Phosphorylation Assays Research Articles

Abstract 27: Identification and characterization of a novel dual FLT3/microtubule polymerization inhibitor through cell-based screening

Abstract Activating mutations in FLT3 receptor tyrosine kinase are found in one-third of acute myeloid leukemia (AML) patients and are associated with disease relapse and bad prognosis. Majority of these mutations are Internal tandem duplications (ITD) in the juxtamembrane domain of FLT3 and have been validated as a therapeutic target. Clinical success of selective inhibitors targeting oncogenic FLT3, however, has been limited due to the acquisition of drug resistance. Here we report the identification of a dual FLT3/microtubule polymerization inhibitor, HH-IA-208, through cell-based screenings for the differential killing of leukemia cell lines expressing FLT3-ITD (MV411) or BCR-ABL (K562) using an MTS proliferation assay and for mitotic inducers using a dot-blot assay for histone H3 phosphorylation in HCT116 cell line. HH-IA-208, a chalcone derivative, inhibits FLT3 in vitro and treatment of FLT3-ITD+ cell lines with HH-IA-208 results in reduced FLT3 signaling. Similarly, HH-IA-208 inhibits tubulin polymerization in a biochemical assay and in cells that results in a mitotic arrest. HH-IA-208 is selectively more potent in leukemia cell lines expressing FLT3-ITD and treatment for 24 and 48 hours with the inhibitor results in apoptotic cell death. Furthermore, HH-IA-208 is able to overcome TKD mutation-mediated acquired resistance to FLT3 inhibitors in a MOLM13 cell line expressing FLT3-ITD with the D835Y mutation. HH-IA-208, therefore, is a promising lead for the discovery of dual target FLT3 inhibitors that can overcome resistance to selective FLT3 inhibitors. Citation Format: Haleema Sadia Malik, Aishah Bilal, Rahim Ullah, Maheen Iqbal, Rahman Shah Saleem, Hidayat Hussain, Amir Faisal. Identification and characterization of a novel dual FLT3/microtubule polymerization inhibitor through cell-based screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 27.

Abstract 4813: Comparative activity profiling of tyrosine kinase inhibitors (TKIs) against exon 20 insertions and the wild-type form of epidermal growth factor receptor (EGFR)

Abstract Exon 20 insertions (Ex20Ins) have been identified in approximately 5% of epidermal growth factor receptor (EGFR)-mutated lung tumours in patients presenting with non-small cell lung cancer (NSCLC). Several small molecule tyrosine kinase inhibitors (TKIs) have been reported to have pre-clinical activity against such insertions including afatinib, poziotinib, osimertinib, nazartinib, AP32788/TAK-788 and TAS6417. However, there remains a lack of approved treatments for patients with Ex20Ins with early approved EGFR agents appearing to be ineffective in this setting. Poziotinib, osimertinib and AP32788/TAK-788 are undergoing clinical evaluation in patients whose tumours carry Ex20Ins and in some cases clinical responses have been reported giving hope that such insertions can be targeted by small molecules. There is however a need for comparable data across such compounds that would enable understanding of the relative activity of these compounds between Ex20Ins and the wild-type form of EGFR. As many of the Exon 20 insertions are not part of the ATP binding pocket achieving selectivity over wild type EGFR is highly challenging and may limit the clinical utility of agents due to dose limiting EGFR wild-type driven toxicity. A selection of TKIs were profiled for Ex20Ins and wild-type EGFR activity using biochemical, in vitro cellular phosphorylation and proliferation assays. This has enabled us to differentiate the Ex20Ins versus wild-type EGFR selectivity profiles of a range of pre-clinical, clinical and proprietary compounds. As part of this evaluation we utilized a CRISPR CAS9 approach in H2073 EGFR wild-type NSCLC cell line, where we have established cellular disease models against the most prevalent insertions including D770-N771insSVD (22%). Finally, we will show anti-tumour efficacy data for a selection of these inhibitors along with a potential combination approach of osimertinib and cetuximab. EGFR D770-N771InsSVD cell phospho IC50 (µM)EGFR WT (H2073) cell phospho IC50 (µM)Fold-EGFR WT margin (cell)afatinib0.0060.00330.5osimertinib0.0940.414.4poziotinib0.00340.00351TAS64170.0230.0672.9AZ62810.0972.223 Citation Format: Richard A. Ward, Ambra Bianco, Nicola Colclough, Darren Cross, Emanuela M. Cuomo, M. Raymond V. Finlay, Martina Fitzek, Nicolas Floc’h, Sladjana Gagrica, Beverley Hammond, Matthew J. Martin, Darren McKerrecher, Daniel J. O’Neill, Jonathan P. Orme, Paul D. Smith, Anna D. Staniszewska, Jelena Urosevic, Nicky Whalley, James W. Yates. Comparative activity profiling of tyrosine kinase inhibitors (TKIs) against exon 20 insertions and the wild-type form of epidermal growth factor receptor (EGFR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4813.

