Backbone NMR resonance assignment of the catalytic subunit of cAMP-dependent protein kinase A in complex with AMP-PNP

The catalytic subunit of protein kinase A is involved with a number of signal transduction pathways and has been used as a benchmark to study the structural biology and biochemistry for the entire kinase family of enzymes. Here, we report the backbone assignment of the intact 41 kDa catalytic subunit bound to AMP-PNP. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The first large duplication of the RSK2 gene identified in a Coffin-Lowry syndrome patient

Heterogeneous mutations in the X-linked gene RPS6KA3, encoding the protein kinase RSK2, are responsible for Coffin-Lowry Syndrome. Here we have further studied a male patient with a highly suggestive clinical diagnosis of CLS but in whom no mutation was found by exon sequencing. Western blot analysis revealed a protein much larger than the normal expected size. Sequencing of the RSK2 cDNA, showed the presence of an in-frame tandem duplication of exons 17–20. The mutated RSK2 protein was found to be inactive in an in-vitro kinase assay. This event, which was the result of a homologous unequal recombination between Alu sequences, is the first reported large duplication of the RPS6KA3 gene. Our finding provides further evidence that immunoblot analysis, or a molecular assay capable to detect large genomic mutational events, is essential for patients with a highly suggestive CLS clinical diagnosis but remaining without mutation after exon sequencing. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Acetylcholine inhibits long-term hypoxia-induced apoptosis by suppressing the oxidative stress-mediated MAPKs activation as well as regulation of Bcl-2, c-IAPs,and caspase-3 in mouse embryonic stem cells

This study examined the effect of acetylcholine (ACh) on the hypoxia-induced apoptosis of mouse embryonic stem (ES) cells. Hypoxia (60 h) decreased both the cell viability and level of [³H] thymidine incorporation, which were prevented by a pretreatment with ACh. However, the atropine (ACh receptor [AChR] inhibitor) treatment blocked the protective effect of ACh. Hypoxia (90 min) increased the intracellular level of reactive oxygen species (ROS). On the other hand, ACh inhibited the hypoxia-induced increase in ROS, which was blocked by an atropine treatment. Subsequently, the hypoxia-induced ROS increased the level of p38 mitogen activated protein kinase (MAPK) and Jun-N-terminal kinase (JNK) phosphorylation, which were inhibited by the ACh pretreatment. Moreover, hypoxic exposure (90 min) increased the level of nuclear factor-κB (NF-κB) phosphorylation, which was blocked by a pretreatment with SB 203580 (p38 MAPK inhibitor) or SP 600125 (JNK inhibitor). However, hypoxia (60 h) decreased the protein levels of Bcl-2 and c-IAPs (cellular inhibitor of apoptosis proteins) but increased the level of caspase-3 activation. All these effects were inhibited by a pretreatment with ACh. In conclusion, ACh prevented the hypoxia-induced apoptosis of mouse ES cells by inhibiting the ROS-mediated p38 MAPK and JNK activation as well as the regulation of Bcl-2, c-IAPs, and caspase-3. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Combined HSP90 and kinase inhibitor therapy: Insights from The Cancer Genome Atlas

The merging of knowledge from genomics, cellular signal transduction and molecular evolution is producing new paradigms of cancer analysis. Protein kinases have long been understood to initiate and promote malignant cell growth and targeting kinases to fight cancer has been a major strategy within the pharmaceutical industry for over two decades. Despite the initial success of kinase inhibitors (KIs), the ability of cancer to evolve resistance and reprogram oncogenic signaling networks has reduced the efficacy of kinase targeting. The molecular chaperone HSP90 physically supports global kinase function while also acting as an evolutionary capacitor. The Cancer Genome Atlas (TCGA) has compiled a trove of data indicating that a large percentage of tumors overexpress or possess mutant kinases that depend on the HSP90 molecular chaperone complex. Moreover, the overexpression or mutation of parallel activators of kinase activity (PAKA) increases the number of components that promote malignancy and indirectly associate with HSP90. Therefore, targeting HSP90 is predicted to complement kinase inhibitors by inhibiting oncogenic reprogramming and cancer evolution. Based on this hypothesis, consideration should be given by both the research and clinical communities towards combining kinase inhibitors and HSP90 inhibitors (H90Ins) in combating cancer. The purpose of this perspective is to reflect on the current understanding of HSP90 and kinase biology as well as promote the exploration of potential synergistic molecular therapy combinations through the utilization of The Cancer Genome Atlas.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-015-0604-1) contains supplementary material, which is available to authorized users.     

