An unexpected twist to the activation of IKKβ: TAK1 primes IKKβ for activation by autophosphorylation.
Ontology highlight
ABSTRACT:
SUBMITTER: Zhang J
PROVIDER: S-EPMC4206954 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Activation of dual-specificity tyrosine-phosphorylation-regulated kinases 1A and 1B (DYRK1A and DYRK1B) requires prolyl hydroxylation by PHD1 prolyl hydroxylase. Prolyl hydroxylation of DYRK1 initiates a cascade of events leading to the release of molecular constraints on von Hippel-Lindau (VHL) ubiquitin ligase tumor suppressor function. However, the proline residue of DYRK1 targeted by hydroxylation and the role of prolyl hydroxylation in tyrosine autophosphorylation of DYRK1 are unknown. We found that a highly conserved proline in the CMGC insert of the DYRK1 kinase domain is hydroxylated by PHD1, and this event precedes tyrosine autophosphorylation. Mutation of the hydroxylation acceptor proline precludes tyrosine autophosphorylation and folding of DYRK1, resulting in a kinase unable to preserve VHL function and lacking glioma suppression activity. The consensus proline sequence is shared by most CMGC kinases, and prolyl hydroxylation is essential for catalytic activation. Thus, formation of prolyl-hydroxylated intermediates is a novel mechanism of kinase maturation and likely a general mechanism of regulation of CMGC kinases in eukaryotes.
| S-EPMC7849370 | biostudies-literature
Project description:IRAK4 kinase plays a critical role in innate immune responses and inflammation by modulating the TLR/IL-1R signaling pathway, yet the mechanism by which it regulates downstream pathways and transcription factors to induce inflammatory cytokines is unclear. IRAK4 can mediate signaling events by mechanisms both dependent and independent of its kinase activity. Understanding this regulation is important for deciphering the role of IRAK4 and for the development of treatments for inflammatory diseases and cancer. Through transcriptomic and biochemical analyses of primary human monocytes treated with a highly potent and selective inhibitor of IRAK4, we show that IRAK4 kinase activity controls the transcription factor IRF5 which in turn induces inflammatory cytokine and type I interferon transcription in myeloid cells. We also show that IRAK4 kinase activity does not control activation of NF-κB. Following TLR stimulation, translocation of IRF5, but not NF-κB, to the nucleus in human monocytes is abolished by IRAK4 kinase inhibition. In addition, binding of IRF5, but not NF-κB p65, to promoters of inflammatory target genes (TNF-α and IP10) is blocked with an IRAK4 kinase inhibitor. IKKβ, a known activator of IRF5, is phosphorylated in response to TLR mediated signaling, and inhibition of IRAK4 kinase blocks IKKβ phosphorylation. Pharmacological inhibition of IKKβ and TAK1, the upstream kinase of IKKβ, in human monocytes blocks IL-1, IL-6 and TNF-α cytokine production, as well as IRF5 translocation to the nucleus. Taken together, our data suggest a novel mechanism by which IRAK4 kinase activity regulates TAK1 and IKKβ activation, leading to the translocation of IRF5 and induction of inflammatory cytokines in human monocytes.
2017-10-12 | GSE86234 | GEO
Project description:The NLRP3 inflammasome plays a central role in antimicrobial defense as well as in the context of sterile inflammatory conditions. NLRP3 activity is governed by two independent signals: the first signal primes NLRP3, rendering it responsive to the second signal, which then triggers inflammasome formation. Our understanding of how NLRP3 priming contributes to inflammasome activation remains limited. Here, we show that IKKβ, a kinase activated during priming, induces recruitment of NLRP3 to phosphatidylinositol-4-phosphate (PI4P), a phospholipid enriched on the trans-Golgi network. NEK7, a mitotic spindle kinase that had previously been thought to be indispensable for NLRP3 activation, was redundant for inflammasome formation when IKKβ recruited NLRP3 to PI4P. Studying iPSC-derived human macrophages revealed that the IKKβ-mediated NEK7-independent pathway constitutes the predominant NLRP3 priming mechanism in human myeloid cells. Our results suggest that PI4P binding represents a primed state into which NLRP3 is brought by IKKβ activity.