LI1035, a putative effector secreted by Lawsonia intracellularis, targets the MAPK pathway and regulates actin organizationin yeast and mammalian cells

Lawsonia intracellularis is an obligate intracellular Gram-negative bacterium that has been identified as the etiological agent of the contagious disease proliferative enteropathy (PE) in a wide range of animals, mainly pigs. The genome sequence of L. intracellularis indicates that this bacterium possess a type III secretion system (T3SS), which may assist the bacterium during cell invasion and host innate immune system evasion and could be a mechanism for inducing cellular proliferation. However, the effectors secreted by the T3SS (T3Es) of L. intracellularis have not been reported. T3Es often target conserved eukaryotic cellular processes, and yeast is an established and robust model system in which to reveal their function. By screening the growth inhibition of an ordered array of Saccharomyces cerevisiae strains expressing the hypothetical genes of L. intracellularis, LI1035 was identified as the first putative effector that inhibits yeast growth. The LI1035-induced growth inhibition was rescued in two of the 14 mitogen-activated protein kinase (MAPK) yeast haploid deletion strains, suggesting that LI1035 interacts with the components of the MAPK pathway in yeast. Phosphorylation assays confirmed that LI1035 inhibits MAPK signaling cascades in yeast and mammalian cells. Actin staining assays revealed that LI1035 regulates actin organization in yeast and mammalian cells. Taken together, these results indicate that LI1035 alters MAPK pathway activity and regulates actin organization in the host. These findings may contribute to the understanding the pathogenesis of L. intracellularis and support the use of yeast as a heterologous system for the functional analysis of pathogen-specific gene products in the laboratory.

Small Molecule Inhibitors of KDM5 Histone Demethylases Increase the Radiosensitivity of Breast Cancer Cells Overexpressing JARID1B.

Background: KDM5 enzymes are H3K4 specific histone demethylases involved in transcriptional regulation and DNA repair. These proteins are overexpressed in different kinds of cancer, including breast, prostate and bladder carcinomas, with positive effects on cancer proliferation and chemoresistance. For these reasons, these enzymes are potential therapeutic targets. Methods: In the present study, we analyzed the effects of three different inhibitors of KDM5 enzymes in MCF-7 breast cancer cells over-expressing one of them, namely KDM5B/JARID1B. In particular we tested H3K4 demethylation (western blot); radio-sensitivity (cytoxicity and clonogenic assays) and damage accumulation (COMET assay and kinetics of H2AX phosphorylation). Results: we show that all three compounds with completely different chemical structures can selectively inhibit KDM5 enzymes and are capable of increasing sensitivity of breast cancer cells to ionizing radiation and radiation-induced damage. Conclusions: These findings confirm the involvement of H3K4 specific demethylases in the response to DNA damage, show a requirement of the catalytic function and suggest new strategies for the therapeutic use of their inhibitors.

Protein kinase D1 induces G1-phase cell-cycle arrest independent of Checkpoint kinases by phosphorylating Cell Division Cycle Phosphatase 25.

Protein Kinase D1 (PrKD1) functions as a tumor and metastasis suppressor in several human cancers by influencing cell-cycle progression. However, the exact mechanism of cell-cycle regulation by PrKD1 is unclear. Overexpression and ectopic expression of PrKD1 induces G1 arrest in cancer cell lines. Because checkpoint kinases (CHEKs) are known to play a role in progression through the G1 phase, we downregulated CHEK1, which did not overcome the G1 arrest induced by PrKD1. Using in vitro phosphorylation and Western blot assays, we showed that PrKD1 phosphorylates all CDC25 isoforms (known substrates of CHEK kinases), independent from CHEK kinases, suggesting that direct phosphorylation of CDC25 by PrKD1 may be an alternate mechanism of G1 arrest. The study has identified a molecular mechanism for the influence of PrKD1 in cell-cycle progression.

