Insights into the evolution of divergent nucleotide-binding mechanisms among pseudokinases revealed by crystal structures of human and mouse MLKL
The pseudokinase, Mixed lineage kinase domain-like (MLKL), was recently identified as an essential checkpoint in the programmed necrosis or “necroptosis” cell death pathway. Here, we present the crystal structure of the human MLKL pseudokinase domain at 1.7 Å resolution and probe its nucleotide-binding mechanism by performing structure-based mutagenesis. By comparing the structures and nucleotide binding determinants of human and mouse MLKL orthologues, our study provides insights into the evolution of nucleotide binding mechanisms amongst pseudokinases and their mechanistic divergence from conventiona...
Source: BJ Signal - November 13, 2013 Category: Biochemistry Authors: J M. Murphy, I S. Lucet, J M. Hildebrand, M C. Tanzer, S N. Young, P Sharma, G Lessene, W S. Alexander, J J. Babon, J Silke, P E. Czabotar Tags: BJ Biomolecules Source Type: research
Characterization of WZ4003 and HTH-01-015 as selective inhibitors of the LKB1 tumour suppressor activated NUAK kinases
We describe the first highly specific protein kinase inhibitors of NUAK kinases namely WZ4003 and HTH-01-015. WZ4003 inhibits both NUAK isoforms, whereas HTH-01-015 inhibits only NUAK1. These compounds display extreme selectivity and do not significantly inhibit the activity of 139 other kinases tested including 10 AMPK family members. WZ4003 and HTH-01-015 inhibit the phosphorylation of the only well-characterised substrate namely MYPT1 that is phosphorylated by NUAK1 at Ser445. We identify a mutation that does not affect basal NUAK1 activity but renders it resistant to both WZ4003 and HTH-01-015. Consistent with NUAK1 me...
Source: BJ Signal - October 30, 2013 Category: Biochemistry Authors: S Banerjee, S J. Buhrlage, H Huang, X Deng, W Zhou, J Wang, R Traynor, A R. Prescott, D R. Alessi, N S. Gray Tags: BJ ChemBio Source Type: research
Multivalent interactions of the SUMO-interaction motifs in the RING-finger protein 4 (RNF4) determine the specificity for chains of the small ubiquitin-related modifier (SUMO)
We describe that the sequence and spacing of the SUMO-interaction motifs (SIMs) in RNF4 regulate the avidity-driven recognition of substrate proteins carrying SUMO chains of variable length. (Source: BJ Signal)
Source: BJ Signal - October 23, 2013 Category: Biochemistry Authors: K Keusekotten, V N. Bade, K Meyer-Teschendorf, A Miriam Sriramachandran, K Fischer-Schrader, A Krause, C Horst, G Schwarz, K Hofmann, R Jürgen Dohmen, G J.K. Praefcke Tags: BJ Signal Source Type: research
A robust methodology to subclassify pseudokinases based on their nucleotide binding properties
Protein kinase-like domains that lack conserved residues known to catalyze phosphoryl transfer, termed pseudokinases, have emerged as important signaling domains across the kingdoms of life. Although predicted to function principally as catalysis-independent protein interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amidst controversy. We established a thermal stability shift assay as a benchmark technique to define the nucleotide binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities o...
Source: BJ Signal - October 10, 2013 Category: Biochemistry Authors: J M Murphy, Q Zhang, S N Young, M L Reese, F P Bailey, P A Eyers, D Ungureanu, H Hammarén, O Silvennoinen, L N Varghese, K Chen, A Tripaydonis, N Jura, K Fukuda, J Qin, Z Nimchuk, M Mudgett, S Elowe, C Lesley Gee, L Liu, R J Daly, G Manning, J J Babon, I Tags: BJ Signal Source Type: research
Positive and Negative Phosphorylation Regulates RIP1 and RIP3-Induced Programmed Necrosis
Programmed necrosis or necroptosis is controlled by the action of two serine/threonine kinases, receptor interacting protein kinase 1 (RIP1) and RIP3. The phosphorylation of RIP1 and RIP3 is critical for assembly of the necrosome, an amyloid-like complex that initiates transmission of the pro-necrotic signal. Here, we used site-directed mutagenesis to systematically examine the effects of putative phosphor acceptor sites on RIP1 and RIP3 on TNF-induced programmed necrosis. We found that mutation of individual serine residues in the kinase domain of RIP1 had little effects on RIP1 kinase activity and TNF-induced programmed ...
