ATP binding to the pseudokinase domain of JAK2 is critical for pathogenic activation

Pseudokinases lack conserved motifs typically required for kinase activity. Nearly half of pseudokinases bind ATP, but only few retain phosphotransfer activity, leaving the functional role of nucleotide binding in most cases unknown. Janus kinases (JAKs) are nonreceptor tyrosine kinases with a tandem pseudokinase–kinase domain configuration, where the pseudokinase domain (JAK homology 2, JH2) has important regulatory functions and harbors mutations underlying hematological and immunological diseases. JH2 of JAK1, JAK2, and TYK2 all bind ATP, but the significance of this is unclear. We characterize the role of nucleotide binding in normal and pathogenic JAK signaling using comprehensive structure-based mutagenesis. Disruption of JH2 ATP binding in wild-type JAK2 has only minor effects, and in the presence of type I cytokine receptors, the mutations do not affect JAK2 activation. However, JH2 mutants devoid of ATP binding ameliorate the hyperactivation of JAK2 V617F. Disrupting ATP binding in JH2 also inhibits the hyperactivity of other pathogenic JAK2 mutants, as well as of JAK1 V658F, and prevents induction of erythrocytosis in a JAK2 V617F myeloproliferative neoplasm mouse model. Molecular dynamic simulations and thermal-shift analysis indicate that ATP binding stabilizes JH2, with a pronounced effect on the C helix region, which plays a critical role in pathogenic activation of JAK2. Taken together, our results suggest that ATP binding to JH2 serves a structural role in JAKs, which is required for aberrant activity of pathogenic JAK mutants. The inhibitory effect of abrogating JH2 ATP binding in pathogenic JAK mutants may warrant novel therapeutic approaches.

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Essential role of IPS-1 in innate immune responses against RNA viruses

Journal of Experimental Medicine ◽

10.1084/jem.20060792 ◽

2006 ◽

Vol 203 (7) ◽

pp. 1795-1803 ◽

Cited By ~ 345

Author(s):

Himanshu Kumar ◽

Taro Kawai ◽

Hiroki Kato ◽

Shintaro Sato ◽

Ken Takahashi ◽

...

Keyword(s):

Rna Viruses ◽

Rna Virus ◽

Critical Role ◽

Nuclear Factor Κb ◽

Type I ◽

Innate Immune Responses ◽

Antiviral Responses ◽

Deficient Mice

IFN-β promoter stimulator (IPS)-1 was recently identified as an adapter for retinoic acid–inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (Mda5), which recognize distinct RNA viruses. Here we show the critical role of IPS-1 in antiviral responses in vivo. IPS-1–deficient mice showed severe defects in both RIG-I– and Mda5-mediated induction of type I interferon and inflammatory cytokines and were susceptible to RNA virus infection. RNA virus–induced interferon regulatory factor-3 and nuclear factor κB activation was also impaired in IPS-1–deficient cells. IPS-1, however, was not essential for the responses to either DNA virus or double-stranded B-DNA. Thus, IPS-1 is the sole adapter in both RIG-I and Mda5 signaling that mediates effective responses against a variety of RNA viruses.

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Role of the chaperone protein 14-3-3ε in the regulation of influenza A virus-activated beta interferon

Journal of Virology ◽

10.1128/jvi.00231-21 ◽

2021 ◽

Author(s):

Ee-Hong Tam ◽

Yen-Chin Liu ◽

Chian-Huey Woung ◽

Helene Minyi Liu ◽

Guan-Hong Wu ◽

...