Coiled Coil Affinity-Based Systems for the Controlled Release of Biofunctionalized Gold Nanoparticles from Alginate Hydrogels.

Affinity-based systems represent a promising solution to control the delivery of therapeutics using hydrogels. Here, we report a hybrid system that is based on the peptidic E/K coiled coil affinity pair to mediate the release of gold nanoparticles (NPs) from alginate scaffolds. On one hand, the gold NPs were functionalized with the Ecoil-tagged epidermal growth factor (EGF). The bioactivity of the grafted EGF and the bioavailability of the Ecoil moiety were confirmed by EGF receptor phosphorylation assays and by capturing the functionalized NPs on a Kcoil-derivatized surface. On the other hand, alginate chains were modified with azido-homoalanine Kcoil (Aha-Kcoil) by azide-alkyne click chemistry. The hybrid system was formed by dispersing NPs functionalized with the Ecoil-tagged EGF in alginate hydrogels containing either 0, 10, or 20% of Kcoil-modified alginate (Alg-Kcoil). With 20% of Alg-Kcoil, the release of Ecoil-functionalized NPs was reduced by half when compared to the release of NPs without Ecoil, whereas little to no differences were noticed with either 0 or 10% of Alg-Kcoil. Differential dynamic microscopy was used to determine the diffusion coefficient of the NPs, and the results showed a decrease in the diffusion coefficient of Ecoil-functionalized NPs, when compared to bare PEGylated NPs. Altogether, our data demonstrated that the E/K coiled coil system can control the release of NPs in a high Kcoil content alginate gel, opening diverse applications in drug delivery.

Long non-coding RNA HOTAIR promotes exosome secretion by regulating RAB35 and SNAP23 in hepatocellular carcinoma

BackgroundEmerging evidence indicates that tumor cells release a large amount of exosomes loaded with cargos during tumorigenesis. Exosome secretion is a multi-step process regulated by certain related molecules. Long non-coding RNAs (lncRNAs) play an important role in hepatocellular carcinoma (HCC) progression. However, the role of lncRNA HOTAIR in regulating exosome secretion in HCC cells remains unclear.MethodsWe analyzed the relationship between HOTAIR expression and exosome secretion-related genes using gene set enrichment analysis (GSEA). Nanoparticle tracking analysis was performed to validate the effect of HOTAIR on exosome secretion. The transport of multivesicular bodies (MVBs) after overexpression of HOTAIR was detected by transmission electron microscopy and confocal microscopy analysis of cluster determinant 63 (CD63) with synaptosome associated protein 23 (SNAP23). The mechanism of HOTAIR’s regulation of Ras-related protein Rab-35 (RAB35), vesicle associated membrane protein 3 (VAMP3), and SNAP23 was assessed using confocal co-localization analysis, phosphorylation assays, and rescue experiments.ResultsWe found an enrichment of exosome secretion-related genes in the HOTAIR high expression group. HOTAIR promoted the release of exosomes by inducing MVB transport to the plasma membrane. HOTAIR regulated RAB35 expression and localization, which controlled the docking process. Moreover, HOTAIR facilitated the final step of fusion by influencing VAMP3 and SNAP23 colocalization. In addition, we validated that HOTAIR induced the phosphorylation of SNAP23 via mammalian target of rapamycin (mTOR) signaling.ConclusionOur study demonstrated a novel function of lncRNA HOTAIR in promoting exosome secretion from HCC cells and provided a new understanding of lncRNAs in tumor cell biology.

Calcium Release from Endoplasmic Reticulum Involves Calmodulin-Mediated NADPH Oxidase-Derived Reactive Oxygen Species Production in Endothelial Cells.

