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Sample records for receptor-like kinase interacts

  1. ERECTA and BAK1 Receptor Like Kinases Interact to Regulate Immune Responses in Arabidopsis.

    PubMed

    Jordá, Lucía; Sopeña-Torres, Sara; Escudero, Viviana; Nuñez-Corcuera, Beatriz; Delgado-Cerezo, Magdalena; Torii, Keiko U; Molina, Antonio

    2016-01-01

    ERECTA (ER) receptor-like kinase (RLK) regulates Arabidopsis thaliana organ growth, and inflorescence and stomatal development by interacting with the ERECTA-family genes (ERf) paralogs, ER-like 1 (ERL1) and ERL2, and the receptor-like protein (RLP) TOO MANY MOUTHS (TMM). ER also controls immune responses and resistance to pathogens such as the bacterium Pseudomonas syringae pv. tomato DC3000 (Pto) and the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM). We found that er null-mutant plants overexpressing an ER dominant-negative version lacking the cytoplasmic kinase domain (ERΔK) showed an enhanced susceptibility to PcBMM, suggesting that ERΔK associates and forms inactive complexes with additional RLKs/RLPs required for PcBMM resistance. Genetic analyses demonstrated that ER acts in a combinatorial specific manner with ERL1, ERL2, and TMM to control PcBMM resistance. Moreover, BAK1 (BRASSINOSTEROID INSENSITIVE 1-associated kinase 1) RLK, which together with ERf/TMM regulates stomatal patterning and resistance to Pto, was also found to have an unequal contribution with ER in regulating immune responses and resistance to PcBMM. Co-immunoprecipitation experiments in Nicotiana benthamiana further demonstrated BAK1-ER protein interaction. The secreted epidermal pattern factor peptides (EPF1 and EPF2), which are perceived by ERf members to specify stomatal patterning, do not seem to regulate ER-mediated immunity to PcBMM, since their inducible overexpression in A. thaliana did not impact on PcBMM resistance. Our results indicate that the multiproteic receptorsome formed by ERf, TMM and BAK1 modulates A. thaliana resistance to PcBMM, and suggest that the cues underlying ERf/TMM/BAK1-mediated immune responses are distinct from those regulating stomatal pattering.

  2. ERECTA and BAK1 Receptor Like Kinases Interact to Regulate Immune Responses in Arabidopsis

    PubMed Central

    Jordá, Lucía; Sopeña-Torres, Sara; Escudero, Viviana; Nuñez-Corcuera, Beatriz; Delgado-Cerezo, Magdalena; Torii, Keiko U.; Molina, Antonio

    2016-01-01

    ERECTA (ER) receptor-like kinase (RLK) regulates Arabidopsis thaliana organ growth, and inflorescence and stomatal development by interacting with the ERECTA-family genes (ERf) paralogs, ER-like 1 (ERL1) and ERL2, and the receptor-like protein (RLP) TOO MANY MOUTHS (TMM). ER also controls immune responses and resistance to pathogens such as the bacterium Pseudomonas syringae pv. tomato DC3000 (Pto) and the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM). We found that er null-mutant plants overexpressing an ER dominant-negative version lacking the cytoplasmic kinase domain (ERΔK) showed an enhanced susceptibility to PcBMM, suggesting that ERΔK associates and forms inactive complexes with additional RLKs/RLPs required for PcBMM resistance. Genetic analyses demonstrated that ER acts in a combinatorial specific manner with ERL1, ERL2, and TMM to control PcBMM resistance. Moreover, BAK1 (BRASSINOSTEROID INSENSITIVE 1-associated kinase 1) RLK, which together with ERf/TMM regulates stomatal patterning and resistance to Pto, was also found to have an unequal contribution with ER in regulating immune responses and resistance to PcBMM. Co-immunoprecipitation experiments in Nicotiana benthamiana further demonstrated BAK1-ER protein interaction. The secreted epidermal pattern factor peptides (EPF1 and EPF2), which are perceived by ERf members to specify stomatal patterning, do not seem to regulate ER-mediated immunity to PcBMM, since their inducible overexpression in A. thaliana did not impact on PcBMM resistance. Our results indicate that the multiproteic receptorsome formed by ERf, TMM and BAK1 modulates A. thaliana resistance to PcBMM, and suggest that the cues underlying ERf/TMM/BAK1-mediated immune responses are distinct from those regulating stomatal pattering. PMID:27446127

  3. Interactions in the pollen-specific receptor-like kinases-containing signaling network.

    PubMed

    Löcke, Susanne; Fricke, Inka; Mucha, Elena; Humpert, Marie-Luise; Berken, Antje

    2010-12-01

    The pollen-specific receptor-like kinases (PRKs) from Solanum lycopersicum, LePRK1 and LePRK2, are believed to be involved in the regulation of pollen germination and pollen tube growth. They appear to be part of a multimeric complex in which the transmembranic LePRKs presumably have a key position in transducing exogenous signals through the plasma membrane. Here, we focused on extra- and intracellular interactions involving the LePRKs. We show in yeast two-hybrid experiments a cross-interaction of putative PRK-ligands, the oligomerization of LePRK2 and a direct contact of LePRKs to activated Rho proteins of plants (ROPs). Moreover, we observed that pollen-specific RopGEFs, which catalyze ROP activation and may be regulated by PRK interaction, are active in vitro while autoinhibition seems to occur in vivo. We suggest that activation of RopGEFs as a checkpoint in PRK signal transduction is a more complex event including further components in planta. Our findings point to some new aspects in PRK-mediated signal transduction implying a LePRK2 complex with different signaling activity and a further direct control of LePRKs by activated ROP.

  4. Receptor-Like Kinase RUPO Interacts with Potassium Transporters to Regulate Pollen Tube Growth and Integrity in Rice

    PubMed Central

    Liu, Lingtong; Zheng, Canhui; Kuang, Baijan; Wei, Liqin; Yan, Longfeng; Wang, Tai

    2016-01-01

    During sexual reproduction of flowering plants, the pollen tube grows fast and over a long distance within the pistil to deliver two sperms for double fertilization. Growing plant cells need to communicate constantly with external stimuli as well as monitor changes in surface tension of the cell wall and plasma membrane to coordinate these signals and internal growth machinery; however, the underlying mechanisms remain largely unknown. Here we show that the rice member of plant-specific receptor-like kinase CrRLK1Ls subfamily, Ruptured Pollen tube (RUPO), is specifically expressed in rice pollen. RUPO localizes to the apical plasma membrane and vesicle of pollen tubes and is required for male gamete transmission. K+ levels were greater in pollen of homozygous CRISPR-knockout lines than wild-type plants, and pollen tubes burst shortly after germination. We reveal the interaction of RUPO with high-affinity potassium transporters. Phosphorylation of RUPO established and dephosphorylation abolished the interaction. These results have revealed the receptor-like kinase as a regulator of high-affinity potassium transporters via phosphorylation-dependent interaction, and demonstrated a novel receptor-like kinase signaling pathway that mediates K+ homeostasis required for pollen tube growth and integrity. PMID:27447945

  5. Receptor-Like Kinase RUPO Interacts with Potassium Transporters to Regulate Pollen Tube Growth and Integrity in Rice.

    PubMed

    Liu, Lingtong; Zheng, Canhui; Kuang, Baijan; Wei, Liqin; Yan, Longfeng; Wang, Tai

    2016-07-01

    During sexual reproduction of flowering plants, the pollen tube grows fast and over a long distance within the pistil to deliver two sperms for double fertilization. Growing plant cells need to communicate constantly with external stimuli as well as monitor changes in surface tension of the cell wall and plasma membrane to coordinate these signals and internal growth machinery; however, the underlying mechanisms remain largely unknown. Here we show that the rice member of plant-specific receptor-like kinase CrRLK1Ls subfamily, Ruptured Pollen tube (RUPO), is specifically expressed in rice pollen. RUPO localizes to the apical plasma membrane and vesicle of pollen tubes and is required for male gamete transmission. K+ levels were greater in pollen of homozygous CRISPR-knockout lines than wild-type plants, and pollen tubes burst shortly after germination. We reveal the interaction of RUPO with high-affinity potassium transporters. Phosphorylation of RUPO established and dephosphorylation abolished the interaction. These results have revealed the receptor-like kinase as a regulator of high-affinity potassium transporters via phosphorylation-dependent interaction, and demonstrated a novel receptor-like kinase signaling pathway that mediates K+ homeostasis required for pollen tube growth and integrity.

  6. Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination1[OPEN

    PubMed Central

    Wang, Yao; Ahsan, Nagib; Biener, Gabriel; Paprocki, Joel

    2017-01-01

    Cell signaling pathways mediated by leucine-rich repeat receptor-like kinases (LRR-RLKs) are essential for plant growth, development, and defense. The EMS1 (EXCESS MICROSPOROCYTES1) LRR-RLK and its small protein ligand TPD1 (TAPETUM DETERMINANT1) play a fundamental role in somatic and reproductive cell differentiation during early anther development in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether other cell surface molecules serve as coregulators of EMS1. Here, we show that SERK1 (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1) and SERK2 LRR-RLKs act redundantly as coregulatory and physical partners of EMS1. The SERK1/2 genes function in the same genetic pathway as EMS1 in anther development. Bimolecular fluorescence complementation, Förster resonance energy transfer, and coimmunoprecipitation approaches revealed that SERK1 interacted biochemically with EMS1. Transphosphorylation of EMS1 by SERK1 enhances EMS1 kinase activity. Among 12 in vitro autophosphorylation and transphosphorylation sites identified by tandem mass spectrometry, seven of them were found to be critical for EMS1 autophosphorylation activity. Furthermore, complementation test results suggest that phosphorylation of EMS1 is required for its function in anther development. Collectively, these data provide genetic and biochemical evidence of the interaction and phosphorylation between SERK1/2 and EMS1 in anther development. PMID:27920157

  7. SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE Directly Interacts with the Cytoplasmic Domain of SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE and Negatively Regulates Leaf Senescence in Arabidopsis1[OPEN

    PubMed Central

    Xiao, Dong; Cui, Yanjiao; Xu, Fan; Xu, Xinxin; Gao, Guanxiao; Wang, Yaxin; Guo, Zhaoxia; Wang, Dan; Wang, Ning Ning

    2015-01-01

    Reversible protein phosphorylation mediated by protein kinases and phosphatases plays an important role in the regulation of leaf senescence. We previously reported that the leucine-rich repeat receptor-like kinase SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE (AtSARK) positively regulates leaf senescence in Arabidopsis (Arabidopsis thaliana). Here, we report the involvement of a protein serine/threonine phosphatase 2C-type protein phosphatase, SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE (SSPP), in the negative regulation of Arabidopsis leaf senescence. SSPP transcript levels decreased greatly during both natural senescence and SARK-induced precocious senescence. Overexpression of SSPP significantly delayed leaf senescence in Arabidopsis. Protein pull-down and bimolecular fluorescence complementation assays demonstrated that the cytosol-localized SSPP could interact with the cytoplasmic domain of the plasma membrane-localized AtSARK. In vitro assays showed that SSPP has protein phosphatase function and can dephosphorylate the cytosolic domain of AtSARK. Consistent with these observations, overexpression of SSPP effectively rescued AtSARK-induced precocious leaf senescence and changes in hormonal responses. All our results suggested that SSPP functions in sustaining proper leaf longevity and preventing early senescence by suppressing or perturbing SARK-mediated senescence signal transduction. PMID:26304848

  8. The receptor-like kinase SOBIR1 interacts with Brassica napus LepR3 and is required for Leptosphaeria maculans AvrLm1-triggered immunity

    PubMed Central

    Ma, Lisong; Borhan, M. Hossein

    2015-01-01

    The fungus Leptosphaeria maculans (L. maculans) is the causal agent of blackleg disease of canola/oilseed rape (Brassica napus) worldwide. We previously reported cloning of the B. napus blackleg resistance gene, LepR3, which encodes a receptor-like protein. LepR3 triggers localized cell death upon recognition of its cognate Avr protein, AvrLm1. Here, we exploited the Nicotiana benthamiana model plant to investigate the recognition mechanism of AvrLm1 by LepR3. Co-expression of the LepR3/AvrLm1 gene pair in N. benthamiana resulted in development of a hypersensitive response (HR). However, a truncated AvrLm1 lacking its indigenous signal peptide was compromised in its ability to induce LepR3-mediated HR, indicating that AvrLm1 is perceived by LepR3 extracellularly. Structure-function analysis of the AvrLm1 protein revealed that the C-terminal region of AvrLm1 was required for LepR3-mediated HR in N. benthamiana and for resistance to L. maculans in B. napus. LepR3 was shown to be physically interacting with the B. napus receptor like kinase, SOBIR1 (BnSOBIR1). Silencing of NbSOBIR1 or NbSERK3 (BAK1) compromised LepR3-AvrLm1-dependent HR in N. benthamiana, suggesting that LepR3-mediated resistance to L. maculans in B. napus requires SOBIR1 and BAK1/SERK3. Using this model system, we determined that BnSOBIR1 and SERK3/BAK1 are essential partners in the LepR3 signaling complex and were able to define the AvrLm1 effector domain. PMID:26579176

  9. GLYCINE-RICH RNA-BINDING PROTEIN1 interacts with RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 and suppresses cell death and defense responses in pepper (Capsicum annuum).

    PubMed

    Kim, Dae Sung; Kim, Nak Hyun; Hwang, Byung Kook

    2015-01-01

    Plants use a variety of innate immune regulators to trigger cell death and defense responses against pathogen attack. We identified pepper (Capsicum annuum) GLYCINE-RICH RNA-BINDING PROTEIN1 (CaGRP1) as a RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 (CaPIK1)-interacting partner, based on bimolecular fluorescence complementation and coimmunoprecipitation analyses as well as gene silencing and transient expression analysis. CaGRP1 contains an N-terminal RNA recognition motif and a glycine-rich region at the C-terminus. The CaGRP1 protein had DNA- and RNA-binding activity in vitro. CaGRP1 interacted with CaPIK1 in planta. CaGRP1 and CaGRP1-CaPIK1 complexes were localized to the nucleus in plant cells. CaPIK1 phosphorylated CaGRP1 in vitro and in planta. Transient coexpression of CaGRP1 with CaPIK1 suppressed the CaPIK1-triggered cell death response, accompanied by a reduced CaPIK1-triggered reactive oxygen species (ROS) burst. The RNA recognition motif region of CaGRP1 was responsible for the nuclear localization of CaGRP1 as well as the suppression of the CaPIK1-triggered cell death response. CaGRP1 silencing in pepper conferred enhanced resistance to Xanthomonas campestris pv vesicatoria (Xcv) infection; however, CaPIK1-silenced plants were more susceptible to Xcv. CaGRP1 interacts with CaPIK1 and negatively regulates CaPIK1-triggered cell death and defense responses by suppressing ROS accumulation.

  10. CAST AWAY, a membrane-associated receptor-like kinase, inhibits organ abscission in Arabidopsis.

    PubMed

    Burr, Christian A; Leslie, Michelle E; Orlowski, Sara K; Chen, Iris; Wright, Catherine E; Daniels, Mark J; Liljegren, Sarah J

    2011-08-01

    Receptor-like kinase-mediated cell signaling pathways play fundamental roles in many aspects of plant growth and development. A pair of Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like kinases (LRR-RLKs), HAESA (HAE) and HAESA-LIKE2 (HSL2), have been shown to activate the cell separation process that leads to organ abscission. Another pair of LRR-RLKs, EVERSHED (EVR) and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1, act as inhibitors of abscission, potentially by modulating HAE/HSL2 activity. Cycling of these RLKs to and from the cell surface may be regulated by NEVERSHED (NEV), a membrane trafficking regulator that is essential for organ abscission. We report here the characterization of CAST AWAY (CST), a receptor-like cytoplasmic kinase that acts as a spatial inhibitor of cell separation. Disruption of CST suppresses the abscission defects of nev mutant flowers and restores the discrete identity of the trans-Golgi network in nev abscission zones. After organ shedding, enlarged abscission zones with obscured boundaries are found in nev cst flowers. We show that CST is a dual-specificity kinase in vitro and that myristoylation at its amino terminus promotes association with the plasma membrane. Using the bimolecular fluorescence complementation assay, we have detected interactions of CST with HAE and EVR at the plasma membrane of Arabidopsis protoplasts and hypothesize that CST negatively regulates cell separation signaling directly and indirectly. A model integrating the potential roles of receptor-like kinase signaling and membrane trafficking during organ separation is presented.

  11. Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases

    PubMed Central

    Shiu, Shin-Han; Bleecker, Anthony B.

    2001-01-01

    Plant receptor-like kinases (RLKs) are proteins with a predicted signal sequence, single transmembrane region, and cytoplasmic kinase domain. Receptor-like kinases belong to a large gene family with at least 610 members that represent nearly 2.5% of Arabidopsis protein coding genes. We have categorized members of this family into subfamilies based on both the identity of the extracellular domains and the phylogenetic relationships between the kinase domains of subfamily members. Surprisingly, this structurally defined group of genes is monophyletic with respect to kinase domains when compared with the other eukaryotic kinase families. In an extended analysis, animal receptor kinases, Raf kinases, plant RLKs, and animal receptor tyrosine kinases form a well supported group sharing a common origin within the superfamily of serine/threonine/tyrosine kinases. Among animal kinase sequences, Drosophila Pelle and related cytoplasmic kinases fall within the plant RLK clade, which we now define as the RLK/Pelle family. A survey of expressed sequence tag records for land plants reveals that mosses, ferns, conifers, and flowering plants have similar percentages of expressed sequence tags representing RLK/Pelle homologs, suggesting that the size of this gene family may have been close to the present-day level before the diversification of land plant lineages. The distribution pattern of four RLK subfamilies on Arabidopsis chromosomes indicates that the expansion of this gene family is partly a consequence of duplication and reshuffling of the Arabidopsis genome and of the generation of tandem repeats. PMID:11526204

  12. Novel receptor-like kinases in cacao contain PR-1 extracellular domains.

    PubMed

    Teixeira, Paulo José Pereira Lima; Costa, Gustavo Gilson Lacerda; Fiorin, Gabriel Lorencini; Pereira, Gonçalo Amarante Guimarães; Mondego, Jorge Maurício Costa

    2013-08-01

    Members of the pathogenesis-related protein 1 (PR-1) family are well-known markers of plant defence responses, forming part of the arsenal of the secreted proteins produced on pathogen recognition. Here, we report the identification of two cacao (Theobroma cacao L.) PR-1s that are fused to transmembrane regions and serine/threonine kinase domains, in a manner characteristic of receptor-like kinases (RLKs). These proteins (TcPR-1f and TcPR-1g) were named PR-1 receptor kinases (PR-1RKs). Phylogenetic analysis of RLKs and PR-1 proteins from cacao indicated that PR-1RKs originated from a fusion between sequences encoding PR-1 and the kinase domain of a LecRLK (Lectin Receptor-Like Kinase). Retrotransposition marks surround TcPR-1f, suggesting that retrotransposition was involved in the origin of PR-1RKs. Genes with a similar domain architecture to cacao PR-1RKs were found in rice (Oryza sativa), barrel medic (Medicago truncatula) and a nonphototrophic bacterium (Herpetosiphon aurantiacus). However, their kinase domains differed from those found in LecRLKs, indicating the occurrence of convergent evolution. TcPR-1g expression was up-regulated in the biotrophic stage of witches' broom disease, suggesting a role for PR-1RKs during cacao defence responses. We hypothesize that PR-1RKs transduce a defence signal by interacting with a PR-1 ligand.

  13. A novel Glycine soja cysteine proteinase inhibitor GsCPI14, interacting with the calcium/calmodulin-binding receptor-like kinase GsCBRLK, regulated plant tolerance to alkali stress.

    PubMed

    Sun, Xiaoli; Yang, Shanshan; Sun, Mingzhe; Wang, Sunting; Ding, Xiaodong; Zhu, Dan; Ji, Wei; Cai, Hua; Zhao, Chaoyue; Wang, Xuedong; Zhu, Yanming

    2014-05-01

    It has been well demonstrated that cystatins regulated plant stress tolerance through inhibiting the cysteine proteinase activity under environmental stress. However, there was limited information about the role of cystatins in plant alkali stress response, especially in wild soybean. Here, in this study, we focused on the biological characterization of a novel Glycine soja cystatin protein GsCPI14, which interacted with the calcium/calmodulin-binding receptor-like kinase GsCBRLK and positively regulated plant alkali stress tolerance. The protein-protein interaction between GsCBRLK and GsCPI14 was confirmed by using split-ubiquitin based membrane yeast two-hybrid analysis and bimolecular fluorescence complementation assay. Expression of GsCPI14 was greatly induced by salt, ABA and alkali stress in G. soja, and GsCBRLK overexpression (OX) in Glycine max promoted the stress induction of GmCPI14 expression under stress conditions. Furthermore, we found that GsCPI14-eGFP fusion protein localized in the entire Arabidopsis protoplast and onion epidermal cell, and GsCPI14 showed ubiquitous expression in different tissues of G. soja. In addition, we gave evidence that the GST-GsCPI14 fusion protein inhibited the proteolytic activity of papain in vitro. At last, we demonstrated that OX of GsCPI14 in Arabidopsis promoted the seed germination under alkali stress, as evidenced by higher germination rates. GsCPI14 transgenic Arabidopsis seedlings also displayed better growth performance and physiological index under alkali stress. Taken together, results presented in this study demonstrated that the G. soja cysteine proteinase inhibitor GsCPI14 interacted with the calcium/calmodulin-binding receptor-like kinase GsCBRLK and regulated plant tolerance to alkali stress.

  14. Expansion of the Receptor-Like Kinase/Pelle Gene Family and Receptor-Like Proteins in Arabidopsis1[w

    PubMed Central

    Shin-Han, Shiu; Bleecker, Anthony B.

    2003-01-01

    Receptor-like kinases (RLKs) are a family of transmembrane proteins with versatile N-terminal extracellular domains and C-terminal intracellular kinases. They control a wide range of physiological responses in plants and belong to one of the largest gene families in the Arabidopsis genome with more than 600 members. Interestingly, this gene family constitutes 60% of all kinases in Arabidopsis and accounts for nearly all transmembrane kinases in Arabidopsis. Analysis of four fungal, six metazoan, and two Plasmodium sp. genomes indicates that the family was represented in all but fungal genomes, indicating an ancient origin for the family with a more recent expansion only in the plant lineages. The RLK/Pelle family can be divided into several subfamilies based on three independent criteria: the phylogeny based on kinase domain sequences, the extracellular domain identities, and intron locations and phases. A large number of receptor-like proteins (RLPs) resembling the extracellular domains of RLKs are also found in the Arabidopsis genome. However, not all RLK subfamilies have corresponding RLPs. Several RLK/Pelle subfamilies have undergone differential expansions. More than 33% of the RLK/Pelle members are found in tandem clusters, substantially higher than the genome average. In addition, 470 of the RLK/Pelle family members are located within the segmentally duplicated regions in the Arabidopsis genome and 268 of them have a close relative in the corresponding regions. Therefore, tandem duplications and segmental/whole-genome duplications represent two of the major mechanisms for the expansion of the RLK/Pelle family in Arabidopsis. PMID:12805585

  15. Immunoprecipitation of Plasma Membrane Receptor-Like Kinases for Identification of Phosphorylation Sites and Associated Proteins.

    PubMed

    Kadota, Yasuhiro; Macho, Alberto P; Zipfel, Cyril

    2016-01-01

    Membrane proteins are difficult to study for numerous reasons. The surface of membrane proteins is relatively hydrophobic and sometimes very unstable, additionally requiring detergents for their extraction from the membrane. This leads to challenges at all levels, including expression, solubilization, purification, identification of associated proteins, and the identification of post-translational modifications. However, recent advances in immunoprecipitation technology allow to isolate membrane proteins efficiently, facilitating the study of protein-protein interactions, the identification of novel associated proteins, and to identify post-translational modifications, such as phosphorylation. Here, we describe an optimized immunoprecipitation protocol for plant plasma membrane receptor-like kinases.

  16. Genome-wide analysis of lectin receptor-like kinases in Populus

    SciTech Connect

    Yang, Yongil; Labbé, Jessy; Muchero, Wellington; Yang, Xiaohan; Jawdy, Sara S.; Kennedy, Megan; Johnson, Jenifer; Sreedasyam, Avinash; Schmutz, Jeremy; Tuskan, Gerald A.; Chen, Jin -Gui

    2016-09-01

    Receptor-like kinases (RLKs) belong to a large protein family with over 600 members in Arabidopsis and over 1000 in rice. Among RLKs, the lectin receptor-like kinases (LecRLKs) possess a characteristic extracellular carbohydrate-binding lectin domain and play important roles in plant development and innate immunity. In addition, there are 75 and 173 LecRLKs in Arabidopsis and rice, respectively. However, little is known about LecRLKs in perennial woody plants.

  17. Genome-wide analysis of lectin receptor-like kinases in Populus

    DOE PAGES

    Yang, Yongil; Labbé, Jessy; Muchero, Wellington; ...

    2016-09-01

    Receptor-like kinases (RLKs) belong to a large protein family with over 600 members in Arabidopsis and over 1000 in rice. Among RLKs, the lectin receptor-like kinases (LecRLKs) possess a characteristic extracellular carbohydrate-binding lectin domain and play important roles in plant development and innate immunity. In addition, there are 75 and 173 LecRLKs in Arabidopsis and rice, respectively. However, little is known about LecRLKs in perennial woody plants.

  18. The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses.

    PubMed

    Kim, Dae Sung; Kim, Nak Hyun; Hwang, Byung Kook

    2015-04-01

    The pepper receptor-like cytoplasmic protein kinase, CaPIK1, which mediates signalling of plant cell death and defence responses was previously identified. Here, the identification of a class IV chitinase, CaChitIV, from pepper plants (Capsicum annuum), which interacts with CaPIK1 and promotes CaPIK1-triggered cell death and defence responses, is reported. CaChitIV contains a signal peptide, chitin-binding domain, and glycol hydrolase domain. CaChitIV expression was up-regulated by Xanthomonas campestris pv. vesicatoria (Xcv) infection. Notably, avirulent Xcv infection rapidly induced CaChitIV expression in pepper leaves. Bimolecular fluorescence complementation and co-immunoprecipitation revealed that CaPIK1 interacts with CaChitIV in planta, and that the CaPIK1-CaChitIV complex is localized mainly in the cytoplasm and plasma membrane. CaChitIV is also localized in the endoplasmic reticulum. Transient co-expression of CaChitIV with CaPIK1 enhanced CaPIK1-triggered cell death response and reactive oxygen species (ROS) and nitric oxide (NO) bursts. Co-silencing of both CaChitIV and CaPIK1 in pepper plants conferred enhanced susceptibility to Xcv infection, which was accompanied by a reduced induction of cell death response, ROS and NO bursts, and defence response genes. Ectopic expression of CaPIK1 in Arabidopsis enhanced basal resistance to Hyaloperonospora arabidopsidis infection. Together, the results suggest that CaChitIV positively regulates CaPIK1-triggered cell death and defence responses through its interaction with CaPIK1.

  19. A receptor-like kinase from Arabidopsis thaliana is a calmodulin-binding protein.

    PubMed Central

    Charpenteau, Martine; Jaworski, Krzysztof; Ramirez, Bertha C; Tretyn, Andrzej; Ranjeva, Raoul; Ranty, Benoît

    2004-01-01

    Screening a cDNA expression library with a radiolabelled calmodulin (CaM) probe led to the isolation of AtCaMRLK, a receptor-like kinase (RLK) of Arabidopsis thaliana. AtCaMRLK polypeptide sequence shows a modular organization consisting of the four distinctive domains characteristic of receptor kinases: an amino terminal signal sequence, a domain containing seven leucine-rich repeats, a single putative membrane-spanning segment and a protein kinase domain. Using truncated versions of the protein and a synthetic peptide, we demonstrated that a region of 23 amino acids, located near the kinase domain of AtCaMRLK, binds CaM in a calcium-dependent manner. Real-time binding experiments showed that AtCaMRLK interacted in vitro with AtCaM1, a canonical CaM, but not with AtCaM8, a divergent isoform of the Ca2+ sensor. The bacterially expressed kinase domain of the protein was able to autophosphorylate and to phosphorylate the myelin basic protein, using Mn2+ preferentially to Mg2+ as an ion activator. Site-directed mutagenesis of the conserved lysine residue (Lys423) to alanine, in the kinase subdomain II, resulted in a complete loss of kinase activity. CaM had no influence on the autophosphorylation activity of AtCaMRLK. AtCaMRLK was expressed in reproductive and vegetative tissues of A. thaliana, except in leaves. Disruption in the AtCaMRLK coding sequence by insertion of a DsG transposable element in an Arabidopsis mutant did not generate a discernible phenotype. The CaM-binding motif of AtCaMRLK was found to be conserved in several other members of the plant RLK family, suggesting a role for Ca2+/CaM in the regulation of RLK-mediated pathways. PMID:14720124

  20. Crystal structure of the plant receptor-like kinase TDR in complex with the TDIF peptide

    PubMed Central

    Morita, Junko; Kato, Kazuki; Nakane, Takanori; Kondo, Yuki; Fukuda, Hiroo; Nishimasu, Hiroshi; Ishitani, Ryuichiro; Nureki, Osamu

    2016-01-01

    In plants, leucine-rich repeat receptor-like kinases (LRR-RKs) perceive ligands, including peptides and small molecules, to regulate various physiological processes. TDIF, a member of the CLE peptide family, specifically interacts with the LRR-RK TDR to inhibit meristem differentiation into tracheary elements, and promotes cell proliferation. Here we report the crystal structure of the extracellular domain of TDR in complex with the TDIF peptide. The extracellular domain of TDR adopts a superhelical structure comprising 22 LRRs, and specifically recognizes TDIF by its inner concave surface. Together with our biochemical and sequence analyses, our structure reveals a conserved TDIF-recognition mechanism of TDR among plant species. Furthermore, a structural comparison of TDR with other plant LRR-RKs suggested the activation mechanism of TDR by TDIF. The structure of this CLE peptide receptor provides insights into the recognition mechanism of the CLE family peptides. PMID:27498761

  1. Evolutionary Dynamics of the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) Subfamily in Angiosperms.

    PubMed

    Fischer, Iris; Diévart, Anne; Droc, Gaetan; Dufayard, Jean-François; Chantret, Nathalie

    2016-03-01

    Gene duplications are an important factor in plant evolution, and lineage-specific expanded (LSE) genes are of particular interest. Receptor-like kinases expanded massively in land plants, and leucine-rich repeat receptor-like kinases (LRR-RLK) constitute the largest receptor-like kinases family. Based on the phylogeny of 7,554 LRR-RLK genes from 31 fully sequenced flowering plant genomes, the complex evolutionary dynamics of this family was characterized in depth. We studied the involvement of selection during the expansion of this family among angiosperms. LRR-RLK subgroups harbor extremely contrasting rates of duplication, retention, or loss, and LSE copies are predominantly found in subgroups involved in environmental interactions. Expansion rates also differ significantly depending on the time when rounds of expansion or loss occurred on the angiosperm phylogenetic tree. Finally, using a dN/dS-based test in a phylogenetic framework, we searched for selection footprints on LSE and single-copy LRR-RLK genes. Selective constraint appeared to be globally relaxed at LSE genes, and codons under positive selection were detected in 50% of them. Moreover, the leucine-rich repeat domains, and specifically four amino acids in them, were found to be the main targets of positive selection. Here, we provide an extensive overview of the expansion and evolution of this very large gene family.