Keyword(s):

Virus Infection ◽

Influenza A Virus ◽

Influenza A ◽

Critical Role ◽

Type I ◽

Type I Ifn ◽

Ns1 Protein ◽

Chaperone Protein ◽

Influenza A Virus Infection

The NS1 protein of the influenza A virus plays a critical role in regulating several biological processes in cells, including the type I interferon (IFN) response. We previously profiled the cellular factors that interact with the NS1 protein of influenza A virus and found that the NS1 protein interacts with proteins involved in RNA splicing/processing, cell cycle regulation, and protein targeting processes, including 14-3-3ε. Since 14-3-3ε plays an important role in RIG-I translocation to MAVS to activate type I IFN expression, the interaction of the NS1 and 14-3-3ε proteins may prevent the RIG-I-mediated IFN response. In this study, we confirmed that the 14-3-3ε protein interacts with the N-terminal domain of the NS1 protein and that the NS1 protein inhibits RIG-I-mediated IFN-β promoter activation in 14-3-3ε-overexpressing cells. In addition, our results showed that knocking down 14-3-3ε can reduce IFN-β expression elicited by influenza A virus and enhance viral replication. Furthermore, we found that threonine in the 49 th amino acid position of the NS1 protein plays a role in the interaction with 14-3-3ε. Influenza A virus expressing C-terminus-truncated NS1 with T49A mutation dramatically increases IFN-β mRNA in infected cells and causes slower replication than that of virus without the T-to-A mutation. Collectively, this study demonstrates that 14-3-3ε is involved in influenza A virus-initiated IFN-β expression and that the interaction of the NS1 protein and 14-3-3ε may be one of the mechanisms for inhibiting type I IFN activation during influenza A virus infection. IMPORTANCE Influenza A virus is an important human pathogen causing severe respiratory disease. The virus has evolved several strategies to dysregulate the innate immune response and facilitate its replication. We demonstrate that the NS1 protein of influenza A virus interacts with the cellular chaperone protein 14-3-3ε, which plays a critical role in RIG-I translocation that induces type I IFN expression, and that NS1 protein prevents RIG-I translocation to mitochondrial membrane. The interaction site for 14-3-3ε is the RNA-binding domain (RBD) of the NS1 protein. Therefore, this research elucidates a novel mechanism by which the NS1 RBD mediates IFN-β suppression to facilitate influenza A viral replication. Additionally, the findings reveal the antiviral role of 14-3-3ε during influenza A virus infection.

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PDGF-AA and its receptor influence early lung branching via an epithelial-mesenchymal interaction

Development ◽

10.1242/dev.121.8.2559 ◽

1995 ◽

Vol 121 (8) ◽

pp. 2559-2567 ◽

Cited By ~ 3

Author(s):

P. Souza ◽

M. Kuliszewski ◽

J. Wang ◽

I. Tseu ◽

A.K. Tanswell ◽

...

Keyword(s):

Dna Synthesis ◽

Critical Role ◽

Polymerase Chain Reaction Analysis ◽

Branching Morphogenesis ◽

Inhibitory Effect ◽

Embryonic Lung ◽

Morphometric Analyses ◽

Terminal Buds ◽

Lung Branching

The biological role of platelet-derived growth factor (PDGF)-AA in lung morphogenesis was investigated by incubating embryonic lung explants with phosphorothioate antisense PDGF-A oligonucleotides, which decreased PDGF-AA but not PDGF-BB protein content. Antisense PDGF-A oligonucleotides inhibited DNA synthesis. This inhibitory effect of antisense PDGF-A was reversed by the addition of exogenous PDGF-AA but not PDGF-BB. Morphometric analyses of antisense-treated cultures showed a significant reduction in lung size. The number of terminal buds of the lung explants was significantly decreased by antisense PDGF-A oligonucleotides. PDGF-AA but not PDGF-BB attenuated the inhibitory effect of antisense PDGF-A on early lung branching. Sense PDGF-A had no effect on DNA synthesis and early lung branching. Reverse transcriptase-polymerase chain reaction analysis revealed PDGF-A mRNA expression in the epithelial component of the embryonic lung, while message for PDGF alpha-receptor was expressed in the mesenchyme. Incubation of explants with neutralizing PDGF-AA antibodies also reduced DNA synthesis and early branching morphogenesis. We conclude that PDGF-AA and its receptor represent an important epithelial-mesenchymal interaction which plays a critical role in early lung branching morphogenesis.