Background: Previous studies demonstrated that calcium/calmodulin (Ca2+/CaM) activates nicotinamide adenine dinucleotide phosphate oxidases (NOX). In endothelial cells, the elevation of intracellular Ca2+ level consists of two components: Ca2+ mobilization from the endoplasmic reticulum (ER) and the subsequent store-operated Ca2+ entry. However, little is known about which component of Ca2+ increase is required to activate NOX in endothelial cells. Here, we investigated the mechanism that regulates NOX-derived reactive oxygen species (ROS) production via a Ca2+/CaM-dependent pathway. Methods: We measured ROS production using a fluorescent indicator in endothelial cells and performed phosphorylation assays. Results: Bradykinin (BK) increased NOX-derived cytosolic ROS. When cells were exposed to BK with either a nominal Ca2+-free or 1 mM of extracellular Ca2+ concentration modified Tyrode’s solution, no difference in BK-induced ROS production was observed; however, chelating of cytosolic Ca2+ by BAPTA/AM or the depletion of ER Ca2+ contents by thapsigargin eliminated BK-induced ROS production. BK-induced ROS production was inhibited by a CaM inhibitor; however, a Ca2+/CaM-dependent protein kinase II (CaMKII) inhibitor did not affect BK-induced ROS production. Furthermore, BK stimulation did not increase phosphorylation of NOX2, NOX4, and NOX5. Conclusions: BK-induced NOX-derived ROS production was mediated via a Ca2+/CaM-dependent pathway; however, it was independent from NOX phosphorylation. This was strictly regulated by ER Ca2+ contents.

Receptor Binding and Phosphorylation of Anabaena Sensory Rhodopsin Transducer towards Signaling via Protein‐Protein Cross Talk

Anabaena sensory rhodopsin transducer, ASRT [125 amino acid sequence] is present under the same promoter of its sensory rhodopsin, ASR in Anabaena PCC 7120 a freshwater, filamentous, photosynthetic cynobacteria. The ASRT exists as tetrameric soluble protein indicated to function as downstream signaling partner to sensory rhodopsin. Both solution and crystal structures has reveled that this primarily beta stranded protein exhibit a helical face at carboxyl terminus. Besides receptor binding, ASRT is been shown to serve as a novel eukaryotic-like interaction with DNA. We have demonstrated that carboxyl helical face of tetramer is involved in unusual tetrameric stability. A blast search matches the ASRT to others of unknown function, with 84% similarity and 74% identity to a protein in the myxobacterium Polyangium cellulosum and with 72% similarity and 58% identity to a protein in the legume symbiont Sinorhizobium meliloti. Often the homologues of this family are termed as “DUF”, Domain of Unknown Function member. This DUF1362 family is wide spread over microbial population including pathogens as well. The gene neighbor hood, along with structural fold analysis termed this family as novel small carbohydrate binding protein. We have observed unique xylan binding via electrostatic interaction. Additional bioinformatics analysis has revealed a phosphor transfer along with unique structural fold that may transform it as a unique carbohydrate binding module. However, the signaling state/mechanism of ASRT is obscure. Our initial data with efficient donor, acetyl phosphate, acP using fluorescence quenching of lone Trp-9 suggests that ASRT is involved in phosphorylation. We noticed two notable consensus phosphor transfer residue motif in ASRT, SDKE [53–56] and TRLD [105–108]. Acetyl phosphate has been used as phosphoryl donor in vitro to numerous response regulators [CheY/PhoB/OmpR]. The extent of quenching in presence of Mg2+by increasing concentration of acP yielded lower KM [~21mM] compared to PhoB [~8mM]. It is likely that proximity of phosphor accepting residue (s) to the lone Trp is not comparable to others. Interestingly the ASRT mutant E56Q and D108N indicate the loss of phosphor transfer. We hypothesize that putative position shown as red in figure is involved in ASRT phosphorylation. This motif is in close proximity to receptor binding deduced by CABS docking and GST-assay using various length of ASR [full length, 1–261, and truncated (1–229, 1–239, 1–249)]. Phosphor transfer data along with plausible impact on tetrameric stability will be discussed in this study. We have identified a Serine Threonine Kinase, STK 3169 gene as a likely candidate in phosphor relay. Recent results of STK 3169 cloning, optimized expression, purification and subsequent characterization in phosphorylation assay will be presented. Support or Funding Information Supported by NIH-NIGMS Support for Competitive Research, SCORE SC3 award GM113803 to VT This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ERK1/2 phosphorylates HIF-2α and regulates its activity by controlling its CRM1-dependent nuclear shuttling.