  2. Evolutionary Dynamics of the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) Subfamily in Angiosperms1[OPEN

    PubMed Central

    Dufayard, Jean-François; Chantret, Nathalie

    2016-01-01

    Gene duplications are an important factor in plant evolution, and lineage-specific expanded (LSE) genes are of particular interest. Receptor-like kinases expanded massively in land plants, and leucine-rich repeat receptor-like kinases (LRR-RLK) constitute the largest receptor-like kinases family. Based on the phylogeny of 7,554 LRR-RLK genes from 31 fully sequenced flowering plant genomes, the complex evolutionary dynamics of this family was characterized in depth. We studied the involvement of selection during the expansion of this family among angiosperms. LRR-RLK subgroups harbor extremely contrasting rates of duplication, retention, or loss, and LSE copies are predominantly found in subgroups involved in environmental interactions. Expansion rates also differ significantly depending on the time when rounds of expansion or loss occurred on the angiosperm phylogenetic tree. Finally, using a dN/dS-based test in a phylogenetic framework, we searched for selection footprints on LSE and single-copy LRR-RLK genes. Selective constraint appeared to be globally relaxed at LSE genes, and codons under positive selection were detected in 50% of them. Moreover, the leucine-rich repeat domains, and specifically four amino acids in them, were found to be the main targets of positive selection. Here, we provide an extensive overview of the expansion and evolution of this very large gene family. PMID:26773008

  3. LysM receptor-like kinases to improve plant defense response against fungal pathogens

    DOEpatents

    Wan, Jinrong [Columbia, MO; Stacey, Gary [Columbia, MO; Stacey, Minviluz [Columbia, MO; Zhang, Xuecheng [Columbia, MO

    2012-01-17

    Perception of chitin fragments (chitooligosaccharides) is an important first step in plant defense response against fungal pathogen. LysM receptor-like kinases (LysM RLKs) are instrumental in this perception process. LysM RLKs also play a role in activating transcription of chitin-responsive genes (CRGs) in plants. Mutations in the LysM kinase receptor genes or the downstream CRGs may affect the fungal susceptibility of a plant. Mutations in LysM RLKs or transgenes carrying the same may be beneficial in imparting resistance against fungal pathogens.

  4. LysM receptor-like kinases to improve plant defense response against fungal pathogens

    SciTech Connect

    Wan, Jinrong; Stacey, Gary; Stacey, Minviluz; Zhang, Xuecheng

    2013-10-15

    Perception of chitin fragments (chitooligosaccharides) is an important first step in plant defense response against fungal pathogen. LysM receptor-like kinases (LysM RLKs) are instrumental in this perception process. LysM RLKs also play a role in activating transcription of chitin-responsive genes (CRGs) in plants. Mutations in the LysM kinase receptor genes or the downstream CRGs may affect the fungal susceptibility of a plant. Mutations in LysM RLKs or transgenes carrying the same may be beneficial in imparting resistance against fungal pathogens.

  5. Spontaneous symbiotic reprogramming of plant roots triggered by receptor-like kinases.

    PubMed

    Ried, Martina Katharina; Antolín-Llovera, Meritxell; Parniske, Martin

    2014-11-25

    Symbiosis Receptor-like Kinase (SYMRK) is indispensable for the development of phosphate-acquiring arbuscular mycorrhiza (AM) as well as nitrogen-fixing root nodule symbiosis, but the mechanisms that discriminate between the two distinct symbiotic developmental fates have been enigmatic. In this study, we show that upon ectopic expression, the receptor-like kinase genes Nod Factor Receptor 1 (NFR1), NFR5, and SYMRK initiate spontaneous nodule organogenesis and nodulation-related gene expression in the absence of rhizobia. Furthermore, overexpressed NFR1 or NFR5 associated with endogenous SYMRK in roots of the legume Lotus japonicus. Epistasis tests revealed that the dominant active SYMRK allele initiates signalling independently of either the NFR1 or NFR5 gene and upstream of a set of genes required for the generation or decoding of calcium-spiking in both symbioses. Only SYMRK but not NFR overexpression triggered the expression of AM-related genes, indicating that the receptors play a key role in the decision between AM- or root nodule symbiosis-development.

  6. Two receptor-like kinases required together for the establishment of Arabidopsis cotyledon primordia.

    PubMed

    Nodine, Michael D; Tax, Frans E

    2008-02-01

    Inter-regional signaling coordinates pattern formation in Arabidopsis thaliana embryos. However, little is known regarding the cells and molecules involved in inter-regional communication. We have characterized two related leucine-rich repeat receptor-like kinases (LRR-RLKs), RECEPTOR-LIKE PROTEIN KINASE1 (RPK1) and TOADSTOOL2 (TOAD2), which are required together for patterning the apical embryonic domain cell types that generate cotyledon primordia. Central domain protoderm patterning defects were always observed subjacent to the defective cotyledon primordia cell types in mutant embryos. In addition, RPK1-GFP and TOAD2-GFP translational fusions were both localized to the central domain protodermal cells when cotyledon primordia were first recognizable. We propose that RPK1 and TOAD2 are primarily required to maintain central domain protoderm cell fate and that the loss of this key embryonic cell type in mutant embryos results in patterning defects in other regions of the embryo including the failure to initiate cotyledon primordia.

  7. Spontaneous symbiotic reprogramming of plant roots triggered by receptor-like kinases

    PubMed Central

    Ried, Martina Katharina; Antolín-Llovera, Meritxell; Parniske, Martin

    2014-01-01

    Symbiosis Receptor-like Kinase (SYMRK) is indispensable for the development of phosphate-acquiring arbuscular mycorrhiza (AM) as well as nitrogen-fixing root nodule symbiosis, but the mechanisms that discriminate between the two distinct symbiotic developmental fates have been enigmatic. In this study, we show that upon ectopic expression, the receptor-like kinase genes Nod Factor Receptor 1 (NFR1), NFR5, and SYMRK initiate spontaneous nodule organogenesis and nodulation-related gene expression in the absence of rhizobia. Furthermore, overexpressed NFR1 or NFR5 associated with endogenous SYMRK in roots of the legume Lotus japonicus. Epistasis tests revealed that the dominant active SYMRK allele initiates signalling independently of either the NFR1 or NFR5 gene and upstream of a set of genes required for the generation or decoding of calcium-spiking in both symbioses. Only SYMRK but not NFR overexpression triggered the expression of AM-related genes, indicating that the receptors play a key role in the decision between AM- or root nodule symbiosis-development. DOI: http://dx.doi.org/10.7554/eLife.03891.001 PMID:25422918

  8. The Arabidopsis thaliana cysteine-rich receptor-like kinases CRK6 and CRK7 protect against apoplastic oxidative stress.

    PubMed

    Idänheimo, Niina; Gauthier, Adrien; Salojärvi, Jarkko; Siligato, Riccardo; Brosché, Mikael; Kollist, Hannes; Mähönen, Ari Pekka; Kangasjärvi, Jaakko; Wrzaczek, Michael

    2014-03-07

    Receptor-like kinases are important regulators of many different processes in plants. Despite their large number only a few have been functionally characterized. One of the largest subgroups of receptor-like kinases in Arabidopsis is the cysteine-rich receptor like kinases (CRKs). High sequence similarity among the CRKs has been suggested as major cause for functional redundancy. The genomic localization of CRK genes in back-to-back repeats has made their characterization through mutant analysis unpractical. Expression profiling has linked the CRKs with reactive oxygen species, important signaling molecules in plants. Here we have investigated the role of two CRKs, CRK6 and CRK7, and analyzed their role in extracellular ROS signaling. CRK6 and CRK7 are active protein kinases with differential preference for divalent cations. Our results suggest that CRK7 is involved in mediating the responses to extracellular but not chloroplastic ROS production.

  9. Arabidopsis receptor-like protein30 and receptor-like kinase suppressor of BIR1-1/EVERSHED mediate innate immunity to necrotrophic fungi.

    PubMed

    Zhang, Weiguo; Fraiture, Malou; Kolb, Dagmar; Löffelhardt, Birgit; Desaki, Yoshitake; Boutrot, Freddy F G; Tör, Mahmut; Zipfel, Cyril; Gust, Andrea A; Brunner, Frédéric

    2013-10-01

    Effective plant defense strategies rely in part on the perception of non-self determinants, so-called microbe-associated molecular patterns (MAMPs), by transmembrane pattern recognition receptors leading to MAMP-triggered immunity. Plant resistance against necrotrophic pathogens with a broad host range is complex and yet not well understood. Particularly, it is unclear if resistance to necrotrophs involves pattern recognition receptors. Here, we partially purified a novel proteinaceous elicitor called sclerotinia culture filtrate elicitor1 (SCFE1) from the necrotrophic fungal pathogen Sclerotinia sclerotiorum that induces typical MAMP-triggered immune responses in Arabidopsis thaliana. Analysis of natural genetic variation revealed five Arabidopsis accessions (Mt-0, Lov-1, Lov-5, Br-0, and Sq-1) that are fully insensitive to the SCFE1-containing fraction. We used a forward genetics approach and mapped the locus determining SCFE1 sensitivity to receptor-like protein30 (RLP30). We also show that SCFE1-triggered immune responses engage a signaling pathway dependent on the regulatory receptor-like kinases brassinosteroid insensitive1-associated receptor kinase1 (BAK1) and Suppressor of BIR1-1/evershed (SOBIR1/EVR). Mutants of RLP30, BAK1, and SOBIR1 are more susceptible to S. sclerotiorum and the related fungus Botrytis cinerea. The presence of an elicitor in S. sclerotiorum evoking MAMP-triggered immune responses and sensed by RLP30/SOBIR1/BAK1 demonstrates the relevance of MAMP-triggered immunity in resistance to necrotrophic fungi.

  10. Receptor-like kinase ACR4 restricts formative cell divisions in the Arabidopsis root.

    PubMed

    De Smet, Ive; Vassileva, Valya; De Rybel, Bert; Levesque, Mitchell P; Grunewald, Wim; Van Damme, Daniël; Van Noorden, Giel; Naudts, Mirande; Van Isterdael, Gert; De Clercq, Rebecca; Wang, Jean Y; Meuli, Nicholas; Vanneste, Steffen; Friml, Jirí; Hilson, Pierre; Jürgens, Gerd; Ingram, Gwyneth C; Inzé, Dirk; Benfey, Philip N; Beeckman, Tom

    2008-10-24

    During the development of multicellular organisms, organogenesis and pattern formation depend on formative divisions to specify and maintain pools of stem cells. In higher plants, these activities are essential to shape the final root architecture because the functioning of root apical meristems and the de novo formation of lateral roots entirely rely on it. We used transcript profiling on sorted pericycle cells undergoing lateral root initiation to identify the receptor-like kinase ACR4 of Arabidopsis as a key factor both in promoting formative cell divisions in the pericycle and in constraining the number of these divisions once organogenesis has been started. In the root tip meristem, ACR4 shows a similar action by controlling cell proliferation activity in the columella cell lineage. Thus, ACR4 function reveals a common mechanism of formative cell division control in the main root tip meristem and during lateral root initiation.

  11. Characterization and expression analysis of somatic embryogenesis receptor-like kinase genes from Phalaenopsis.

    PubMed

    Huang, Y W; Tsai, Y J; Chen, F C

    2014-12-18

    Somatic embryogenesis receptor-like kinase (SERK) genes have been found to be involved in the somatic embryogenesis of several plant species. We identified and characterized 5 PhSERK genes in the Phalaenopsis orchid. The amino acid sequences of PhSERKs and other SERK proteins are highly conserved, with the highest homology observed in the leucine-rich repeat-receptor-like kinase domain. All 5 PhSERKs were expressed in all Phalaenopsis organs examined (root, leaf, shoot apical meristem, and flower), with the strongest expression, particularly for PhSERK1 and 3, in the shoot apical meristem of mature plants. Expression of all PhSERKs was downregulated during early floral bud development and was upregulated gradually until the semi-open flower stage was reached. All 5 PhSERKs were expressed during both seed germination and protocorm-like-body (PLB) development. In germinated seeds, quantitative real-time PCR revealed upregulation of all PhSERKs except PhSERK4 at 1 week and downregulation after 4 weeks. The 5 PhSERKs were differentially expressed in the early stage of PLB development and maintained substantial levels during PLB formation, with PhSERK1 and 5 upregulated 1 week after culture and PhSERK2, 3, and 4 downregulated over this period. Because physical wounding of PLB stimulates secondary PLB formation, the PhSERK5 expression peak at week 3 coincided with visible and fully developed secondary PLBs. PhSERK5 may be important in PLB induction and subsequent development. Our PhSERK expression analysis revealed that these genes have a broad role during orchid plant development.

  12. Analysis of Phosphorylation of the Receptor-Like Protein Kinase HAESA during Arabidopsis Floral Abscission

    PubMed Central

    Taylor, Isaiah; Wang, Ying; Seitz, Kati; Baer, John; Bennewitz, Stefan; Mooney, Brian P.; Walker, John C.

    2016-01-01

    Receptor-like protein kinases (RLKs) are the largest family of plant transmembrane signaling proteins. Here we present functional analysis of HAESA, an RLK that regulates floral organ abscission in Arabidopsis. Through in vitro and in vivo analysis of HAE phosphorylation, we provide evidence that a conserved phosphorylation site on a region of the HAE protein kinase domain known as the activation segment positively regulates HAE activity. Additional analysis has identified another putative activation segment phosphorylation site common to multiple RLKs that potentially modulates HAE activity. Comparative analysis suggests that phosphorylation of this second activation segment residue is an RLK specific adaptation that may regulate protein kinase activity and substrate specificity. A growing number of RLKs have been shown to exhibit biologically relevant dual specificity toward serine/threonine and tyrosine residues, but the mechanisms underlying dual specificity of RLKs are not well understood. We show that a phospho-mimetic mutant of both HAE activation segment residues exhibits enhanced tyrosine auto-phosphorylation in vitro, indicating phosphorylation of this residue may contribute to dual specificity of HAE. These results add to an emerging framework for understanding the mechanisms and evolution of regulation of RLK activity and substrate specificity. PMID:26784444

  13. Functional analysis of related CrRLK1L receptor-like kinases in pollen tube reception.

    PubMed

    Kessler, Sharon A; Lindner, Heike; Jones, Daniel S; Grossniklaus, Ueli

    2015-01-01

    The Catharanthus roseus Receptor-Like Kinase 1-like (CrRLK1L) family of 17 receptor-like kinases (RLKs) has been implicated in a variety of signaling pathways in Arabidopsis, ranging from pollen tube (PT) reception and tip growth to hormonal responses. The extracellular domains of these RLKs have malectin-like domains predicted to bind carbohydrate moieties. Domain swap analysis showed that the extracellular domains of the three members analyzed (FER, ANX1, HERK1) are not interchangeable, suggesting distinct upstream components, such as ligands and/or co-factors. In contrast, their intercellular domains are functionally equivalent for PT reception, indicating that they have common downstream targets in their signaling pathways. The kinase domain is necessary for FER function, but kinase activity itself is not, indicating that other kinases may be involved in signal transduction during PT reception.

  14. Cloning and Characterization of a Receptor-Like Protein Kinase Gene Associated with Senescence

    PubMed Central

    Hajouj, Taleb; Michelis, Regina; Gepstein, Shimon

    2000-01-01

    Senescence-associated genes are up-regulated during plant senescence and many have been implicated in encoding enzymes involved in the metabolism of senescing tissues. Using the differential display technique, we identified a SAG in bean (Phaseolus vulgaris) leaf that was exclusively expressed during senescence and was designated senescence-associated receptor-like kinase (SARK). The deduced SARK polypeptide consists of a signal peptide, a leucine-rich repeat in the extracellular region, a single membrane-spanning domain, and the characteristic serine/threonine protein kinase domain. The mRNA level for SARK increased prior to the loss of chlorophyll and the decrease of chlorophyll a/b-binding protein mRNA. Detached mature bean leaves, which senesce at an accelerated rate compared with leaves on intact plants, showed a similar temporal pattern of SARK message accumulation. Light and cytokinin, which delayed the initiation of leaf senescence, also delayed SARK gene expression; in contrast, darkness and ethylene, which accelerated senescence, advanced the initial appearance of the SARK transcript. SARK protein accumulation exhibited a temporal pattern similar to that of its mRNA. A possible role for SARK in the regulation of leaf senescence was considered. PMID:11080306

  15. Activin receptor-like kinases: a diverse family playing an important role in cancer

    PubMed Central

    Loomans, Holli A; Andl, Claudia D

    2016-01-01

    The role and function of the members of the TGFβ superfamily has been a substantial area of research focus for the last several decades. During that time, it has become apparent that aberrations in TGFβ family signaling, whether through the BMP, Activin, or TGFβ arms of the pathway, can result in tumorigenesis or contribute to its progression. Downstream signaling regulates cellular growth under normal physiological conditions yet induces diverse processes during carcinogenesis, ranging from epithelial- to-mesenchymal transition to cell migration and invasion to angiogenesis. Due to these observations, the question has been raised how to utilize and target components of these signaling pathways in cancer therapy. Given that these cascades include both ligands and receptors, there are multiple levels at which to interfere. Activin receptor-like kinases (ALKs) are a group of seven type I receptors responsible for TGFβ family signal transduction and are utilized by many ligands within the superfamily. The challenge lies in specifically targeting the often-overlapping functional effects of BMP, Activin, or TGFβ signaling during cancer progression. This review focuses on the characteristic function of the individual receptors within each subfamily and their recognized roles in cancer. We next explore the clinical utility of therapeutically targeting ALKs as some have shown partial responses in Phase I clinical trials but disappointing outcomes when used in Phase II studies. Finally, we discuss the challenges and future directions of this body of work. PMID:27904762

  16. A nonRD receptor-like kinase prevents nodule early senescence and defense-like reactions during symbiosis.

    PubMed

    Berrabah, Fathi; Bourcy, Marie; Eschstruth, Alexis; Cayrel, Anne; Guefrachi, Ibtissem; Mergaert, Peter; Wen, Jiangqi; Jean, Viviane; Mysore, Kirankumar S; Gourion, Benjamin; Ratet, Pascal

    2014-09-01

    Rhizobia and legumes establish symbiotic interactions leading to the production of root nodules, in which bacteria fix atmospheric nitrogen for the plant's benefit. This symbiosis is efficient because of the high rhizobia population within nodules. Here, we investigated how legumes accommodate such bacterial colonization. We used a reverse genetic approach to identify a Medicago truncatula gene, SymCRK, which encodes a cysteine-rich receptor-like kinase that is required for rhizobia maintenance within the plant cells, and performed detailed phenotypic analyses of the corresponding mutant. The Medicago truncatula symCRK mutant developed nonfunctional and necrotic nodules. A nonarginine asparate (nonRD) motif, typical of receptors involved in innate immunity, is present in the SymCRK kinase domain. Similar to the dnf2 mutant, bacteroid differentiation defect, defense-like reactions and early senescence were observed in the symCRK nodules. However, the dnf2 and symCRK nodules differ by their degree of colonization, which is higher in symCRK. Furthermore, in contrast to dnf2, symCRK is not a conditional mutant. These results suggest that in M. truncatula at least two genes are involved in the symbiotic control of immunity. Furthermore, phenotype differences between the two mutants suggest that two distinct molecular mechanisms control suppression of plant immunity during nodulation.

  17. xopAC-triggered Immunity against Xanthomonas Depends on Arabidopsis Receptor-Like Cytoplasmic Kinase Genes PBL2 and RIPK

    PubMed Central

    Guy, Endrick; Lautier, Martine; Chabannes, Matthieu; Roux, Brice; Lauber, Emmanuelle; Arlat, Matthieu; Noël, Laurent D.

    2013-01-01

    Xanthomonas campestris pv. campestris (Xcc) colonizes the vascular system of Brassicaceae and ultimately causes black rot. In susceptible Arabidopsis plants, XopAC type III effector inhibits by uridylylation positive regulators of the PAMP-triggered immunity such as the receptor-like cytoplasmic kinases (RLCK) BIK1 and PBL1. In the resistant ecotype Col-0, xopAC is a major avirulence gene of Xcc. In this study, we show that both the RLCK interaction domain and the uridylyl transferase domain of XopAC are required for avirulence. Furthermore, xopAC can also confer avirulence to both the vascular pathogen Ralstonia solanacearum and the mesophyll-colonizing pathogen Pseudomonas syringae indicating that xopAC-specified effector-triggered immunity is not specific to the vascular system. In planta, XopAC-YFP fusions are localized at the plasma membrane suggesting that XopAC might interact with membrane-localized proteins. Eight RLCK of subfamily VII predicted to be localized at the plasma membrane and interacting with XopAC in yeast two-hybrid assays have been isolated. Within this subfamily, PBL2 and RIPK RLCK genes but not BIK1 are important for xopAC-specified effector-triggered immunity and Arabidopsis resistance to Xcc. PMID:23951354

  18. Specific activin receptor-like kinase 3 inhibitors enhance liver regeneration.

    PubMed

    Tsugawa, Daisuke; Oya, Yuki; Masuzaki, Ryota; Ray, Kevin; Engers, Darren W; Dib, Martin; Do, Nhue; Kuramitsu, Kaori; Ho, Karen; Frist, Audrey; Yu, Paul B; Bloch, Kenneth D; Lindsley, Craig W; Hopkins, Corey R; Hong, Charles C; Karp, Seth J

    2014-12-01

    Pharmacologic agents to enhance liver regeneration after injury would have wide therapeutic application. Based on previous work suggesting inhibition of bone morphogenetic protein (BMP) signaling stimulates liver regeneration, we tested known and novel BMP inhibitors for their ability to accelerate regeneration in a partial hepatectomy (PH) model. Compounds were produced based on the 3,6-disubstituted pyrazolo[1,5-a] pyrimidine core of the BMP antagonist dorsomorphin and evaluated for their ability to inhibit BMP signaling and enhance liver regeneration. Antagonists of the BMP receptor activin receptor-like kinase 3 (ALK3), including LDN-193189 (LDN; 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), DMH2 (4-(2-(4-(3-(quinolin-4-yl)pyrazolo[1,5-a]pyrimidin-6-yl)phenoxy)ethyl)morpholine; VU0364849), and the novel compound VU0465350 (7-(4-isopropoxyphenyl)-3-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridine; VU5350), blocked SMAD phosphorylation in vitro and in vivo, and enhanced liver regeneration after PH. In contrast, an antagonist of the BMP receptor ALK2, VU0469381 (5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone; 1LWY), did not affect liver regeneration. LDN did not affect liver synthetic or metabolic function. Mechanistically, LDN increased serum interleukin-6 levels and signal transducer and activator of transcription 3 phosphorylation in the liver, and modulated other factors known to be important for liver regeneration, including suppressor of cytokine signaling 3 and p53. These findings suggest that inhibition of ALK3 may be part of a therapeutic strategy for treating human liver disease.

  19. A rice lectin receptor-like kinase that is involved in innate immune responses also contributes to seed germination.

    PubMed

    Cheng, Xiaoyan; Wu, Yan; Guo, Jianping; Du, Bo; Chen, Rongzhi; Zhu, Lili; He, Guangcun

    2013-11-01

    Seed germination and innate immunity both have significant effects on plant life spans because they control the plant's entry into the ecosystem and provide defenses against various external stresses, respectively. Much ecological evidence has shown that seeds with high vigor are generally more tolerant of various environmental stimuli in the field than those with low vigor. However, there is little genetic evidence linking germination and immunity in plants. Here, we show that the rice lectin receptor-like kinase OslecRK contributes to both seed germination and plant innate immunity. We demonstrate that knocking down the OslecRK gene depresses the expression of α-amylase genes, reducing seed viability and thereby decreasing the rate of seed germination. Moreover, it also inhibits the expression of defense genes, and so reduces the resistance of rice plants to fungal and bacterial pathogens as well as herbivorous insects. Yeast two-hybrid and co-immunoprecipitation experiments revealed that OslecRK interacts with an actin-depolymerizing factor (ADF) in vivo via its kinase domain. Moreover, the rice adf mutant exhibited a reduced seed germination rate due to the suppression of α-amylase gene expression. This mutant also exhibited depressed immune responses and reduced resistance to biotic stresses. Our results thus provide direct genetic evidence for a common physiological pathway connecting germination and immunity in plants. They also partially explain the common observation that high-vigor seeds often perform well in the field. The dual effects of OslecRK may be indicative of progressive adaptive evolution in rice.

  20. Specificity and Structure of a High Affinity Activin Receptor-like Kinase 1 (ALK1) Signaling Complex

    PubMed Central

    Townson, Sharon A.; Martinez-Hackert, Erik; Greppi, Chloe; Lowden, Patricia; Sako, Dianne; Liu, June; Ucran, Jeffrey A.; Liharska, Katia; Underwood, Kathryn W.; Seehra, Jasbir; Kumar, Ravindra; Grinberg, Asya V.

    2012-01-01

    Activin receptor-like kinase 1 (ALK1), an endothelial cell-specific type I receptor of the TGF-β superfamily, is an important regulator of normal blood vessel development as well as pathological tumor angiogenesis. As such, ALK1 is an important therapeutic target. Thus, several ALK1-directed agents are currently in clinical trials as anti-angiogenic cancer therapeutics. Given the biological and clinical importance of the ALK1 signaling pathway, we sought to elucidate the biophysical and structural basis underlying ALK1 signaling. The TGF-β family ligands BMP9 and BMP10 as well as the three type II TGF-β family receptors ActRIIA, ActRIIB, and BMPRII have been implicated in ALK1 signaling. Here, we provide a kinetic and thermodynamic analysis of BMP9 and BMP10 interactions with ALK1 and type II receptors. Our data show that BMP9 displays a significant discrimination in type II receptor binding, whereas BMP10 does not. We also report the crystal structure of a fully assembled ternary complex of BMP9 with the extracellular domains of ALK1 and ActRIIB. The structure reveals that the high specificity of ALK1 for BMP9/10 is determined by a novel orientation of ALK1 with respect to BMP9, which leads to a unique set of receptor-ligand interactions. In addition, the structure explains how BMP9 discriminates between low and high affinity type II receptors. Taken together, our findings provide structural and mechanistic insights into ALK1 signaling that could serve as a basis for novel anti-angiogenic therapies. PMID:22718755

  1. A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity

    PubMed Central

    Lu, Dongping; Wu, Shujing; Gao, Xiquan; Zhang, Yulan; Shan, Libo; He, Ping

    2010-01-01

    Plants and animals rely on innate immunity to prevent infections by detection of microbe-associated molecular patterns (MAMPs) through pattern-recognition receptors (PRRs). The plant PRR FLS2, a leucine-rich repeat-receptor kinase, recognizes bacterial flagellin and initiates immune signaling by association with another leucine-rich repeat-receptor-like kinase, BAK1. It remains unknown how the FLS2/BAK1 receptor complex activates intracellular signaling cascades. Here we identified the receptor-like cytoplasmic kinase BIK1 that is rapidly phosphorylated upon flagellin perception, depending on both FLS2 and BAK1. BIK1 associates with FLS2 and BAK1 in vivo and in vitro. BIK1 is phosphorylated by BAK1, and BIK1 also directly phosphorylates BAK1 and FLS2 in vitro. The flagellin phosphorylation site Thr237 of BIK1 is required for its phosphorylation on BAK1 and FLS2, suggesting that BIK1 is likely first phosphorylated upon flagellin perception and subsequently transphosphorylates FLS2/BAK1 to propagate flagellin signaling. Importantly, bik1 mutants are compromised in diverse flagellin-mediated responses and immunity to the nonpathogenic bacterial infection. Thus, BIK1 is an essential component in MAMP signal transduction, which links the MAMP receptor complex to downstream intracellular signaling. PMID:20018686

  2. Ligand-induced dynamics of heterotrimeric G protein-coupled receptor-like kinase complexes

    PubMed Central

    Tunc-Ozdemir, Meral; Jones, Alan M.

    2017-01-01

    Background Arabidopsis, 7-transmembrane Regulator of G signaling protein 1 (AtRGS1) modulates canonical G protein signaling by promoting the inactive state of heterotrimeric G protein complex on the plasma membrane. It is known that plant leucine-rich repeat receptor–like kinases (LRR RLKs) phosphorylate AtRGS1 in vitro but little is known about the in vivo interaction, molecular dynamics, or the cellular consequences of this interaction. Methods Therefore, a subset of the known RLKs that phosphorylate AtRGS1 were selected for elucidation, namely, BAK1, BIR1, FLS2. Several microscopies for both static and dynamic protein-protein interactions were used to follow in vivo interactions between the RLKs and AtRGS1 after the presentation of the Pathogen-associated Molecular Pattern, Flagellin 22 (Flg22). These microscopies included Förster Resonance Energy Transfer, Bimolecular Fluoresence Complementation, and Cross Number and Brightness Fluorescence Correlation Spectroscopy. In addition, reactive oxygen species and calcium changes in living cells were quantitated using luminometry and R-GECO1 microscopy. Results The LRR RLKs BAK1 and BIR1, interact with AtRGS1 at the plasma membrane. The RLK ligand flg22 sets BAK1 in motion toward AtRGS1 and BIR1 away, both returning to the baseline orientations by 10 minutes. The C-terminal tail of AtRGS1 is important for the interaction with BAK1 and for the tempo of the AtRGS1/BIR1 dynamics. This window of time corresponds to the flg22-induced transient production of reactive oxygen species and calcium release which are both attenuated in the rgs1 and the bak1 null mutants. Conclusions A temporal model of these interactions is proposed. flg22 binding induces nearly instantaneous dimerization between FLS2 and BAK1. Phosphorylated BAK1 interacts with and enables AtRGS1 to move away from BIR1 and AtRGS1 becomes phosphorylated leading to its endocytosis thus leading to de-repression by permitting AtGPA1 to exchange GDP for GTP

  3. Evolution of S-domain receptor-like kinases in land plants and origination of S-locus receptor kinases in Brassicaceae

    PubMed Central

    2013-01-01

    Background The S-domain serine/threonine receptor-like kinases (SRLKs) comprise one of the largest and most rapidly expanding subfamilies in the plant receptor-like/Pelle kinase (RLKs) family. The founding member of this subfamily, the S-locus receptor kinase (SRK), functions as the female determinant of specificity in the self-incompatibility (SI) responses of crucifers. Two classes of proteins resembling the extracellular S domain (designated S-domain receptor-like proteins, SRLPs) or the intracellular kinase domain (designated S-domain receptor-like cytoplasmic kinases, SRLCKs) of SRK are also ubiquitous in land plants, indicating that the SRLKs are composite molecules that originated by domain fusion of the two component proteins. Here, we explored the origin and diversification of SRLKs by phylogenomic methods. Results Based on the distribution patterns of SRLKs and SRLCKs in a reconciled species-domain tree, a maximum parsimony model was then established for simultaneously inferring and dating gene duplication/loss and fusion /fission events in SRLK evolution. Various SRK alleles from crucifer species were then included in our phylogenetic analyses to infer the origination of SRKs by identifying the proper outgroups. Conclusions Two gene fusion events were inferred and the major gene fusion event occurred in the common ancestor of land plants generated almost all of extant SRLKs. The functional diversification of duplicated SRLKs was illustrated by molecular evolution analyses of SRKs. Our findings support that SRKs originated as two ancient haplotypes derived from a pair of tandem duplicate genes through random regulatory neo-/sub- functionalization in the common ancestor of the Brassicaceae. PMID:23510165

  4. Knowing your friends and foes--plant receptor-like kinases as initiators of symbiosis or defence.

    PubMed

    Antolín-Llovera, Meritxell; Petutsching, Elena Kristin; Ried, Martina Katharina; Lipka, Volker; Nürnberger, Thorsten; Robatzek, Silke; Parniske, Martin

    2014-12-01

    The decision between defence and symbiosis signalling in plants involves alternative and modular plasma membrane-localized receptor complexes. A critical step in their activation is ligand-induced homo- or hetero-oligomerization of leucine-rich repeat (LRR)- and/or lysin motif (LysM) receptor-like kinases (RLKs). In defence signalling, receptor complexes form upon binding of pathogen-associated molecular patterns (PAMPs), including the bacterial flagellin-derived peptide flg22, or chitin. Similar mechanisms are likely to operate during the perception of microbial symbiont-derived (lipo)-chitooligosaccharides. The structurally related chitin-oligomer ligands chitooctaose and chitotetraose trigger defence and symbiosis signalling, respectively, and their discrimination involves closely related, if not identical, LysM-RLKs. This illustrates the demand for and the challenges imposed on decision mechanisms that ensure appropriate signal initiation. Appropriate signalling critically depends on abundance and localization of RLKs at the cell surface. This is regulated by internalization, which also provides a mechanism for the removal of activated signalling RLKs. Abundance of the malectin-like domain (MLD)-LRR-RLK Symbiosis Receptor-like Kinase (SYMRK) is additionally controlled by cleavage of its modular ectodomain, which generates a truncated and rapidly degraded RLK fragment. This review explores LRR- and LysM-mediated signalling, the involvement of MLD-LRR-RLKs in symbiosis and defence, and the role of endocytosis in RLK function.