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Cutting Edge: Critical Role of IκB Kinase α in TLR7/9-Induced Type I IFN Production by Conventional Dendritic Cells

The Journal of Immunology ◽

10.4049/jimmunol.0901648 ◽

2010 ◽

Vol 184 (7) ◽

pp. 3341-3345 ◽

Cited By ~ 26

Author(s):

Katsuaki Hoshino ◽

Izumi Sasaki ◽

Takahiro Sugiyama ◽

Takahiro Yano ◽

Chihiro Yamazaki ◽

...

Keyword(s):

Dendritic Cells ◽

Critical Role ◽

Cutting Edge ◽

Type I ◽

Type I Ifn ◽

Iκb Kinase

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Role of phosphoinositide 3-kinase β in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways

Biochemical Journal ◽

10.1042/bj20100166 ◽

2010 ◽

Vol 429 (2) ◽

pp. 369-377 ◽

Cited By ~ 64

Author(s):

Analia Garcia ◽

Soochong Kim ◽

Kamala Bhavaraju ◽

Simone M. Schoenwaelder ◽

Satya P. Kunapuli

Keyword(s):

Platelet Aggregation ◽

Critical Role ◽

Thromboxane A2 ◽

Inhibitory Effect ◽

Human Platelets ◽

P2y12 Receptor ◽

Functional Responses ◽

Induce Platelet Aggregation ◽

Erk Phosphorylation

PI3Ks (phosphoinositide 3-kinases) play a critical role in platelet functional responses. PI3Ks are activated upon P2Y12 receptor stimulation and generate pro-aggregatory signals. P2Y12 receptor has been shown to play a key role in the platelet aggregation and thromboxane A2 generation caused by co-stimulation with Gq or Gz, or super-stimulation of Gi pathways. In the present study, we evaluated the role of specific PI3K isoforms α, β, γ and δ in platelet aggregation, thromboxane A2 generation and ERK (extracellular-signal-regulated kinase) activation. Our results show that loss of the PI3K signal impaired the ability of ADP to induce platelet aggregation, ERK phosphorylation and thromboxane A2 generation. We also show that Gq plus Gi- or Gi plus Gz-mediated platelet aggregation, ERK phosphorylation and thromboxane A2 generation in human platelets was inhibited by TGX-221, a PI3Kβ-selective inhibitor, but not by PIK75 (a PI3Kα inhibitor), AS252424 (a PI3Kγ inhibitor) or IC87114 (a PI3Kδ inhibitor). TGX-221 also showed a similar inhibitory effect on the Gi plus Gz-mediated platelet responses in platelets from P2Y1−/− mice. Finally, 2MeSADP (2-methyl-thio-ADP)-induced Akt phosphorylation was significantly inhibited in the presence of TGX-221, suggesting a critical role for PI3Kβ in Gi-mediated signalling. Taken together, our results demonstrate that PI3Kβ plays an important role in ADP-induced platelet aggregation. Moreover, PI3Kβ mediates ADP-induced thromboxane A2 generation by regulating ERK phosphorylation.

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A critical role for IRAK4 kinase activity in Toll-like receptor–mediated innate immunity

Journal of Experimental Medicine ◽

10.1084/jem.20061825 ◽

2007 ◽

Vol 204 (5) ◽

pp. 1025-1036 ◽

Cited By ~ 174

Author(s):

Tae Whan Kim ◽

Kirk Staschke ◽

Katarzyna Bulek ◽

Jianhong Yao ◽

Kristi Peters ◽

...