Hypoxia-inducible factor 2 (HIF-2) is a principal component of the cellular response to oxygen deprivation (hypoxia). Its inducible subunit, HIF-2α (also known as EPAS1), is controlled by oxygen-dependent as well as oxygen-independent mechanisms, such as phosphorylation. We show here that HIF-2α is phosphorylated under hypoxia (1% O2) by extracellular signal-regulated protein kinases 1 and 2 (ERK1/2; also known as MAPK3 and MAPK1, respectively) at serine residue 672, as identified by in vitro phosphorylation assays. Mutation of this site to an alanine residue or inhibition of the ERK1/2 pathway decreases HIF-2 transcriptional activity and causes HIF-2α to mislocalize to the cytoplasm without changing its protein expression levels. Localization, reporter gene and immunoprecipitation experiments further show that HIF-2α associates with the exportin chromosomal maintenance 1 (CRM1, also known as XPO1) in a phosphorylation-sensitive manner and identify two critical leucine residues as part of an atypical CRM1-dependent nuclear export signal (NES) neighboring serine 672. Inhibition of CRM1 or mutation of these residues restores nuclear accumulation and activity of HIF-2α lacking the ERK1/2-mediated modification. In summary, we reveal a novel regulatory mechanism of HIF-2, involving ERK1/2-dependent phosphorylation of HIF-2α, which controls its nucleocytoplasmic shuttling and the HIF-2 transcriptional activity.This article has an associated First Person interview with the first author of the paper.

Role of RGS Homology and Pleckstrin Homology Domains in GPCR Kinase 2 Activation

G protein‐coupled receptors (GPCRs) are transmembrane proteins that act through multi‐step pathways to regulate metabolic and synaptic signaling, as well as many other processes. GPCR kinases (GRKs) regulate GPCR signaling via phosphorylation. The seven vertebrate GRKs map to three subfamilies: GRK1 (GRK1/7), GRK4 (GRK4/5/6), and GRK2 (GRK2/3). GRK2 is a modular protein composed of an AGC kinase domain (KD), a pleckstrin homology domain (PHD) that binds Gβγ subunits and phospholipids, and a regulator of G protein signaling homology domain (RHD). The RHD is the central domain and makes contacts with both PHD and KD. Although GRK2 is activated by Gβγ and membrane phospholipids, it is its interaction with an activated GPCR that most dramatically promotes kinase domain activation. We seek to understand how GPCRs activate GRK2. Previously we used site‐directed mutagenesis to test the hypothesis that the release of inter‐domain contacts might artificially activate GRK2. Our hypothesis was not supported: mutations at the RH/KD small lobe interface disrupted the structural integrity of GRK2, while a mutation at the RH/KD large lobe interface (E96A) did not impact rhodopsin phosphorylation (Lodowski et al., 2005). This contrasts recent in vitro kinetic studies and molecular dynamic simulations with GRK4 family members (Komolov et al., 2017; Yao et al., 2017) that suggest that disruption of the RHD/KD interface (“ionic lock”) leads to RHD separation from the kinase domain and allows the KD to acquire an active conformation. The PH domain, only found in the GRK2 subfamily of GRKs, is essential for localizing GRK2 to the site of an activated receptor. Comparison of crystal structures of monomeric GRK2 versus the GRK2–Gβγ complex suggests that Gβγ binding causes slight rotation of the RHD away from the KD (Lodowski et al., 2005). Single time‐point kinase assays showed that mutations at the RHD/PHD interface, particularly P638D, impaired GRK2 function. It is possible that the PHD in GRK2 subfamily plays a large role in receptor mediated KD activation. In this work, we use an intact cell phosphorylation assay as well as more detailed kinetic studies in combination with expanded mutagenesis to assess the role of the RHD and the RHD/PHD interface in GRK2 activation.Support or Funding InformationSiena College Center for Undergraduate Research and Creative ActivityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of a novel scaffold for a small molecule GPR139 receptor agonist

GPR139 is an orphan G protein-coupled receptor (GPCR) that is primarily expressed in the brain in regions known to regulate motor control and metabolism. Here, we screened a diverse 4,000 compound library in order to identify GPR139 agonists. We identified 11 initial hits in a calcium mobilization screen, including one compound, AC4, which contains a different chemical scaffold to what has previously been described for GPR139 agonists. Our mutagenesis data shows that AC4 interacts with the same hotspots in the binding site of GPR139 as those reported to interact with the reference agonists 1a and 7c. We additionally tested and validated 160 analogs in a calcium mobilization assay and found 5 compounds with improved potency compared to AC4. In total, we identified 36 GPR139 agonists with potencies in the nanomolar range (90–990 nM). The most potent compounds were confirmed as GPR139 agonists using an orthogonal ERK phosphorylation assay where they displayed a similar rank order of potency. Accordingly, we herein introduce multiple novel GPR139 agonists, including one with a novel chemical scaffold, which can be used as tools for future pharmacological and medicinal chemistry exploration of GPR139.