  5. Genomic and Post-Translational Modification Analysis of Leucine-Rich-Repeat Receptor-Like Kinases in Brassica rapa

    PubMed Central

    Dhandapani, Vignesh; Yu, Xiaona; Choi, Su Ryun; Oh, Man-Ho; Lim, Yong Pyo

    2015-01-01

    Among several receptor-like kinases (RLKs), leucine-rich-repeat receptor-like kinases (LRR-RLKs) are a major group of genes that play crucial roles in growth, development and stress responses in plant systems. Given that they have several functional roles, it is important to investigate their roles in Brassica rapa. In the present study, 303 LRR-RLKs were identified in the genome of B. rapa and comparative phylogenetic analysis of 1213 combined LRR-RLKs of B. rapa, Arabidopsis thaliana, Oryza sativa and Populus trichocarpa helped us to categorize the gene family into 15 subfamilies based on their sequence and structural similarities. The chromosome localizations of 293 genes allowed the prediction of duplicates, and motif conservation and intron/exon patterns showed differences among the B. rapa LRR-RLK (BrLRR-RLK) genes. Additionally, computational function annotation and expression analysis was used to predict their possible functional roles in the plant system. Biochemical results for 11 selected genes showed variations in phosphorylation activity. Interestingly, BrBAK1 showed strong auto-phosphorylation and trans-phosphorylation on its tyrosine and threonine residues compared with AtBAK1 in previous studies. The AtBAK1 receptor kinase is involved in plant growth and development, plant innate immunity, and programmed cell death, and our results suggest that BrBAK1 might also be involved in the same functions. Another interesting result was that BrBAK1, BrBRI1, BrPEPR1 and BrPEPR2 showed activity with both anti-phosphotyrosine and anti-phosphothreonine antibodies, indicating that they might have dual-specificity kinase activity. This study provides comprehensive results for the BrLRR-RLKs, revealing expansion of the gene family through gene duplications, structural similarities and variations among the genes, and potential functional roles according to gene ontology, transcriptome profiling and biochemical analysis. PMID:26588465

  6. The Medicago truncatula lysin [corrected] motif-receptor-like kinase gene family includes NFP and new nodule-expressed genes.

    PubMed

    Arrighi, Jean-François; Barre, Annick; Ben Amor, Besma; Bersoult, Anne; Soriano, Lidia Campos; Mirabella, Rossana; de Carvalho-Niebel, Fernanda; Journet, Etienne-Pascal; Ghérardi, Michèle; Huguet, Thierry; Geurts, René; Dénarié, Jean; Rougé, Pierre; Gough, Clare

    2006-09-01

    Rhizobial Nod factors are key symbiotic signals responsible for starting the nodulation process in host legume plants. Of the six Medicago truncatula genes controlling a Nod factor signaling pathway, Nod Factor Perception (NFP) was reported as a candidate Nod factor receptor gene. Here, we provide further evidence for this by showing that NFP is a lysin [corrected] motif (LysM)-receptor-like kinase (RLK). NFP was shown both to be expressed in association with infection thread development and to be involved in the infection process. Consistent with deviations from conserved kinase domain sequences, NFP did not show autophosphorylation activity, suggesting that NFP needs to associate with an active kinase or has unusual functional characteristics different from classical kinases. Identification of nine new M. truncatula LysM-RLK genes revealed a larger family than in the nonlegumes Arabidopsis (Arabidopsis thaliana) or rice (Oryza sativa) of at least 17 members that can be divided into three subfamilies. Three LysM domains could be structurally predicted for all M. truncatula LysM-RLK proteins, whereas one subfamily, which includes NFP, was characterized by deviations from conserved kinase sequences. Most of the newly identified genes were found to be expressed in roots and nodules, suggesting this class of receptors may be more extensively involved in nodulation than was previously known.

  7. Overexpression of the leucine-rich receptor-like kinase gene LRK2 increases drought tolerance and tiller number in rice.

    PubMed

    Kang, Junfang; Li, Jianmin; Gao, Shuang; Tian, Chao; Zha, Xiaojun

    2017-02-09

    Drought represents a key limiting factor of global crop distribution. Receptor-like kinases play major roles in plant development and defense responses against stresses such as drought. In this study, LRK2, which encodes a leucine-rich receptor-like kinase, was cloned and characterized and found to be localized on the plasma membrane in rice. Promoter-GUS analysis revealed strong expression in tiller buds, roots, nodes and anthers. Transgenic plants overexpressing LRK2 exhibited enhanced tolerance to drought stress due to an increased number of lateral roots compared to the wild-type at the vegetative stage. Moreover, ectopic expression of LRK2 seedlings resulted in increased tiller development. Yeast two-hybrid screening and bimolecular fluorescence complementation (BiFC) indicated a possible interaction between LRK2 and elongation factor 1 alpha (OsEF1A) in vitro. These results suggest that LRK2 functions as a positive regulator of the drought stress response and tiller development via increased branch development in rice. These findings will aid our understanding of branch regulation in other grasses and support improvements in rice genetics. This article is protected by copyright. All rights reserved.

  8. The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2.

    PubMed

    Sklodowski, Kamil; Riedelsberger, Janin; Raddatz, Natalia; Riadi, Gonzalo; Caballero, Julio; Chérel, Isabelle; Schulze, Waltraud; Graf, Alexander; Dreyer, Ingo

    2017-03-16

    The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H(+)-ATPase-energized K(+) uptake. Moreover, through reversible post-translational modifications it can also function as an open, K(+)-selective channel, which taps a 'potassium battery', providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals.

  9. The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2

    PubMed Central

    Sklodowski, Kamil; Riedelsberger, Janin; Raddatz, Natalia; Riadi, Gonzalo; Caballero, Julio; Chérel, Isabelle; Schulze, Waltraud; Graf, Alexander; Dreyer, Ingo

    2017-01-01

    The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H+-ATPase-energized K+ uptake. Moreover, through reversible post-translational modifications it can also function as an open, K+-selective channel, which taps a ‘potassium battery’, providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals. PMID:28300158

  10. The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2

    NASA Astrophysics Data System (ADS)

    Sklodowski, Kamil; Riedelsberger, Janin; Raddatz, Natalia; Riadi, Gonzalo; Caballero, Julio; Chérel, Isabelle; Schulze, Waltraud; Graf, Alexander; Dreyer, Ingo

    2017-03-01

    The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H+-ATPase-energized K+ uptake. Moreover, through reversible post-translational modifications it can also function as an open, K+-selective channel, which taps a ‘potassium battery’, providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals.

  11. Genomic Dissection and Expression Profiling Revealed Functional Divergence in Triticum aestivum Leucine Rich Repeat Receptor Like Kinases (TaLRRKs)

    PubMed Central

    Shumayla; Sharma, Shailesh; Kumar, Rohit; Mendu, Venugopal; Singh, Kashmir; Upadhyay, Santosh K.

    2016-01-01

    The leucine rich repeat receptor like kinases (LRRK) constitute the largest subfamily of receptor like kinases (RLK), which play critical roles in plant development and stress responses. Herein, we identified 531 TaLRRK genes in Triticum aestivum (bread wheat), which were distributed throughout the A, B, and D sub-genomes and chromosomes. These were clustered into 233 homologous groups, which were mostly located on either homeologous chromosomes from various sub-genomes or in proximity on the same chromosome. A total of 255 paralogous genes were predicted which depicted the role of duplication events in expansion of this gene family. Majority of TaLRRKs consisted of trans-membrane region and localized on plasma-membrane. The TaLRRKs were further categorized into eight phylogenetic groups with numerous subgroups on the basis of sequence homology. The gene and protein structure in terms of exon/intron ratio, domains, and motifs organization were found to be variably conserved across the different phylogenetic groups/subgroups, which indicated a potential divergence and neofunctionalization during evolution. High-throughput transcriptome data and quantitative real time PCR analyses in various developmental stages, and biotic and abiotic (heat, drought, and salt) stresses provided insight into modus operandi of TaLRRKs during these conditions. Distinct expression of majority of stress responsive TaLRRKs homologous genes suggested their specified role in a particular condition. These results provided a comprehensive analysis of various characteristic features including functional divergence, which may provide the way for future functional characterization of this important gene family in bread wheat. PMID:27713749

  12. GsCBRLK, a calcium/calmodulin-binding receptor-like kinase, is a positive regulator of plant tolerance to salt and ABA stress.

    PubMed

    Yang, Liang; Ji, Wei; Zhu, Yanming; Gao, Peng; Li, Yong; Cai, Hua; Bai, Xi; Guo, Dianjing

    2010-05-01

    Calcium/calmodulin-dependent kinases play vital roles in protein phosphorylation in eukaryotes, yet little is known about the phosphorylation process of calcium/calmodulin-dependent protein kinase and its role in stress signal transduction in plants. A novel plant-specific calcium-dependent calmodulin-binding receptor-like kinase (GsCBRLK) has been isolated from Glycine soja. A subcellular localization study using GFP fusion protein indicated that GsCBRLK is localized in the plasma membrane. Binding assays demonstrated that calmodulin binds to GsCBRLK with an affinity of 25.9 nM in a calcium-dependent manner and the binding motif lies between amino acids 147 to169 within subdomain II of the kinase domain. GsCBRLK undergoes autophosphorylation and Myelin Basis Protein phosphorylation in the presence of calcium. It was also found that calcium/calmodulin positively regulates GsCBRLK kinase activity through direct interaction between the calmodulin-binding domain and calmodulin. So, it is likely that GsCBRLK responds to an environmental stimulus in two ways: by increasing the protein expression level and by regulating its kinase activity through the calcium/calmodulin complex. Furthermore, cold, salinity, drought, and ABA stress induce GsCBRLK gene transcripts. Over-expression of GsCBRLK in transgenic Arabidopsis resulted in enhanced plant tolerance to high salinity and ABA and increased the expression pattern of a number of stress gene markers in response to ABA and high salt. These results identify GsCBRLK as a molecular link between the stress- and ABA-induced calcium/calmodulin signal and gene expression in plant cells.

  13. Conserved Roles of CrRLK1L Receptor-Like Kinases in Cell Expansion and Reproduction from Algae to Angiosperms

    PubMed Central

    Galindo-Trigo, Sergio; Gray, Julie E.; Smith, Lisa M.

    2016-01-01

    Receptor-like kinases (RLKs) are regulators of plant development through allowing cells to sense their extracellular environment. They facilitate detection of local endogenous signals, in addition to external biotic and abiotic stimuli. The Catharanthus roseus RLK1-like (CrRLK1L) protein kinase subfamily, which contains FERONIA, plays a central role in regulating fertilization and in cell expansion mechanisms such as cell elongation and tip growth, as well as having indirect links to plant–pathogen interactions. Several components of CrRLK1L signaling pathways have been identified, including an extracellular ligand, coreceptors, and downstream signaling elements. The presence and abundance of the CrRLK1L proteins in the plant kingdom suggest an origin within the Streptophyta lineage, with a notable increase in prevalence in the seeded land plants. Given the function of the sole CrRLK1L protein in a charophycean alga, the possibility of a conserved role in detection and/or regulation of cell wall integrity throughout the Strephtophytes is discussed. Orthologs of signaling pathway components are also present in extant representatives of non-vascular land plants and early vascular land plants including the liverwort Marchantia polymorpha, the moss Physcomitrella patens and the lycophyte Selaginella moellendorffii. Deciphering the roles in development of the CrRLK1L protein kinases in early diverging land plants will provide insights into their ancestral function, furthering our understanding of this diversified subfamily of receptors in higher plants. PMID:27621737

  14. Genome-wide identification, characterization and expression analysis of populus leucine-rich repeat receptor-like protein kinase genes

    PubMed Central

    2013-01-01

    Background Leucine-rich repeat receptor-like kinases (LRR-RLKs) comprise the largest group within the receptor-like kinase (RLK) superfamily in plants. This gene family plays critical and diverse roles in plant growth, development and stress response. Although the LRR-RLK families in Arabidopsis and rice have been previously analyzed, no comprehensive studies have been performed on this gene family in tree species. Results In this work, 379 LRR-RLK genes were retrieved from the Populus trichocarpa genome and further grouped into 14 subfamilies based on their structural and sequence similarities. Approximately 82% (312 out of 379) of the PtLRR-RLK genes are located in segmental duplication blocks indicating the role of duplication process in the expansion of this gene family. The conservation and variation in motif composition and intron/exon arrangement among PtLRR-RLK subfamilies were analyzed to provide additional support for their phylogenetic relationship and more importantly to indicate the potential divergence in their functions. Expression profiling of PtLRR-RLKs showed that they were differentially expressed in different organs and tissues and some PtLRR-RLKs were specifically expressed in meristem tissues, which indicated their potential involvement in tissue development and differentiation. For most AtLRR-RLKs with defined functions, Populus homologues exhibiting similar expression patterns could be identified, which might indicate the functional conservation during evolution. Among 12 types of environmental cues analyzed by the genome-wide microarray data, PtLRR-RLKs showed specific responses to shoot organogenesis, wounding, low ammonium feeding, hypoxia and seasonal dormancy, but not to drought, re-watering after drought, flooding, AlCl3 treatment and bacteria or fungi treatments. Conclusions This study provides the first comprehensive genomic analysis of the Populus LRR-RLK gene family. Segmental duplication contributes significantly to the expansion

  15. Overexpression of GbRLK, a putative receptor-like kinase gene, improved cotton tolerance to Verticillium wilt.

    PubMed

    Jun, Zhao; Zhang, Zhiyuan; Gao, Yulong; Zhou, Lei; Fang, Lei; Chen, Xiangdong; Ning, Zhiyuan; Chen, Tianzi; Guo, Wangzhen; Zhang, Tianzhen

    2015-10-08

    Verticillium dahliae is a causative fungal pathogen and only a few genes have been identified that exhibit critical roles in disease resistance and few has shown positive effects on the resistance to Verticillium wilt in transgenic cotton. We cloned a receptor-like kinase gene (GbRLK) induced by Verticillium dahliae (VD) in the disease-resistant cotton Gossypium barbadense cv. Hai7124. Northern blotting revealed that the GbRLK was induced by VD at 96 h after inoculation. The functional GbRLK is from D subgenome since a single base deletion results in a frameshift or dysfunctional homologue in the A subgenome in tetraploid cotton. To verify the function of GbRLK, we developed the overexpression transgenic GbRLK cotton and Arabidopsis lines, and found that they all showed the higher resistance to Verticillium in the greenhouse and field trial. The results of the expression profile using transgenic and non-transgenic Arabidopsis thaliana revealed that the GbRLK regulated expressions of a series genes associated with biotic and abiotic stresses. Therefore, we propose that the increased resistance to Verticillium dahliae infection in transgnic plants could result from reduction in the damage of water loss and regulation of defense gene expression.

  16. Overproduction of the membrane-bound receptor-like protein kinase 1, RPK1, enhances abiotic stress tolerance in Arabidopsis.

    PubMed

    Osakabe, Yuriko; Mizuno, Shinji; Tanaka, Hidenori; Maruyama, Kyonoshin; Osakabe, Keishi; Todaka, Daisuke; Fujita, Yasunari; Kobayashi, Masatomo; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2010-03-19

    RPK1 (receptor-like protein kinase 1) localizes to the plasma membrane and functions as a regulator of abscisic acid (ABA) signaling in Arabidopsis. In our current study, we investigated the effect of RPK1 disruption and overproduction upon plant responses to drought stress. Transgenic Arabidopsis overexpressing the RPK1 protein showed increased ABA sensitivity in their root growth and stomatal closure and also displayed less transpirational water loss. In contrast, a mutant lacking RPK1 function, rpk1-1, was found to be resistant to ABA during these processes and showed increased water loss. RPK1 overproduction in these transgenic plants thus increased their tolerance to drought stress. We performed microarray analysis of RPK1 transgenic plants and observed enhanced expression of several stress-responsive genes, such as Cor15a, Cor15b, and rd29A, in addition to H(2)O(2)-responsive genes. Consistently, the expression levels of ABA/stress-responsive genes in rpk1-1 had decreased compared with wild type. The results suggest that the overproduction of RPK1 enhances both the ABA and drought stress signaling pathways. Furthermore, the leaves of the rpk1-1 plants exhibit higher sensitivity to oxidative stress upon ABA-pretreatment, whereas transgenic plants overproducing RPK1 manifest increased tolerance to this stress. Our current data suggest therefore that RPK1 overproduction controls reactive oxygen species homeostasis and enhances both water and oxidative stress tolerance in Arabidopsis.

  17. Local and Systemic Regulation of Plant Root System Architecture and Symbiotic Nodulation by a Receptor-Like Kinase

    PubMed Central

    Huault, Emeline; Laffont, Carole; Wen, Jiangqi; Mysore, Kirankumar S.; Ratet, Pascal; Duc, Gérard; Frugier, Florian

    2014-01-01

    In plants, root system architecture is determined by the activity of root apical meristems, which control the root growth rate, and by the formation of lateral roots. In legumes, an additional root lateral organ can develop: the symbiotic nitrogen-fixing nodule. We identified in Medicago truncatula ten allelic mutants showing a compact root architecture phenotype (cra2) independent of any major shoot phenotype, and that consisted of shorter roots, an increased number of lateral roots, and a reduced number of nodules. The CRA2 gene encodes a Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) that primarily negatively regulates lateral root formation and positively regulates symbiotic nodulation. Grafting experiments revealed that CRA2 acts through different pathways to regulate these lateral organs originating from the roots, locally controlling the lateral root development and nodule formation systemically from the shoots. The CRA2 LRR-RLK therefore integrates short- and long-distance regulations to control root system architecture under non-symbiotic and symbiotic conditions. PMID:25521478

  18. A calcium/calmodulin-regulated member of the receptor-like kinase family confers cold tolerance in plants.

    PubMed

    Yang, Tianbao; Chaudhuri, Shubho; Yang, Lihua; Du, Liqun; Poovaiah, B W

    2010-03-05

    Cold is a limiting environmental factor that adversely affects plant growth and productivity. Calcium/calmodulin-mediated signaling is believed to play a pivotal role in plant response to cold stress, but its exact role is not clearly understood. Here, we report that CRLK1, a novel calcium/calmodulin-regulated receptor-like kinase, is crucial for cold tolerance in plants. CRLK1 has two calmodulin-binding sites with different affinities as follows: one located at residues 369-390 with a K(d) of 25 nm, and the other located at residues 28-112 with a K(d) of 160 nm. Calcium/calmodulin stimulated the kinase activity, but the addition of chlorpromazine, a calmodulin antagonist, blocked its stimulation. CRLK1 is mainly localized in the plasma membrane, and its expression is stimulated by cold and hydrogen peroxide treatments. Under normal growth conditions, there is no noticeable phenotypic difference between wild-type and crlk1 knock-out mutant plants. However, as compared with wild-type plants, the crlk1 knock-out mutants exhibited an increased sensitivity to chilling and freezing temperatures. Northern analysis showed that the induction of cold-responsive genes, including CBF1, RD29A, COR15a, and KIN1 in crlk1 mutants, is delayed as compared with wild-type plants. These results indicate that CRLK1 is a positive regulator of cold tolerance in plants. Furthermore, our results suggest that CRLK1 plays a role in bridging calcium/calmodulin signaling and cold signaling.

  19. Expression and functional analysis of genes encoding cytokinin receptor-like histidine kinase in maize (Zea mays L.).

    PubMed

    Wang, Bo; Chen, Yanhong; Guo, Baojian; Kabir, Muhammad Rezaul; Yao, Yingyin; Peng, Huiru; Xie, Chaojie; Zhang, Yirong; Sun, Qixin; Ni, Zhongfu

    2014-08-01

    Cytokinin signaling is vital for plant growth and development which function via the two-component system (TCS). As one of the key component of TCS, transmembrane histidine kinases (HK) are encoded by a small gene family in plants. In this study, we focused on expression and functional analysis of cytokinin receptor-like HK genes (ZmHK) in maize. Firstly, bioinformatics analysis revealed that seven cloned ZmHK genes have different expression patterns during maize development. Secondly, ectopic expression by CaMV35S promoter in Arabidopsis further revealed that functional differentiation exists among these seven members. Among them, the ZmHK1a2-OX transgenic line has the lowest germination rate in the dark, ZmHK1-OX and ZmHK2a2-OX can delay leaf senescence, and seed size of ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX was obviously reduced as compared to wild type. Additionally, ZmHK genes play opposite roles in shoot and root development; all ZmHK-OX transgenic lines display obvious shorter root length and reduced number of lateral roots, but enhanced shoot development compared with the wild type. Most notably, Arabidopsis response regulator ARR5 gene was up-regulated in ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX as compared to wild type. Although the causal link between ZmHK genes and cytokinin signaling pathway is still an area to be further elucidated, these findings reflected that the diversification of ZmHK genes expression patterns and functions occurred in the course of maize evolution, indicating that some ZmHK genes might play different roles during maize development.

  20. Cysteine-rich receptor-like kinase CRK5 as a regulator of growth, development, and ultraviolet radiation responses in Arabidopsis thaliana.

    PubMed

    Burdiak, Paweł; Rusaczonek, Anna; Witoń, Damian; Głów, Dawid; Karpiński, Stanisław

    2015-06-01

    In plants, receptor-like protein kinases play essential roles in signal transduction by recognizing extracellular stimuli and activating the downstream signalling pathways. Cysteine-rich receptor-like kinases (CRKs) constitute a large subfamily of receptor-like protein kinases, with 44 members in Arabidopsis thaliana. They are distinguished by the novel C-X8-C-X2-C motif (DUF26) in the extracellular domains. One of them, CRK5, is an important component of the biochemical machinery involved in the regulation of essential physiological processes. Functional characterization of crk5 mutant plants showed their clear phenotype, manifested by impaired stomatal conductance and accelerated senescence. This phenotype correlated with accumulation of reactive oxygen species, higher foliar levels of ethylene and salicylic acid, and increased transcript abundance for genes associated with signalling pathways corresponding to these hormones. Moreover, the crk5 plants displayed enhanced cell death and oxidative damage in response to ultraviolet radiation. Complementation of CRK5 mutation managed to recover the wild-type phenotype, indicating an essential role of this gene in the regulation of growth, development, and acclimatory responses.

  1. A Genetic Screen Identifies a Requirement for Cysteine-Rich–Receptor-Like Kinases in Rice NH1 (OsNPR1)-Mediated Immunity

    PubMed Central

    Chern, Mawsheng; Xu, Qiufang; Bart, Rebecca S.; Bai, Wei; Ruan, Deling; Sze-To, Wing Hoi; Canlas, Patrick E.; Jain, Rashmi; Chen, Xuewei; Ronald, Pamela C.

    2016-01-01

    Systemic acquired resistance, mediated by the Arabidopsis NPR1 gene and the rice NH1 gene, confers broad-spectrum immunity to diverse pathogens. NPR1 and NH1 interact with TGA transcription factors to activate downstream defense genes. Despite the importance of this defense response, the signaling components downstream of NPR1/NH1 and TGA proteins are poorly defined. Here we report the identification of a rice mutant, snim1, which suppresses NH1-mediated immunity and demonstrate that two genes encoding previously uncharacterized cysteine-rich-receptor-like kinases (CRK6 and CRK10), complement the snim1 mutant phenotype. Silencing of CRK6 and CRK10 genes individually in the parental genetic background recreates the snim1 phenotype. We identified a rice mutant in the Kitaake genetic background with a frameshift mutation in crk10; this mutant also displays a compromised immune response highlighting the important role of crk10. We also show that elevated levels of NH1 expression lead to enhanced CRK10 expression and that the rice TGA2.1 protein binds to the CRK10 promoter. These experiments demonstrate a requirement for CRKs in NH1-mediated immunity and establish a molecular link between NH1 and induction of CRK10 expression. PMID:27176732

  2. Modes of Action and Functions of ERECTA-family Receptor-like Kinases in Plant Organ Growth and Development

    SciTech Connect

    TORII, Keiko U.

    2012-05-01

    Higher plants constitute the central resource for renewable lignocellulose biomass that can supplement for the world's depleting stores of fossil fuels. As such, understanding the molecular and genetic mechanisms of plant organ growth will provide key knowledge and genetic resources that enables manipulation of plant biomass feedstock for better growth and productivity. The goal of this proposal is to understand how cell proliferation and growth are coordinated during aboveground organ morphogenesis, and how cell-cell signaling mediated by a family of receptor kinases coordinates plant organogenesis. The well-established model plant Arabidopsis thaliana is used for our research to facilitate rapid progress. Specifically, we focus on how ERECTA-family leucine-rich repeat receptor kinases (LRR-RLKs) interact in a synergistic manner to promote organogenesis and pattern formation in Arabidopsis. This project was highly successful, resulted in fourteen publications including nine peer-reviewed original research articles. One provisional US patent has been filed through this DOE funding. We have addressed the critical roles for a family of receptor kinases in coordinating proliferation and differentiation of plants, and we successfully elucidated the downstream targets of this signaling pathway in specifying stomatal patterning.

  3. A LysM receptor-like kinase plays a critical role in chitin signaling and fungal resistance in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chitin, a polymer of N-acetyl-D-glucosamine, is found in fungal cell walls, but not in plants. Plant cells are capable of perceiving chitin fragments (chitooligosaccharides) to trigger plant defense. We identified a LysM receptor-like protein (AtLysM RLK1) that is required for the perception of chit...

  4. A LysM Receptor-like Kinase Plays a Critical Role in Chitin Signaling and Fungal Resistance in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chitin, a polymer of N-acetyl-D-glucosamine, is found in fungal cell walls, but not in plants. Plant cells are capable of perceiving chitin fragments (chitooligosaccharides) to trigger plant defense. We identified a LysM receptor-like protein (AtLysM RLK1) that is required for the perception of chit...

  5. Molecular characterisation of the STRUBBELIG-RECEPTOR FAMILY of genes encoding putative leucine-rich repeat receptor-like kinases in Arabidopsis thaliana

    PubMed Central

    Eyüboglu, Banu; Pfister, Karen; Haberer, Georg; Chevalier, David; Fuchs, Angelika; Mayer, Klaus FX; Schneitz, Kay

    2007-01-01

    Background Receptor-like kinases are a prominent class of surface receptors that regulate many aspects of the plant life cycle. Despite recent advances the function of most receptor-like kinases remains elusive. Therefore, it is paramount to investigate these receptors. The task is complicated by the fact that receptor-like kinases belong to a large monophyletic family with many sub-clades. In general, functional analysis of gene family members by reverse genetics is often obscured by several issues, such as redundancy, subtle or difficult to detect phenotypes in mutants, or by decision problems regarding suitable biological and biochemical assays. Therefore, in many cases additional strategies have to be employed to allow inference of hypotheses regarding gene function. Results We approached the function of genes encoding the nine-member STRUBBELIG-RECEPTOR FAMILY (SRF) class of putative leucine-rich repeat receptor-like kinases. Sequence comparisons show overall conservation but also divergence in predicted functional domains among SRF proteins. Interestingly, SRF1 undergoes differential splicing. As a result, SRF1 is predicted to exist in a standard receptor configuration and in a membrane-anchored receptor-like version that lacks most of the intracellular domain. Furthermore, SRF1 is characterised by a high degree of polymorphism between the Ler and Col accessions. Two independent T-DNA-based srf4 mutants showed smaller leaves while 35S::SRF4 plants displayed enlarged leaves. This is in addition to the strubbelig phenotype which has been described before. Additional single and several key double mutant combinations did not reveal obvious mutant phenotypes. Ectopic expression of several SRF genes, using the 35S promoter, resulted in male sterility. To gain possible insights into SRF gene function we employed a computational analysis of publicly available microarray data. We performed global expression profiling, coexpression analysis, and an analysis of the

  6. Overexpression of an Arabidopsis cysteine-rich receptor-like protein kinase, CRK5, enhances abscisic acid sensitivity and confers drought tolerance

    PubMed Central

    Lu, Kai; Liang, Shan; Wu, Zhen; Bi, Chao; Yu, Yong-Tao; Wang, Xiao-Fang; Zhang, Da-Peng

    2016-01-01

    Receptor-like kinases (RLKs) have been reported to regulate many developmental and defense process, but only a few members have been functionally characterized. In the present study, our observations suggest that one of the RLKs, a membrane-localized cysteine-rich receptor-like protein kinase, CRK5, is involved in abscisic acid (ABA) signaling in Arabidopsis thaliana. Overexpression of CRK5 increases ABA sensitivity in ABA-induced early seedling growth arrest and promotion of stomatal closure and inhibition of stomatal opening. Interestingly, and importantly, overexpression of CRK5 enhances plant drought tolerance without affecting plant growth at the mature stages and plant productivity. Transgenic lines overexpressing a mutated form of CRK5, CRK5 K372E with the change of the 372nd conserved amino acid residue from lysine to glutamic acid in its kinase domain, result in wild-type ABA and drought responses, supporting the role of CRK5 in ABA signaling. The loss-of-function mutation of the CRK5 gene does not affect the ABA response, while overexpression of two homologs of CRK5, CRK4 and CRK19, confers ABA responses, suggesting that these CRK members function redundantly. We further showed that WRKY18, WRKY40 and WRKY60 transcription factors repress the expression of CRK5, and that CRK5 likely functions upstream of ABI2 in ABA signaling. These findings help in understanding the complex ABA signaling network. PMID:27406784

  7. A Glycine soja ABA-responsive receptor-like cytoplasmic kinase, GsRLCK, positively controls plant tolerance to salt and drought stresses.