The Evolution of Guanylyl Cyclases as Multidomain Proteins: Conserved Features of Kinase-Cyclase Domain Fusions

Guanylyl cyclases (GCs) are enzymes that generate cyclic GMP and regulate different physiologic and developmental processes in a number of organisms. GCs possess sequence similarity to class III adenylyl cyclases (ACs) and are present as either membrane-bound receptor GCs or cytosolic soluble GCs. We sought to determine the evolution of GCs using a large-scale bioinformatic analysis and found multiple lineage-specific expansions of GC genes in the genomes of many eukaryotes. Moreover, a few GC-like proteins were identified in prokaryotes, which come fused to a number of different domains, suggesting allosteric regulation of nucleotide cyclase activity. Eukaryotic receptor GCs are associated with a kinase homology domain (KHD), and phylogenetic analysis of these proteins suggest coevolution of the KHD and the associated cyclase domain as well as a conservation of the sequence and the size of the linker region between the KHD and the associated cyclase domain. Finally, we also report the existence of mimiviral proteins that contain putative active kinase domains associated with a cyclase domain, which could suggest early evolution of the fusion of these two important domains involved in signal transduction. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The VEGFR2 and PKA pathways converge at MEK/ERK1/2 to promote survival in serum deprived neuronal cells

Identifying prosurvival mechanisms in stressed neuronal cells would provide protective strategies to hinder neurodegeneration. Recent evidence shows that vascular endothelial growth factor (VEGF), a well-established mitogen in endothelial cells, can mediate neuroprotection against damaging insults through the activation of its cognate receptor VEGFR2. In addition, growth factor receptor signaling pathways have been shown to crosstalk with cAMP-dependent Protein Kinase A (PKA) to protect neuronal cells from harmful stimuli. Whether a relationship exists between VEGFR2 and PKA in mediating neuroprotection under stressful conditions is unknown. Using SK-N-SH neuronal cells as a model system, we show that serum deprivation induces an upregulation in VEGF and VEGFR2 that concomitantly serves as a prosurvival signaling pathway. Inhibitor studies revealed that PKA functioned concurrently with VEGFR2 pathway to signal the activation of the extracellular signal-regulated protein kinases (ERK1/2) as protection against caspase-3/7 activation and a subsequent cell death. The loss in cell viability induced by VEGFR2 and PKA inhibition was prevented by caspase inhibition or overexpression of ERK1. Overexpression of the antiapoptotic protein Bcl-xL also promoted survival when VEGFR2 function was blocked. However, the protection elicited by all three treatments were prevented by the inclusion of a selective inhibitor of mitogen-activated protein kinase kinase (MEK), the upstream kinase that activates ERK1/2. Taken together, these findings suggested that PKA and VEGFR2 converge at the MEK/ERK1/2 pathway to protect serum starved neuronal cells from a caspase-dependent cell death. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An approach for determining and measuring network hierarchy applied to comparing the phosphorylome and the regulome

Many biological networks naturally form a hierarchy with a preponderance of downward information flow. In this study, we define a score to quantify the degree of hierarchy in a network and develop a simulated-annealing algorithm to maximize the hierarchical score globally over a network. We apply our algorithm to determine the hierarchical structure of the phosphorylome in detail and investigate the correlation between its hierarchy and kinase properties. We also compare it to the regulatory network, finding that the phosphorylome is more hierarchical than the regulome.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0624-2) contains supplementary material, which is available to authorized users.     