Molecular determinants on extracellular loop domains that dictate interaction between β-arrestin and human APJ receptor.

The human APJ receptor (APJR), activated by apelin isoforms, regulates cardiovascular functions and fluid homeostasis. Understanding its structure-function relationship is crucial for a comprehensive knowledge of signalling aberrations that cause several physiological disorders. Here, we demonstrate the influence of extracellular loop (ECL) domains in the mechanism of β-arrestin-mediated signalling from human APJR: Apelin system. Alanine mutations of evolutionarily conserved residues were characterized using receptor internalization, β-arrestin pull-down, Akt phosphorylation and cell migration assay. C281A and 268 KTL270 -AAA in ECL3 were deficient in all assays, whereas 183 MDYS186 -AAAA mutant in ECL2 showed impaired β-arrestin-mediated signalling but demonstrated Gi -dependent cell migration. Our findings establish that conserved residues in the extracellular domain play a prominent role in modulating receptor interactions with the β-arrestin signalling cascade.

Impact of cardiac myosin light chain kinase gene mutation on development of dilated cardiomyopathy.

AimsCardiac myosin light chain kinase (cMLCK) phosphorylates ventricular myosin regulatory light chain 2 (MLC2v) and regulates sarcomere and cardiomyocyte organization. However, few data exist regarding the relationship between cMLCK mutations and MLC2v phosphorylation, particularly in terms of developing familial dilated cardiomyopathy (DCM) in whom cMLCK gene mutations were identified. The purpose of the present study was to investigate functional consequences of cMLCK mutations in DCM patients.Methods and resultsThe diagnosis of DCM was based on the patients' history and on echocardiography. We screened cMLCK gene mutations in DCM probands with high resolution melting analysis. Known DCM‐causing genes mutations were excluded by exome sequencing of family members. MLC2v phosphorylation was analysed by Phos‐tag sodium dodecyl sulfate–polyacrylamide gel electrophoresis assays. We also performed ADP‐Glo assays for determining the total amount of adenosine triphosphate used in the kinase reaction. Unrelated DCM probands (109 males and 40 females) were enrolled in this study, of which 16 were familial and 133 sporadic. By mutation screening, a truncation variant of c1915‐1 g>t (p.Pro639Valfs*15) was identified, which was not detected in 400 chromosomes of 200 healthy volunteers; it is listed in the Human Genetic Variation Database with an allele frequency < 0.001. In the proband, the presence of mutations in known DCM‐causing genes was excluded with exome analysis. Familial analysis identified a 19‐year‐old male carrier who manifested slight left ventricular dilation with preserved systolic function. Phosphorylation assays analysed by Phos‐tag SDS‐PAGE revealed that the identified p.Pro639Valfs*15 mutation results in a complete lack of kinase activity, although it did not affect wild‐type cMLCK activity. ADP‐Glo assays confirmed that the mutant cMLCK had no kinase activity, whereas wild‐type cMLCK had a Km value of 5.93 ± 1.47 μM and a V max of 1.28 ± 0.03 mol/min/mol kinase.ConclusionsThese results demonstrate that a truncation mutation in the cMLCK gene p.Pro639Valfs*15 can be associated with significant impairment of MLC2v phosphorylation and possibly with development of DCM, although a larger study of DCM patients is required to determine the prevalence of this mutation and further strengthen its association with disease development.

Structural Analysis of the Hanks-Type Protein Kinase YabT From Bacillus subtilis Provides New Insights in its DNA-Dependent Activation.

YabT is a serine/threonine kinase of the Hanks family from Bacillus subtilis, which lacks the canonical extracellular signal receptor domain but is anchored to the membrane through a C-terminal transmembrane helix. A previous study demonstrated that a basic juxtamembrane region corresponds to a DNA-binding motif essential for the activation of YabT trans-autophosphorylation. YabT is expressed during spore development and localizes to the asymmetric septum where it specifically phosphorylates essential proteins involved in genome maintenance, such as RecA, SsbA, and YabA. YabT has also been shown to phosphorylate proteins involved in protein synthesis, such as AbrB and Ef-Tu, suggesting a possible regulatory role in the progressive metabolic quiescence of the forespore. Finally, cross phosphorylations with other protein kinases implicate YabT in the regulation of numerous other cellular processes. Using an artificial protein scaffold as crystallization helper, we determined the first crystal structure of this DNA-dependent bacterial protein kinase. This allowed us to trap the active conformation of the kinase domain of YabT. Using NMR, we showed that the basic juxtamembrane region of YabT is disordered in the absence of DNA in solution, just like it is in the crystal, and that it is stabilized upon DNA binding. In comparison with its closest structural homolog, the mycobacterial kinase PknB allowed us to discuss the dimerization mode of YabT. Together with phosphorylation assays and DNA-binding experiments, this structural analysis helped us to gain new insights into the regulatory activation mechanism of YabT.