| S-EPMC7614333 | biostudies-literature
Project description:Lambda-like double-stranded (ds) DNA bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. Capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm of pressure during genome packaging. The extensive integration between subunits in capsids requires the formation of an intermediate complex, termed a procapsid, from which individual subunits can undergo the necessary refolding and structural rearrangements needed to transition to the more stable capsid. Although various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage. Here we present a procapsid X-ray structure at 3.65 A resolution, termed prohead II, of the lambda-like bacteriophage HK97, the mature capsid structure of which was previously solved to 3.44 A (ref. 2). A comparison of the two largely different capsid forms has unveiled an unprecedented expansion mechanism that describes the transition. Crystallographic and hydrogen/deuterium exchange data presented here demonstrate that the subunit tertiary structures are significantly different between the two states, with twisting and bending motions occurring in both helical and beta-sheet regions. We also identified subunit interactions at each three-fold axis of the capsid that are maintained throughout maturation. The interactions sustain capsid integrity during subunit refolding and provide a fixed hinge from which subunits undergo rotational and translational motions during maturation. Previously published calorimetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90 kJ mol(-1) of energy. We propose that the major tertiary changes presented in this study reveal a structural basis for an exothermic maturation process probably present in many dsDNA bacteriophage and possibly viruses such as herpesvirus, which share the HK97 subunit fold.
| S-EPMC2765791 | biostudies-literature
Project description:Vinculin phosphorylation has been implicated as a potential mechanism for focal adhesion growth and maturation. Four vinculin residues-Y100, S1033, S1045, and Y1065-are phosphorylated by kinases during focal adhesion maturation. In this study, phosphorylation at each of these residues is simulated using molecular dynamics models. The simulations demonstrate that once each phosphorylated vinculin structure is at equilibrium, significant local conformational changes result that may impact either vinculin activation or vinculin binding to actin and PIP2. Simulation of vinculin activation after phosphorylation shows that the added phosphoryl groups can prime vinculin for activation. It remains to be seen if vinculin can be phosphorylated at S1033 in vivo, but these simulations highlight that in the event of a S1033 phophorylation vinculin will likely be primed for activation.
| S-EPMC3341567 | biostudies-literature
Project description:I kappa B kinase β (IKKβ) is one of the primary targets to regulate canonical NF-κB activity. The misregulation of NF-κB is associated with various diseases, including chronic inflammation and cancers. Most of the known IKKβ inhibitors target its active form and suffer from poor selectivity. In the present study, we aim to design inhibitors that can bind to the IKKβ inactive form and block its activation. We identified a potential allosteric site between the kinase domain (KD) and ubiquitin-like domain (ULD) of human IKKβ and used it to virtually screen a chemical library for allosteric inhibitors. Among the 133 compounds tested, 16 inhibited NF-κB activity by over 50% at 50 μM in a reporter gene assay. Further quantitative measurements and cytotoxicity study gave one compound 124 (3,4-dichloro-2-ethoxy-N-(2,2,6,6-tetramethylpiperidin-4-yl)benzenesulfonamide) which specifically targets the IKKβ inactive form. In cells, 124 inhibited IκBα phosphorylation and NF-κB transcriptional activity for the reporter gene with an IC50 of 35 μM by decreasing the phosphorylation level of Ser177/181 on IKKβ and blocking its activation upon TNFα stimulation. Molecular dynamics simulations demonstrated that 124 binds to the pocket between KD and ULD in the inactive conformation of IKKβ rather than the active conformation. As the first allosteric inhibitor that prevents IKKβ activation, 124 provides a good starting point for further inhibitor discovery and a probe for IKKβ enzyme cycle and regulatory mechanism study.