Source: BJ Signal - September 24, 2013 Category: Biochemistry Authors: T McQuade, Y Cho, F Ka-Ming Chan Tags: BJ Signal Source Type: research
Dynamic conformational switching in the chemokine ligand is essential for G Protein coupled-receptor activation
Chemokines mediate diverse functions from organogenesis to mobilizing leukocytes, and are unusual agonists for class-A GPCRs because of their large size and multi-domain structure. The current model for receptor activation, which involves interactions between chemokine N-loop and receptor N-terminal residues (Site-I) and between chemokine N-terminal and receptor extracellular loop/transmembrane residues (Site-II), fails to describe differences in ligand/receptor selectivity and the activation of multiple signaling pathways. Here, we show in neutrophil-activating chemokine CXCL8 that the highly conserved Gly-Pro (GP) motif ...
Source: BJ Signal - September 16, 2013 Category: Biochemistry Authors: P B. Joseph, K V. Sawant, A Isley, M Pedroza, R P. Garofalo, R M. Richardson, K Rajarathnam Tags: BJ Signal Source Type: research
Crystal Structure of the TRIM25 B30.2 (PRYSPRY) domain: A Key Component of Antiviral Signaling
Tripartite motif (TRIM) proteins primarily function as ubiquitin E3 ligases that regulate the innate immune response to infection. TRIM25 (also known as estrogen-responsive finger protein (Efp)) has been implicated in the regulation of estrogen receptor α signaling, and in the regulation of innate immune signaling via retinoic acid-inducible gene-I (RIG-I). RIG-I senses cytosolic viral RNA, and is subsequently ubiquitinated by TRIM25 at its N-terminal CARD domains, leading to type I interferon production. The interaction with RIG-I is dependent on the TRIM25 B30.2 domain, a protein interaction domain composed of the...
Source: BJ Signal - September 10, 2013 Category: Biochemistry Authors: A Antonio D'Cruz, N J Kershaw, J J Chiang, M K Wang, N A Nicola, J J Babon, M U Gack, S E Nicholson Tags: BJ Biomolecules Source Type: research
Chemical library screening for WNK signaling inhibitors by using fluorescent correlation spectroscopy
With-no-lysine kinases (WNK) are the causative genes of a hereditary hypertensive disease, pseudohypoaldosteronism type II (PHA II), and form a signal cascade with oxidative stress-responsive 1 (OSR1)/STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and Slc12a transporters. We have shown that this signal cascade regulates blood pressure by controlling vascular tone as well as renal NaCl excretion. Therefore, agents that inhibit this signal cascade could be a new class of antihypertensive drugs. Since the binding of WNK to OSR1/SPAK kinases was postulated to be important for signal transduction, we sought to di...
Source: BJ Signal - August 27, 2013 Category: Biochemistry Authors: T Mori, E Kikuchi, Y Watanabe, S Fujii, M Ishigami-Yuasa, H Kagechika, E Sohara, T Rai, S Sasaki, S Uchida Tags: BJ ChemBio Source Type: research
Phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) is an AMPK target participating in contraction-stimulated glucose uptake in skeletal muscle
Phosphatidylinositol 3-phosphate 5-kinase (PIKfyve), the lipid kinase that phosphorylates PtdIns3P to PtdIns(3,5)P2, has been implicated in insulin-stimulated glucose uptake. We investigated whether PIKfyve could also be involved in contraction/AMP-activated protein kinase (AMPK)-stimulated glucose uptake in skeletal muscle. Incubation of rat epitrochlearis muscles with YM201636, a selective PIKfyve inhibitor, reduced contraction- and AICA riboside (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside)-stimulated glucose uptake. Consistently, PIKfyve knockdown in C2C12 myotubes reduced AICA riboside-stimulated glucos...
Source: BJ Signal - August 1, 2013 Category: Biochemistry Authors: Y Liu, Y Lai, E V Hill, D Tyteca, S Carpentier, A Ingvaldsen, D Vertommen, L Lantier, M Foretz, F Dequiedt, P J Courtoy, C Erneux, B Viollet, P R Shepherd, J M Tavare, J Jensen, M H Rider Tags: BJ Signal Source Type: research
Phosphoproteomics of Collagen Receptor Networks Reveals SHP-2 Phosphorylation Downstream of Wildtype DDR2 and its Lung Cancer Mutants
Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the β1 integrins and the Discoidin Domain Receptors (DDRs). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 ty...