Phosphorylation Properties of the N-Terminal Region of Twitchin from Molluscan Catch Muscle

Molluscan smooth muscles, such as the bivalve adductor muscles and the mussel anterior byssus retractor muscles (ABRM), exhibit a unique contraction called “catch”. Catch contraction is regulated through twitchin phosphorylation and dephosphorylation. Twitchin from the ABRM of the Mediterranean mussel, Mytilus galloprovincialis, is phosphorylated by cAMP-dependent protein kinase (PKA), and PKA phosphorylation sites are located in both the N- and C-terminal regions of the twitchin molecule. The D2 site, which is adjacently located to the C-terminus, participates in forming a myosin, actin, and twitchin complex that is thought to contribute towards the maintenance of tension in the catch state. In contrast, although it has been reported to interact with thin-filaments, the molecular function of the region including the D1 site has remained largely unstudied. Three additional PKA consensus sequences were identified near the D1 site; however, it was not known if these sites could be directly phosphorylated by PKA. Here, we performed phosphorylation assays to identify phosphorylation sites near the D1 site using recombinant protein variants (TWD1-SSSS, TWD1-AAAS, TWD1-AASA, TWD1-ASAA, TWD1-SAAA, and TWD1-AAAA). All variants, except TWD1-AAAA (where all phosphorylatable serine residues were replaced by alanines), were phosphorylated by PKA. The four phosphorylation sites were named D1-1, D1-2, D1-3, and D1-4 (the originally identified D1) in order from the N-terminus. Phosphorylation assays using a 1/12.5 weight ratio of PKA to each TWD1 variant revealed that D1-4 was the most rapidly phosphorylated, closely followed by D1-1. However, D1-2 and D1-3 were phosphorylated at a lower level under equivalent conditions and were not phosphorylated when PKA was incubated with each TWD1 variant at a 1/100 weight ratio. Furthermore, we observed that TWD1-SSSS was phosphorylated in a stepwise fashion. These findings contribute towards the elucidation of the function of the twitchin D1 region in the regulatory system of catch contraction.

Purification of Recombinant ESCRT-III Proteins and Their Use in Atomic Force Microscopy and In Vitro Binding and Phosphorylation Assays.

The endosomal sorting complex required for transport (ESCRT)-III proteins are known to assemble into filaments that mediate membrane remodeling and fission in various biological processes, including the formation of endosomal multivesicular bodies, viral budding, cytokinesis, plasma membrane repair, nuclear pore quality control, nuclear envelope reformation, and neuron pruning. The study of the regulation and function of ESCRT-III proteins is therefore crucial to understand these events and requires a combination of in vivo and in vitro experimental techniques. Here we describe two protocols for the purification of human and Drosophila ESCRT-III proteins from bacteria and their use in in vitro phosphorylation assays and atomic force microscopy experiments on membrane lipid bilayers. These protocols can also be applied for the purification of other proteins that are insoluble when expressed in bacteria.

The relation between ABO blood types and clinical and platelet function parameters in patients who underwent percutaneous coronary intervention.