Mitogen-activated protein kinase OsMPK6 negatively regulates rice disease resistance to bacterial pathogens

Mitogen-activated protein kinase (MAPK) cascades play important roles in diverse developmental and physiological processes of plants, including pathogen-induced defense responses. Although at least 17 rice MAPKs have been identified and more than half of these MAPK genes have been shown to be pathogen or elicitor responsive, the exact role of most of the MAPKs in host-pathogen interaction is unknown. Here we report that OsMPK6 is an important regulator in rice disease resistance. Suppressing OsMPK6 or knocking out of OsMPK6 enhanced rice resistance to different races of Xanthomonas oryzae pv. oryzae, causing bacterial blight, one of the most devastating diseases of rice worldwide. The resistant plants showed increased expression of a subset of defense-responsive genes functioning in the NH1 (an Arabidopsis NPR1 orthologue)-involved defense signal transduction pathway. These results suggest that OsMPK6 functions as a repressor to regulate rice defense responses upon bacterial invasion. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Midostaurin preferentially attenuates proliferation of triple-negative breast cancer cell lines through inhibition of Aurora kinase family

Background

Breast cancer is classified into three subtypes by the expression of biomarker receptors such as hormone receptors and human epidermal growth factor receptor 2. Triple-negative breast cancer (TNBC) expresses none of these receptors and has an aggressive phenotype with a poor prognosis, which is insensitive to the drugs that target the hormone receptors and human epidermal growth factor receptor 2. It is, thus, required to develop an effective therapeutic reagent to treat TNBC.

Results

The study using a panel of 19 breast cancer cell lines revealed that midostaurin, a multi-target protein kinase inhibitor, suppresses preferentially the growth of TNBC cells comparing with non-TNBC cells. Clustering analysis of the drug activity data for the panel of cancer cell lines predicted that midostaurin shares the target with Aurora kinase inhibitors. Following studies indicated that midostaurin attenuates the phosphorylation reaction mediated by Aurora kinase in the cells and directly inhibits this protein kinase in vitro, and that this reagent induces apoptosis accompanying accumulation of 4N and 8N DNA cells in TNBC cells.

Conclusion

Midostaurin suppresses the proliferation of TNBC cells among the breast cancer cell lines presumably through the inhibition of the Aurora kinase family. The precise study of midostaurin on cell growth will contribute to the development of the drug for the treatment of TNBC.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0150-2) contains supplementary material, which is available to authorized users.     

A unique highly thermostable 2-phosphoglycerate forming glycerate kinase from the hyperthermophilic archaeon <Emphasis Type="Italic">Pyrococcus horikoshii</Emphasis>: gene cloning,expression and characterization

A glycerate kinase (GK) gene (PH0495) from the hyperthermophilic archaeon Pyrococcus horikoshii, was cloned and expressed in Escherichia coli. The recombinant protein was purified to homogeneity by affinity chromatography and ion exchange chromatography. The enzyme was likely a homodimer based on SDS-PAGE (47 kDa) and gel filtration chromatography (100 kDa) analysis. A radioisotope-labeling examination method was initially used for the enzymatic activity detection, and the enzyme (GK_ph) was found to catalyze the formation of 2-phosphoglycerate using d-glycerate as the substrate. The enzyme exhibited unique phosphoryl donor specificity with maximal activity towards pyrophosphate. The temperature and pH optima of the enzyme were 45°C and 7.0, respectively, and about half of the maximal activity remained at 100°C. The enzyme was highly thermostable with almost no loss of activity at 90°C for 12 h. Based on sequence alignment and structural comparison it was assigned to group I of the trichotomy of GKs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

1H, 13C and 15N resonance assignment of 6aJL2(R25G), a highly fibrillogenic λVI light chain variable domain

An allotypic variation at position 25 influences the fibrillogenicity of λVI light chains, which are related to humoral immune response and have been associated with AL amyloidosis. The full resonance assignment and a preliminary structural characterization of 6aJL2(R25G) are reported. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of the codon use frequency of AMPK family genes from different species

In mammals, AMP-activated protein kinase (AMPK) is involved in the regulation of cellular energy homeostasis and, on the whole animal level, in regulating energy balance and food intake. In this paper, the relative synonymous codon use frequency of 40 AMPK family genes from seven mammal species (Bos taurus, Homo sapiens, Macaca mulatta, Mus musculus, Pan troglodytes, Rattus norvegicus, Sus scrofa) were analyzed using correspondence analysis and hierarchical cluster method. The result suggests that gene function is the dominant factor that determines codon usage bias in AMPK family genes, while species is a minor factor that determines further difference in codon usage bias for genes with similar functions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fast co-evolution of sevenless and bride of sevenless in endopterygote insects