| S-EPMC6083782 | biostudies-literature
Project description:Lipases are enzymes necessary for the proper distribution and utilization of lipids in the human body. Lipoprotein lipase (LPL) is active in capillaries, where it plays a crucial role in preventing dyslipidemia by hydrolyzing triglycerides from packaged lipoproteins. Thirty years ago, the existence of a condensed and inactive LPL oligomer was proposed. Although recent work has shed light on the structure of the LPL monomer, the inactive oligomer remained opaque. Here we present a cryo-EM reconstruction of a helical LPL oligomer at 3.8-Å resolution. Helix formation is concentration-dependent, and helices are composed of inactive dihedral LPL dimers. Heparin binding stabilizes LPL helices, and the presence of substrate triggers helix disassembly. Superresolution fluorescent microscopy of endogenous LPL revealed that LPL adopts a filament-like distribution in vesicles. Mutation of one of the helical LPL interaction interfaces causes loss of the filament-like distribution. Taken together, this suggests that LPL is condensed into its inactive helical form for storage in intracellular vesicles.
| S-EPMC7229681 | biostudies-literature
Project description:By combining biochemical experiments with computer modelling of biochemical reactions we elucidated some of the currently unresolved aspects of calcium-calmodulin-dependent protein kinase II (CaMKII) activation and autophosphorylation that might be relevant for its physiological function and provided a model that incorporates in detail the mechanism of CaMKII activation and autophosphorylation at T286 that is based on experimentally determined binding constants and phosphorylation rates. To this end, we developed a detailed state model of CaMKII activation and autophosphorylation based on the currently available literature, and constrained it with data from CaMKII autophosphorylation essays. Our model takes exact phosphorylation patterns of CaMKII holoenzymes into account, and is valid at physiologically relevant conditions where the concentrations of calcium and calmodulin are not saturating. Our results strongly suggest that even when bound to less than fully calcium-bound calmodulin, CaMKII is in the active state, and indicate that the autophosphorylation of T286 by an active non-phosphorylated CaMKII subunit is significantly faster than by an autophosphorylated CaMKII subunit. These results imply that CaMKII can be efficiently activated at significantly lower calcium concentrations than previously thought, which may explain how CaMKII gets activated at calcium concentrations existing at synapses in vivo. We also investigated the significance of CaMKII holoenzyme structure on CaMKII autophosphorylation and obtained estimates of previously unknown binding constants.
| S-EPMC2801814 | biostudies-literature
Project description:ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.
| S-EPMC1538573 | biostudies-literature
Project description:TAK1 kinase is an indispensable intermediate in several cytokine signaling pathways including tumor necrosis factor, interleukin-1, and transforming growth factor-beta signaling pathways. TAK1 also participates in stress-activated intracellular signaling pathways such as osmotic stress signaling pathway. TAK1-binding protein 1 (TAB1) is constitutively associated with TAK1 through its C-terminal region. Although TAB1 is known to augment TAK1 catalytic activity when it is overexpressed, the role of TAB1 under physiological conditions has not yet been identified. In this study, we determined the role of TAB1 in TAK1 signaling by analyzing TAB1-deficient mouse embryonic fibroblasts (MEFs). Tumor necrosis factor- and interleukin-1-induced activation of TAK1 was entirely normal in Tab1-deficient MEFs and could activate both mitogen-activated protein kinases and NF-kappaB. In contrast, we found that osmotic stress-induced activation of TAK1 was largely impaired in Tab1-deficient MEFs. Furthermore, we showed that the C-terminal 68 amino acids of TAB1 were sufficient to mediate osmotic stress-induced TAK1 activation. Finally, we attempted to determine the mechanism by which TAB1 activates TAK1. We found that TAK1 is spontaneously activated when the concentration is increased and that it is totally dependent on TAB1. Cell shrinkage under the osmotic stress condition increases the concentration of TAB1-TAK1 and may oligomerize and activate TAK1 in a TAB1-dependent manner. These results demonstrate that TAB1 mediates TAK1 activation only in a subset of TAK1 pathways that are mediated through spontaneous oligomerization of TAB1-TAK1.
| S-EPMC2586273 | biostudies-other