The Structure of the SlrP-hTrx1 Complex Sheds Light on the Autoinhibition Mechanism of the Type III Secretion System Effectors of the NEL Family
Salmonella infections are a leading cause of bacterial foodborne illness in the United States and the European Union. Antimicrobial therapy is often administered to treat the infection but increasing isolates are being detected that demonstrate resistance to multiple antibiotics. Salmonella enterica contains two virulence related type III secretion systems (T3SS): one promotes invasion of the intestine and the other one mediates systemic disease. Both of them secrete the SlrP protein acting as E3 ubiquitin ligase in human host cells where it targets thioredoxin-1 (Trx1). SlrP belongs to the NEL family of bacterial E3 ubiqu...
Source: BJ Signal - September 3, 2014 Category: Biochemistry Authors: S Zouhir, J Bernal-Bayard, M Cordero-Alba, E Cardenal-Muñoz, B Guimaraes, N Lazar, F Ramos-Morales, S Nessler Tags: BJ Biomolecules Source Type: research
Characterisation of VPS34-IN1, a selective inhibitor of Vps34 reveals that the phosphatidylinositol 3-phosphate binding SGK3 protein kinase is a downstream target of Class III PI-3 kinase
We describe a inhibitor of Vps34, termed VPS34-IN1 (25 nM IC50) that does not significantly inhibit the activity of 340 protein kinases or 25 lipid kinases tested that include all isoforms of Class I and II PI3Ks. VPS34-IN1 induces a rapid dose dependent dispersal of a specific PtdIns(3)P binding probe from endosome membranes. We explored whether SGK3, the only protein kinase known to interact specifically with PtdIns(3)P, might be controlled by Vps34. Mutations disrupting PtdIns(3)P-binding, ablated SGK3 kinase activity by suppressing phosphorylation of the T-loop and hydrophobic-motif residues. VPS34-IN1 induced a rapid ...
Source: BJ Signal - September 1, 2014 Category: Biochemistry Authors: R Bago, N Malik, M J Munson, A Prescott, P Davies, E M Sommer, N Shpiro, R Ward, D Cross, I G Ganley, D R Alessi Tags: BJ ChemBio Source Type: research
CYP-13A12 of the nematode C. elegans is a PUFA-epoxygenase involved in behavioural response to reoxygenation
A specific behavioural response of Caenorhabditis elegans, the rapid increase of locomotion in response to anoxia/reoxygenation called the O2-ON response, has been used to model key aspects of ischemia/reperfusion injury. A genetic suppressor screen demonstrated a direct causal role of CYP-13A12 in this response and suggested that CYP-eicosanoids, which in mammals influence the contractility of cardiomyocytes and vascular smooth muscle cells, might function in C. elegans as specific regulators of the body muscle cell activity. Here we show that co-expression of CYP-13A12 with the NADPH-CYP-reductase EMB-8 in insect cells r...
Source: BJ Signal - August 20, 2014 Category: Biochemistry Authors: J Keller, A Ellieva, D K. Ma, J Ju, E Nehk, A Konkel, J R. Falck, W Schunck, R Menzel Tags: BJ Signal Source Type: research
Crystal structure of the mouse interleukin-3 {beta}-receptor: insights into interleukin-3 binding and receptor activation
Interleukin-3 (IL-3) is a cytokine secreted by mast cells and activated T-cells known to be an important regulator of differentiation, survival, proliferation and activation of a range of hematopoietic lineages. The effects of IL-3 on target cells are mediated by a transmembrane receptor system composed of a cytokine-specific α-subunit and a β-subunit, the principal signalling entity. In the mouse, two β-subunits have co-evolved: a common β-subunit (βc) shared between IL-3 and the related cytokines, IL-5 and GM-CSF; and an IL-3-specific β-subunit (βIL-3). βIL‑3...
Source: BJ Signal - August 19, 2014 Category: Biochemistry Authors: P D Carr, C L Ewens, J Dai, D L Ollis, J M Murphy, C J Jackson, I G Young Tags: BJ Biomolecules Source Type: research
A new tumor suppression mechanism by p27Kip1: EGFR down-regulation mediated by JNK/c-Jun pathway inhibition
This study provides new insights into p27Kip1 suppression of cancer cell growth, migration and metastasis. (Source: BJ Signal)
Source: BJ Signal - August 14, 2014 Category: Biochemistry Authors: Y Fang, Y Wang, Y Wang, Y Meng, J Zhu, H Jin, J Li, D Zhang, Y Yu, X Wu, C Huang Tags: BJ Signal Source Type: research
A new calmodulin binding motif for inositol 1,4,5-trisphosphate 3-kinase regulation
Inositol 1,4,5-triphoshate 3-kinase (IP3 3-K) is a key enzyme that catalyses the synthesis of inositol 1,3,4,5-tetrakisphosphate (IP4), using IP3 and ATP as substrates. Both inositides, substrate and product, present crucial roles in the cell. IP3 is a key point in Ca2+ metabolism that promotes Ca2+ release from intracellular stores and together with IP4 regulates Ca2+ homeostasis. In addition, IP4 is involved in the immune cell development. It has been proved that Ca2+/calmodulin (Ca2+/CaM) regulates the activity of IP3 3-K, via direct interaction between both enzymes. Although we have e...