    PubMed

    Sun, XiaoLi; Sun, Mingzhe; Luo, Xiao; Ding, XiaoDong; Ji, Wei; Cai, Hua; Bai, Xi; Liu, XiaoFei; Zhu, YanMing

    2013-06-01

    Receptor such as protein kinases are proposed to work as sensors to initiate signaling cascades in higher plants. However, little is known about the precise functions of receptor such as protein kinases in abiotic stress response in plants, especially in wild soybean. Here, we focused on characterization of the biological functions of a receptor-like cytoplasmic serine/threonine protein kinase gene, GsRLCK, which was previously identified as a putative salt-alkali stress-related gene from the transcriptome profiles of Glycine soja. Bioinformatic analysis showed that GsRLCK protein contained a conserved kinase catalytic domain and two transmembrane domains at the N-terminus, but no typical extracellular domain. Consistently, GsRLCK-eGFP fusion protein was observed on the plasma membrane, but eGFP alone was distributing throughout the cytoplasm in onion epidermal cells. Quantitative real-time PCR analysis revealed the induced expression of GsRLCK by ABA, salt, alkali, and drought stresses. However, the expression levels of GsRLCK seemed to be similar in different tissues, except soybean pod. Phenotypic assays demonstrated that GsRLCK overexpression decreased ABA sensitivity and altered expression levels of ABA-responsive genes. Furthermore, we also found that GsRLCK conferred increased tolerance to salt and drought stresses and increased expression levels of a handful of stress-responsive genes, when overexpressing in Arabidopsis. In a word, we gave exact evidence that GsRLCK was a novel receptor-like cytoplasmic protein kinase and played a crucial role in plant responses to ABA, salt, and drought stresses.

  8. Molecular cloning and characterization of two novel genes from hexaploid wheat that encode double PR-1 domains coupled with a receptor-like protein kinase.

    PubMed

    Lu, Shunwen; Faris, Justin D; Edwards, Michael C

    2017-04-01

    Hexaploid wheat (Triticum aestivum L.) contains at least 23 TaPr-1 genes encoding the group 1 pathogenesis-related (PR-1) proteins as identified in our previous work. Here, we report the cloning and characterization of TaPr-1-rk1 and TaPr-1-rk2, two novel genes closely related to the wheat PR-1 family. The two TaPr-1-rk genes are located on homoeologous chromosomes 3D and 3A, respectively, and each contains a large open reading frame (7385 or 6060 bp) that is interrupted by seven introns and subjected to alternative splicing (AS) with five or six isoforms of mRNA transcripts. The deduced full-length TaPR-1-RK1 and TaPR-1-RK2 proteins (95% identity) contain two repeat PR-1 domains, the second of which is fused via a transmembrane helix to a serine/threonine kinase catalytic (STKc) domain characteristic of receptor-like protein kinases. Phylogenetic analysis indicated that the two PR-1 domains of the TaPR-1-RK proteins form sister clades with their homologues identified in other monocot plants and are well separated from stand-alone PR-1 proteins, whereas the STKc domains may have originated from cysteine-rich receptor-like kinases (CRKs). Reverse-transcriptase-PCR analysis revealed that the TaPr-1-rk genes are predominantly expressed in wheat leaves and their expression levels are elevated in response to pathogen attack, such as infection by barley stripe mosaic virus (BSMV), and also to stress conditions, most obviously, to soil salinity. This is the first report of PR-1-CRK hybrid proteins in wheat. The data may shed new insights into the function/evolutionary origin of the PR-1 family and the STKc-mediated defense/stress response pathways in plants.

  9. Duplication and Divergence of Leucine-Rich Repeat Receptor-Like Protein Kinase (LRR-RLK) Genes in Basal Angiosperm Amborella trichopoda

    PubMed Central

    Liu, Ping-Li; Xie, Lu-Lu; Li, Peng-Wei; Mao, Jian-Feng; Liu, Hui; Gao, Shu-Min; Shi, Peng-Hao; Gong, Jun-Qing

    2016-01-01

    Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are the largest group of receptor-like kinases, which are one of the largest protein superfamilies in plants, and play crucial roles in development and stress responses. Although the evolution of LRR-RLK families has been investigated in some eudicot and monocot plants, no comprehensive evolutionary studies have been performed for these genes in basal angiosperms like Amborella trichopoda. In this study, we identified 94 LRR-RLK genes in the genome of A. trichopoda. The number of LRR-RLK genes in the genome of A. trichopoda is only 17–50% of that of several eudicot and monocot species. Tandem duplication and whole-genome duplication have made limited contributions to the expansion of LRR-RLK genes in A. trichopoda. According to the phylogenetic analysis, all A. trichopoda LRR-RLK genes can be organized into 18 subfamilies, which roughly correspond to the LRR-RLK subfamilies defined in Arabidopsis thaliana. Most LRR-RLK subfamilies are characterized by highly conserved protein structures, motif compositions, and gene structures. The unique gene structure, protein structures, and protein motif compositions of each subfamily provide evidence for functional divergence among LRR-RLK subfamilies. Moreover, the expression data of LRR-RLK genes provided further evidence for the functional diversification of them. In addition, selection analyses showed that most LRR-RLK protein sites are subject to purifying selection. Our results contribute to a better understanding of the evolution of LRR-RLK gene family in angiosperm and provide a framework for further functional investigation on A. trichopoda LRR-RLKs. PMID:28066499

  10. Duplication and Divergence of Leucine-Rich Repeat Receptor-Like Protein Kinase (LRR-RLK) Genes in Basal Angiosperm Amborella trichopoda.

    PubMed

    Liu, Ping-Li; Xie, Lu-Lu; Li, Peng-Wei; Mao, Jian-Feng; Liu, Hui; Gao, Shu-Min; Shi, Peng-Hao; Gong, Jun-Qing

    2016-01-01

    Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are the largest group of receptor-like kinases, which are one of the largest protein superfamilies in plants, and play crucial roles in development and stress responses. Although the evolution of LRR-RLK families has been investigated in some eudicot and monocot plants, no comprehensive evolutionary studies have been performed for these genes in basal angiosperms like Amborella trichopoda. In this study, we identified 94 LRR-RLK genes in the genome of A. trichopoda. The number of LRR-RLK genes in the genome of A. trichopoda is only 17-50% of that of several eudicot and monocot species. Tandem duplication and whole-genome duplication have made limited contributions to the expansion of LRR-RLK genes in A. trichopoda. According to the phylogenetic analysis, all A. trichopoda LRR-RLK genes can be organized into 18 subfamilies, which roughly correspond to the LRR-RLK subfamilies defined in Arabidopsis thaliana. Most LRR-RLK subfamilies are characterized by highly conserved protein structures, motif compositions, and gene structures. The unique gene structure, protein structures, and protein motif compositions of each subfamily provide evidence for functional divergence among LRR-RLK subfamilies. Moreover, the expression data of LRR-RLK genes provided further evidence for the functional diversification of them. In addition, selection analyses showed that most LRR-RLK protein sites are subject to purifying selection. Our results contribute to a better understanding of the evolution of LRR-RLK gene family in angiosperm and provide a framework for further functional investigation on A. trichopoda LRR-RLKs.

  11. A Receptor-Like Kinase Mediates Ammonium Homeostasis and Is Important for the Polar Growth of Root Hairs in Arabidopsis[W

    PubMed Central

    Bai, Ling; Ma, Xiaonan; Zhang, Guozeng; Song, Shufei; Zhou, Yun; Gao, Lijie; Miao, Yuchen; Song, Chun-Peng

    2014-01-01

    Ammonium (NH4+) is both a necessary nutrient and an important signal in plants, but can be toxic in excess. Ammonium sensing and regulatory mechanisms in plant cells have not been fully elucidated. To decipher the complex network of NH4+ signaling, we analyzed [Ca2+]cyt-associated protein kinase (CAP) genes, which encode signaling components that undergo marked changes in transcription levels in response to various stressors. We demonstrated that CAP1, a tonoplast-localized receptor-like kinase, regulates root hair tip growth by maintaining cytoplasmic Ca2+ gradients. A CAP1 knockout mutant (cap1-1) produced elevated levels of cytoplasmic NH4+. Furthermore, root hair growth of cap1-1 was inhibited on Murashige and Skoog medium, but NH4+ depletion reestablished the Ca2+ gradient necessary for normal growth. The lower net NH4+ influx across the vacuolar membrane and relatively alkaline cytosolic pH of cap1-1 root hairs implied that mutation of CAP1 increased NH4+ accumulation in the cytoplasm. Furthermore, CAP1 functionally complemented the npr1 (nitrogen permease reactivator protein) kinase yeast mutant, which is defective in high-affinity NH4+ uptake via MEP2 (methylammonium permease 2), distinguishing CAP1 as a cytosolic modulator of NH4+ levels that participates in NH4+ homeostasis-regulated root hair growth by modulating tip-focused cytoplasmic Ca2+ gradients. PMID:24769480

  12. A cell-free method for expressing and reconstituting membrane proteins enables functional characterization of the plant receptor-like protein kinase FERONIA.

    PubMed

    Minkoff, Benjamin B; Makino, Shin-Ichi; Haruta, Miyoshi; Beebe, Emily T; Wrobel, Russell L; Fox, Brian G; Sussman, Michael R

    2017-04-07

    There are more than 600 receptor-like kinases (RLKs) in Arabidopsis, but due to challenges associated with the characterization of membrane proteins, only a few have known biological functions. The plant RLK FERONIA is a peptide receptor and has been implicated in plant growth regulation, but little is known about its molecular mechanism of action. To investigate the properties of this enzyme, we used a cell-free wheat germ-based expression system in which mRNA encoding FERONIA was co-expressed with mRNA encoding the membrane scaffold protein variant MSP1D1. With the addition of the lipid cardiolipin, assembly of these proteins into nanodiscs was initiated. FERONIA protein kinase activity in nanodiscs was higher than that of soluble protein and comparable with other heterologously expressed protein kinases. Truncation experiments revealed that the cytoplasmic juxtamembrane domain is necessary for maximal FERONIA activity, whereas the transmembrane domain is inhibitory. An ATP analogue that reacts with lysine residues inhibited catalytic activity and labeled four lysines; mutagenesis demonstrated that two of these, Lys-565 and Lys-663, coordinate ATP in the active site. Mass spectrometric phosphoproteomic measurements further identified phosphorylation sites that were examined using phosphomimetic mutagenesis. The results of these experiments are consistent with a model in which kinase-mediated phosphorylation within the C-terminal region is inhibitory and regulates catalytic activity. These data represent a step further toward understanding the molecular basis for the protein kinase catalytic activity of FERONIA and show promise for future characterization of eukaryotic membrane proteins.

  13. Cloning of a Conserved Receptor-Like Protein Kinase Gene and Its Use as a Functional Marker for Homoeologous Group-2 Chromosomes of the Triticeae Species

    PubMed Central

    Qin, Bi; Chen, Tingting; Cao, Aizhong; Wang, Haiyan; Xing, Liping; Ling, Hongqing; Wang, Daowen; Yu, Chunmei; Xiao, Jin; Ji, Jianhui; Chen, Xueluan; Chen, Peidu; Liu, Dajun; Wang, Xiue

    2012-01-01

    Receptor-like kinases (RLKs) play broad biological roles in plants. We report on a conserved receptor-like protein kinase (RPK) gene from wheat and other Triticeae species. The TaRPK1 was isolated from the Triticum aestivum cv. Prins - Triticum timopheevii introgression line IGVI-465 carrying the powdery mildew resistance gene Pm6. The TaRPK1 was mapped to homoeologous chromosomes 2A (TaRPK1-2A), 2D (TaRPK1-2D) and the Pm6-carrier chromosome 2G (TaRPK1-2G) of IGVI-465. Under the tested conditions, only the TaRPK1-2G allele was actively transcribed, producing two distinct transcripts via alternative splicing. The predicted 424-amino acid protein of TaRPK1-2G contained a signal peptide, a transmembrane domain and an intracellular serine/threonine kinase domain, but lacked a typical extracellular domain. The expression of TaRPK1-2G gene was up-regulated upon the infection by Blumeria graminis f.sp. tritici (Bgt) and treatment with methyl jasmonate (MeJA), but down-regulated in response to treatments of SA and ABA. Over-expression of TaRPK1-2G in the powdery mildew susceptible wheat variety Prins by a transient expression assay showed that it slightly reduced the haustorium index of the infected Bgt. These data indicated that TaRPK1-2G participated in the defense response to Bgt infection and in the JA signaling pathway. Phylogenetic analysis indicated that TaRPK1-2G was highly conserved among plant species, and the amino acid sequence similarity of TaRPK1-2G among grass species was more than 86%. Based on its conservation, the RPK gene-based STS primers were designed, and used to amplify the RPK orthologs from the homoeologous group-2 chromosomes of all the tested Triticeae species, such as chromosome 2G of T. timopheevii, 2R of Secale cereale, 2H of Hordeum vulgare, 2S of Aegilops speltoides, 2Sl of Ae. longissima, 2Mg of Ae. geniculata, 2Sp and 2Up of Ae. peregrina. The developed STS markers serve as conserved functional markers for the identification of

  14. Pea lectin receptor-like kinase functions in salinity adaptation without yield penalty, by alleviating osmotic and ionic stresses and upregulating stress-responsive genes.

    PubMed

    Vaid, Neha; Pandey, Prashant; Srivastava, Vineet Kumar; Tuteja, Narendra

    2015-05-01

    Lectin receptor-like kinases (LecRLKs) are members of RLK family composed of lectin-like extracellular recognition domain, transmembrane domain and cytoplasmic kinase domain. LecRLKs are plasma membrane proteins believed to be involved in signal transduction. However, most of the members of the protein family even in plants have not been functionally well characterized. Herein, we show that Pisum sativum LecRLK (PsLecRLK) localized in plasma membrane systems and/or other regions of the cell and its transcript upregulated under salinity stress. Overexpression of PsLecRLK in transgenic tobacco plants confers salinity stress tolerance by alleviating both the ionic as well the osmotic component of salinity stress. The transgenic plants show better tissue compartmentalization of Na(+) and higher ROS scavenging activity which probably results in lower membrane damage, improved growth and yield maintenance even under salinity stress. Also, expression of several genes involved in cellular homeostasis is perturbed by PsLecRLK overexpression. Alleviation of osmotic and ionic components of salinity stress along with reduced oxidative damage and upregulation of stress-responsive genes in transgenic plants under salinity stress conditions could be possible mechanism facilitating enhanced stress tolerance. This study presents PsLecRLK as a promising candidate for crop improvement and also opens up new avenue to investigate its signalling pathway.

  15. Pea lectin receptor-like kinase promotes high salinity stress tolerance in bacteria and expresses in response to stress in planta.

    PubMed

    Joshi, Amita; Dang, Hung Quang; Vaid, Neha; Tuteja, Narendra

    2010-01-01

    The plant lectin receptor-like kinases (LecRLKs) are involved in various signaling pathways but their role in salinity stress tolerance has not heretofore been well described. Salinity stress negatively affects plant growth/productivity and threatens food security worldwide. Based on functional gene-mining assay, we have isolated 34 salinity tolerant genes out of one million Escherichia coli (SOLR) transformants containing pea cDNAs grown in 0.8 M NaCl. Sequence analysis of one of these revealed homology to LecRLK, which possesses N-myristilation and N-glycosylation sites thus corroborating the protein to be a glycoconjugate. The homology based computational modeling of the kinase domain suggested high degree of conservation with the protein already known to be stress responsive in plants. The NaCl tolerance provided by PsLecRLK to the above bacteria was further confirmed in E. coli (DH5alpha). In planta studies showed that the expression of PsLecRLK cDNA was significantly upregulated in response to NaCl as compared to K(+) and Li(+) ions, suggesting the Na(+) ion specific response. Transcript of the PsLecRLK gene accumulates mainly in roots and shoots. The purified 47 kDa recombinant PsLecRLK-KD (kinase domain) protein has been shown to phosphorylate general substrates like MBP and casein. This study not only suggests the conservation of the cellular response to high salinity stress across prokaryotes and plant kingdom but also provides impetus to develop novel concepts for better understanding of mechanism of stress tolerance in bacteria and plants. It also opens up new avenues for studying practical aspects of plant salinity tolerance for enhanced agricultural productivity.

  16. An LRR/Malectin Receptor-Like Kinase Mediates Resistance to Non-adapted and Adapted Powdery Mildew Fungi in Barley and Wheat

    PubMed Central

    Rajaraman, Jeyaraman; Douchkov, Dimitar; Hensel, Götz; Stefanato, Francesca L.; Gordon, Anna; Ereful, Nelzo; Caldararu, Octav F.; Petrescu, Andrei-Jose; Kumlehn, Jochen; Boyd, Lesley A.; Schweizer, Patrick

    2016-01-01

    Pattern recognition receptors (PRRs) belonging to the multigene family of receptor-like kinases (RLKs) are the sensing devices of plants for microbe- or pathogen-associated molecular patterns released from microbial organisms. Here we describe Rnr8 (for Required for non-host resistance 8) encoding HvLEMK1, a LRR-malectin domain-containing transmembrane RLK that mediates non-host resistance of barley to the non-adapted wheat powdery mildew fungus Blumeria graminis f.sp. tritici. Transgenic barley lines with silenced HvLEMK1 allow entry and colony growth of the non-adapted pathogen, although sporulation was reduced and final colony size did not reach that of the adapted barley powdery mildew fungus B. graminis f.sp. hordei. Transient expression of the barley or wheat LEMK1 genes enhanced resistance in wheat to the adapted wheat powdery mildew fungus while expression of the same genes did not protect barley from attack by the barley powdery mildew fungus. The results suggest that HvLEMK1 is a factor mediating non-host resistance in barley and quantitative host resistance in wheat to the wheat powdery mildew fungus. PMID:28018377

  17. The phenome analysis of mutant alleles in Leucine-Rich Repeat Receptor-Like Kinase genes in rice reveals new potential targets for stress tolerant cereals.

    PubMed

    Dievart, Anne; Perin, Christophe; Hirsch, Judith; Bettembourg, Mathilde; Lanau, Nadège; Artus, Florence; Bureau, Charlotte; Noel, Nicolas; Droc, Gaétan; Peyramard, Matthieu; Pereira, Serge; Courtois, Brigitte; Morel, Jean-Benoit; Guiderdoni, Emmanuel

    2016-01-01

    Plants are constantly exposed to a variety of biotic and abiotic stresses that reduce their fitness and performance. At the molecular level, the perception of extracellular stimuli and the subsequent activation of defense responses require a complex interplay of signaling cascades, in which protein phosphorylation plays a central role. Several studies have shown that some members of the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) family are involved in stress and developmental pathways. We report here a systematic analysis of the role of the members of this gene family by mutant phenotyping in the monocotyledon model plant rice, Oryza sativa. We have then targeted 176 of the ∼320 LRR-RLK genes (55.7%) and genotyped 288 mutant lines. Position of the insertion was confirmed in 128 lines corresponding to 100 LRR-RLK genes (31.6% of the entire family). All mutant lines harboring homozygous insertions have been screened for phenotypes under normal conditions and under various abiotic stresses. Mutant plants have been observed at several stages of growth, from seedlings in Petri dishes to flowering and grain filling under greenhouse conditions. Our results show that 37 of the LRR-RLK rice genes are potential targets for improvement especially in the generation of abiotic stress tolerant cereals.

  18. Molecular basis of lipo-chitooligosaccharide recognition by the lysin motif receptor-like kinase LYR3 in legumes.

    PubMed

    Malkov, Nikita; Fliegmann, Judith; Rosenberg, Charles; Gasciolli, Virginie; Timmers, Antonius C J; Nurisso, Alessandra; Cullimore, Julie; Bono, Jean-Jacques

    2016-05-15

    LYR3 [LysM (lysin motif) receptor-like kinase 3] of Medicago truncatula is a high-affinity binding protein for symbiotic LCO (lipo-chitooligosaccharide) signals, produced by rhizobia bacteria and arbuscular mycorrhizal fungi. The present study shows that LYR3 from several other legumes, but not from two Lupinus species which are incapable of forming the mycorrhizal symbiosis, bind LCOs with high affinity and discriminate them from COs (chitooligosaccharides). The biodiversity of these proteins and the lack of binding to the Lupinus proteins were used to identify features required for high-affinity LCO binding. Swapping experiments between each of the three LysMs of the extracellular domain of the M. truncatula and Lupinus angustifolius LYR3 proteins revealed the crucial role of the third LysM in LCO binding. Site-directed mutagenesis identified a tyrosine residue, highly conserved in all LYR3 LCO-binding proteins, which is essential for high-affinity binding. Molecular modelling suggests that it may be part of a hydrophobic tunnel able to accommodate the LCO acyl chain. The lack of conservation of these features in the binding site of plant LysM proteins binding COs provides a mechanistic explanation of how LCO recognition might differ from CO perception by structurally related LysM receptors.

  19. GATA6 Promotes Angiogenic Function and Survival in Endothelial Cells by Suppression of Autocrine Transforming Growth Factor β/Activin Receptor-like Kinase 5 Signaling*

    PubMed Central

    Froese, Natali; Kattih, Badder; Breitbart, Astrid; Grund, Andrea; Geffers, Robert; Molkentin, Jeffery D.; Kispert, Andreas; Wollert, Kai C.; Drexler, Helmut; Heineke, Joerg

    2011-01-01

    Understanding the transcriptional regulation of angiogenesis could lead to the identification of novel therapeutic targets. We showed here that the transcription factor GATA6 is expressed in different human primary endothelial cells as well as in vascular endothelial cells of mice in vivo. Activation of endothelial cells was associated with GATA6 nuclear translocation, chromatin binding, and enhanced GATA6-dependent transcriptional activation. siRNA-mediated down-regulation of GATA6 after growth factor stimulation led to a dramatically reduced capacity of macro- and microvascular endothelial cells to proliferate, migrate, or form capillary-like structures on Matrigel. Adenoviral overexpression of GATA6 in turn enhanced angiogenic function, especially in cardiac endothelial microvascular cells. Furthermore, GATA6 protected endothelial cells from undergoing apoptosis during growth factor deprivation. Mechanistically, down-regulation of GATA6 in endothelial cells led to increased expression of transforming growth factor (TGF) β1 and TGFβ2, whereas enhanced GATA6 expression, accordingly, suppressed Tgfb1 promoter activity. High TGFβ1/β2 expression in GATA6-depleted endothelial cells increased the activation of the activin receptor-like kinase 5 (ALK5) and SMAD2, and suppression of this signaling axis by TGFβ neutralizing antibody or ALK5 inhibition restored angiogenic function and survival in endothelial cells with reduced GATA6 expression. Together, these findings indicate that GATA6 plays a crucial role for endothelial cell function and survival, at least in part, by suppressing autocrine TGFβ expression and ALK5-dependent signaling. PMID:21127043

  20. An LRR/Malectin Receptor-Like Kinase Mediates Resistance to Non-adapted and Adapted Powdery Mildew Fungi in Barley and Wheat.

    PubMed

    Rajaraman, Jeyaraman; Douchkov, Dimitar; Hensel, Götz; Stefanato, Francesca L; Gordon, Anna; Ereful, Nelzo; Caldararu, Octav F; Petrescu, Andrei-Jose; Kumlehn, Jochen; Boyd, Lesley A; Schweizer, Patrick

    2016-01-01

    Pattern recognition receptors (PRRs) belonging to the multigene family of receptor-like kinases (RLKs) are the sensing devices of plants for microbe- or pathogen-associated molecular patterns released from microbial organisms. Here we describe Rnr8 (for Required for non-host resistance 8) encoding HvLEMK1, a LRR-malectin domain-containing transmembrane RLK that mediates non-host resistance of barley to the non-adapted wheat powdery mildew fungus Blumeria graminis f.sp. tritici. Transgenic barley lines with silenced HvLEMK1 allow entry and colony growth of the non-adapted pathogen, although sporulation was reduced and final colony size did not reach that of the adapted barley powdery mildew fungus B. graminis f.sp. hordei. Transient expression of the barley or wheat LEMK1 genes enhanced resistance in wheat to the adapted wheat powdery mildew fungus while expression of the same genes did not protect barley from attack by the barley powdery mildew fungus. The results suggest that HvLEMK1 is a factor mediating non-host resistance in barley and quantitative host resistance in wheat to the wheat powdery mildew fungus.

  1. Evaluation of the Role of the LysM Receptor-Like Kinase, OsNFR5/OsRLK2 for AM Symbiosis in Rice.

    PubMed

    Miyata, Kana; Hayafune, Masahiro; Kobae, Yoshihiro; Kaku, Hanae; Nishizawa, Yoko; Masuda, Yoshiki; Shibuya, Naoto; Nakagawa, Tomomi

    2016-11-01

    In legume-specific rhizobial symbiosis, host plants perceive rhizobial signal molecules, Nod factors, by a pair of LysM receptor-like kinases, NFR1/LYK3 and NFR5/NFP, and activate symbiotic responses through the downstream signaling components also required for arbuscular mycorrhizal (AM) symbiosis. Recently, the rice NFR1/LYK3 ortholog, OsCERK1, was shown to play crucial roles for AM symbiosis. On the other hand, the roles of the NFR5/NFP ortholog in rice have not been elucidated, while it has been shown that NFR5/NFP orthologs, Parasponia PaNFR5 and tomato SlRLK10, engage in AM symbiosis. OsCERK1 also triggers immune responses in combination with a receptor partner, OsCEBiP, against fungal or bacterial infection, thus regulating opposite responses against symbiotic and pathogenic microbes. However, it has not been elucidated how OsCERK1 switches these opposite functions. Here, we analyzed the function of the rice NFR5/NFP ortholog, OsNFR5/OsRLK2, as a possible candidate of the OsCERK1 partner for symbiotic signaling. Inoculation of AM fungi induced the expression of OsNFR5 in the rice root, and the chimeric receptor consisting of the extracellular domain of LjNFR5 and the intracellular domain of OsNFR5 complemented the Ljnfr5 mutant for rhizobial symbiosis, indicating that the intracellular kinase domain of OsNFR5 could activate symbiotic signaling in Lotus japonicus. Although these data suggested the possible involvement of OsNFR5 in AM symbiosis, osnfr5 knockout mutants were colonized by AM fungi similar to the wild-type rice. These observations suggested several possibilities including the presence of functionally redundant genes other than OsNFR5 or involvement of novel ligands, which do not require OsNFR5 for recognition.

  2. Targeting activin receptor-like kinase 1 inhibits angiogenesis and tumorigenesis through a mechanism of action complementary to anti-VEGF therapies.

    PubMed

    Hu-Lowe, Dana D; Chen, Enhong; Zhang, Lianglin; Watson, Katherine D; Mancuso, Patrizia; Lappin, Patrick; Wickman, Grant; Chen, Jeffrey H; Wang, Jianying; Jiang, Xin; Amundson, Karin; Simon, Ronald; Erbersdobler, Andreas; Bergqvist, Simon; Feng, Zheng; Swanson, Terri A; Simmons, Brett H; Lippincott, John; Casperson, Gerald F; Levin, Wendy J; Stampino, Corrado Gallo; Shalinsky, David R; Ferrara, Katherine W; Fiedler, Walter; Bertolini, Francesco

    2011-02-15

    Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1) plays an important role in vascular development, remodeling, and pathologic angiogenesis. Here we investigated the role of ALK1 in angiogenesis in the context of common proangiogenic factors [PAF; VEGF-A and basic fibroblast growth factor (bFGF)]. We observed that PAFs stimulated ALK1-mediated signaling, including Smad1/5/8 phosphorylation, nuclear translocation and Id-1 expression, cell spreading, and tubulogenesis of endothelial cells (EC). An antibody specifically targeting ALK1 (anti-ALK1) markedly inhibited these events. In mice, anti-ALK1 suppressed Matrigel angiogenesis stimulated by PAFs and inhibited xenograft tumor growth by attenuating both blood and lymphatic vessel angiogenesis. In a human melanoma model with acquired resistance to a VEGF receptor kinase inhibitor, anti-ALK1 also delayed tumor growth and disturbed vascular normalization associated with VEGF receptor inhibition. In a human/mouse chimera tumor model, targeting human ALK1 decreased human vessel density and improved antitumor efficacy when combined with bevacizumab (anti-VEGF). Antiangiogenesis and antitumor efficacy were associated with disrupted co-localization of ECs with desmin(+) perivascular cells, and reduction of blood flow primarily in large/mature vessels as assessed by contrast-enhanced ultrasonography. Thus, ALK1 may play a role in stabilizing angiogenic vessels and contribute to resistance to anti-VEGF therapies. Given our observation of its expression in the vasculature of many human tumor types and in circulating ECs from patients with advanced cancers, ALK1 blockade may represent an effective therapeutic opportunity complementary to the current antiangiogenic modalities in the clinic.

  3. High expression of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE coincides with initiation of various developmental pathways in in vitro culture of Trifolium nigrescens.

    PubMed

    Pilarska, Maria; Malec, Przemysław; Salaj, Jan; Bartnicki, Filip; Konieczny, Robert

    2016-03-01

    The aim of this study was to identify and examine the expression pattern of the ortholog of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE gene from Trifolium nigrescens (TnSERK) in embryogenic and non-regenerative cultures of immature cotyledonary-stage zygotic embryos (CsZEs). In the presence of 1-naphthaleneacetic acid and N(6)-[2-isopentenyl]-adenine, the CsZE regenerated embryoids directly and in a lengthy culture produced callus which was embryogenic or remained non-regenerative. As revealed by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), the TnSERK was expressed in both embryogenic and non-regenerative cultures, but the expression level was significantly higher in embryogenic ones. An in situ RNA hybridization assay revealed that the expression of TnSERK preceded the induction of cell division in explants, and then, it was maintained exclusively in actively dividing cells from which embryoids, embryo-like structures (ELSs), callus or tracheary elements were produced. However, the cells involved in different morphogenic events differed in intensity of hybridization signal which was the highest in embryogenic cells. The TnSERK was up-regulated during the development of embryoids, but in cotyledonary embryos, it was preferentially expressed in the regions of the apical meristems. The occurrence of morphological and anatomical abnormalities in embryoid development was preceded by a decline in TnSERK expression, and this coincided with the parenchymatization of the ground tissue in developing ELSs. TnSERK was also down-regulated during the maturation of parenchyma and xylem elements in CsZE and callus. Altogether, these data suggest the involvement of TnSERK in the induction of various developmental programs related to differentiation/transdifferentiation and totipotent state of cell(s).

  4. Endoglin and activin receptor-like kinase 1 heterozygous mice have a distinct pulmonary and hepatic angiogenic profile and response to anti-VEGF treatment.

    PubMed

    Ardelean, Daniela S; Jerkic, Mirjana; Yin, Melissa; Peter, Madonna; Ngan, Bo; Kerbel, Robert S; Foster, F Stuart; Letarte, Michelle

    2014-01-01

    Hereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia associated with dysregulated angiogenesis and arteriovascular malformations. The disease is caused by mutations in endoglin (ENG; HHT1) or activin receptor-like kinase 1 (ALK1; HHT2) genes, coding for transforming growth factor β (TGF-β) superfamily receptors. Vascular endothelial growth factor (VEGF) has been implicated in HHT and beneficial effects of anti-VEGF treatment were recently reported in HHT patients. To investigate the systemic angiogenic phenotype of Endoglin and Alk1 mutant mice and their response to anti-VEGF therapy, we assessed microvessel density (MVD) in multiple organs after treatment with an antibody to mouse VEGF or vehicle. Lungs were the only organ showing an angiogenic defect, with reduced peripheral MVD and secondary right ventricular hypertrophy (RVH), yet distinctly associated with a fourfold increase in thrombospondin-1 (TSP-1) in Eng (+/-) versus a rise in angiopoietin-2 (Ang-2) in Alk1 (+/-) mice. Anti-VEGF treatment did reduce lung VEGF levels but interestingly, led to an increase in peripheral pulmonary MVD and attenuation of RVH; it also normalized TSP-1 and Ang-2 expression. Hepatic MVD, unaffected in mutant mice, was reduced by anti-VEGF therapy in heterozygous and wild type mice, indicating a liver-specific effect of treatment. Contrast-enhanced micro-ultrasound demonstrated a reduction in hepatic microvascular perfusion after anti-VEGF treatment only in Eng (+/-) mice. Our findings indicate that the mechanisms responsible for the angiogenic imbalance and the response to anti-VEGF therapy differ between Eng and Alk1 heterozygous mice and raise the need for systemic monitoring of anti-angiogenic therapy effects in HHT patients.

