binding the ATP-binding site of the catalytic domain of protein kinases are well established as being effective drugs for the treatments of chronic myeloid leukemia (CML). However, the off-target kinase activities of these non-specific TKIs are associated with adverse events that can limit their suitability for the treatment of patients and can negatively impact quality of life. Therefore, a new drug combining high efficacy with minimal side-effects could provide substantial therapeutic advantages. Asciminib is a new investigational agent that at concentrations <10 micromolar has very little effect (residual activities ≥66%) on the transphosphorylation activity of a large panel comprising of 335wild-typeprotein kinases (including ABL1 and ABL2). In this study (33PanQinase® Activity Assay, ProQinase GmbH, Freiburg, Germany) the radiometric kinase assay employed recombinant kinase constructs of the catalytic domains including the ATP-binding site. However, in isothermal calorimetry studies asciminib showed strong binding affinity (KD 0.5 nM) to a much larger construct ofwild-typeABL1 that contains the catalytic, SH2 and SH3 domains (residues 46-534, ABL46-534), with thermodynamic parameters (ΔH = -72.8 kJ/mol, ΔS = -65.3 J/mol/K, resulting in ΔG = -53.2 kJ/mol) indicating strong enthalpy-driven, entropically unfavorable binding. This binding translates to inhibition of tyrosine phosphorylation catalysed by the ABL64-515 construct with a mean IC50 value of 2.6 ± 0.8 nM (radiometric filter binding assay) and 0.5 ± 0.1 nM (fluorescence resonance energy transfer assay). This data shows that asciminib inhibits the kinase activity of ABL1 by an allosteric mechanism which does not involve direct interaction with the ATP-binding pocket. X-ray crystallographic studies of a ternary complex between asciminib, nilotinib and the ABL146-534 protein possessing Thr315Ile and Asp382Asn substitutions show that asciminib binds to ABL1 in a deep-pocket on the C-lobe of the kinase domain, referred to as the myristate (or myristoyl) pocket. Nuclear magnetic resonance studies confirm that asciminib can also form asciminib-ABL1-TKI ternary complexes with thewild-typeABL83-534 protein in solution. Native ABL1 kinase is post-translationally myristoylated at the N-terminal glycine residue and this myristate group plays an important role in autoregulating the kinase by binding to a pocket (myristate pocket) on the catalytic domain of the protein to induce the formation of an inactive conformation of the enzyme. This regulatory mechanism is lost in BCR-ABL1 since the N-terminal region is replaced in the fusion protein with a fragment of the BCR protein, thus rendering the ABL1 kinase constitutively active. The binding of asciminib in this pocket therefore mimics that of myristate, thus stabilizing the inactive state of the ABL1 kinase. Consistent with this binding mode to the ABL1 kinase, asciminib potently inhibits BCR-ABL1 driven proliferation of leukemia-derived cell lines, while having little effect on cells that do not express BCR-ABL1 (Figure 1). Thein vitrodata translates into anti-tumor activity in mouse models of CML where asciminib dose-dependently inhibited the growth of subcutaneous KCL22 cell xenografts, with 30 mg/kg administered twice-daily by oral gavage affording 92% tumor regression (Figure 2). Asciminib, the first-in-class STAMP (Specifically Targeting the ABL Myristoyl Pocket) inhibitor, has several important features as a potential treatment of CML. At physiologically achievable concentrations asciminib can overcome mutations on the ATP-binding site of BCR-ABL1 that impede the binding of TKIs which lead to drug resistance in patients with CML. Secondly, by not inhibiting kinases such as EGFR, KIT, CSF1R, PDGFR or the sSRC-family kinases that are associated with off-target activities of TKIs such as bosutinib, dasatinib, imatinib, nilotinib and ponatinib, asciminib is not expected to be associated with cross-intolerance. Thirdly, asciminib can bind to the ABL1 kinase domain together with ATP-competitive TKIs to form ternary complexes, such that appropriate drug combinations should greatly impede the emergence of drug resistant kinase mutations. Disclosures Schuld: Novartis Pharma AG:Current Employment, Current equity holder in publicly-traded company.Grzesiek:Novartis Pharma:Research Funding.Jahnke:Novartis:Current Employment, Current equity holder in publicly-traded company, Patents & Royalties, Research Funding.Barys:Novartis Pharma AG:Current Employment.Cowan-Jacob:Novartis Pharma AG:Current Employment.Loo:Novartis Pharma AG:Current Employment.Wiget:Novartis Pharma AG:Current Employment.Manley:Novartis Pharma AG:Current Employment.