Source: BJ Signal - August 7, 2014 Category: Biochemistry Authors: E Franco-Echevarria, J Ignacio Baños-Sanz, B Monterroso, A Round, J Sanz-Aparicio, B Gonzalez Tags: BJ Biomolecules Source Type: research
Influence of association state and DNA-binding on the O2-reactivity of [4Fe-4S] Fumarate and Nitrate Reduction (FNR) regulator
The Fumarate and Nitrate Reduction (FNR) regulator is the master switch for the transition between anaerobic and aerobic respiration in Escherichia coli. Reaction of dimeric [4Fe-4S] FNR with O2 results in conversion of the cluster to a [2Fe-2S] form, via a [3Fe-4S] intermediate, leading to the loss of DNA-binding through dissociation of the dimer into monomers. Here we report studies of two previously identified variants of FNR, D154A and I151A, in which the form of the cluster is decoupled from the association state. In vivo studies of permanently dimeric D154A FNR show that DNA-binding does not affect the rate of cluste...
Source: BJ Signal - July 14, 2014 Category: Biochemistry Authors: J C. Crack, M R. Stapleton, J Green, A J. Thomson, N E. Le Brun Tags: BJ Biomolecules Source Type: research
Osteopontin O-glycosylation contributes to its phosphorylation and cell adhesion properties
In this study, we demonstrated that OPN O-glycosylation self-regulates its biological activities and also affects its phosphorylation status. We prepared two recombinant OPNs, wild-type (WT)-OPN and mutant OPN (ΔO-OPN), lacking five O-glycosylation sites at a threonine–proline rich region. O-glycan defects in OPN increased its phosphorylation level, as observed by dephosphorylation assays. Moreover, compared with WT-OPN, ΔO-OPN exhibited enhanced cell spreading and adhesion activities and decreased associations with β1 integrins. This suggested that defects in O-glycans in OPN altered these acti...
Source: BJ Signal - July 7, 2014 Category: Biochemistry Authors: Y Kariya, M Kanno, K Matsumoto-Morita, M Konno, Y Yamaguchi, Y Hashimoto Tags: BJ Biomolecules Source Type: research
Regulation of PKA activity by an autophosphorylation mechanism in Saccharomyces cerevisiae.
The cAMP dependent Protein Kinase activity, as well as other AGC members, is regulated by multiple phosphorylations of its catalytic subunits. In Saccharomyces cerevisiae PKA regulatory subunit is encoded by the gene BCY1, and the catalytic subunits are encoded by three genes: TPK1, TPK2 and TPK3. Previously we have reported that following cAMP-PKA pathway activation, Tpk1 increases its phosphorylation status. Now, in vivo genetic and in vitro experiments indicate an autophosphorylation mechanism for Tpk1. Using array peptides derived from Tpk1 we identified Ser179 as a target residue. Tpk1 is phosphorylated on Ser179 in v...
Source: BJ Signal - June 20, 2014 Category: Biochemistry Authors: C Andrea Solari, V Tudisca, M Pugliessi, A Daniel Nadra, S Moreno, P Portela Tags: BJ Signal Source Type: research
An unexpected twist to the activation of IKK{beta}: TAK1 primes IKK{beta} for activation by autophosphorylation
The IκB kinase β (IKKβ) is required to activate the transcription factor NF-κB, but how IKKβ itself is activated in vivo is still unclear. It was found to require phosphorylation by one or more “upstream” protein kinases in some reports but by auto-phosphorylation in others. Here, we resolve this controversy by demonstrating that the activation of IKKβ induced by IL-1 or TNF in embryonic fibroblasts, or by ligands that activate Toll-Like receptors in macrophages, requires two distinct phosphorylation events; first, the TAK1-catalysed phosphorylation of Ser177 and seco...