Source: BJ Signal - July 4, 2013 Category: Biochemistry Authors: L K Iwai, L S Payne, M T Luczynski, F Chang, H Xu, R W Clinton, A Paul, E A Esposito, S Gridley, B Leitinger, K M Naegle, P H Huang Tags: BJ Signal Source Type: research
Structural basis for phosphorylation-triggered autophagic clearance of Salmonella
Selective autophagy is mediated by the interaction of autophagy modifiers and autophagy receptors that also bind to ubiquitinated cargo. Optineurin is an autophagy receptor that plays a role in the clearance of cytosolic Salmonella. The interaction between receptors and modifiers is often relatively weak, with typical values for the dissociation constant in the low micromolar range. The interaction of optineurin with autophagy modifiers is even weaker but can be significantly enhanced through phosphorylation by the TANK binding kinase 1. Here we present the NMR and crystal structures of the autophagy modifier LC3B in compl...
Source: BJ Signal - June 28, 2013 Category: Biochemistry Authors: V V Rogov, H Suzuki, E Fiskin, P Wild, A Kniss, A Rozenknop, R Kato, M Kawasaki, D G McEwan, F Löhr, P Güntert, I Dikic, S Wakatsuki, V Dötsch Tags: BJ Signal Source Type: research
Tamoxifen inhibits macrophage FABP4 expression through the combined effects of GR and PPAR{gamma} pathways
In this study, we determined if the anti-atherogenic property of tamoxifen was related to its inhibition of macrophage FABP4 expression. We initially observed that tamoxifen inhibited macrophage/foam cell formation but the inhibition was attenuated when FABP4 expression was selectively inhibited by siRNA. We then observed that tamoxifen and 4-hydroxytamoxifen inhibited FABP4 protein expression in primary macrophages isolated from both the male and female wild type mice suggesting the inhibition is sex-independent. Tamoxifen and 4-hydroxytamoxifen inhibited macrophage FABP4 protein expression induced either by activation of...
Source: BJ Signal - June 28, 2013 Category: Biochemistry Authors: M Jiang, L Zhang, X Ma, W Hu, Y Chen, M Yu, Q Wang, X Li, Z Yin, Y Zhu, X Gao, D P Hajjar, Y Duan, J Han Tags: BJ Metabolism Source Type: research
ROS inhibitor N-acetyl-L-cysteine antagonizes the activity of proteasome inhibitors
In this study, we identified inhibition of proteasome inhibitors as a novel activity of NAC. Both NAC and catalase, another known scavenger of ROS, similarly inhibited ROS levels and apoptosis associated with H2O2. However, only NAC, but not catalase or another ROS scavenger Trolox was able to prevent effects linked to proteasome inhibition, such as protein stabilization, apoptosis and accumulation of ubiquitin conjugates. These observations suggest that NAC has a dual activity as an inhibitor of ROS and proteasome inhibitors. Recently, NAC was used as a ROS inhibitor to functionally characterize a novel anticancer compoun...
Source: BJ Signal - June 18, 2013 Category: Biochemistry Authors: M Halasi, M Wang, T S Chavan, V Gaponenko, N Hay, A L Gartel Tags: BJ ChemBio Source Type: research
Diacylglycerol Kinase-{theta} Couples FXR-Dependent Bile Acid Signaling to Akt Activation and Glucose Homeostasis in Hepatocytes
Diacylglycerol kinases (DGKs) catalyze the conversion of diacylglycerol into phosphatidic acid (PA), a positive modulator of mammalian target of rapamycin (mTOR). We have found that chenodeoxycholic acid and the synthetic farnesoid X receptor (FXR) ligand GW4064 induce the mRNA and protein expression of DGKQ in the HepG2 cell line and in primary human hepatocytes. Reporter gene studies using 1.5 kB of the DGKQ promoter fused to the luciferase gene revealed that bile acids increase DGKQ transcriptional activity. Mutation of putative FXR binding sites attenuated the ability of GW4046 to increase DGKQ luciferase activity. Con...
Source: BJ Signal - June 17, 2013 Category: Biochemistry Authors: K Cai, M B Sewer Tags: BJ Signal Source Type: research
Metabolic phenotypes of Saccharomyces cerevisiae mutants with altered trehalose-6-phosphate dynamics
In Saccharomyces cerevisiae, synthesis of trehalose-6-phosphate (T6P) is essential for growth on most fermentable carbon sources. The metabolic response to glucose was analysed in mutants with different capacity to accumulate T6P. A mutant carrying a deletion in the T6P synthase encoding gene, TPS1, which had no measurable T6P exhibited impaired ethanol production, showed diminished plasma membrane H+-ATPase activation, and became rapidly depleted of nearly all adenine nucleotides which were irreversibly converted to inosine. Deletion of the AMP deaminase encoding gene, AMD1, in the tps1 strain prevented inosine for...
Source: BJ Signal - June 14, 2013 Category: Biochemistry Authors: T Walther, N Mtimet, C Alkim, A Vax, M Loret, A Ullah, C Gancedo, G J Smits, J François Tags: BJ Metabolism Source Type: research