Activation of Ras signaling by the receptor tyrosine kinase Sevenless plays important roles during retinal patterning and male germline development in Drosophila. Sevenless is orthologous to the vertebrate receptor tyrosine kinase c-ros. Remarkably, vertebrate ligands of c-Ros as well as non-Drosophila orthologs of the Sevenless ligand Bride of sevenless have remained elusive. Using newly available insect genome sequence information, we investigated the evolutionary conservation of the seven transmembrane domain protein gene bride of sevenless. Single orthologs were identified in the genomes of mosquito, flour beetle, and honeybee due to strong sequence conservation in the seven transmembrane domain. The extracellular region, however, is only detectably conserved within but not outside Diptera. Analysis of domain-specific substitution rates demonstrates correlated fast rates of evolutionary change in the extracellular domains of both bride of sevenless and sevenless. The rapid pace of sequence change explains why Sevenless ligands are difficult to detect by sequence similarity in distantly related phyla. Second, the conservation of bride of sevenless in flour beetle and honeybee raises the possibility of conserved Sevenless signaling controlled patterning processes in endopterygote insects. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The use of isobaric tag peptide labeling (iTRAQ) and mass spectrometry to examine rare,primitive hematopoietic cells from patients with chronic myeloid leukemia

Chronic Myeloid Leukemia (CML) is a hematopoietic stem cell disease, associated with a t(9, 22) chromosomal translocation leading to formation of the BCR/ABL chimeric protein, which has an intrinsic tyrosine kinase activity. Recently, the BCR/ABL tyrosine kinase inhibitor imatinib mesylate (imatinib) has been successfully used clinically, although, disease relapse can still occur. The precise detail of the mechanism by which CML cells respond to imatinib is still unclear. We therefore systematically examined the effects of imatinib on the primitive CML cell proteome, having first established that the drug inhibits proliferation and induces increased apoptosis and differentiation. To define imatinib-induced effects on the CML proteome, we employed isobaric tag peptide labeling (iTRAQ) coupled to two-dimensional liquid chromatography/tandem mass spectrometry. Given the limited clinical material available, the isobaric tag approach identified a large population of proteins and provided relative quantification on four samples at once. Novel consequences of the action of imatinib were identified using this mass spectrometric approach. DEAD-box protein 3, heat shock protein 105 kDa, and peroxiredoxin-3 were identified as potential protein markers for response to imatinib. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Stephen D. Griffiths and John Burthem contributed equally to this publication. This work is supported by The Leukaemia Research Fund (UK).     

Amino acid partitioning using a Fiedler vector model

This paper presents a new Fiedler vector model for categorising amino acids, which is based on the Miyazawa-Jernigan matrix. The model splits the amino acid residues into two hydrophobic groups (LFI) and (MVWCY) and two polar groups (HATGP) and (RQSNEDK). In so doing, it independently confirms the findings of Wang and Wang and Cieplak et al. and demonstrates the validity of using eigenvectors to partition amino acid groups. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Anti-proliferative effects of recombinant iron superoxide dismutase on HepG2 cells via a redox-dependent PI3k/Akt pathway

The coding sequence for an iron superoxide dismutase (fe-sod) was amplified from the Nostoc commune genome. Recombinant Fe-SOD was overexpressed in Escherichia coli, accounting for ∼76% of total bacterial protein. Fe-SOD was purified from bacterial lysate by Ni-NTA column chromatography and used to generate an anti-SOD antibody. The purified Fe-SOD was encapsulated in liposomes and delivered to HepG2 liver tumor cells to eliminate cellular superoxide anions. The SOD-loaded cells exhibited lower reactive oxygen species (ROS) levels and higher reduced glutathione (GSH) levels. In Fe-SOD-treated cells, the cell cycle was delayed in the G₁ phase, and HepG2 cell growth slowed in association with dephosphorylation of the serine–threonine kinase Akt. Low-dose H₂O₂ stimulated Akt phosphorylation, implying that Akt activation in HepG2 cells is redox-sensitive. Akt phosphorylation was abrogated by phosphatidylinositol 3-kinase (PI3K) inhibitors, suggesting that PI3K is an upstream mediator of Akt activation in HepG2 cells. This study provides insight into recombinant Fe-SOD-induced signaling mechanisms in liver tumor cells and suggests the feasibility of using Fe-SOD as an antitumor agent. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