Source: BJ Signal - June 9, 2014 Category: Biochemistry Authors: J Zhang, K Clark, T Lawrence, M W Peggie, P Cohen Tags: BJ Signal Source Type: research
N-Terminus of the Protein Kinase CLK1 Induces SR Protein Hyper-Phosphorylation
SR proteins are essential splicing factors that are regulated through multisite phosphorylation of their RS (arginine-serine-rich) domains by two major families of protein kinases. The SRPKs efficiently phosphorylate the arginine-serine dipeptides in the RS domain using a conserved docking groove in the kinase domain. In contrast, CLKs lack a docking groove and phosphorylate both arginine-serine and serine-proline dipeptides, modifications that generate a hyper-phosphorylated state important for unique SR protein-dependent splicing activities. All CLKs contain long, flexible N-terminal extensions (140-300 residues) that re...
Source: BJ Signal - May 29, 2014 Category: Biochemistry Authors: B E. Aubol, R M. Plocinik, M Keshwani, M L. McGlone, J C. Hagopian, G Ghosh, X Fu, J A. Adams Tags: BJ Biomolecules Source Type: research
Structural Analysis of poly-SUMO Chain Recognition by RNF4-SIMs Domain
The E3 ubiquitin-protein ligase RNF4 contains four tandem SUMO interacting motif (SIM) repeats for selective interaction with poly-SUMO modified proteins, which it targets for degradation. We employed a multifaceted approach to characterise structures of the RNF4-SIMs domain and tetra-SUMO2 chain to elucidate the interaction between them. In solution, the SIMs domain was intrinsically disordered and the linkers of the tetra-SUMO2 were highly flexible. Individual SIMs of RNF4-SIMs domains bind to SUMO2 in the groove between the β2 strand and the α1-helix parallel to the β2 strand. SIM2 and SIM3 bound to...
Source: BJ Signal - May 20, 2014 Category: Biochemistry Authors: C C.-H. Kung, M T. Naik, S Wang, H Shih, C Chang, L Lin, C Chen, C Ma, C Chang, T Huang Tags: BJ Biomolecules Source Type: research
Interplay between Polo kinase, LKB1-activated NUAK1 kinase, PP1{beta}MYPT1 phosphatase complex and the SCF{beta}TrCP E3 ubiquitin ligase
NUAK1 and NUAK2 protein kinases are activated by the LKB1 tumour suppressor and have been implicated in regulating multiple processes such as cell survival, senescence, adhesion and polarity. Here we present evidence that expression of NUAK1 is controlled by cyclin dependent kinase (CDK), Polo kinase (PLK) and the Skp, Cullin, F-boxβTrCP (SCFβTrCP) E3 ubiquitin ligase complex. Our data indicate that CDK phosphorylates NUAK1 at Ser445, triggering binding to PLK, which subsequently phosphorylates NUAK1 at two conserved non-catalytic Ser residues (Ser476 and Ser480). This induces binding of NUAK1 to bTrCP, the s...
Source: BJ Signal - May 2, 2014 Category: Biochemistry Authors: S Banerjee, A Zagorska, M Deak, D G Campbell, A Prescott, D R Alessi Tags: BJ Signal Source Type: research
Inositol phosphates induce DAPI fluorescence shift
The polymer inorganic polyphosphate (polyP) and inositol phosphates, such as phytic acid (IP6), share many biophysical features. These similarities must be attributed to the phosphate groups present in these molecules. Given the ability of polyP to modify the excitation-emission spectra of DAPI (4',6-diamidino-2-phenylindole) we decided to investigate if inositol phosphates possess the same property. We discovered that DAPI-IP6 complexes emit at around 550 nm when excited with light of wavelength 410-420 nm. Inositol pentakisphosphate (IP5) is also able to induce a similar shift in DAPI fluorescence. Conversely, inositol t...
Source: BJ Signal - March 26, 2014 Category: Biochemistry Authors: B Kolozsvari, F Parisi, A Saiardi Tags: BJ ChemBio Source Type: research
Myoplasmic resting Ca2{+} regulation by RyR is under control of a novel Ca2{+}-binding region of the ryanodine receptor
Passive SR Ca2+-leak through the ryanodine receptor (RyR) plays a critical role in the mechanisms that regulate intracellular resting Ca2+ concentration ([Ca2+]rest) in muscle. This process appears to be isoform specific as expression of either RyR1 or RyR3 confers myotubes with different [Ca2+]rest. Using chimeric RyR3/RyR1 receptors expressed in dyspedic myotubes here we show that isoform dependent regulation of [Ca2+]rest is primarily defined by a small region of the receptor encompassing amino acid 3770-4007 of RyR1 (3620-3859 of RyR3) named as the Ca2+ Leak Regulatory (CLR) re...
Source: BJ Signal - March 18, 2014 Category: Biochemistry Authors: Y Chen, S Xue, J Zou, J R Lopez, J J Yang, C F Perez Tags: BJ ChemBio Source Type: research