  5. The FERONIA Receptor-like Kinase Mediates Male-Female Interactions During Pollen Tube Reception

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fertilization in higher plants requires intricate signaling between the male and female. The molecular details underlying this communication are of great interest, not only to understand plant reproduction but also to guide efforts in crossing plant species to generate new hybrids. On page 656 of th...

  6. Receptor-like Molecules on Human Intestinal Epithelial Cells Interact with an Adhesion Factor from Lactobacillus reuteri.

    PubMed

    Matsuo, Yosuke; Miyoshi, Yukihiro; Okada, Sanae; Satoh, Eiichi

    2012-01-01

    A surface protein of Lactobacillus reuteri, mucus adhesion-promoting protein (MapA), is considered to be an adhesion factor. MapA is expressed in L. reuteri strains and adheres to piglet gastric mucus, collagen type I, and human intestinal epithelial cells such as Caco-2. The aim of this study was to identify molecules that mediate the attachment of MapA from L. reuteri to the intestinal epithelial cell surface by investigating the adhesion of MapA to receptor-like molecules on Caco-2 cells. MapA-binding receptor-like molecules were detected in Caco-2 cell lysates by 2D-PAGE. Two proteins, annexin A13 (ANXA13) and paralemmin (PALM), were identified by MALDI TOF-MS. The results of a pull-down assay showed that MapA bound directly to ANXA13 and PALM. Fluorescence microscopy studies confirmed that MapA binding to ANXA13 and PALM was colocalized on the Caco-2 cell membrane. To evaluate whether ANXA13 and PALM are important for MapA adhesion, ANXA13 and PALM knockdown cell lines were established. The adhesion of MapA to the abovementioned cell lines was reduced compared with that to wild-type Caco-2 cells. These knockdown experiments established the importance of these receptor-like molecules in MapA adhesion.

  7. Characterization of Drosophila GDNF Receptor-Like and Evidence for Its Evolutionarily Conserved Interaction with Neural Cell Adhesion Molecule (NCAM)/FasII

    PubMed Central

    Kallijärvi, Jukka; Stratoulias, Vassilis; Virtanen, Kristel; Hietakangas, Ville; Heino, Tapio I.; Saarma, Mart

    2012-01-01

    Background Glial cell line-derived neurotrophic factor (GDNF) family ligands are secreted growth factors distantly related to the TGF-β superfamily. In mammals, they bind to the GDNF family receptor α (Gfrα) and signal through the Ret receptor tyrosine kinase. In order to gain insight into the evolution of the Ret-Gfr-Gdnf signaling system, we have cloned and characterized the first invertebrate Gfr-like cDNA (DmGfrl) from Drosophila melanogaster and generated a DmGfrl mutant allele. Results We found that DmGfrl encodes a large GPI-anchored membrane protein with four GFR-like domains. In line with the fact that insects lack GDNF ligands, DmGfrl mediated neither Drosophila Ret phosphorylation nor mammalian RET phosphorylation. In situ hybridization analysis revealed that DmGfrl is expressed in the central and peripheral nervous systems throughout Drosophila development, but, surprisingly, DmGfrl and DmRet expression patterns were largely non-overlapping. We generated a DmGfrl null allele by genomic FLP deletion and found that both DmGfrl null females and males are viable but display fertility defects. The female fertility defect manifested as dorsal appendage malformation, small size and reduced viability of eggs laid by mutant females. In male flies DmGfrl interacted genetically with the Drosophila Ncam (neural cell adhesion molecule) homolog FasII to regulate fertility. Conclusion Our results suggest that Ret and Gfrl did not function as an in cis receptor-coreceptor pair before the emergence of GDNF family ligands, and that the Ncam-Gfr interaction predated the in cis Ret-Gfr interaction in evolution. The fertility defects that we describe in DmGfrl null flies suggest that GDNF receptor-like has an evolutionarily ancient role in regulating male fertility and a previously unrecognized role in regulating oogenesis. Significance These results shed light on the evolutionary aspects of the structure, expression and function of Ret-Gfrα and Ncam-Gfrα signaling

  8. GsLRPK, a novel cold-activated leucine-rich repeat receptor-like protein kinase from Glycine soja, is a positive regulator to cold stress tolerance.

    PubMed

    Yang, Liang; Wu, Kangcheng; Gao, Peng; Liu, Xiaojuan; Li, Guangpu; Wu, Zujian

    2014-02-01

    Plant LRR-RLKs serve as protein interaction platforms, and as regulatory modules of protein activation. Here, we report the isolation of a novel plant-specific LRR-RLK from Glycine soja (termed GsLRPK) by differential screening. GsLRPK expression was cold-inducible and shows Ser/Thr protein kinase activity. Subcellular localization studies using GFP fusion protein indicated that GsLRPK is localized in the plasma membrane. Real-time PCR analysis indicated that temperature, salt, drought, and ABA treatment can alter GsLRPK gene transcription in G. soja. However, just protein induced by cold stress not by salinity and ABA treatment in tobacco was found to possess kinase activity. Furthermore, we found that overexpression of GsLRPK in yeast and Arabidopsis can enhance resistance to cold stress and increase the expression of a number of cold responsive gene markers.

  9. Novel type of receptor-like protein kinase from a higher plant (Catharanthus roseus). cDNA, gene, intramolecular autophosphorylation, and identification of a threonine important for auto- and substrate phosphorylation.

    PubMed

    Schulze-Muth, P; Irmler, S; Schröder, G; Schröder, J

    1996-10-25

    We characterize CrRLK1, a novel type of receptor-like kinase (RLK), from the plant Catharanthus roseus (Madagascar periwinkle). The protein (90.2 kDa) deduced from the complete genomic and cDNA sequences is a RLK by predicting a N-terminal signal peptide, a large extracytoplasmic domain, a membrane-spanning hydrophobic region followed by a transfer-stop signal, and a C-terminal cytoplasmic protein kinase with all 11 conserved subdomains. It is a novel RLK type because the predicted extracytoplasmic region shares no similarity with other RLKs. The autophosphorylation was investigated with affinity-purified proteins expressed in Escherichia coli. The activity was higher with Mn2+ than with Mg2+ and achieved half-maximal rates at 2-2.5 microM ATP. The phosphorylation was predominantly on Thr, less on Ser, and not on Tyr. In contrast to other plant RLK, the kinase used an intra- rather than an intermolecular phosphorylation mechanism. After protein cleavage with formic acid, most of the radioactivity was in a 14.1-kDa peptide located at the end of the kinase domain. Mutagenesis of the four Thr residues in this peptide identified Thr-720 in the subdomain XI as important for autophosphorylation and for phosphorylation of beta-casein. This Thr is conserved in other related kinases, suggesting a subfamily sharing common autophosphorylation mechanisms.

  10. Glutaredoxin GrxC2 catalyzes the glutathionylation and inactivation of Arabidopsis BRI1-ASSOCIATED RECEPTOR-LIKE KINASE 1 (BAK1) in vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Reversible protein phosphorylation, catalyzed by protein kinases, is the most widely studied post-translational modification (PTM) both in terms of its occurrence and the regulatory consequences of phosphorylation events on phosphorylated proteins. In addition to reversible phosphorylation, many pro...

  11. A Receptor-Like Kinase, Related to Cell Wall Sensor of Higher Plants, is Required for Sexual Reproduction in the Unicellular Charophycean Alga, Closterium peracerosum-strigosum-littorale Complex.

    PubMed

    Hirano, Naoko; Marukawa, Yuka; Abe, Jun; Hashiba, Sayuri; Ichikawa, Machiko; Tanabe, Yoichi; Ito, Motomi; Nishii, Ichiro; Tsuchikane, Yuki; Sekimoto, Hiroyuki

    2015-07-01

    Here, we cloned the CpRLK1 gene, which encodes a receptor-like protein kinase expressed during sexual reproduction, from the heterothallic Closterium peracerosum-strigosum-littorale complex, one of the closest unicellular alga to land plants. Mating-type plus (mt(+)) cells with knockdown of CpRLK1 showed reduced competence for sexual reproduction and formed an abnormally enlarged conjugation papilla after pairing with mt(-) cells. The knockdown cells were unable to release a naked gamete, which is indispensable for zygote formation. We suggest that the CpRLK1 protein is an ancient cell wall sensor that now functions to regulate osmotic pressure in the cell to allow proper gamete release.

  12. Natural variation in host-specific nodulation of pea is associated with a haplotype of the SYM37 LysM-type receptor-like kinase.

    PubMed

    Li, Ronghui; Knox, Maggie R; Edwards, Anne; Hogg, Bridget; Ellis, T H Noel; Wei, Gehong; Downie, J Allan

    2011-11-01

    Rhizobium leguminosarum bv. viciae, which nodulates pea and vetch, makes a mixture of secreted nodulation signals (Nod factors) carrying either a C18:4 or a C18:1 N-linked acyl chain. Mutation of nodE blocks the formation of the C18:4 acyl chain, and nodE mutants, which produce only C18:1-containing Nod factors, are less efficient at nodulating pea. However, there is significant natural variation in the levels of nodulation of different pea cultivars by a nodE mutant of R. leguminosarum bv. viciae. Using recombinant inbred lines from two pea cultivars, one which nodulated relatively well and one very poorly by the nodE mutant, we mapped the nodE-dependent nodulation phenotype to a locus on pea linkage group I. This was close to Sym37 and PsK1, predicted to encode LysM-domain Nod-factor receptor-like proteins; the Sym2 locus that confers Nod-factor-specific nodulation is also in this region. We confirmed the map location using an introgression line carrying this region. Our data indicate that the nodE-dependent nodulation is not determined by the Sym2 locus. We identified several pea lines that are nodulated very poorly by the R. leguminosarum bv. viciae nodE mutant, sequenced the DNA of the predicted LysM-receptor domains of Sym37 and PsK1, and compared the sequences with those derived from pea cultivars that were relatively well nodulated by the nodE mutant. This revealed that one haplotype (encoding six conserved polymorphisms) of Sym37 is associated with very poor nodulation by the nodE mutant. There was no such correlation with polymorphisms at the PsK1 locus. We conclude that the natural variation in nodE-dependent nodulation in pea is most probably determined by the Sym37 haplotype.

  13. Cell wall-associated ROOT HAIR SPECIFIC 10, a proline-rich receptor-like kinase, is a negative modulator of Arabidopsis root hair growth

    PubMed Central

    Hwang, Youra; Lee, Hyodong; Lee, Young-Sook; Cho, Hyung-Taeg

    2016-01-01

    Plant cell growth is restricted by the cell wall, and cell wall dynamics act as signals for the cytoplasmic and nuclear events of cell growth. Among various receptor kinases, ROOT HAIR SPECIFIC 10 (RHS10) belongs to a poorly known receptor kinase subfamily with a proline-rich extracellular domain. Here, we report that RHS10 defines the root hair length of Arabidopsis thaliana by negatively regulating hair growth. RHS10 modulates the duration of root hair growth rather than the growth rate. As poplar and rice RHS10 orthologs also showed a root hair-inhibitory function, this receptor kinase-mediated function appears to be conserved in angiosperms. RHS10 showed a strong association with the cell wall, most probably through its extracellular proline-rich domain (ECD). Deletion analysis of the ECD demonstrated that a minimal extracellular part, which includes a few proline residues, is required for RHS10-mediated root hair inhibition. RHS10 suppressed the accumulation of reactive oxygen species (ROS) in the root, which are necessary for root hair growth. A yeast two-hybrid screening identified an RNase (RNS2) as a putative downstream target of RHS10. Accordingly, RHS10 overexpression decreased and RHS10 loss increased RNA levels in the hair-growing root region. Our results suggest that RHS10 mediates cell wall-associated signals to maintain proper root hair length, at least in part by regulating RNA catabolism and ROS accumulation. PMID:26884603

  14. A Rice Kinase-Protein Interaction Map1[W][OA

    PubMed Central

    Ding, Xiaodong; Richter, Todd; Chen, Mei; Fujii, Hiroaki; Seo, Young Su; Xie, Mingtang; Zheng, Xianwu; Kanrar, Siddhartha; Stevenson, Rebecca A.; Dardick, Christopher; Li, Ying; Jiang, Hao; Zhang, Yan; Yu, Fahong; Bartley, Laura E.; Chern, Mawsheng; Bart, Rebecca; Chen, Xiuhua; Zhu, Lihuang; Farmerie, William G.; Gribskov, Michael; Zhu, Jian-Kang; Fromm, Michael E.; Ronald, Pamela C.; Song, Wen-Yuan

    2009-01-01

    Plants uniquely contain large numbers of protein kinases, and for the vast majority of the 1,429 kinases predicted in the rice (Oryza sativa) genome, little is known of their functions. Genetic approaches often fail to produce observable phenotypes; thus, new strategies are needed to delineate kinase function. We previously developed a cost-effective high-throughput yeast two-hybrid system. Using this system, we have generated a protein interaction map of 116 representative rice kinases and 254 of their interacting proteins. Overall, the resulting interaction map supports a large number of known or predicted kinase-protein interactions from both plants and animals and reveals many new functional insights. Notably, we found a potential widespread role for E3 ubiquitin ligases in pathogen defense signaling mediated by receptor-like kinases, particularly by the kinases that may have evolved from recently expanded kinase subfamilies in rice. We anticipate that the data provided here will serve as a foundation for targeted functional studies in rice and other plants. The application of yeast two-hybrid and TAPtag analyses for large-scale plant protein interaction studies is also discussed. PMID:19109415

  15. PnLRR-RLK27, a novel leucine-rich repeats receptor-like protein kinase from the Antarctic moss Pohlia nutans, positively regulates salinity and oxidation-stress tolerance.

    PubMed

    Wang, Jing; Liu, Shenghao; Li, Chengcheng; Wang, Tailin; Zhang, Pengying; Chen, Kaoshan

    2017-01-01

    Leucine-rich repeats receptor-like kinases (LRR-RLKs) play important roles in plant growth and development as well as stress responses. Here, 56 LRR-RLK genes were identified in the Antarctic moss Pohlia nutans transcriptome, which were further classified into 11 subgroups based on their extracellular domain. Of them, PnLRR-RLK27 belongs to the LRR II subgroup and its expression was significantly induced by abiotic stresses. Subcellular localization analysis showed that PnLRR-RLK27 was a plasma membrane protein. The overexpression of PnLRR-RLK27 in Physcomitrella significantly enhanced the salinity and ABA tolerance in their gametophyte growth. Similarly, PnLRR-RLK27 heterologous expression in Arabidopsis increased the salinity and ABA tolerance in their seed germination and early root growth as well as the tolerance to oxidative stress. PnLRR-RLK27 overproduction in these transgenic plants increased the expression of salt stress/ABA-related genes. Furthermore, PnLRR-RLK27 increased the activities of reactive oxygen species (ROS) scavengers and reduced the levels of malondialdehyde (MDA) and ROS. Taken together, these results suggested that PnLRR-RLK27 as a signaling regulator confer abiotic stress response associated with the regulation of the stress- and ABA-mediated signaling network.

  16. Prosystemin overexpression induces transcriptional modifications of defense-related and receptor-like kinase genes and reduces the susceptibility to Cucumber mosaic virus and its satellite RNAs in transgenic tomato plants

    PubMed Central

    Bubici, Giovanni; Carluccio, Anna Vittoria; Stavolone, Livia

    2017-01-01

    Systemin is a plant signal peptide hormone involved in the responses to wounding and insect damage in the Solanaceae family. It works in the same signaling pathway of jasmonic acid (JA) and enhances the expression of proteinase inhibitors. With the aim of studying a role for systemin in plant antiviral responses, a tomato (Solanum lycopersicum) transgenic line overexpressing the prosystemin cDNA, i.e. the systemin precursor, was inoculated with Cucumber mosaic virus (CMV) strain Fny supporting either a necrogenic or a non-necrogenic satellite RNA (satRNA) variant. Transgenic plants showed reduced susceptibility to both CMV/satRNA combinations. While symptoms of the non-necrogenic inoculum were completely suppressed, a delayed onset of lethal disease occurred in about half of plants challenged with the necrogenic inoculum. RT-qPCR analysis showed a correlation between the systemin-mediated reduced susceptibility and the JA biosynthetic and signaling pathways (e.g. transcriptional alteration of lipoxygenase D and proteinase inhibitor II). Moreover, transgenically overexpressed systemin modulated the expression of a selected set of receptor-like protein kinase (RLK) genes, including some playing a known role in plant innate immunity. A significant correlation was found between the expression profiles of some RLKs and the systemin-mediated reduced susceptibility to CMV/satRNA. These results show that systemin can increase plant defenses against CMV/satRNA through transcriptional reprogramming of diverse signaling pathways. PMID:28182745

  17. PnLRR-RLK27, a novel leucine-rich repeats receptor-like protein kinase from the Antarctic moss Pohlia nutans, positively regulates salinity and oxidation-stress tolerance

    PubMed Central

    Wang, Jing; Liu, Shenghao; Li, Chengcheng; Wang, Tailin; Chen, Kaoshan

    2017-01-01

    Leucine-rich repeats receptor-like kinases (LRR-RLKs) play important roles in plant growth and development as well as stress responses. Here, 56 LRR-RLK genes were identified in the Antarctic moss Pohlia nutans transcriptome, which were further classified into 11 subgroups based on their extracellular domain. Of them, PnLRR-RLK27 belongs to the LRR II subgroup and its expression was significantly induced by abiotic stresses. Subcellular localization analysis showed that PnLRR-RLK27 was a plasma membrane protein. The overexpression of PnLRR-RLK27 in Physcomitrella significantly enhanced the salinity and ABA tolerance in their gametophyte growth. Similarly, PnLRR-RLK27 heterologous expression in Arabidopsis increased the salinity and ABA tolerance in their seed germination and early root growth as well as the tolerance to oxidative stress. PnLRR-RLK27 overproduction in these transgenic plants increased the expression of salt stress/ABA-related genes. Furthermore, PnLRR-RLK27 increased the activities of reactive oxygen species (ROS) scavengers and reduced the levels of malondialdehyde (MDA) and ROS. Taken together, these results suggested that PnLRR-RLK27 as a signaling regulator confer abiotic stress response associated with the regulation of the stress- and ABA-mediated signaling network. PMID:28241081

  18. Glycosylation of a Fasciclin-Like Arabinogalactan-Protein (SOS5) Mediates Root Growth and Seed Mucilage Adherence via a Cell Wall Receptor-Like Kinase (FEI1/FEI2) Pathway in Arabidopsis

    PubMed Central

    Basu, Debarati; Tian, Lu; Debrosse, Tayler; Poirier, Emily; Emch, Kirk; Herock, Hayley; Travers, Andrew; Showalter, Allan M.

    2016-01-01

    Fundamental processes that underpin plant growth and development depend crucially on the action and assembly of the cell wall, a dynamic structure that changes in response to both developmental and environmental cues. While much is known about cell wall structure and biosynthesis, much less is known about the functions of the individual wall components, particularly with respect to their potential roles in cellular signaling. Loss-of-function mutants of two arabinogalactan-protein (AGP)-specific galactosyltransferases namely, GALT2 and GALT5, confer pleiotropic growth and development phenotypes indicating the important contributions of carbohydrate moieties towards AGP function. Notably, galt2galt5 double mutants displayed impaired root growth and root tip swelling in response to salt, likely as a result of decreased cellulose synthesis. These mutants phenocopy a salt-overly sensitive mutant called sos5, which lacks a fasciclin-like AGP (SOS5/FLA4) as well as a fei1fei2 double mutant, which lacks two cell wall-associated leucine-rich repeat receptor-like kinases. Additionally, galt2gal5 as well as sos5 and fei2 showed reduced seed mucilage adherence. Quintuple galt2galt5sos5fei1fei2 mutants were produced and provided evidence that these genes act in a single, linear genetic pathway. Further genetic and biochemical analysis of the quintuple mutant demonstrated involvement of these genes with the interplay between cellulose biosynthesis and two plant growth regulators, ethylene and ABA, in modulating root cell wall integrity. PMID:26731606

  19. Crystallization and preliminary X-ray analysis of ginkbilobin-2 from Ginkgo biloba seeds: a novel antifungal protein with homology to the extracellular domain of plant cysteine-rich receptor-like kinases

    SciTech Connect

    Miyakawa, Takuya; Sawano, Yoriko; Miyazono, Ken-ichi; Hatano, Ken-ichi; Tanokura, Masaru

    2007-09-01

    Purification and crystallization of ginkbilobin-2 and its selenomethionine derivative allowed the collection of complete data to 2.38 Å resolution and multiwavelength anomalous diffraction data sets, respectively. The antifungal protein ginkbilobin-2 (Gnk2) from Ginkgo biloba seeds does not show homology to other pathogenesis-related proteins, but does show homology to the extracellular domain of plant cysteine-rich receptor-like kinases. Native Gnk2 purified from ginkgo nuts and the selenomethionine derivative of recombinant Gnk2 (SeMet-rGnk2) were crystallized by the sitting-drop vapour-diffusion method using different precipitants. X-ray diffraction data were collected from Gnk2 at 2.38 Å resolution and from SeMet-rGnk2 at 2.79 Å resolution using a synchrotron-radiation source. The crystals of both proteins belonged to the primitive cubic space group P2{sub 1}3, with unit-cell parameters a = b = c = 143.2 Å.

  20. Synergistic interaction of CLAVATA1, CLAVATA2, and RECEPTOR-LIKE PROTEIN KINASE 2 in cyst nematode parasitism of Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant-parasitic cyst nematodes secrete CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (ESR) (CLE)-like effector proteins. These proteins act as ligand mimics of plant CLE peptides and are required for successful nematode infection. Previously, we showed that CLV2 and CORYNE (CRN), a heterodimer recept...

  1. Downregulation of a barley (Hordeum vulgare) leucine-rich repeat, non-arginine-aspartate receptor-like protein kinase reduces expression of numerous genes involved in plant pathogen defense.

    PubMed

    Parrott, David L; Huang, Li; Fischer, Andreas M

    2016-03-01

    Pattern recognition receptors represent a first line of plant defense against pathogens. Comparing the flag leaf transcriptomes of barley (Hordeum vulgare L.) near-isogenic lines varying in the allelic state of a locus controlling senescence, we have previously identified a leucine-rich repeat receptor-like protein kinase gene (LRR-RLK; GenBank accession: AK249842), which was strongly upregulated in leaves of early-as compared to late-senescing germplasm. Bioinformatic analysis indicated that this gene codes for a subfamily XII, non-arginine-aspartate (non-RD) LRR-RLK. Virus-induced gene silencing resulted in a two-fold reduction of transcript levels as compared to controls. Transcriptomic comparison of leaves from untreated plants, from plants treated with virus only without any plant sequences (referred to as 'empty virus' control), and from plants in which AK249842 expression was knocked down identified numerous genes involved in pathogen defense. These genes were strongly induced in 'empty virus' as compared to untreated controls, but their expression was significantly reduced (again compared to 'empty virus' controls) when AK249842 was knocked down, indicating that their expression partially depends on the LRR-RLK investigated here. Expression analysis, using datasets from BarleyBase/PLEXdb, demonstrated that AK249842 transcript levels are heavily influenced by the allelic state of the well-characterized mildew resistance a (Mla) locus, and that the gene is induced after powdery mildew and stem rust infection. Together, our data suggest that AK249842 is a barley pattern recognition receptor with a tentative role in defense against fungal pathogens, setting the stage for its full functional characterization.

  2. Activin Receptor-Like Kinase Receptors ALK5 and ALK1 Are Both Required for TGFβ-Induced Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    de Kroon, Laurie M. G.; Narcisi, Roberto; Blaney Davidson, Esmeralda N.; Cleary, Mairéad A.; van Beuningen, Henk M.; Koevoet, Wendy J. L. M.; van Osch, Gerjo J. V. M.; van der Kraan, Peter M.

    2015-01-01

    Introduction Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor β (TGFβ) is crucial for inducing chondrogenic differentiation of BMSCs and is known to signal via Activin receptor-Like Kinase (ALK) receptors ALK5 and ALK1. Since the specific role of these two TGFβ receptors in chondrogenesis is unknown, we investigated whether ALK5 and ALK1 are expressed in BMSCs and whether both receptors are required for chondrogenic differentiation of BMSCs. Materials & Methods ALK5 and ALK1 gene expression in human BMSCs was determined with RT-qPCR. To induce chondrogenesis, human BMSCs were pellet-cultured in serum-free chondrogenic medium containing TGFβ1. Chondrogenesis was evaluated by aggrecan and collagen type IIα1 RT-qPCR analysis, and histological stainings of proteoglycans and collagen type II. To overexpress constitutively active (ca) receptors, BMSCs were transduced either with caALK5 or caALK1. Expression of ALK5 and ALK1 was downregulated by transducing BMSCs with shRNA against ALK5 or ALK1. Results ALK5 and ALK1 were expressed in in vitro-expanded as well as in pellet-cultured BMSCs from five donors, but mRNA levels of both TGFβ receptors did not clearly associate with chondrogenic induction. TGFβ increased ALK5 and decreased ALK1 gene expression in chondrogenically differentiating BMSC pellets. Neither caALK5 nor caALK1 overexpression induced cartilage matrix formation as efficient as that induced by TGFβ. Moreover, short hairpin-mediated downregulation of either ALK5 or ALK1 resulted in a strong inhibition of TGFβ-induced chondrogenesis. Conclusion ALK5 as well as ALK1 are required for TGFβ-induced chondrogenic differentiation of BMSCs, and TGFβ not only directly induces chondrogenesis, but also modulates ALK5 and ALK1 receptor signaling in BMSCs. These results imply that optimizing cartilage formation by

  3. The protein interaction landscape of the human CMGC kinase group.

    PubMed

    Varjosalo, Markku; Keskitalo, Salla; Van Drogen, Audrey; Nurkkala, Helka; Vichalkovski, Anton; Aebersold, Ruedi; Gstaiger, Matthias

    2013-04-25

    Cellular information processing via reversible protein phosphorylation requires tight control of the localization, activity, and substrate specificity of protein kinases, which to a large extent is accomplished by complex formation with other proteins. Despite their critical role in cellular regulation and pathogenesis, protein interaction information is available for only a subset of the 518 human protein kinases. Here we present a global proteomic analysis of complexes of the human CMGC kinase group. In addition to subgroup-specific functional enrichment and modularity, the identified 652 high-confidence kinase-protein interactions provide a specific biochemical context for many poorly studied CMGC kinases. Furthermore, the analysis revealed a kinase-kinase subnetwork and candidate substrates for CMGC kinases. Finally, the presented interaction proteome uncovered a large set of interactions with proteins genetically linked to a range of human diseases, including cancer, suggesting additional routes for analyzing the role of CMGC kinases in controlling human disease pathways.

  4. The tomato leucine-rich repeat receptor-like kinases SlSERK3A and SlSERK3B have overlapping functions in bacterial and nematode innate immunity.

    PubMed

    Peng, Hsuan-Chieh; Kaloshian, Isgouhi

    2014-01-01

    The Somatic Embryogenesis Receptor Kinase 3 (SERK3)/Brassinosteroid (BR) Insensitive 1-Associated Kinase 1 (BAK1) is required for pattern-triggered immunity (PTI) in Arabidopsis thaliana and Nicotiana benthamiana. Tomato (Solanum lycopersicum) has three SlSERK members. Two of them exhibit particularly high levels of sequence similarity to AtSERK3 and, therefore, were named SlSERK3A and SlSERK3B. To characterize a role for SlSERK3A and SlSERK3B in defense, we suppressed each gene individually or co-silenced both using virus-induced gene silencing (VIGS) in the tomato cv. Moneymaker. Co-silencing SlSERK3A and SlSERK3B resulted in spontaneous necrotic lesions and reduced sensitivity to exogenous BR treatment. Silencing either SlSERK3A or SlSERK3B resulted in enhanced susceptibility to root knot-nematode and to non-pathogenic Pseudomonas syringae pv. tomato (Pst) DC3000 hrcC indicating that both SlSERK3s are positive regulators of defense. Interestingly, silencing SlSERK3B, but not SlSERK3A, resulted in enhanced susceptibility to the pathogenic strain Pst DC3000 indicating distinct roles for these two SlSERK3 paralogs. SlSERK3A and SlSERK3B are active kinases, localized to the plasma membrane, and interact in vivo with the Flagellin Sensing 2 receptor in a flg22-dependent manner. Complementation of the Atserk3/bak1-4 mutant with either SlSERK3A or SlSERK3B partially rescued the mutant phenotype. Thus, SlSERK3A and SlSERK3B are likely to constitute tomato orthologs of BAK1.