13C and 15N chemical shift assignments and secondary structure of the B3 immunoglobulin-binding domain of streptococcal protein G by magic-angle spinning solid-state NMR spectroscopy

Complete ¹³C and ¹⁵N assignments of the B3 IgG-binding domain of protein G (GB3) in the microcrystalline solid phase, obtained using 2D and 3D MAS NMR, are presented. The chemical shifts are used to predict the protein backbone conformation and compared with solution-state shifts. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Airborne sperm of Conocephalum conicum (Conocephalaceae)

We have obtained the first momentary photographs of sperms just as they are discharged from the antheridium of a liverwort, Conocephalum conicum, and have succeeded in monitoring the airborne sperms of bryophytes under field conditions. Airborne sperm of liverworts seems to be an effective strategy for raising the efficiency of fertilization between male and female plants separated in a drought environment. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and characterization of PiORP1, a Petunia oxysterol-binding-protein related protein involved in receptor-kinase mediated signaling in pollen, and analysis of the ORP gene family in Arabidopsis

Oxysterol-binding proteins (OSBPs) and oxysterol-binding-protein related proteins (ORPs) are encoded by most eukaryotic genomes examined to date; however, they have not yet been characterized in plants. Here we report the identification and characterization of PiORP1, an ORP of Petunia inflata that interacts with the cytoplasmic kinase domain of a receptor-like kinase, named PRK1, of P. inflata. PiORP1 is phosphorylated by PRK1 in vitro and therefore may be involved in PRK1 signaling during pollen development and growth. RNA gel blot analysis showed that PiORP1 and PRK1 had very similar expression patterns in developing pollen, mature pollen and pollen tubes. GFP fusion proteins of PiORP1 localized in the plasma membrane of pollen tubes at distinct foci and its PH domain alone was sufficient to mediate this localization. The sequence for the oxysterol-binding domain of PiORP1 was used to search the genome of Arabidopsis; 12 ORPs were identified and phylogenetic analysis revealed that they fell into two distinct clades, consistent with the ORPs of other eukaryotes. RT-PCR analysis showed that all 12 Arabidopsis ORPs were expressed; 10 were expressed in most of the tissues examined under normal growth conditions, but only three were expressed in pollen. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users. GenBank accession number for PiORP1: DQ241801     

Identification of proteins that interact with catalytically active calcium-dependent protein kinases from <Emphasis Type="Italic">Arabidopsis</Emphasis>

Calcium-dependent protein kinases (CDPKs) are essential sensor-transducers of calcium signaling pathways in plants. Functional characterization of CDPKs is of great interest because they play important roles during growth, development, and in response to a wide range of environmental stimuli. The Arabidopsis genome encodes 34 CDPKs, but very few substrates of these enzymes have been identified. In this study, we exploited the unique characteristics of CDPKs to develop an efficient approach for the discovery of CDPK-interacting proteins. High-throughput, semi-automated yeast two-hybrid interaction screens with two different cDNA libraries each containing 18 million prey clones were performed using catalytically impaired and constitutively active AtCPK4 and AtCPK11 variants as baits. The use of the constitutively active versions of the CPK baits improved the recovery of positive interacting proteins relative to the wild type kinase. Titration of interaction strength by growth under increasing concentrations of 3-aminotriazole (3-AT), a histidine analog and competitive inhibitor of the His3 gene product, confirmed these results. Possible mechanisms for this observed improvement are discussed. The reproducibility of this approach was assessed by the overlap of several interacting proteins of AtCPK4 and AtCPK11 and the recovery of several putative substrates and indicated that yeast two-hybrid screens using constitutively active and/or catalytically impaired forms of CDPK provides a useful tool to identify potential substrates of the CDPK family and potentially the entire protein kinase superfamily. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.