TBK1 regulates the induction of innate immune response against GCRV by phosphorylating IRF3 in rare minnow (Gobiocypris rarus)

Rare minnow (Gobiocypris rarus), a small cyprinid species that is highly sensitive to the grass carp reovirus (GCRV), is regarded as an ideal model to study the mechanisms of innate immunity in fish. In the present study, a TBK1 homologue from rare minnow (GrTBK1) was identified and its roles in defence against viral infection were investigated. Sequence analysis showed that GrTBK1 encoded a 727-amino acid peptide which shared 98% and 72% identity to the black carp (Mylopharyngodon piceus) and human (Homo sapiens) orthologues, respectively. The amino acid sequence analysis demonstrated that GrTBK1 contains a conserved Serine/Threonine protein kinases catalytic domain (S_TKc) at the N-terminus. Furthermore, cellular distribution proved that GrTBK1 was located in the cytoplasm region. Quantitative real-time PCR analysis revealed that GrTBK1 was ubiquitously expressed in all examined organs, but especially highly in liver. Temporal expression analysis in vivo showed that the expression levels of GrTBK1 were obviously up-regulated in response to GCRV infection. Meanwhile, qRT-PCR assay revealed that the levels of S7 RNA, an important segment of GCRV genome, were higher in the liver than in other tissues. This indicates that GrTBK1 might play a crucial role in responses to GCRV infection in fish. In addition, GrTBK1 activated several type I interferon (IFN) promoters and induced the expression of downstream type I IFN-stimulated genes (ISGs). Furthermore, GrTBK1 obviously phosphorylated the interferon regulatory factor 3 (IRF3). Furthermore, overexpression of GrTBK1 remarkably decreased the GCRV proliferation. In summary, we systematically characterized GrTBK1 and illustrated its role in the innate immune response to GCRV infections.

Molecular characterization of a cyprinid fish (Ancherythroculter nigrocauda) TBK1 and its kinase activity in IFN regulation

TANK-binding kinase 1 (TBK1) plays a vital role in activating interferon (IFN) production and positively regulating antiviral response in mammals. Research on more species of fish is necessary to clarify whether the function of fish TBK1 is conserved compared to that in mammals. Here, a cyprinid fish (Ancherythroculter nigrocauda) TBK1 (AnTBK1) was functionally identified and characterized. The full-length open reading frame (ORF) of AnTBK1 consists of 2184 nucleotides encoding 727 amino acids and contains a conserved Serine/Threonine protein kinase catalytic domain (S_TKc) in the N-terminal, similar to TBK1 in other species. The transcripts of AnTBK1 were found in all the tissues evaluated and the cellular distribution indicated that AnTBK1 was localized in the cytoplasm. In terms of functional identification, AnTBK1 induced a variety of IFN promoter activities as well as the expression of downstream IFN-stimulated genes (ISGs). In addition, AnTBK1 interacted with and significantly phosphorylated IFN regulatory factor 3 (IRF3), exhibiting the canonical kinase activity of TBK1. Finally, AnTBK1 presented strong antiviral activity against spring viremia of carp virus (SVCV) infection. Taken together, our research on the features and functions of AnTBK1 demonstrated that AnTBK1 plays a central role in IFN induction against SVCV infection.