Keyword(s):

Immune Responses ◽

Kinase Activity ◽

Critical Role ◽

Nuclear Factor Κb ◽

Type I ◽

Toll Like Receptor ◽

Chemokine Production ◽

Cytokines And Chemokines ◽

Plasmacytoid Dcs

IRAK4 is a member of IL-1 receptor (IL-1R)–associated kinase (IRAK) family and has been shown to play an essential role in Toll-like receptor (TLR)–mediated signaling. We recently generated IRAK4 kinase-inactive knock-in mice to examine the role of kinase activity of IRAK4 in TLR-mediated signaling pathways. The IRAK4 kinase–inactive knock-in mice were completely resistant to lipopolysaccharide (LPS)- and CpG-induced shock, due to impaired TLR-mediated induction of proinflammatory cytokines and chemokines. Although inactivation of IRAK4 kinase activity did not affect the levels of TLR/IL-1R–mediated nuclear factor κB activation, a reduction of LPS-, R848-, and IL-1–mediated mRNA stability contributed to the reduced cytokine and chemokine production in bone marrow–derived macrophages from IRAK4 kinase–inactive knock-in mice. Both TLR7- and TLR9-mediated type I interferon production was abolished in plasmacytoid dendritic cells isolated from IRAK4 knock-in mice. In addition, influenza virus–induced production of interferons in plasmacytoid DCs was also dependent on IRAK4 kinase activity. Collectively, our results indicate that IRAK4 kinase activity plays a critical role in TLR-dependent immune responses.

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Type I interferons induce peripheral T regulatory cell differentiation under tolerogenic conditions

International Immunology ◽

10.1093/intimm/dxaa058 ◽

2020 ◽

Cited By ~ 1

Author(s):

Sara Vitale ◽

Valentina Russo ◽

Beatrice Dettori ◽

Cecilia Palombi ◽

Denis Baev ◽

...

Keyword(s):

Cell Differentiation ◽

T Cell ◽

Dominant Role ◽

Critical Role ◽

Peripheral Tolerance ◽

Type I Interferons ◽

Type I ◽

T Regulatory Cell ◽

Cell Conversion

Abstract The type I interferons are central to a vast array of immunological functions. The production of these immune-modulatory molecules is initiated at the early stages of the innate immune responses and, therefore, plays a dominant role in shaping downstream events in both innate and adaptive immunity. Indeed, the major role of IFN-α/β is the induction of priming states, relevant for the functional differentiation of T lymphocyte subsets. Among T-cell subtypes, the CD4+CD25+Foxp3+ T regulatory cells (Tregs) represent a specialized subset of CD4+ T cells with a critical role in maintaining peripheral tolerance and immune homeostasis. Although the role of type I interferons in maintaining the function of thymus-derived Tregs has been previously described, the direct contribution of these innate factors to peripheral Treg (pTreg) and induced Treg (iTreg) differentiation and suppressive function is still unclear. We now show that, under tolerogenic conditions, IFN-α/β play a critical role in antigen-specific and also polyclonal naive CD4+ T-cell conversion into peripheral antigen-specific CD4+CD25+Foxp3+ Tregs and inhibit CD4+ T helper (Th) cell expansion in mice. While type I interferons sustain the expression and the activation of the transcription master regulators Foxp3, Stat3 and Stat5, these innate molecules reciprocally inhibit Th17 cell differentiation. Altogether, these results indicate a new pivotal role of IFN-α/β on pTreg differentiation and induction of peripheral tolerance, which may have important implications in the therapeutic control of inflammatory disorders, such as of autoimmune diseases.

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A probable role of dihydropyridine receptors in repression of Ca2+ sparks demonstrated in cultured mammalian muscle

AJP Cell Physiology ◽

10.1152/ajpcell.00592.2004 ◽

2006 ◽

Vol 290 (2) ◽

pp. C539-C553 ◽

Cited By ~ 56

Author(s):

Jingsong Zhou ◽

Jianxun Yi ◽

Leandro Royer ◽

Bradley S. Launikonis ◽

Adom González ◽

...