  5. Nuclear Receptor-Like Structure and Interaction of Congenital Heart Disease-Associated Factors GATA4 and NKX2-5

    PubMed Central

    Tölli, Marja; Wohlfahrt, Gerd; Darwich, Rami; Komati, Hiba; Nemer, Mona; Ruskoaho, Heikki

    2015-01-01

    Aims Transcription factor GATA4 is a dosage sensitive regulator of heart development and alterations in its level or activity lead to congenital heart disease (CHD). GATA4 has also been implicated in cardiac regeneration and repair. GATA4 action involves combinatorial interaction with other cofactors such as NKX2-5, another critical cardiac regulator whose mutations also cause CHD. Despite its critical importance to the heart and its evolutionary conservation across species, the structural basis of the GATA4-NKX2-5 interaction remains incompletely understood. Methods and Results A homology model was constructed and used to identify surface amino acids important for the interaction of GATA4 and NKX2-5. These residues were subjected to site-directed mutagenesis, and the mutant proteins were characterized for their ability to bind DNA and to physically and functionally interact with NKX2-5. The studies identify 5 highly conserved amino acids in the second zinc finger (N272, R283, Q274, K299) and its C-terminal extension (R319) that are critical for physical and functional interaction with the third alpha helix of NKX2-5 homeodomain. Integration of the experimental data with computational modeling suggests that the structural arrangement of the zinc finger-homeodomain resembles the architecture of the conserved DNA binding domain of nuclear receptors. Conclusions The results provide novel insight into the structural basis for protein-protein interactions between two important classes of transcription factors. The model proposed will help to elucidate the molecular basis for disease causing mutations in GATA4 and NKX2-5 and may be relevant to other members of the GATA and NK classes of transcription factors. PMID:26642209

  6. Interaction of SNF1 Protein Kinase with Its Activating Kinase Sak1▿

    PubMed Central

    Liu, Yang; Xu, Xinjing; Carlson, Marian

    2011-01-01

    The Saccharomyces cerevisiae SNF1 protein kinase, a member of the SNF1/AMP-activated protein kinase (AMPK) family, is activated by three kinases, Sak1, Tos3, and Elm1, which phosphorylate the Snf1 catalytic subunit on Thr-210 in response to glucose limitation and other stresses. Sak1 is the primary Snf1-activating kinase and is associated with Snf1 in a complex. Here we examine the interaction of Sak1 with SNF1. We report that Sak1 coimmunopurifies with the Snf1 catalytic subunit from extracts of both glucose-replete and glucose-limited cultures and that interaction occurs independently of the phosphorylation state of Snf1 Thr-210, Snf1 catalytic activity, and other SNF1 subunits. Sak1 interacts with the Snf1 kinase domain, and nonconserved sequences C terminal to the Sak1 kinase domain mediate interaction with Snf1 and augment the phosphorylation and activation of Snf1. The Sak1 C terminus is modified in response to glucose depletion, dependent on SNF1 activity. Replacement of the C terminus of Elm1 (or Tos3) with that of Sak1 enhanced the ability of the Elm1 kinase domain to interact with and phosphorylate Snf1. These findings indicate that the C terminus of Sak1 confers its function as the primary Snf1-activating kinase and suggest that the physical association of Sak1 with SNF1 facilitates responses to environmental change. PMID:21216941

  7. Kinase-interacting substrate screening is a novel method to identify kinase substrates

    PubMed Central

    Amano, Mutsuki; Hamaguchi, Tomonari; Shohag, Md. Hasanuzzaman; Kozawa, Kei; Kato, Katsuhiro; Zhang, Xinjian; Yura, Yoshimitsu; Matsuura, Yoshiharu; Kataoka, Chikako; Nishioka, Tomoki

    2015-01-01

    Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 proteins were identified as candidate substrates for Rho-associated kinase (Rho-kinase/ROCK2), including known substrates. The KISS method was also applied to additional kinases, including PKA, MAPK1, CDK5, CaMK1, PAK7, PKN, LYN, and FYN, and a lot of candidate substrates and their phosphorylation sites were determined, most of which have not been reported previously. Among the candidate substrates for Rho-kinase, several functional clusters were identified, including the polarity-associated proteins, such as Scrib. We found that Scrib plays a crucial role in the regulation of subcellular contractility by assembling into a ternary complex with Rho-kinase and Shroom2 in a phosphorylation-dependent manner. We propose that the KISS method is a comprehensive and useful substrate screen for various kinases. PMID:26101221

  8. Glycogen Synthase KinaseInteraction Protein Functions as an A-kinase Anchoring Protein*

    PubMed Central

    Hundsrucker, Christian; Skroblin, Philipp; Christian, Frank; Zenn, Hans-Michael; Popara, Viola; Joshi, Mangesh; Eichhorst, Jenny; Wiesner, Burkhard; Herberg, Friedrich W.; Reif, Bernd; Rosenthal, Walter; Klussmann, Enno

    2010-01-01

    A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinaseinteraction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3β (glycogen synthase kinase 3β). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3β by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3β and thereby provides a mechanism for the integration of PKA and GSK3β signaling pathways. PMID:20007971

  9. Kinase Pathway Database: An Integrated Protein-Kinase and NLP-Based Protein-Interaction Resource

    PubMed Central

    Koike, Asako; Kobayashi, Yoshiyuki; Takagi, Toshihisa

    2003-01-01

    Protein kinases play a crucial role in the regulation of cellular functions. Various kinds of information about these molecules are important for understanding signaling pathways and organism characteristics. We have developed the Kinase Pathway Database, an integrated database involving major completely sequenced eukaryotes. It contains the classification of protein kinases and their functional conservation, ortholog tables among species, protein–protein, protein–gene, and protein–compound interaction data, domain information, and structural information. It also provides an automatic pathway graphic image interface. The protein, gene, and compound interactions are automatically extracted from abstracts for all genes and proteins by natural-language processing (NLP).The method of automatic extraction uses phrase patterns and the GENA protein, gene, and compound name dictionary, which was developed by our group. With this database, pathways are easily compared among species using data with more than 47,000 protein interactions and protein kinase ortholog tables. The database is available for querying and browsing at http://kinasedb.ontology.ims.u-tokyo.ac.jp/. PMID:12799355

  10. The Two Faces of Receptor Interacting Protein Kinase-1

    PubMed Central

    Weinlich, Ricardo; Green, Douglas R.

    2014-01-01

    Receptor Interacting Protein Kinase-1 (RIPK1), a key player in inflammation and cell death, assumes opposite functions depending on the cellular context and its posttranslational modifications. Genetic evidence supported by biochemical and cellular biology approaches shed light on the circumstances in which RIPK1 promotes or inhibits these processes. PMID:25459879

  11. KLIFS: a structural kinase-ligand interaction database

    PubMed Central

    Kooistra, Albert J.; Kanev, Georgi K.; van Linden, Oscar P.J.; Leurs, Rob; de Esch, Iwan J.P.; de Graaf, Chris

    2016-01-01

    Protein kinases play a crucial role in cell signaling and are important drug targets in several therapeutic areas. The KLIFS database contains detailed structural kinase-ligand interaction information derived from all (>2900) structures of catalytic domains of human and mouse protein kinases deposited in the Protein Data Bank in order to provide insights into the structural determinants of kinase-ligand binding and selectivity. The kinase structures have been processed in a consistent manner by systematically analyzing the structural features and molecular interaction fingerprints (IFPs) of a predefined set of 85 binding site residues with bound ligands. KLIFS has been completely rebuilt and extended (>65% more structures) since its first release as a data set, including: novel automated annotation methods for (i) the assessment of ligand-targeted subpockets and the analysis of (ii) DFG and (iii) αC-helix conformations; improved and automated protocols for (iv) the generation of sequence/structure alignments, (v) the curation of ligand atom and bond typing for accurate IFP analysis and (vi) weekly database updates. KLIFS is now accessible via a website (http://klifs.vu-compmedchem.nl) that provides a comprehensive visual presentation of different types of chemical, biological and structural chemogenomics data, and allows the user to easily access, compare, search and download the data. PMID:26496949

  12. Overexpression of the tomato pollen receptor kinase LePRK1 rewires pollen tube growth to a blebbling mode

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The tubular growth of a pollen tube cell is crucial for the sexual reproduction of flowering plants. LePRK1 is a pollen-specific and plasma membrane–localized receptor-like kinase from tomato (Solanum lycopersicum). LePRK1 interacts with another receptor, LePRK2, and with KINASE PARTNER PROTEIN (KPP...

  13. Phosphorylation of the Kinase Interaction Motif in Mitogen-activated Protein (MAP) Kinase Phosphatase-4 Mediates Cross-talk between Protein Kinase A and MAP Kinase Signaling Pathways*

    PubMed Central

    Dickinson, Robin J.; Delavaine, Laurent; Cejudo-Marín, Rocío; Stewart, Graeme; Staples, Christopher J.; Didmon, Mark P.; Trinidad, Antonio Garcia; Alonso, Andrés; Pulido, Rafael; Keyse, Stephen M.

    2011-01-01

    MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site 55RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo. PMID:21908610

  14. Plant Aurora kinases interact with and phosphorylate transcription factors.

    PubMed

    Takagi, Mai; Sakamoto, Takuya; Suzuki, Ritsuko; Nemoto, Keiichirou; Obayashi, Takeshi; Hirakawa, Takeshi; Matsunaga, Tomoko M; Kurihara, Daisuke; Nariai, Yuko; Urano, Takeshi; Sawasaki, Tatsuya; Matsunaga, Sachihiro

    2016-11-01

    Aurora kinase (AUR) is a well-known mitotic serine/threonine kinase that regulates centromere formation, chromosome segregation, and cytokinesis in eukaryotes. In addition to regulating mitotic events, AUR has been shown to regulate protein dynamics during interphase in animal cells. In contrast, there has been no identification and characterization of substrates and/or interacting proteins during interphase in plants. The Arabidopsis thaliana genome encodes three AUR paralogues, AtAUR1, AtAUR2, and AtAUR3. Among them, AtAUR1 and AtAUR2 are considered to function redundantly. Here, we confirmed that both AtAUR1 and AtAUR3 are localized in the nucleus and cytoplasm during interphase, suggesting that they have functions during interphase. To identify novel interacting proteins, we used AlphaScreen to target 580 transcription factors (TFs) that are mainly functional during interphase, using recombinant A. thaliana TFs and AtAUR1 or AtAUR3. We found 133 and 32 TFs had high potential for interaction with AtAUR1 and AtAUR3, respectively. The highly AtAUR-interacting TFs were involved in various biological processes, suggesting the functions of the AtAURs during interphase. We found that AtAUR1 and AtAUR3 showed similar interaction affinity to almost all TFs. However, in some cases, the interaction affinity differed substantially between the two AtAUR homologues. These results suggest that AtAUR1 and AtAUR3 have both redundant and distinct functions through interactions with TFs. In addition, database analysis revealed that most of the highly AtAUR-interacting TFs contained a detectable phosphopeptide that was consistent with the consensus motifs for human AURs, suggesting that these TFs are substrates of the AtAURs. The AtAURs phosphorylated several highly interacting TFs in the AlphaScreen in vitro. Overall, in line with the regulation of TFs through interaction, our results indicate the possibility of phosphoregulation of several TFs by the AtAURs (280/300).

  15. Targeting chk2 kinase: molecular interaction maps and therapeutic rationale.

    PubMed

    Pommier, Yves; Sordet, Olivier; Rao, V Ashutosh; Zhang, Hongliang; Kohn, Kurt W

    2005-01-01

    Most anticancer drugs presently used clinically target genomic DNA. The selectivity of these anticancer drugs for tumor tissues is probably due to tumor-specific defects suppressing cell cycle checkpoints and DNA repair, and enhancing apoptotic response in the tumor. We will review the molecular interactions within the ATM-Chk2 pathway implicating the DNA damage sensor kinases (ATM, ATR and DNA-PK), the adaptor BRCT proteins (Nbs1, Brca1, 53BP1, MDC1) and the effector kinases (Chk2, Chk1, Plk3, JNK, p38). The molecular interaction map convention (MIM) will be used for presenting this molecular network (http://discover.nci.nih.gov/mim/). A characteristic of the ATM-Chk2 pathway is its redundancy. First, ATM and Chk2 phosphorylate common substrates including p53, E2F1, BRCA1, and Chk2 itself, which suggests that Chk2 (also known as CHECK2, Cds1 in fission yeast, and Dmchk2 or Dmnk or Loki in the fruit fly) acts as a relay for ATM and/or as a salvage pathway when ATM is inactivated. Secondly, redundancy is apparent for the substrates, which can be phosphorylated/activated at similar residues by Chk2, Chk1, and the polo kinases (Plk's). Functionally, Chk2 can activate both apoptosis (via p53, E2F1 and PML) and cell cycle checkpoint (via Cdc25A and Cdc25C, p53, and BRCA1). We will review the short list of published Chk2 inhibitors. We will also propose a novel paradigm for screening interfacial inhibitors of Chk2. Chk2 inhibitors might be used to enhance the tumor selectivity of DNA targeted agents in p53-deficient tumors, and for the treatment of tumors whose growth depends on enhanced Chk2 activity.

  16. Lectin Receptor Kinases Participate in Protein-Protein Interactions to Mediate Plasma Membrane-Cell Wall Adhesions in Arabidopsis1

    PubMed Central

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces. PMID:16361528

  17. Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis.

    PubMed

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces.

  18. Protein interaction network of the mammalian Hippo pathway reveals mechanisms of kinase-phosphatase interactions.

    PubMed

    Couzens, Amber L; Knight, James D R; Kean, Michelle J; Teo, Guoci; Weiss, Alexander; Dunham, Wade H; Lin, Zhen-Yuan; Bagshaw, Richard D; Sicheri, Frank; Pawson, Tony; Wrana, Jeffrey L; Choi, Hyungwon; Gingras, Anne-Claude

    2013-11-19

    The Hippo pathway regulates organ size and tissue homeostasis in response to multiple stimuli, including cell density and mechanotransduction. Pharmacological inhibition of phosphatases can also stimulate Hippo signaling in cell culture. We defined the Hippo protein-protein interaction network with and without inhibition of serine and threonine phosphatases by okadaic acid. We identified 749 protein interactions, including 599 previously unrecognized interactions, and demonstrated that several interactions with serine and threonine phosphatases were phosphorylation-dependent. Mutation of the T-loop of MST2 (mammalian STE20-like protein kinase 2), which prevented autophosphorylation, disrupted its association with STRIPAK (striatin-interacting phosphatase and kinase complex). Deletion of the amino-terminal forkhead-associated domain of SLMAP (sarcolemmal membrane-associated protein), a component of the STRIPAK complex, prevented its association with MST1 and MST2. Phosphatase inhibition produced temporally distinct changes in proteins that interacted with MOB1A and MOB1B (Mps one binder kinase activator-like 1A and 1B) and promoted interactions with upstream Hippo pathway proteins, such as MST1 and MST2, and with the trimeric protein phosphatase 6 complex (PP6). Mutation of three basic amino acids that are part of a phospho-serine- and phospho-threonine-binding domain in human MOB1B prevented its interaction with MST1 and PP6 in cells treated with okadaic acid. Collectively, our results indicated that changes in phosphorylation orchestrate interactions between kinases and phosphatases in Hippo signaling, providing a putative mechanism for pathway regulation.

  19. Zipper-interacting protein kinase interacts with human cell division cycle 14A phosphatase.

    PubMed

    Wu, Wei; Hu, Haiying; Ye, Zi; Leong, Mancheong; He, Min; Li, Qin; Hu, Renming; Zhang, Shuo

    2015-04-01

    Zipper‑interacting protein kinase (ZIPK) is a novel serine/threonine protein kinase and a member of a large family of protein kinases, known as the death‑associated protein kinases. However, the function of ZIPK has yet to be fully elucidated, as few physiological substrates have currently been identified. In the present study, a yeast two‑hybrid screen was used and the human cell division cycle 14A (HsCdc14A) phosphatase was identified as a novel ZIPK binding protein. To the best of our knowledge, this is the first study to report the interaction between these proteins. The interaction between ZIPK and HsCdc14A was confirmed by in vitro experiments. In addition, ZIPK‑mediated phosphorylation was shown to activate the phosphatase activity of HsCdc14A. These findings indicated that ZIPK may also be involved in the regulation of the cell cycle in human cells, by interacting with HsCdc14A.

  20. Cytoskeletal Modulation of Lipid Interactions Regulates Lck Kinase Activity*

    PubMed Central

    Chichili, Gurunadh R.; Cail, Robert C.; Rodgers, William

    2012-01-01

    The actin cytoskeleton promotes clustering of proteins associated with cholesterol-dependent rafts, but its effect on lipid interactions that form and maintain rafts is not understood. We addressed this question by determining the effect of disrupting the cytoskeleton on co-clustering of dihexadecyl-(C16)-anchored DiO and DiI, which co-enrich in ordered lipid environments such as rafts. Co-clustering was assayed by fluorescence resonance energy transfer (FRET) in labeled T cells, where rafts function in the phosphoregulation of the Src family kinase Lck. Our results show that probe co-clustering was sensitive to depolymerization of actin filaments with latrunculin B (Lat B), inhibition of myosin II with blebbistatin, and treatment with neomycin to sequester phosphatidylinositol 4,5-bisphosphate. Cytoskeletal effects on lipid interactions were not restricted to order-preferring label because co-clustering of C16-anchored DiO with didodecyl (C12)-anchored DiI, which favors disordered lipids, was also reduced by Lat B and blebbistatin. Furthermore, conditions that disrupted probe co-clustering resulted in activation of Lck. These data show that the cytoskeleton globally modulates lipid interactions in the plasma membrane, and this property maintains rafts that function in Lck regulation. PMID:22613726

  1. AOP-1 interacts with cardiac-specific protein kinase TNNI3K and down-regulates its kinase activity.

    PubMed

    Feng, Yan; Liu, Dong-Qing; Wang, Zhen; Liu, Zhao; Cao, Hui-Qing; Wang, Lai-Yuan; Shi, Na; Meng, Xian-Min

    2007-11-01

    In the present study, a yeast two-hybrid screening system was used to identify the interaction partners of cardiac troponin I-interacting kinase (TNNI3K) that might serve as regulators or targets, and thus in turn to gain some insights on the roles of TNNI3K. After screening the adult heart cDNA library with a bait construct encoding the ANK motif of TNNI3K, antioxidant protein 1 (AOP-1) was isolated. The interaction between TNNI3K and AOP-1 was confirmed by the in vitro binding assay and coexpression experiments in vivo. The colocalization of TNNI3K and AOP-1 was clarified by confocal immunofluorescence. Moreover, coexpression of AOP-1 inhibited TNNI3K kinase activity in the in vitro kinase assay.

  2. A double-mutant collection targeting MAP kinase related genes in Arabidopsis for studying genetic interactions.

    PubMed

    Su, Shih-Heng; Krysan, Patrick J

    2016-12-01

    Mitogen-activated protein kinase cascades are conserved in all eukaryotes. In Arabidopsis thaliana there are approximately 80 genes encoding MAP kinase kinase kinases (MAP3K), 10 genes encoding MAP kinase kinases (MAP2K), and 20 genes encoding MAP kinases (MAPK). Reverse genetic analysis has failed to reveal abnormal phenotypes for a majority of these genes. One strategy for uncovering gene function when single-mutant lines do not produce an informative phenotype is to perform a systematic genetic interaction screen whereby double-mutants are created from a large library of single-mutant lines. Here we describe a new collection of 275 double-mutant lines derived from a library of single-mutants targeting genes related to MAP kinase signaling. To facilitate this study, we developed a high-throughput double-mutant generating pipeline using a system for growing Arabidopsis seedlings in 96-well plates. A quantitative root growth assay was used to screen for evidence of genetic interactions in this double-mutant collection. Our screen revealed four genetic interactions, all of which caused synthetic enhancement of the root growth defects observed in a MAP kinase 4 (MPK4) single-mutant line. Seeds for this double-mutant collection are publicly available through the Arabidopsis Biological Resource Center. Scientists interested in diverse biological processes can now screen this double-mutant collection under a wide range of growth conditions in order to search for additional genetic interactions that may provide new insights into MAP kinase signaling.

  3. Interaction of phospholipase D1 with a casein-kinase-2-like serine kinase.

    PubMed Central

    Ganley, I G; Walker, S J; Manifava, M; Li, D; Brown, H A; Ktistakis, N T

    2001-01-01

    Phospholipase D (PLD)1 was phosphorylated in vivo and by an associated kinase in vitro following immunoprecipitation. Both phosphorylation events were greatly reduced in a catalytically inactive point mutant in which the serine residue at position 911 was converted into alanine (S911A). The kinase could be enriched from detergent-extracted brain membranes and bind and phosphorylate PLD1 that was immunoprecipitated from COS-7 cells. Using in-gel kinase assays we determined that the size of the kinase is approximately 40 kDa and that PLD1 is more effective than S911A in binding the kinase. Preliminary analysis of the phosphorylation sites on PLD1 suggested that the kinase belongs to the casein kinase 2 (CK2) family. Consistent with this, we found that the kinase could utilize GTP, and could be inhibited by heparin and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Membrane fractions from Chinese hamster ovary (CHO) cell lines that inducibly express PLD1 contained an endogenous kinase activity that phosphorylated PLD1 using GTP and was inhibited by DRB. Direct evidence that the kinase is CK2 came from observations that immunoprecipitates using PLD1 antibodies contained immunoreactive CK2alpha, and immunoprecipitates using CK2alpha antibodies contained immunoreactive PLD1. Co-expression of PLD1 in COS-7 cells with the two recombinant CK2 subunits, alpha or beta, suggests that the association of PLD1 with the kinase is through the beta subunit. Supporting this, phosphorylation of PLD1 by purified recombinant CK2alpha was enhanced by purified recombinant CK2beta. Assays measuring PLD1 catalytic activity following phosphorylation by CK2 suggest that this phosphorylation event does not influence PLD1-mediated hydrolysis of phosphatidylcholine in vitro. PMID:11171116

  4. Protein-protein interactions of tandem affinity purification-tagged protein kinases in rice.

    PubMed

    Rohila, Jai S; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald; Dardick, Chris; Canlas, Patrick; Xu, Xia; Gribskov, Michael; Kanrar, Siddhartha; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E

    2006-04-01

    Forty-one rice cDNAs encoding protein kinases were fused to the tandem affinity purification (TAP) tag and expressed in transgenic rice plants. The TAP-tagged kinases and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by mass spectrometry. Ninety-five percent of the TAP-tagged kinases were recovered. Fifty-six percent of the TAP-tagged kinases were found to interact with other rice proteins. A number of these interactions were consistent with known protein complexes found in other species, validating the TAP-tag method in rice plants. Phosphorylation sites were identified on four of the kinases that interacted with either 14-3-3 proteins or cyclins.

  5. Recognition of a PP2C interaction motif in several plant protein kinases.

    PubMed

    Chakraborty, Niranjan; Ohta, Masaru; Zhu, Jian-Kang

    2007-01-01

    Protein phosphatase 2Cs (PP2Cs) constitute a major class of phosphatases in plants. PP2Cs play important roles in many signaling pathways by countering the action of specific protein kinases. In addition to their role in several environmental stress-related signal transduction pathways, they are also involved in plant metabolism. Protein phosphatases often physically associate with their protein kinase counterparts. One approach to understanding PP2C function is to identify their interacting protein kinases. We describe a yeast two-hybrid assay system used in our lab to determine the interaction between members of the PP2C family and protein kinases in the SOS2 family. This chapter and the cited articles describing related work might be of help in discovering interactions between other protein phosphatases and kinases.

  6. Do Src Kinase and Caveolin Interact Directly with Na,K-ATPase?

    PubMed

    Yosef, Eliyahu; Katz, Adriana; Peleg, Yoav; Mehlman, Tevie; Karlish, Steven J D

    2016-05-27

    Much evidence points to a role of Na,K-ATPase in ouabain-dependent signal transduction. Based on experiments with different cell lines and native tissue membranes, a current hypothesis postulates direct interactions between the Na,K-ATPase and Src kinase (non-receptor tyrosine kinase). Na,K-ATPase is proposed to bind Src kinase and inhibit its activity, whereas ouabain, the specific Na,K-ATPase inhibitor, binds and stabilizes the E2 conformation, thus exposing the Src kinase domain and its active site Tyr-418 for activation. Ouabain-dependent signaling is thought to be mediated within caveolae by a complex consisting of Na,K-ATPase, caveolin, and Src kinase. In the current work, we have looked for direct interactions utilizing purified recombinant Na,K-ATPase (human α1β1FXYD1 or porcine α1D369Nβ1FXYD1) and purified human Src kinase and human caveolin 1 or interactions between these proteins in native membrane vesicles isolated from rabbit kidney. By several independent criteria and techniques, no stable interactions were detected between Na,K-ATPase and purified Src kinase. Na,K-ATPase was found to be a substrate for Src kinase phosphorylation at Tyr-144. Clear evidence for a direct interaction between purified human Na,K-ATPase and human caveolin was obtained, albeit with a low molar stoichiometry (1:15-30 caveolin 1/Na,K-ATPase). In native renal membranes, a specific caveolin 14-5 oligomer (95 kDa) was found to be in direct interaction with Na,K-ATPase. We inferred that a small fraction of the renal Na,K-ATPase molecules is in a ∼1:1 complex with a caveolin 14-5 oligomer. Thus, overall, whereas a direct caveolin 1/Na,K-ATPase interaction is confirmed, the lack of direct Src kinase/Na,K-ATPase binding requires reassessment of the mechanism of ouabain-dependent signaling.

  7. Functional Significance of Aurora Kinases-p53 Protein Family Interactions in Cancer.

    PubMed

    Sasai, Kaori; Treekitkarnmongkol, Warapen; Kai, Kazuharu; Katayama, Hiroshi; Sen, Subrata

    2016-01-01

    Aurora kinases play critical roles in regulating spindle assembly, chromosome segregation, and cytokinesis to ensure faithful segregation of chromosomes during mitotic cell division cycle. Molecular and cell biological studies have revealed that Aurora kinases, at physiological levels, orchestrate complex sequential cellular processes at distinct subcellular locations through functional interactions with its various substrates. Aberrant expression of Aurora kinases, on the other hand, cause defects in mitotic spindle assembly, checkpoint response activation, and chromosome segregation leading to chromosomal instability. Elevated expression of Aurora kinases correlating with chromosomal instability is frequently detected in human cancers. Recent genomic profiling of about 3000 human cancer tissue specimens to identify various oncogenic signatures in The Cancer Genome Atlas project has reported that recurrent amplification and overexpression of Aurora kinase-A characterize distinct subsets of human tumors across multiple cancer types. Besides the well-characterized canonical pathway interactions of Aurora kinases in regulating assembly of the mitotic apparatus and chromosome segregation, growing evidence also supports the notion that deregulated expression of Aurora kinases in non-canonical pathways drive transformation and genomic instability by antagonizing tumor suppressor and exacerbating oncogenic signaling through direct interactions with critical proteins. Aberrant expression of the Aurora kinases-p53 protein family signaling axes appears to be critical in the abrogation of p53 protein family mediated tumor suppressor pathways frequently deregulated during oncogenic transformation process. Recent findings reveal the existence of feedback regulatory loops in mRNA expression and protein stability of these protein families and their consequences on downstream effectors involved in diverse physiological functions, such as mitotic progression, checkpoint response

  8. Drug-drug interactions with tyrosine-kinase inhibitors: a clinical perspective.

    PubMed

    van Leeuwen, Roelof W F; van Gelder, Teun; Mathijssen, Ron H J; Jansman, Frank G A

    2014-07-01

    In the past decade, many tyrosine-kinase inhibitors have been introduced in oncology and haemato-oncology. Because this new class of drugs is extensively used, serious drug-drug interactions are an increasing risk. In this Review, we give a comprehensive overview of known or suspected drug-drug interactions between tyrosine-kinase inhibitors and other drugs. We discuss all haemato-oncological and oncological tyrosine-kinase inhibitors that had been approved by Aug 1, 2013, by the US Food and Drug Administration or the European Medicines Agency. Various clinically relevant drug interactions with tyrosine-kinase inhibitors have been identified. Most interactions concern altered bioavailability due to altered stomach pH, metabolism by cytochrome P450 isoenzymes, and prolongation of the QTc interval. To guarantee the safe use of tyrosine-kinase inhibitors, a drugs review for each patient is needed. This Review provides specific recommendations to guide haemato-oncologists, oncologists, and clinical pharmacists, through the process of managing drug-drug interactions during treatment with tyrosine-kinase inhibitors in daily clinical practice.

  9. BDNF stimulation of protein synthesis in cortical neurons requires the MAP kinase-interacting kinase MNK1.

    PubMed

    Genheden, Maja; Kenney, Justin W; Johnston, Harvey E; Manousopoulou, Antigoni; Garbis, Spiros D; Proud, Christopher G

    2015-01-21

    Although the MAP kinase-interacting kinases (MNKs) have been known for >15 years, their roles in the regulation of protein synthesis have remained obscure. Here, we explore the involvement of the MNKs in brain-derived neurotrophic factor (BDNF)-stimulated protein synthesis in cortical neurons from mice. Using a combination of pharmacological and genetic approaches, we show that BDNF-induced upregulation of protein synthesis requires MEK/ERK signaling and the downstream kinase, MNK1, which phosphorylates eukaryotic initiation factor (eIF) 4E. Translation initiation is mediated by the interaction of eIF4E with the m(7)GTP cap of mRNA and with eIF4G. The latter interaction is inhibited by the interactions of eIF4E with partner proteins, such as CYFIP1, which acts as a translational repressor. We find that BDNF induces the release of CYFIP1 from eIF4E, and that this depends on MNK1. Finally, using a novel combination of BONCAT and SILAC, we identify a subset of proteins whose synthesis is upregulated by BDNF signaling via MNK1 in neurons. Interestingly, this subset of MNK1-sensitive proteins is enriched for functions involved in neurotransmission and synaptic plasticity. Additionally, we find significant overlap between our subset of proteins whose synthesis is regulated by MNK1 and those encoded by known FMRP-binding mRNAs. Together, our data implicate MNK1 as a key component of BDNF-mediated translational regulation in neurons.

  10. Isoform-dependent interaction of BRDG1 with Tec kinase.

    PubMed

    Yokohari, K; Yamashita, Y; Okada, S; Ohya, K; Oda, S; Hatano, M; Mano, H; Hirasawa, H; Tokuhisa, T

    2001-11-30

    Tec is the prototype of an emerging family of protein-tyrosine kinases. Tec and Btk, another member of this family, together participate in the development of B-cell immune system. We previously identified one of the downstream messengers for human Tec kinase, BRDG1. BRDG1 is associated with Tec and becomes tyrosine-phosphorylated in B-cells by the engagement of B-cell antigen receptor (BCR). Here we show that overexpression of BRDG1 strongly augments BCR-mediated activation of cAMP-response element binding protein (CREB) but not that of c-Jun and the promoters of c-MYC and BCL-xL genes. Furthermore, we isolated the murine orthologue of BRDG1. Three isoforms of BRDG1 are generated by alternative splicing of the message. Two of them have a deletion of 33 amino acids in a Pleckstrin homology (PH) domain of BRDG1. Both the tyrosine-phosphorylation and CREB-activating ability of BRDG1 were isoform-dependent, suggesting a role of the PH domain of BRDG1. These data have identified a novel regulatory mechanism of CREB family of transcriptional factors.

  11. Tamoxifen Dependent Interaction Between the Estrogen Receptor and a Novel P21 Activated Kinase

    DTIC Science & Technology

    2002-06-01

    AD Award Number: DAMDl7-01-1-0149 TITLE: Tamoxifen Dependent Interaction Between the Estrogen Receptor and a Novel P21 Activated Kinase PRINCIPAL...Tamoxifen Dependent Interaction Between the DAMD17-00-1-0114 Estrogen Receptor and a Novel P21 Activated Kinase 6. AUTHOR(S) Steven P. Balk, M.D., Ph.D. 7...Z, Karas RH, nisms of androgen receptor activation and function. J Mendelsohn ME, Shaul PW 1999 Estrogen receptor a Steroid Biochem Mol Biol 69:307

  12. Direct interactions with the integrin β1 cytoplasmic tail activate the Abl2/Arg kinase.

    PubMed

    Simpson, Mark A; Bradley, William D; Harburger, David; Parsons, Maddy; Calderwood, David A; Koleske, Anthony J

    2015-03-27

    Integrins are heterodimeric α/β extracellular matrix adhesion receptors that couple physically to the actin cytoskeleton and regulate kinase signaling pathways to control cytoskeletal remodeling and adhesion complex formation and disassembly. β1 integrins signal through the Abl2/Arg (Abl-related gene) nonreceptor tyrosine kinase to control fibroblast cell motility, neuronal dendrite morphogenesis and stability, and cancer cell invasiveness, but the molecular mechanisms by which integrin β1 activates Arg are unknown. We report here that the Arg kinase domain interacts directly with a lysine-rich membrane-proximal segment in the integrin β1 cytoplasmic tail, that Arg phosphorylates the membrane-proximal Tyr-783 in the β1 tail, and that the Arg Src homology domain then engages this phosphorylated region in the tail. We show that these interactions mediate direct binding between integrin β1 and Arg in vitro and in cells and activate Arg kinase activity. These findings provide a model for understanding how β1-containing integrins interact with and activate Abl family kinases.

  13. Dataset of integrin-linked kinase protein: Protein interactions in cardiomyocytes identified by mass spectrometry.

    PubMed

    Traister, Alexandra; Lu, Mingliang; Coles, John G; Maynes, Jason T

    2016-06-01

    Using hearts from mice overexpressing integrin linked kinase (ILK) behind the cardiac specific promoter αMHC, we have performed immunoprecipitation and mass spectrometry to identify novel ILK protein:protein interactions that regulate cardiomyocyte activity and calcium flux. Integrin linked kinase complexes were captured from mouse heart lysates using a commercial antibody, with subsequent liquid chromatography tandem mass spectral analysis. Interacting partners were identified using the MASCOT server, and important interactions verified using reverse immunoprecipitation and mass spectrometry. All ILK interacting proteins were identified in a non-biased manner, and are stored in the ProteomeXchange Consortium via the PRIDE partner repository (reference ID PRIDE: PXD001053). The functional role of identified ILK interactions in cardiomyocyte function and arrhythmia were subsequently confirmed in human iPSC-cardiomyocytes.