ABO blood groups have been associated with venous thromboembolism and arterial thrombosis. Although platelets play key roles in thrombogenesis, the relation between ABO groups and platelets is not well known and was investigated in this study. ABO blood type information was retrospectively obtained for 206 patients who underwent percutaneous coronary intervention (PCI) and received dual antiplatelet therapy with aspirin and clopidogrel. Platelet function was measured using VerifyNow system, light transmission aggregometry, thromboxane B2, urinary 11-dehydrothromboxane B2, and vasodilator-stimulated phosphoprotein phosphorylation assays. Samples were also tested following treatment with 10 and 30 µmol/l of aspirin or 30 and 100 µmol/l of P2Y12 inhibitor 2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate (2-MeSAMP). Forty-four clinical and 30 platelet function parameters were analyzed. Patients were categorized as aspirin or clopidogrel poor responder (PR) according to cutoff levels of each test. Blood type A was significantly associated with myocardial infarction (MI) history [odds ratio (OR)=2.50, 95% confidence interval (CI)=1.37-4.58, P=0.003], higher baseline troponin T and creatine kinase-MB (CK-MB) index, post-PCI CK-MB index, and platelet reactivity index (PRI), and being PR against 2-MeSAMP (OR=5.75, 95% CI=1.51-21.90, P=0.010). Blood type O was associated with higher arachidonic acid-induced platelet aggregation and negatively associated with MI history (OR=0.47, 95% CI=0.26-0.84, P=0.010), PRI and being PR against clopidogrel (OR=0.54, 95% CI=0.30-0.99, P=0.043) and 2-MeSAMP (OR=0.16, 95% CI=0.03-0.76, P=0.021). Blood type A was found as a risk factor for MI. Higher arachidonic acid-induced aggregation in group O and higher PRI in group A against aspirin and P2Y12 inhibitor treatment, respectively, may suggest alternative antiplatelet therapies for PRs with these blood types.

Phosphorylation-dependent ribonuclease activity of Fra a 1 proteins

Abiotic and biotic stress situations cause the upregulation of the transcription of a number of plant defence genes. They code for so-called pathogenesis-related (PR) proteins such as PR proteins of class-10 (PR-10), whose biological functions are still unclear. PR10 proteins are members of the Bet v 1 (major birch pollen allergen) superfamily including related proteins from the cultivated strawberry Fragaria × ananassa (Fra a 1 proteins). Here, we analyzed the expression of 21 Fra a 1 genes in different tissues of the strawberry plant by quantitative real-time PCR. Thirteen members were mainly expressed in roots, three in stems, two in red fruits and leaves, and one in flowers. Five genes (Fra a 1.04–1.08) were selected based on their expression profiles, heterologously expressed in Escherichia coli, and their recombinant proteins functionally characterized. Ribonuclease activity, demonstrated by in-solution and in-gel RNA degradation assays, indicated complete hydrolysis of RNA only by Fra a 1.06. Moreover, phosphorylation assays showed that except for Fra a 1.06, the remaining four recombinant proteins were phosphorylated. Consequently, we investigated whether the phosphorylation status of the proteins affects their ribonuclease activity. Using an in-solution as well as an in-gel RNase activity assay, results demonstrated that the four recombinant proteins, dephosphorylated with phosphatases, exhibited ribonucleolytic activity against total RNA. Thus, the PR10 related proteins characterized in this study harbour a phosphorylation-dependent RNase activity. The results shed new light on the assumed function of PR10 proteins in plant defence.

TaCIPK10 interacts with and phosphorylates TaNH2 to activate wheat defense responses to stripe rust

SummaryCalcineurin B‐like interacting protein kinase (CIPKs) has been shown to be required for biotic stress tolerance of plants in plant‐pathogen interactions. However, the roles of CIPKs in immune signalling of cereal crops and an in‐depth knowledge of substrates of CIPKs in response to biotic stress are under debate. In this study, we identified and cloned a CIPK homologue gene TaCIPK10 from wheat. TaCIPK10 was rapidly induced by Puccinia striiformis f. sp. tritici (Pst) inoculation and salicylic acid (SA) treatment. In vitro phosphorylation assay demonstrated that the kinase activity of TaCIPK10 is regulated by Ca2+ and TaCBL4. Knockdown TaCIPK10 significantly reduced wheat resistance to Pst, whereas TaCIPK10 overexpression resulted in enhanced wheat resistance to Pst by the induction of defense response in different aspects, including hypersensitive cell death, ROS accumulation and pathogenesis‐relative genes expression. Moreover, TaCIPK10 physically interacted with and phosphorylated TaNH2, which was homologous to AtNPR3/4. Silencing of TaNH2 in wheat resulted in enhanced susceptibility to the avirulent Pst race, CYR23, indicating its positive role in wheat resistance. Our results demonstrate that TaCIPK10 positively regulate wheat resistance to Pst as molecular links between of Ca2+ and downstream components of defense response and TaCIPK10 interacts with and phosphorylates TaNH2 to regulate wheat resistance to Pst.