Keyword(s):

Ryanodine Receptors ◽

Critical Role ◽

Primary Cultures ◽

Inhibitory Effect ◽

Wild Type ◽

Dihydropyridine Receptors ◽

Skeletal Muscle Contraction ◽

Probable Role ◽

Emission Ratios

To activate skeletal muscle contraction, action potentials must be sensed by dihydropyridine receptors (DHPRs) in the T tubule, which signal the Ca2+ release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) to open. We demonstrate here an inhibitory effect of the T tubule on the production of sparks of Ca2+ release. Murine primary cultures were confocally imaged for Ca2+ detection and T tubule visualization. After 72 h of differentiation, T tubules extended from the periphery for less than one-third of the myotube radius. Spontaneous Ca2+ sparks were found away from the region of cells where tubules were found. Immunostaining showed RyR1 and RyR3 isoforms in all areas, implying inhibition of both isoforms by a T tubule component. To test for a role of DHPRs in this inhibition, we imaged myotubes from dysgenic mice ( mdg) that lack DHPRs. These exhibited T tubule development similar to that of normal myotubes, but produced few sparks, even in regions where tubules were absent. To increase spark frequency, a high-Ca2+ saline with 1 mM caffeine was used. Wild-type cells in this saline plus 50 μM nifedipine retained the topographic suppression pattern of sparks, but dysgenic cells in high-Ca2+ saline did not. Shifted excitation and emission ratios of indo-1 in the cytosol or mag-indo-1 in the SR were used to image [Ca2+] in these compartments. Under the conditions of interest, wild-type and mdg cells had similar levels of free [Ca2+] in cytosol and SR. These data suggest that DHPRs play a critical role in reducing the rate of spontaneous opening of Ca2+ release channels and/or their susceptibility to Ca2+-induced activation, thereby suppressing the production of Ca2+ sparks.

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Influence of genetic background on albuminuria and kidney injury in Ins2+/C96Y (Akita) mice

AJP Renal Physiology ◽

10.1152/ajprenal.90515.2008 ◽

2010 ◽

Vol 298 (3) ◽

pp. F788-F795 ◽

Cited By ~ 66

Author(s):

Susan B. Gurley ◽

Carrie L. Mach ◽

Johannes Stegbauer ◽

Jihong Yang ◽

Kamie P. Snow ◽

...

Keyword(s):

Genetic Background ◽

Type I Diabetes ◽

Critical Role ◽

Kidney Injury ◽

Strain Differences ◽

Type I ◽

Naturally Occurring ◽

Akita Mice ◽

Renal Phenotype

Previous studies have shown that Akita mice bearing the Ins2 +/C96Y mutation have significant advantages as a type I diabetes platform for developing models of diabetic nephropathy (DN; Gurley SB, Clare SE, Snow KP, Hu A, Meyer TW, Coffman TM. Am J Physiol Renal Physiol 290: F214–F222, 2006). In view of the critical role for genetic factors in determining susceptibility to DN in humans, we investigated the role of genetic background on kidney injury in Akita mice. To generate a series of inbred Akita mouse lines, we back-crossed the Ins2 C96Y mutation more than six generations onto the 129/SvEv and DBA/2 backgrounds and compared the extent of hyperglycemia and renal disease with the standard C57BL/6- Ins2 +/C96Y line. Male mice from all three Akita strains developed marked and equivalent hyperglycemia. However, there were significant differences in the level of albuminuria among the lines with a hierarchy of DBA/2 > 129/SvEv > C57BL/6. Renal and glomerular hypertrophy was seen in all of the lines, but significant increases in mesangial matrix compared with baseline nondiabetic controls were observed only in the 129 and C57BL/6 backgrounds. In F1(DBA/2 x C57BL/6)- Ins2 +/C96Y mice, the extent of albuminuria was similar to the parental DBA/2- Ins2+/ C96Y line; they also developed marked hyperfiltration. These studies identify strong effects of genetic background to modify the renal phenotype associated with the Ins2 C96Y mutation. Identification of these naturally occurring strain differences should prove useful for nephropathy modeling and may be exploited to allow identification of novel susceptibility alleles for albuminuria in diabetes.

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