  14. Interaction between protein kinase C and protein kinase A can modulate transmitter release at the rat neuromuscular synapse.

    PubMed

    Santafé, M M; Garcia, N; Lanuza, M A; Tomàs, M; Tomàs, J

    2009-02-15

    We used intracellular recording to investigate the functional interaction between protein kinase C (PKC) and protein kinase A (PKA) signal transduction cascades in the control of transmitter release in the neuromuscular synapses from adult rats. Our results indicate that: 1) PKA and PKC are independently involved in asynchronous release. 2) Evoked acetylcholine (ACh) release is enhanced with the PKA agonist Sp-8-BrcAMP and the PKC agonist phorbol ester (PMA). 3) PKA has a constitutive role in promoting a component of normal evoked transmitter release because, when the kinase is inhibited with H-89, the release diminishes. However, the PKC inhibitor calphostin C (CaC) does not affect ACh release. 4) PKA regulates neurotransmission without PKC involvement because, after PMA or CaC modulation of the PKC activity, coupling to the ACh release of PKA can normally be stimulated with Sp-8-BrcAMP or inhibited with H-89. 5) After PKA inhibition with H-89, PKC stimulation with PMA (or inhibition with CaC) does not lead to any change in evoked ACh release. However, in PKA-stimulated preparations with Sp-8-BrcAMP, PKC becomes tonically active, thus potentiating a component of release that can now be blocked with CaC. In normal conditions, therefore, PKA was able to modulate ACh release independently of PKC activity, whereas PKA stimulation caused the PKC coupling to evoked release. In contrast, PKA inhibition prevent PKC stimulation (with the phorbol ester) and coupling to ACh output. There was therefore some dependence of PKC on PKA activity in the fine control of the neuromuscular synaptic functionalism and ACh release.

  15. Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the Sordaria macrospora Striatin-Interacting Phosphatase and Kinase (STRIPAK) Complex

    PubMed Central

    Frey, Stefan; Reschka, Eva J.; Pöggeler, Stefanie

    2015-01-01

    The striatin-interacting phosphatase and kinase (STRIPAK) complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian germinal center kinases (GCKs) MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H) interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP). In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain. PMID:26418262

  16. SARS-CoV nucleocapsid protein interacts with cellular pyruvate kinase protein and inhibits its activity.

    PubMed

    Wei, Wei-Yen; Li, Hui-Chun; Chen, Chiung-Yao; Yang, Chee-Hing; Lee, Shen-Kao; Wang, Chia-Wen; Ma, Hsin-Chieh; Juang, Yue-Li; Lo, Shih-Yen

    2012-04-01

    The pathogenesis of SARS-CoV remains largely unknown. To study the function of the SARS-CoV nucleocapsid protein, we have conducted a yeast two-hybrid screening experiment to identify cellular proteins that may interact with the SARS-CoV nucleocapsid protein. Pyruvate kinase (liver) was found to interact with SARS-CoV nucleocapsid protein in this experiment. The binding domains of these two proteins were also determined using the yeast two-hybrid system. The physical interaction between the SARS-CoV nucleocapsid and cellular pyruvate kinase (liver) proteins was further confirmed by GST pull-down assay, co-immunoprecipitation assay and confocal microscopy. Cellular pyruvate kinase activity in hepatoma cells was repressed by SARS-CoV nucleocapsid protein in either transiently transfected or stably transfected cells. PK deficiency in red blood cells is known to result in human hereditary non-spherocytic hemolytic anemia. It is reasonable to assume that an inhibition of PKL activity due to interaction with SARS-CoV N protein is likely to cause the death of the hepatocytes, which results in the elevation of serum alanine aminotransferase and liver dysfunction noted in most SARS patients. Thus, our results suggest that SARS-CoV could reduce pyruvate kinase activity via its nucleocapsid protein, and this may in turn cause disease.

  17. Activation of G Protein-Coupled Receptor Kinase 1 Involves Interactions between Its N-Terminal Region and Its Kinase Domain

    SciTech Connect

    Huang, Chih-chin; Orban, Tivadar; Jastrzebska, Beata; Palczewski, Krzysztof; Tesmer, John J.G.

    2012-03-16

    G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors (GPCRs) to initiate receptor desensitization. In addition to the canonical phosphoacceptor site of the kinase domain, activated receptors bind to a distinct docking site that confers higher affinity and activates GRKs allosterically. Recent mutagenesis and structural studies support a model in which receptor docking activates a GRK by stabilizing the interaction of its 20-amino acid N-terminal region with the kinase domain. This interaction in turn stabilizes a closed, more active conformation of the enzyme. To investigate the importance of this interaction for the process of GRK activation, we first validated the functionality of the N-terminal region in rhodopsin kinase (GRK1) by site-directed mutagenesis and then introduced a disulfide bond to cross-link the N-terminal region of GRK1 with its specific binding site on the kinase domain. Characterization of the kinetic and biophysical properties of the cross-linked protein showed that disulfide bond formation greatly enhances the catalytic efficiency of the peptide phosphorylation, but receptor-dependent phosphorylation, Meta II stabilization, and inhibition of transducin activation were unaffected. These data indicate that the interaction of the N-terminal region with the kinase domain is important for GRK activation but does not dictate the affinity of GRKs for activated receptors.

  18. The A-Kinase Anchoring Protein (AKAP) Glycogen Synthase KinaseInteraction Protein (GSKIP) Regulates β-Catenin through Its Interactions with Both Protein Kinase A (PKA) and GSK3β.

    PubMed

    Dema, Alessandro; Schröter, Micha Friedemann; Perets, Ekaterina; Skroblin, Philipp; Moutty, Marie Christine; Deàk, Veronika Anita; Birchmeier, Walter; Klussmann, Enno

    2016-09-09

    The A-kinase anchoring protein (AKAP) GSK3β interaction protein (GSKIP) is a cytosolic scaffolding protein binding protein kinase A (PKA) and glycogen synthase kinase 3β (GSK3β). Here we show that both the AKAP function of GSKIP, i.e. its direct interaction with PKA, and its direct interaction with GSK3β are required for the regulation of β-catenin and thus Wnt signaling. A cytoplasmic destruction complex targets β-catenin for degradation and thus prevents Wnt signaling. Wnt signals cause β-catenin accumulation and translocation into the nucleus, where it induces Wnt target gene expression. GSKIP facilitates control of the β-catenin stabilizing phosphorylation at Ser-675 by PKA. Its interaction with GSK3β facilitates control of the destabilizing phosphorylation of β-catenin at Ser-33/Ser-37/Thr-41. The influence of GSKIP on β-catenin is explained by its scavenger function; it recruits the kinases away from the destruction complex without forming a complex with β-catenin. The regulation of β-catenin by GSKIP is specific for this AKAP as AKAP220, which also binds PKA and GSK3β, did not affect Wnt signaling. We find that the binding domain of AKAP220 for GSK3β is a conserved GSK3β interaction domain (GID), which is also present in GSKIP. Our findings highlight an essential compartmentalization of both PKA and GSK3β by GSKIP, and ascribe a function to a cytosolic AKAP-PKA interaction as a regulatory factor in the control of canonical Wnt signaling. Wnt signaling controls different biological processes, including embryonic development, cell cycle progression, glycogen metabolism, and immune regulation; deregulation is associated with diseases such as cancer, type 2 diabetes, inflammatory, and Alzheimer's and Parkinson's diseases.

  19. SOcK, MiSTs, MASK and STicKs: the GCKIII (germinal centre kinase III) kinases and their heterologous protein-protein interactions.

    PubMed

    Sugden, Peter H; McGuffin, Liam J; Clerk, Angela

    2013-08-15

    The GCKIII (germinal centre kinase III) subfamily of the mammalian Ste20 (sterile 20)-like group of serine/threonine protein kinases comprises SOK1 (Ste20-like/oxidant-stress-response kinase 1), MST3 (mammalian Ste20-like kinase 3) and MST4. Initially, GCKIIIs were considered in the contexts of the regulation of mitogen-activated protein kinase cascades and apoptosis. More recently, their participation in multiprotein heterocomplexes has become apparent. In the present review, we discuss the structure and phosphorylation of GCKIIIs and then focus on their interactions with other proteins. GCKIIIs possess a highly-conserved, structured catalytic domain at the N-terminus and a less-well conserved C-terminal regulatory domain. GCKIIIs are activated by tonic autophosphorylation of a T-loop threonine residue and their phosphorylation is regulated primarily through protein serine/threonine phosphatases [especially PP2A (protein phosphatase 2A)]. The GCKIII regulatory domains are highly disorganized, but can interact with more structured proteins, particularly the CCM3 (cerebral cavernous malformation 3)/PDCD10 (programmed cell death 10) protein. We explore the role(s) of GCKIIIs (and CCM3/PDCD10) in STRIPAK (striatin-interacting phosphatase and kinase) complexes and their association with the cis-Golgi protein GOLGA2 (golgin A2; GM130). Recently, an interaction of GCKIIIs with MO25 has been identified. This exhibits similarities to the STRADα (STE20-related kinase adaptor α)-MO25 interaction (as in the LKB1-STRADα-MO25 heterotrimer) and, at least for MST3, the interaction may be enhanced by cis-autophosphorylation of its regulatory domain. In these various heterocomplexes, GCKIIIs associate with the Golgi apparatus, the centrosome and the nucleus, as well as with focal adhesions and cell junctions, and are probably involved in cell migration, polarity and proliferation. Finally, we consider the association of GCKIIIs with a number of human diseases, particularly

  20. Protein-protein interactions of tandem affinity purified protein kinases from rice.

    PubMed

    Rohila, Jai S; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald L; Dardick, Christopher; Canlas, Patrick; Fujii, Hiroaki; Gribskov, Michael; Kanrar, Siddhartha; Knoflicek, Lucas; Stevenson, Becky; Xie, Mingtang; Xu, Xia; Zheng, Xianwu; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E

    2009-08-19

    Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex.

  1. Protein-Protein Interactions of Tandem Affinity Purified Protein Kinases from Rice

    PubMed Central

    Rohila, Jai S.; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald L.; Dardick, Christopher; Canlas, Patrick; Fujii, Hiroaki; Gribskov, Michael; Kanrar, Siddhartha; Knoflicek, Lucas; Stevenson, Becky; Xie, Mingtang; Xu, Xia; Zheng, Xianwu; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E.

    2009-01-01

    Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex. PMID:19690613

  2. Aurora kinase A interacts with H-Ras and potentiates Ras-MAPK signaling.

    PubMed

    Umstead, MaKendra; Xiong, Jinglin; Qi, Qi; Du, Yuhong; Fu, Haian

    2017-02-03

    In cancer, upregulated Ras promotes cellular transformation and proliferation in part through activation of oncogenic Ras-MAPK signaling. While directly inhibiting Ras has proven challenging, new insights into Ras regulation through protein-protein interactions may offer unique opportunities for therapeutic intervention. Here we report the identification and validation of Aurora kinase A (Aurora A) as a novel Ras binding protein. We demonstrate that the kinase domain of Aurora A mediates the interaction with the N-terminal domain of H-Ras. Further more, the interaction of Aurora A and H-Ras exists in a protein complex with Raf-1. We show that binding of H-Ras to Raf-1 and subsequent MAPK signaling is enhanced by Aurora A, and requires active H-Ras. Thus, the functional linkage between Aurora A and the H-Ras/Raf-1 protein complex may provide a mechanism for Aurora A's oncogenic activity through direct activation of the Ras/MAPK pathway.

  3. Targeting the interaction of Aurora kinases and SIRT1 mediated by Wnt signaling pathway in colorectal cancer: A critical review.

    PubMed

    Subramaniyan, Boopathi; Jagadeesan, Kaviya; Ramakrishnan, Sabitha; Mathan, Ganeshan

    2016-08-01

    The Aurora kinases belong to the family of serine/threonine kinase, a central regulator of mitosis and their expression increased during G2/M phase. It is classified into Aurora A, B and C, each has distinct roles in cellular processes, which includes regulation of spindle assembly, function of centrosomes, cytoskeleton and cytokinesis. During cancer growth, their rapid increase makes most attractive marker for cancer treatment at present. However Aurora A kinase is known to be a marker for cancer therapy, the most important serine/threonine kinase of Aurora B kinase involvement in cancer is still inadequate. Subsequently, the recent findings revealed that the class III histone deacetylase of SIRT1 is a key regulator to activate Aurora kinases from S phase damaged DNA through Wnt signaling pathway. Even if both Aurora A kinase and SIRT1 serve as a marker for cancer therapy, the present review reveals it is interaction in Wnt signaling pathway that solely for colorectal cancer.

  4. Runx2 Trans-Activation Mediated by the Msx2-Interacting Nuclear Target Requires Homeodomain Interacting Protein Kinase-3

    PubMed Central

    Sierra, Oscar L.; Towler, Dwight A.

    2010-01-01

    Runt-related transcription factor 2 (Runx2) and muscle segment homeobox homolog 2-interacting nuclear target (MINT) (Spen homolog) are transcriptional regulators critical for mammalian development. MINT enhances Runx2 activation of osteocalcin (OC) fibroblast growth factor (FGF) response element in an FGF2-dependent fashion in C3H10T1/2 cells. Although the MINT N-terminal RNA recognition motif domain contributes, the muscle segment homeobox homolog 2-interacting domain is sufficient for Runx2 activation. Intriguingly, Runx1 cannot replace Runx2 in this assay. To better understand this Runx2 signaling cascade, we performed structure-function analysis of the Runx2-MINT trans-activation relationship. Systematic truncation and domain swapping in Runx1:Runx2 chimeras identified that the unique Runx2 activation domain 3 (AD3), encompassed by residues 316–421, conveys MINT+FGF2 trans-activation in transfection assays. Ala mutagenesis of Runx2 Ser/Thr residues identified that S301 and T326 in AD3 are necessary for full MINT+FGF2 trans-activation. Conversely, phosphomimetic Asp substitution of these AD3 Ser/Thr residues enhanced activation by MINT. Adjacent Pro residues implicated regulation by a proline-directed protein kinase (PDPK). Systematic screening with PDPK inhibitors identified that the casein kinase-2/homeodomain-interacting protein kinase (HIPK)/dual specificity tyrosine phosphorylation regulated kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), but not ERK, c-Jun N-terminal kinase, p38MAPK, or other casein kinase-2 inhibitors, abrogated Runx2-, MINT-, and FGF2-activation. Systematic small interfering RNA-mediated silencing of DMAT-inhibited PDPKs revealed that HIPK3 depletion reduced MINT+FGF2-dependent activation of Runx2. HIPK3 and Runx2 coprecipitate after in vitro transcription-translation, and recombinant HIPK3 recognizes Runx2 AD3 as kinase substrate. Furthermore, DMAT treatment and HIPK3 RNAi inhibited MINT+FGF2 activation of

  5. Conformation-selective ATP-competitive inhibitors control regulatory interactions and noncatalytic functions of mitogen-activated protein kinases.

    PubMed

    Hari, Sanjay B; Merritt, Ethan A; Maly, Dustin J

    2014-05-22

    Most potent protein kinase inhibitors act by competing with ATP to block the phosphotransferase activity of their targets. However, emerging evidence demonstrates that ATP-competitive inhibitors can affect kinase interactions and functions in ways beyond blocking catalytic activity. Here, we show that stabilizing alternative ATP-binding site conformations of the mitogen-activated protein kinases (MAPKs) p38α and Erk2 with ATP-competitive inhibitors differentially, and in some cases divergently, modulates the abilities of these kinases to interact with upstream activators and deactivating phosphatases. Conformation-selective ligands are also able to modulate Erk2's ability to allosterically activate the MAPK phosphatase DUSP6, highlighting how ATP-competitive ligands can control noncatalytic kinase functions. Overall, these studies underscore the relationship between the ATP-binding and regulatory sites of MAPKs and provide insight into how ATP-competitive ligands can be designed to confer graded control over protein kinase function.

  6. Exact solutions to a spatially extended model of kinase-receptor interaction.

    PubMed

    Szopa, Piotr; Lipniacki, Tomasz; Kazmierczak, Bogdan

    2011-10-01

    B and Mast cells are activated by the aggregation of the immune receptors. Motivated by this phenomena we consider a simple spatially extended model of mutual interaction of kinases and membrane receptors. It is assumed that kinase activates membrane receptors and in turn the kinase molecules bound to the active receptors are activated by transphosphorylation. Such a type of interaction implies positive feedback and may lead to bistability. In this study we apply the Steklov eigenproblem theory to analyze the linearized model and find exact solutions in the case of non-uniformly distributed membrane receptors. This approach allows us to determine the critical value of receptor dephosphorylation rate at which cell activation (by arbitrary small perturbation of the inactive state) is possible. We found that cell sensitivity grows with decreasing kinase diffusion and increasing anisotropy of the receptor distribution. Moreover, these two effects are cooperating. We showed that the cell activity can be abruptly triggered by the formation of the receptor aggregate. Since the considered activation mechanism is not based on receptor crosslinking by polyvalent antigens, the proposed model can also explain B cell activation due to receptor aggregation following binding of monovalent antigens presented on the antigen presenting cell.

  7. Exact solutions to a spatially extended model of kinase-receptor interaction

    NASA Astrophysics Data System (ADS)

    Szopa, Piotr; Lipniacki, Tomasz; Kazmierczak, Bogdan

    2011-10-01

    B and Mast cells are activated by the aggregation of the immune receptors. Motivated by this phenomena we consider a simple spatially extended model of mutual interaction of kinases and membrane receptors. It is assumed that kinase activates membrane receptors and in turn the kinase molecules bound to the active receptors are activated by transphosphorylation. Such a type of interaction implies positive feedback and may lead to bistability. In this study we apply the Steklov eigenproblem theory to analyze the linearized model and find exact solutions in the case of non-uniformly distributed membrane receptors. This approach allows us to determine the critical value of receptor dephosphorylation rate at which cell activation (by arbitrary small perturbation of the inactive state) is possible. We found that cell sensitivity grows with decreasing kinase diffusion and increasing anisotropy of the receptor distribution. Moreover, these two effects are cooperating. We showed that the cell activity can be abruptly triggered by the formation of the receptor aggregate. Since the considered activation mechanism is not based on receptor crosslinking by polyvalent antigens, the proposed model can also explain B cell activation due to receptor aggregation following binding of monovalent antigens presented on the antigen presenting cell.

  8. Conformational transitions and interactions underlying the function of membrane embedded receptor protein kinases.

    PubMed

    Bocharov, Eduard V; Sharonov, Georgy V; Bocharova, Olga V; Pavlov, Konstantin V

    2017-01-25

    Among membrane receptors, the single-span receptor protein kinases occupy a broad but specific functional niche determined by distinctive features of the underlying transmembrane signaling mechanisms that are briefly overviewed on the basis of some of the most representative examples, followed by a more detailed discussion of several hierarchical levels of organization and interactions involved. All these levels, including single-molecule interactions (e.g., dimerization, liganding, chemical modifications), local processes (e.g. lipid membrane perturbations, cytoskeletal interactions), and larger scale phenomena (e.g., effects of membrane surface shape or electrochemical potential gradients) appear to be closely integrated to achieve the observed diversity of the receptor functioning. Different species of receptor protein kinases meet their specific functional demands through different structural features defining their responses to stimulation, but certain common patterns exist. Signaling by receptor protein kinases is typically associated with the receptor dimerization and clustering, ligand-induced rearrangements of receptor domains through allosteric conformational transitions with involvement of lipids, release of the sequestered lipids, restriction of receptor diffusion, cytoskeleton and membrane shape remodeling. Understanding of complexity and continuity of the signaling processes can help identifying currently neglected opportunities for influencing the receptor signaling with potential therapeutic implications. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

  9. Physical and functional interactions between ZIP kinase and UbcH5

    SciTech Connect

    Ohbayashi, Norihiko; Okada, Katsuya; Kawakami, Shiho; Togi, Sumihito; Sato, Noriko; Ikeda, Osamu; Kamitani, Shinya; Muromoto, Ryuta; Sekine, Yuichi; Kawai, Taro; Akira, Shizuo; Matsuda, Tadashi

    2008-08-08

    Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase that has been implicated in cell death and transcriptional regulation, but its mechanism of regulation remains unknown. In our previous study, we showed that leukemia inhibitory factor stimulated threonine-265 phosphorylation of ZIPK, thereby leading to phosphorylation and activation of signal transducer and activator of transcription 3. Here, we identified UbcH5c as a novel ZIPK-binding partner by yeast two-hybrid screening. Importantly, we found that UbcH5c induced ubiquitination of ZIPK. Small-interfering RNA-mediated reduction of endogenous UbcH5 expression decreased ZIPK ubiquitination. Furthermore, coexpression of UbcH5c with ZIPK influenced promyelocytic leukemia protein nuclear body (PML-NB) formation. These results suggest that UbcH5 regulates ZIPK accumulation in PML-NBs by interacting with ZIPK and stimulating its ubiquitination.

  10. Making the Auroras glow: regulation of Aurora A and B kinase function by interacting proteins

    PubMed Central

    Carmena, Mar; Ruchaud, Sandrine; Earnshaw, William C

    2009-01-01

    The conserved Aurora family of protein kinases have emerged as crucial regulators of mitosis and cytokinesis. Despite their high degree of homology, Aurora A and B have very distinctive localisations and functions: Aurora A associates with the spindle poles to regulate entry into mitosis, centrosome maturation and spindle assembly; Aurora B is a member of the Chromosomal Passenger Complex (CPC) that transfers from the inner centromere in early mitosis to the spindle midzone, equatorial cortex and midbody in late mitosis and cytokinesis. Aurora B functions include regulation of chromosome–microtubule interactions, cohesion, spindle stability and cytokinesis. This review will focus on how interacting proteins make this functional diversity possible by targeting the kinases to different subcellular locations and regulating their activity. PMID:19836940

  11. Homeodomain-interacting protein kinase (Hipk) phosphorylates the small SPOC family protein Spenito.

    PubMed

    Dewald, D N; Steinmetz, E L; Walldorf, U

    2014-12-01

    The Drosophila homeodomain-interacting protein kinase (Hipk) is a versatile regulator involved in a variety of pathways, such as Notch and Wingless signalling, thereby acting in processes including the promotion of eye development or control of cell numbers in the nervous system. In vertebrates, extensive studies have related its homologue HIPK2 to important roles in the control of p53-mediated apoptosis and tumour suppression. Spenito (Nito) belongs to the group of small SPOC family proteins and has a role, amongst others, as a regulator of Wingless signalling downstream of Armadillo. In the present study, we show that both proteins have an enzyme-substrate relationship, adding a new interesting component to the broad range of Hipk interactions, and we map several phosphorylation sites of Nito. Furthermore, we were able to define a preliminary consensus motif for Hipk target sites, which will simplify the identification of new substrates of this kinase.

  12. Making the Auroras glow: regulation of Aurora A and B kinase function by interacting proteins.

    PubMed

    Carmena, Mar; Ruchaud, Sandrine; Earnshaw, William C

    2009-12-01

    The conserved Aurora family of protein kinases have emerged as crucial regulators of mitosis and cytokinesis. Despite their high degree of homology, Aurora A and B have very distinctive localisations and functions: Aurora A associates with the spindle poles to regulate entry into mitosis, centrosome maturation and spindle assembly; Aurora B is a member of the Chromosomal Passenger Complex (CPC) that transfers from the inner centromere in early mitosis to the spindle midzone, equatorial cortex and midbody in late mitosis and cytokinesis. Aurora B functions include regulation of chromosome-microtubule interactions, cohesion, spindle stability and cytokinesis. This review will focus on how interacting proteins make this functional diversity possible by targeting the kinases to different subcellular locations and regulating their activity.

  13. Phosphorylation by casein kinase II affects the interaction of caldesmon with smooth muscle myosin and tropomyosin.

    PubMed Central

    Bogatcheva, N V; Vorotnikov, A V; Birukov, K G; Shirinsky, V P; Gusev, N B

    1993-01-01

    Smooth muscle caldesmon was phosphorylated by casein kinase II, and the effects of phosphorylation on the interaction of caldesmon and its chymotryptic peptides with myosin and tropomyosin were investigated. The N-terminal chymotryptic peptide of caldesmon of molecular mass 27 kDa interacted with myosin. Phosphorylation of Ser-73 catalysed by casein kinase II resulted in a 2-fold decrease in the affinity of the native caldesmon (or its 27 kDa N-terminal peptide) for smooth muscle myosin. At low ionic strength, caldesmon and its N-terminal peptides of molecular masses 25 and 27 kDa were retarded on a column of immobilized tropomyosin. Phosphorylation of Ser-73 led to a 2-4-fold decrease in the affinity of caldesmon (or its N-terminal peptides) for tropomyosin. Thus phosphorylation of Ser-73 catalysed by casein kinase II affects the interaction of caldesmon with both smooth muscle myosin and tropomyosin. Images Figure 1 Figure 2 Figure 3 PMID:8452532

  14. Inhibitory effects of homeodomain-interacting protein kinase 2 on the aorta-gonad-mapharsen hematopoiesis

    SciTech Connect

    Ohtsu, Naoki; Nobuhisa, Ikuo; Mochita, Miyuki; Taga, Tetsuya . E-mail: taga@kaiju.medic.kumamoto-u.ac.jp

    2007-01-01

    Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region of the mouse embryo. Our previous studies revealed that STAT3, a gp130 downstream transcription factor, is required for AGM hematopoiesis and that homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates serine-727 of STAT3. HIPK2 is a serine/threonine kinase known to be involved in transcriptional repression and apoptosis. In the present study, we examined the role of HIPK2 in hematopoiesis in mouse embryo. HIPK2 transcripts were found in fetal hematopoietic tissues such as the mouse AGM region and fetal liver. In cultured AGM cells, HIPK2 protein was detected in adherent cells. Functional analyses of HIPK2 were carried out by introducing wild-type and mutant HIPK2 constructs into AGM cultures. Production of CD45{sup +} hematopoietic cells was suppressed by forced expression of HIPK2 in AGM cultures. This suppression required the kinase domain and nuclear localization signals of HIPK2, but the kinase activity was dispensable. HIPK2-overexpressing AGM-derived nonadherent cells did not form cobblestone-like colonies in cultures with stromal cells. Furthermore, overexpression of HIPK2 in AGM cultures impeded the expansion of CD45{sup low}c-Kit{sup +} cells, which exhibit the immature hematopoietic progenitor phenotype. These data indicate that HIPK2 plays a negative regulatory role in AGM hematopoiesis in the mouse embryo.

  15. EphB4 cellular kinase activity assayed using an enzymatic protein interaction system.

    PubMed

    Wehrman, Tom; Nguyen, Mimi; Feng, Wei; Bader, Benjamin

    2013-05-01

    Receptor tyrosine kinases (RTKs) are important players in various cellular processes, including proliferation, migration, metabolism, and neuronal development. EphB4 RTK is essential for the development of a functional arterial-venous network in embryonic and adult neoangiogenesis. To develop novel inhibitors of EphB4 that might have applications in severe diseases like cancer and retinopathies, assays need to be in place that resemble, in a most physiological fashion, the activation and downstream function of the kinase. In addition, such assays need to be amenable to high-throughput screening to serve efficiently the modern drug discovery processes in the pharmaceutical industry. The authors have developed an enzyme fragment complementation assay that measures the interaction of a downstream docking protein to the activated and phosphorylated full-length EphB4 kinase in cells. The assay is specific, robust, and amenable to miniaturization and high-throughput screening. It covers most steps in the activation process of EphB4, including ligand binding, autophosphorylation, and docking of a downstream interactor. This assay format can be transferred to other RTKs and adds an important cell-based kinase assay option to researchers in the field.

  16. Discovery of Mer kinase inhibitors by Virtual Screening using Structural Protein-Ligand Interaction Fingerprints

    PubMed Central

    Da, C.; Stashko, M.; Jayakody, C.; Wang, X.; Janzen, W.; Frye, S.; Kireev, D.

    2015-01-01

    Mer is a receptor tyrosine kinase implicated in acute lymphoblastic leukemia (ALL), the most common malignancy in children. The currently available data provide a rationale for development of Mer kinase inhibitors as cancer therapeutics that can target both cell autologous and immune-modulatory anti-tumor effects. We have previously reported several series of potent Mer inhibitors and the objective of the current report is to identify a chemically dissimilar back-up series that might circumvent potential, but currently unknown, flaws inherent to the lead series. To this end, we virtually screened a database of ∼3.8 million commercially available compounds using high-throughput docking followed by a filter involving Structural Protein-Ligand Interaction Fingerprints (SPLIF). SPLIF permits a quantitative assessment of whether a docking pose interacts with the protein target similarly to an endogenous or known synthetic ligand, and therefore helps to improve both sensitivity and specificity with respect to the docking score alone. Of the total of 62 experimentally tested compounds, 15 demonstrated reliable dose-dependent responses in the Mer in vitro kinase activity assay with inhibitory potencies ranging from 0.46 μM to 9.9 μM. PMID:25638502

  17. Discovery of Mer kinase inhibitors by virtual screening using Structural Protein-Ligand Interaction Fingerprints.

    PubMed

    Da, C; Stashko, M; Jayakody, C; Wang, X; Janzen, W; Frye, S; Kireev, D

    2015-03-01

    Mer is a receptor tyrosine kinase implicated in acute lymphoblastic leukemia (ALL), the most common malignancy in children. The currently available data provide a rationale for development of Mer kinase inhibitors as cancer therapeutics that can target both cell autologous and immune-modulatory anti-tumor effects. We have previously reported several series of potent Mer inhibitors and the objective of the current report is to identify a chemically dissimilar back-up series that might circumvent potential, but currently unknown, flaws inherent to the lead series. To this end, we virtually screened a database of ∼3.8million commercially available compounds using high-throughput docking followed by a filter involving Structural Protein-Ligand Interaction Fingerprints (SPLIF). SPLIF permits a quantitative assessment of whether a docking pose interacts with the protein target similarly to an endogenous or known synthetic ligand, and therefore helps to improve both sensitivity and specificity with respect to the docking score alone. Of the total of 62 experimentally tested compounds, 15 demonstrated reliable dose-dependent responses in the Mer in vitro kinase activity assay with inhibitory potencies ranging from 0.46μM to 9.9μM.

  18. Insights into protein interaction networks reveal non-receptor kinases as significant druggable targets for psoriasis.

    PubMed

    Sundarrajan, Sudharsana; Lulu, Sajitha; Arumugam, Mohanapriya

    2015-07-25

    Psoriasis is a chronic disease of the skin characterized by hyper proliferation and inflammation of the epidermis and dermal components of the skin. T-cell-dependent inflammatory process in skin governs the pathogenesis of psoriasis. An in-silico search strategy was utilized to identify psoriatic therapeutic drug targets. The gene expression profiling of psoriatic skin identified a total of 427 differentially expressed genes (DEGs). Gene ontology investigation of DEGs identified genes involved in calcium binding, apoptosis, keratinisation, lipid transportation and homeostasis apart from immune mediated processes. The protein interaction networks identified proteins involved in various signaling mechanisms with high degree of interconnections. The gene modules derived from the main network were enriched with rich kinome. These sub-networks were dominated by the presence of non-receptor kinase family members which are major signal transmitters in immune response. The computational approach has aided in the identification of non-receptor kinases as potential targets for psoriasis drug development.

  19. Catalytic mechanism and kinase interactions of ABA-signaling PP2C phosphatases.

    PubMed

    Zhou, X Edward; Soon, Fen-Fen; Ng, Ley-Moy; Kovach, Amanda; Suino-Powell, Kelly M; Li, Jun; Yong, Eu-Leong; Zhu, Jian-Kang; Xu, H Eric; Melcher, Karsten

    2012-05-01

    Abscisic acid (ABA) is an essential hormone that controls plant growth, development and responses to abiotic stresses. ABA signaling is mediated by type 2C protein phosphatases (PP2Cs), including HAB1 and ABI2, which inhibit stress-activated SnRK2 kinases and whose activity is regulated by ABA and ABA receptors. Based on biochemical data and our previously determined crystal structures of ABI2 and the SnRK2.6-HAB1 complex, we present the catalytic mechanism of PP2C and provide new insight into PP2C-SnRK2 interactions and possible roles of other SnRK2 kinases in ABA signaling.

  20. An autophosphorylation site database for leucine-rich repeat receptor-like kinases in Arabidopsis thaliana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We conducted a family-wide study to identify and characterize sites of autophosphorylation in 73 representative LRR RLKs of the 223 member LRR RLK family in Arabidopsis thaliana. His-tagged constructs of intact cytoplasmic domains (CDs) for 73 of 223 A. thaliana LRR RLKs were cloned into E. coli BL-...

  1. Leucine leucine-37 uses formyl peptide receptor-like 1 to activate signal transduction pathways, stimulate oncogenic gene expression, and enhance the invasiveness of ovarian cancer cells.

    PubMed

    Coffelt, Seth B; Tomchuck, Suzanne L; Zwezdaryk, Kevin J; Danka, Elizabeth S; Scandurro, Aline B

    2009-06-01

    Emerging evidence suggests that the antimicrobial peptide, leucine leucine-37 (LL-37), could play a role in the progression of solid tumors. LL-37 is expressed as the COOH terminus of human cationic antimicrobial protein-18 (hCAP-18) in ovarian, breast, and lung cancers. Previous studies have shown that the addition of LL-37 to various cancer cell lines in vitro stimulates proliferation, migration, and invasion. Similarly, overexpression of hCAP-18/LL-37 in vivo accelerates tumor growth. However, the receptor or receptors through which these processes are mediated have not been thoroughly examined. In the present study, expression of formyl peptide receptor-like 1 (FPRL1) was confirmed on ovarian cancer cells. Proliferation assays indicated that LL-37 does not signal through a G protein-coupled receptor, such as FPRL1, to promote cancer cell growth. By contrast, FPRL1 was required for LL-37-induced invasion through Matrigel. The peptide stimulated mitogen-activated protein kinase and Janus-activated kinase/signal transducers and activators of transcription signaling cascades and led to the significant activation of several transcription factors, through both FPRL1-dependent and FPRL1-independent pathways. Likewise, expression of some LL-37-stimulated genes was attenuated by the inhibition of FPRL1. Increased expression of CXCL10, EGF, and PDGF-BB as well as other soluble factors was confirmed from conditioned medium of LL-37-treated cells. Taken together, these data suggest that LL-37 potentiates a more aggressive behavior from ovarian cancer cells through its interaction with FPRL1.

  2. The Potential for Signal Integration and Processing in Interacting Map Kinase Cascades

    PubMed Central

    Schwacke, John H.; Voit, Eberhard O.

    2009-01-01

    The cellular response to environmental stimuli requires biochemical information processing through which sensory inputs and cellular status are integrated and translated into appropriate responses by way of interacting networks of enzymes. One such network, the Mitogen Activated Protein (MAP) kinase cascade is a highly conserved signal transduction module that propagates signals from cell surface receptors to various cytosolic and nuclear targets by way of a phosphorylation cascade. We have investigated the potential for signal processing within a network of interacting feed-forward kinase cascades typified by the MAP kinase cascade. A genetic algorithm was used to search for sets of kinetic parameters demonstrating representative key input-output patterns of interest. We discuss two of the networks identified in our study, one implementing the exclusive-or function (XOR) and another implementing what we refer to as an in-band detector (IBD) or two-sided threshold. These examples confirm the potential for logic and amplitude-dependent signal processing in interacting MAP kinase cascades demonstrating limited cross-talk. Specifically, the XOR function allows the network to respond to either one, but not both signals simultaneously, while the IBD permits the network to respond exclusively to signals within a given range of strength, and to suppress signals below as well as above this range. The solution to the XOR problem is interesting in that it requires only two interacting pathways, crosstalk at only one layer, and no feedback or explicit inhibition. These types of responses are not only biologically relevant but constitute signal processing modules that can be combined to create other logical functions and that, in contrast to amplification, cannot be achieved with a single cascade or with two non-interacting cascades. Our computational results revealed surprising similarities between experimental data describing the JNK/MKK4/MKK7 pathway and the solution for

  3. Leucine-rich repeat kinase 2 interacts with p21-activated kinase 6 to control neurite complexity in mammalian brain.

    PubMed

    Civiero, Laura; Cirnaru, Maria Daniela; Beilina, Alexandra; Rodella, Umberto; Russo, Isabella; Belluzzi, Elisa; Lobbestael, Evy; Reyniers, Lauran; Hondhamuni, Geshanthi; Lewis, Patrick A; Van den Haute, Chris; Baekelandt, Veerle; Bandopadhyay, Rina; Bubacco, Luigi; Piccoli, Giovanni; Cookson, Mark R; Taymans, Jean-Marc; Greggio, Elisa

    2015-12-01

    Leucine-rich repeat kinase 2 (LRRK2) is a causative gene for Parkinson's disease, but the physiological function and the mechanism(s) by which the cellular activity of LRRK2 is regulated are poorly understood. Here, we identified p21-activated kinase 6 (PAK6) as a novel interactor of the GTPase/ROC domain of LRRK2. p21-activated kinases are serine-threonine kinases that serve as targets for the small GTP binding proteins Cdc42 and Rac1 and have been implicated in different morphogenetic processes through remodeling of the actin cytoskeleton such as synapse formation and neuritogenesis. Using an in vivo neuromorphology assay, we show that PAK6 is a positive regulator of neurite outgrowth and that LRRK2 is required for this function. Analyses of post-mortem brain tissue from idiopathic and LRRK2 G2019S carriers reveal an increase in PAK6 activation state, whereas knock-out LRRK2 mice display reduced PAK6 activation and phosphorylation of PAK6 substrates. Taken together, these results support a critical role of LRRK2 GTPase domain in cytoskeletal dynamics in vivo through the novel interactor PAK6, and provide a valuable platform to unravel the mechanism underlying LRRK2-mediated pathophysiology. We propose p21-activated kinase 6 (PAK6) as a novel interactor of leucine-rich repeat kinase 2 (LRRK2), a kinase involved in Parkinson's disease (PD). In health, PAK6 regulates neurite complexity in the brain and LRRK2 is required for its function, (a) whereas PAK6 is aberrantly activated in LRRK2-linked PD brain (b) suggesting that LRRK2 toxicity is mediated by PAK6.

  4. Protein tyrosine kinase signaling in the mouse oocyte cortex during sperm-egg interactions and anaphase resumption.

    PubMed

    McGinnis, Lynda K; Luo, Jinping; Kinsey, William H

    2013-04-01

    Fertilization triggers activation of a series of pre-programmed signal transduction pathways in the oocyte that establish a block to polyspermy, induce meiotic resumption, and initiate zygotic development. Fusion between sperm and oocyte results in rapid changes in oocyte intracellular free-calcium levels, which in turn activate multiple protein kinase cascades in the ooplasm. The present study examined the possibility that sperm-oocyte interaction involves localized activation of oocyte protein tyrosine kinases, which could provide an alternative signaling mechanism to that triggered by the fertilizing sperm. Confocal immunofluorescence analysis with antibodies to phosphotyrosine and phosphorylated protein tyrosine kinases allowed detection of minute signaling events localized to the site of sperm-oocyte interaction that were not amenable to biochemical analysis. The results provide evidence for localized accumulation of phosphotyrosine at the site of sperm contact, binding, or fusion, which suggests active protein tyrosine kinase signaling prior to and during sperm incorporation. The PYK2 kinase was found to be concentrated and activated at the site of sperm-oocyte interaction, and likely participates in this response. Widespread activation of PYK2 and FAK kinases was subsequently observed within the oocyte cortex, indicating that sperm incorporation is followed by more global signaling via these kinases during meiotic resumption. The results demonstrate an alternate signaling pathway triggered in mammalian oocytes by sperm contact, binding, or fusion with the oocyte.

  5. Reduced formation of advanced glycation endproducts via interactions between glutathione peroxidase 3 and dihydroxyacetone kinase 1.

    PubMed

    Lee, Hana; Chi, Seung Wook; Lee, Phil Young; Kang, Sunghyun; Cho, Sayeon; Lee, Chong-Kil; Bae, Kwang-Hee; Park, Byoung Chul; Park, Sung Goo

    2009-11-06

    Dihydroxyacetone (DHA) induces the formation of advanced glycation endproducts (AGEs), which are involved in several diseases. Earlier, we identified dihydroxyacetone kinase 1 (Dak1) as a candidate glutathione peroxidase 3 (Gpx3)-interacting protein in Saccharomyces cerevisiae. This finding is noteworthy, as no clear evidence on the involvement of oxidative stress systems in DHA-induced AGE formation has been found to date. Here, we demonstrate that Gpx3 interacts with Dak1, alleviates DHA-mediated stress by upregulating Dak activity, and consequently suppresses AGE formation. Based on these results, we propose that defense systems against oxidative stress and DHA-induced AGE formation are related via interactions between Gpx3 and Dak1.

  6. Identification of Polo-like kinase 1 interaction inhibitors using a novel cell-based assay

    PubMed Central

    Normandin, Karine; Lavallée, Jean-François; Futter, Marie; Beautrait, Alexandre; Duchaine, Jean; Guiral, Sébastien; Marinier, Anne; Archambault, Vincent

    2016-01-01

    Polo-like kinase 1 (Plk1) plays several roles in cell division and it is a recognized cancer drug target. Plk1 levels are elevated in cancer and several types of cancer cells are hypersensitive to Plk1 inhibition. Small molecule inhibitors of the kinase domain (KD) of Plk1 have been developed. Their selectivity is limited, which likely contributes to their toxicity. Polo-like kinases are characterized by a Polo-Box Domain (PBD), which mediates interactions with phosphorylation substrates or regulators. Inhibition of the PBD could allow better selectivity or result in different effects than inhibition of the KD. In vitro screens have been used to identify PBD inhibitors with mixed results. We developed the first cell-based assay to screen for PBD inhibitors, using Bioluminescence Resonance Energy Transfer (BRET). We screened through 112 983 compounds and characterized hits in secondary biochemical and biological assays. Subsequent Structure-Activity Relationship (SAR) analysis on our most promising hit revealed that it requires an alkylating function for its activity. In addition, we show that the previously reported PBD inhibitors thymoquinone and Poloxin are also alkylating agents. Our cell-based assay is a promising tool for the identification of new PBD inhibitors with more drug-like profiles using larger and more diverse chemical libraries. PMID:27874094

  7. Interacting factors and cellular localization of SR protein-specific kinase Dsk1

    SciTech Connect

    Tang, Zhaohua; Luca, Maria; Taggart-Murphy, Laura; Portillio, Jessica; Chang, Cathey; Guven, Ayse; Lin, Ren-Jang; Murray, Johanne; Carr, Antony

    2012-10-01

    Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A){sup +} RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G{sub 2} phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.

  8. Decoding the Interactions Regulating the Active State Mechanics of Eukaryotic Protein Kinases

    PubMed Central

    Meharena, Hiruy S.; Fan, Xiaorui; Ahuja, Lalima G.; Keshwani, Malik M.; McClendon, Christopher L.; Chen, Angela M.; Adams, Joseph A.; Taylor, Susan S.

    2016-01-01

    Eukaryotic protein kinases regulate most cellular functions by phosphorylating targeted protein substrates through a highly conserved catalytic core. In the active state, the catalytic core oscillates between open, intermediate, and closed conformations. Currently, the intramolecular interactions that regulate the active state mechanics are not well understood. Here, using cAMP-dependent protein kinase as a representative model coupled with biochemical, biophysical, and computational techniques, we define a set of highly conserved electrostatic and hydrophobic interactions working harmoniously to regulate these mechanics. These include the previously identified salt bridge between a lysine from the β3-strand and a glutamate from the αC-helix as well as an electrostatic interaction between the phosphorylated activation loop and αC-helix and an ensemble of hydrophobic residues of the Regulatory spine and Shell. Moreover, for over three decades it was thought that the highly conserved β3-lysine was essential for phosphoryl transfer, but our findings show that the β3-lysine is not required for phosphoryl transfer but is essential for the active state mechanics. PMID:27902690

  9. A novel microfluidic assay reveals a key role for protein kinase C δ in regulating human neutrophil-endothelium interaction.

    PubMed

    Soroush, Fariborz; Zhang, Ting; King, Devon J; Tang, Yuan; Deosarkar, Sudhir; Prabhakarpandian, Balabhaskar; Kilpatrick, Laurie E; Kiani, Mohammad F

    2016-11-01

    A key step in neutrophil-mediated tissue damage is the migration of activated neutrophils across the vascular endothelium. Previously, we identified protein kinase C δ as a critical regulator of neutrophil migration in sepsis but did not identify specific steps in migration. In this study, we used our novel biomimetic microfluidic assay to delineate systematically the mechanism by which protein kinase C δ regulates individual steps in human neutrophil-endothelial interaction during inflammation. The biomimetic microfluidic assay includes a network of vascular channels, produced from in vivo images connected to a tissue compartment through a porous barrier. HUVECs cultured in vascular channels formed a complete lumen under physiologic shear flow. HUVECs were pretreated with TNF-α ± a protein kinase C δ inhibitor, and the tissue compartment was filled with a chemoattractant (fMLP or IL-8). Under physiologic shear flow, the role of protein kinase C δ on spatial and temporal neutrophil adherence/migration was quantified. Protein kinase C δ inhibition significantly reduced neutrophil adhesion in response to fMLP and IL-8 only under low shear rate and near bifurcations. Protein kinase C δ inhibition also decreased adherence to nonactivated HUVECs in response to fMLP or IL-8. Protein kinase C δ inhibition reduced neutrophil migration into the tissue compartment in response to fMLP and to a lesser degree, to IL-8. Antibody-coated microparticles demonstrated that protein kinase C δ inhibition down-regulated E-selectin and ICAM-1 but not VCAM-1 expression. With the use of a physiologically relevant in vitro model system, we demonstrate that protein kinase C δ plays an important role in the regulation of neutrophil adherence/migration during inflammation and identifies key steps regulated by protein kinase C δ in neutrophil-endothelial interactions.

  10. A family of plasmodesmal proteins with receptor-like properties for plant viral movement proteins.

    PubMed

    Amari, Khalid; Boutant, Emmanuel; Hofmann, Christina; Schmitt-Keichinger, Corinne; Fernandez-Calvino, Lourdes; Didier, Pascal; Lerich, Alexander; Mutterer, Jérome; Thomas, Carole L; Heinlein, Manfred; Mély, Yves; Maule, Andrew J; Ritzenthaler, Christophe

    2010-09-23

    Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement.

  11. Therapeutic potential of mitotic interaction between the nucleoporin Tpr and aurora kinase A.

    PubMed

    Kobayashi, Akiko; Hashizume, Chieko; Dowaki, Takayuki; Wong, Richard W

    2015-01-01

    Spindle poles are defined by centrosomes; therefore, an abnormal number or defective structural organization of centrosomes can lead to loss of spindle bipolarity and genetic integrity. Previously, we showed that Tpr (translocated promoter region), a component of the nuclear pore complex (NPC), interacts with Mad1 and dynein to promote proper chromosome segregation during mitosis. Tpr also associates with p53 to induce autophagy. Here, we report that Tpr depletion induces mitotic catastrophe and enhances the rate of tetraploidy and polyploidy. Mechanistically, Tpr interacts, via its central domain, with Aurora A but not Aurora B kinase. In Tpr-depleted cells, the expression levels, centrosomal localization and phosphorylation of Aurora A were all reduced. Surprisingly, an Aurora A inhibitor, Alisertib (MLN8237), also disrupted centrosomal localization of Tpr and induced mitotic catastrophe and cell death in a time- and dose-dependent manner. Strikingly, over-expression of Aurora A disrupted Tpr centrosomal localization only in cells with supernumerary centrosomes but not in bipolar cells. Our results highlight the mutual regulation between Tpr and Aurora A and further confirm the importance of nucleoporin function in spindle pole organization, bipolar spindle assembly, and mitosis; functions that are beyond the conventional nucleocytoplasmic transport and NPC structural roles of nucleoporins. Furthermore, the central coiled-coil domain of Tpr binds to and sequesters extra Aurora A to safeguard bipolarity. This Tpr domain merits further investigation for its ability to inhibit Aurora kinase and as a potential therapeutic agent in cancer treatment.

  12. A kinase interacting protein (AKIP1) is a key regulator of cardiac stress

    PubMed Central

    Sastri, Mira; Haushalter, Kristofer J.; Panneerselvam, Mathivadhani; Chang, Philip; Fridolfsson, Heidi; Finley, J. Cameron; Ng, Daniel; Schilling, Jan M.; Miyanohara, Atsushi; Day, Michele E.; Hakozaki, Hiro; Petrosyan, Susanna; Koller, Antonius; King, Charles C.; Darshi, Manjula; Blumenthal, Donald K.; Ali, Sameh Saad; Roth, David M.; Patel, Hemal H.; Taylor, Susan S.

    2013-01-01

    cAMP-dependent protein kinase (PKA) regulates a myriad of functions in the heart, including cardiac contractility, myocardial metabolism, and gene expression. However, a molecular integrator of the PKA response in the heart is unknown. Here, we show that the PKA adaptor A-kinase interacting protein 1 (AKIP1) is up-regulated in cardiac myocytes in response to oxidant stress. Mice with cardiac gene transfer of AKIP1 have enhanced protection to ischemic stress. We hypothesized that this adaptation to stress was mitochondrial-dependent. AKIP1 interacted with the mitochondrial localized apoptosis inducing factor (AIF) under both normal and oxidant stress. When cardiac myocytes or whole hearts are exposed to oxidant and ischemic stress, levels of both AKIP1 and AIF were enhanced. AKIP1 is preferentially localized to interfibrillary mitochondria and up-regulated in this cardiac mitochondrial subpopulation on ischemic injury. Mitochondria isolated from AKIP1 gene-transferred hearts showed increased mitochondrial localization of AKIP1, decreased reactive oxygen species generation, enhanced calcium tolerance, decreased mitochondrial cytochrome C release, and enhance phosphorylation of mitochondrial PKA substrates on ischemic stress. These observations highlight AKIP1 as a critical molecular regulator and a therapeutic control point for stress adaptation in the heart. PMID:23319652

  13. Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39.

    PubMed

    Mai, Sanyue; Qu, Xiuhua; Li, Ping; Ma, Qingjun; Liu, Xuan; Cao, Cheng

    2016-04-22

    RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-β. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERα and PRβ in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39.

  14. Molecular dynamics analysis of conserved hydrophobic and hydrophilic bond-interaction networks in ErbB family kinases.

    PubMed

    Shih, Andrew J; Telesco, Shannon E; Choi, Sung-Hee; Lemmon, Mark A; Radhakrishnan, Ravi

    2011-06-01

    The EGFR (epidermal growth factor receptor)/ErbB/HER (human EGFR) family of kinases contains four homologous receptor tyrosine kinases that are important regulatory elements in key signalling pathways. To elucidate the atomistic mechanisms of dimerization-dependent activation in the ErbB family, we have performed molecular dynamics simulations of the intracellular kinase domains of three members of the ErbB family (those with known kinase activity), namely EGFR, ErbB2 (HER2) and ErbB4 (HER4), in different molecular contexts: monomer against dimer and wild-type against mutant. Using bioinformatics and fluctuation analyses of the molecular dynamics trajectories, we relate sequence similarities to correspondence of specific bond-interaction networks and collective dynamical modes. We find that in the active conformation of the ErbB kinases, key subdomain motions are co-ordinated through conserved hydrophilic interactions: activating bond-networks consisting of hydrogen bonds and salt bridges. The inactive conformations also demonstrate conserved bonding patterns (albeit less extensive) that sequester key residues and disrupt the activating bond network. Both conformational states have distinct hydrophobic advantages through context-specific hydrophobic interactions. We show that the functional (activating) asymmetric kinase dimer interface forces a corresponding change in the hydrophobic and hydrophilic interactions that characterize the inactivating bond network, resulting in motion of the αC-helix through allostery. Several of the clinically identified activating kinase mutations of EGFR act in a similar fashion to disrupt the inactivating bond network. The present molecular dynamics study reveals a fundamental difference in the sequence of events in EGFR activation compared with that described for the Src kinase Hck.

  15. Direct interaction between sensor kinase proteins mediates acute and chronic disease phenotypes in a bacterial pathogen

    PubMed Central

    Goodman, Andrew L.; Merighi, Massimo; Hyodo, Mamoru; Ventre, Isabelle; Filloux, Alain; Lory, Stephen

    2009-01-01

    The genome of the opportunistic pathogen Pseudomonas aeruginosa encodes over 60 two-component sensor kinases and uses several (including RetS and GacS) to reciprocally regulate the production of virulence factors involved in the development of acute or chronic infections. We demonstrate that RetS modulates the phosphorylation state of GacS by a direct and specific interaction between these two membrane-bound sensors. The RetS–GacS interaction can be observed in vitro, in heterologous systems in vivo, and in P. aeruginosa. This function does not require the predicted RetS phosphorelay residues and provides a mechanism for integrating multiple signals without cross-phosphorylation from sensors to noncognate response regulators. These results suggest that multiple two-component systems found in a single bacterium can form multisensor signaling networks while maintaining specific phosphorelay pathways that remain insulated from detrimental cross-talk. PMID:19171785

  16. Interactions between beta-enolase and creatine kinase in the cytosol of skeletal muscle cells.

    PubMed Central

    Foucault, G; Vacher, M; Cribier, S; Arrio-Dupont, M

    2000-01-01

    We studied interactions in vivo between the cytosolic muscle isoform of creatine kinase (M-CK) and the muscle isoform of 2-phospho-D-glycerate hydrolyase (beta-enolase) in muscle sarcoplasm by incubating glycerol-skinned fibres with FITC-labelled beta-enolase in the presence or absence of free CK. A small amount of bound beta-enolase was observed in the presence of large concentrations of CK. The mobility of enolase was measured in cultured satellite cells by modulated-fringe-pattern photobleaching. FITC-labelled beta-enolase was totally mobile in both the presence and the absence of CK but its diffusion coefficient was slightly lower in the presence of CK. This suggests a weak interaction in vivo between enolase and CK. PMID:10657248

  17. Protein-Protein Interactions in the Yeast Pheromone Response Pathway: Ste5p Interacts with All Members of the Map Kinase Cascade

    PubMed Central

    Printen, J. A.; Sprague-Jr., G. F.

    1994-01-01

    We have used the two-hybrid system of Fields and Song to identify protein-protein interactions that occur in the pheromone response pathway of the yeast Saccharomyces cerevisiae. Pathway components Ste4p, Ste5p, Ste7p, Ste11p, Ste12p, Ste20p, Fus3p and Kss1p were tested in all pairwise combinations. All of the interactions we detected involved at least one member of the MAP kinase cascade that is a central element of the response pathway. Ste5p, a protein of unknown biochemical function, interacted with protein kinases that operate at each step of the MAP kinase cascade, specifically with Ste11p (an MEKK), Ste7p (an MEK), and Fus3p (a MAP kinase). This finding suggests that one role of Ste5p is to serve as a scaffold to facilitate interactions among members of the kinase cascade. In this role as facilitator, Ste5p may make both signal propagation and signal attenuation more efficient. Ste5p may also help minimize cross-talk with other MAP kinase cascades and thus ensure the integrity of the pheromone response pathway. We also found that both Ste11p and Ste7p interact with Fus3p and Kss1p. Finally, we detected an interaction between one of the MAP kinases, Kss1p, and a presumptive target, the transcription factor Ste12p. We failed to detect interactions of Ste4p or Ste20p with any other component of the response pathway. PMID:7851759

  18. [Interaction of 8-substituted derivatives and adenosine-3',5'-cyclophosphate esters with protein kinase from pig brain].

    PubMed

    Guliaev, N N; Tunitskaia, V L; Nesterova, M V; Mazurova, L A; Murtuzaev, I M

    1977-11-01

    A synthesis of previously unknown 8-substituted derivatives and alkyl esters of cyclic adenosine-3',5'-monophosphate, containing reactive groups, was carried out. The interaction of the compounds obtained with a homogeneous preparation of protein kinase from pig brain was studied. It was found that all compounds, with the exception of neutral esters of 3',5'-AMP, activate the enzyme and competitively inhibit 3H-labelled 3',5'-cAMP binding by the regulatory subunit of protein kinase. The activating effect and affinity of 8-(beta-aminoethylamino)-3',5'-cAMP for protein kinase was 10 times lower than that for 3',5'-cAMP and other 8-substituted derivatives of the cyclic nucleotide. It was found that 8-(N-chloroacetylaminoethylamino)-3',5'-cAMP interaction with the enzyme is of irreversible type, which suggest covalent blocking of the nucleophilic group of the 3',5'-cAMP binding site of protein kinase. The data obtained indicate that the 3',5'-cAMP molecule is bound to the regulatory site of protein kinase in the syn-conformation. The previously made assumption on the crucial importance of the negative charge in the 3',5'-cyclophosphate system for the interaction of cyclic AMP with the regulatory subunit of protein kinase has been thus confirmed.

  19. The tomato I gene for Fusarium wilt resistance encodes an atypical leucine-rich repeat receptor-like protein whose function is nevertheless dependent on SOBIR1 and SERK3/BAK1.

    PubMed

    Catanzariti, Ann-Maree; Do, Huong T T; Bru, Pierrick; de Sain, Mara; Thatcher, Louise F; Rep, Martijn; Jones, David A

    2017-03-01

    We have identified the tomato I gene for resistance to the Fusarium wilt fungus Fusarium oxysporum f. sp. lycopersici (Fol) and show that it encodes a membrane-anchored leucine-rich repeat receptor-like protein (LRR-RLP). Unlike most other LRR-RLP genes involved in plant defence, the I gene is not a member of a gene cluster and contains introns in its coding sequence. The I gene encodes a loopout domain larger than those in most other LRR-RLPs, with a distinct composition rich in serine and threonine residues. The I protein also lacks a basic cytosolic domain. Instead, this domain is rich in aromatic residues that could form a second transmembrane domain. The I protein recognises the Fol Avr1 effector protein, but, unlike many other LRR-RLPs, recognition specificity is determined in the C-terminal half of the protein by polymorphic amino acid residues in the LRRs just preceding the loopout domain and in the loopout domain itself. Despite these differences, we show that I/Avr1-dependent necrosis in Nicotiana benthamiana depends on the LRR receptor-like kinases (RLKs) SERK3/BAK1 and SOBIR1. Sequence comparisons revealed that the I protein and other LRR-RLPs involved in plant defence all carry residues in their last LRR and C-terminal LRR capping domain that are conserved with SERK3/BAK1-interacting residues in the same relative positions in the LRR-RLKs BRI1 and PSKR1. Tyrosine mutations of two of these conserved residues, Q922 and T925, abolished I/Avr1-dependent necrosis in N. benthamiana, consistent with similar mutations in BRI1 and PSKR1 preventing their interaction with SERK3/BAK1.

  20. SUMOylation regulates polo-like kinase 1-interacting checkpoint helicase (PICH) during mitosis.

    PubMed

    Sridharan, Vinidhra; Park, Hyewon; Ryu, Hyunju; Azuma, Yoshiaki

    2015-02-06

    Mitotic SUMOylation has an essential role in faithful chromosome segregation in eukaryotes, although its molecular consequences are not yet fully understood. In Xenopus egg extract assays, we showed that poly(ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that its enzymatic activity could be regulated by SUMOylation. To determine the molecular consequence of mitotic SUMOylation, we analyzed SUMOylated PARP1-specific binding proteins. We identified Polo-like kinase 1-interacting checkpoint helicase (PICH) as an interaction partner of SUMOylated PARP1 in Xenopus egg extract. Interestingly, PICH also bound to SUMOylated topoisomerase IIα (TopoIIα), a major centromeric small ubiquitin-like modifier (SUMO) substrate. Purified recombinant human PICH interacted with SUMOylated substrates, indicating that PICH directly interacts with SUMO, and this interaction is conserved among species. Further analysis of mitotic chromosomes revealed that PICH localized to the centromere independent of mitotic SUMOylation. Additionally, we found that PICH is modified by SUMO2/3 on mitotic chromosomes and in vitro. PICH SUMOylation is highly dependent on protein inhibitor of activated STAT, PIASy, consistent with other mitotic chromosomal SUMO substrates. Finally, the SUMOylation of PICH significantly reduced its DNA binding capability, indicating that SUMOylation might regulate its DNA-dependent ATPase activity. Collectively, our findings suggest a novel SUMO-mediated regulation of the function of PICH at mitotic centromeres.

  1. Interaction between two rice mitogen activated protein kinases and its possible role in plant defense

    PubMed Central

    2013-01-01

    Background The canonical mitogen activated protein kinase (MAPK) signaling pathway plays a vital role in carrying out the normal growth and development of the plant. The pathway, connecting the upstreams signal with the downstream target is considered to be linear, mostly starting with a MAPKKK and ending in a MAPK. Results Here we report a novel interaction between two rice MAPKs, OsMPK20-4 and OsMPK3 suggesting the complex nature of the pathway rather than a linear one at individual steps. The interaction between OsMPK20-4 and OsMPK3 found by yeast two-hybrid analysis was confirmed in planta by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) assays. The interaction is specific and is phosphorylation independent. The results suggest a role of the interaction between OsMPK20-4 and OsMPK3 in basic plant defense. Conclusions The current novel work showing the physical interaction between two plant MAPKs, OsMPK20-4 and OsMPK3 is the diversion from the dogma of a typical MAPK cascade thereby opening a new dimension to the MAPK signal transduction. PMID:23984709

  2. Enzyme- and transporter-mediated drug interactions with small molecule tyrosine kinase inhibitors.

    PubMed

    Shao, Jie; Markowitz, John S; Bei, Di; An, Guohua

    2014-12-01

    Among the novel and target-specific classes of anticancer drugs, small molecule tyrosine kinase inhibitors (TKIs) represent an extremely promising and rapidly expanding group. TKIs attack cancer-specific targets and therefore have a favorable safety profile. However, as TKIs are taken orally along with other medications on a daily basis, there is an elevated risk of potentially significant drug-drug interactions. Most TKIs are metabolized primarily through CYP3A4. In addition, many TKIs are also CYP3A4 inhibitors at the same time. In addition to drug metabolizing enzymes (DMEs), another determinant of TKI disposition are drug transporters. There is accumulating evidence showing that the majority of currently marketed TKIs interact with ATP-binding cassette transporters, particularly P-glycoprotein as well as Breast Cancer Resistance Protein and serve as both substrates and inhibitors. Considering the dual roles of TKIs on both DMEs and drug transporters, and the importance of these enzyme and transporters in drug disposition, the potential for enzyme- and transporter-mediated TKI-drug interactions in patients with cancer is an important consideration. This review provides a comprehensive overview of drug interactions with small molecule TKIs mediated by DMEs and drug transporters. The TKI-drug interactions with TKIs being victims and/or perpetrators are summarized.

  3. PfIRR Interacts with HrIGF-I and Activates the MAP-kinase and PI3-kinase Signaling Pathways to Regulate Glycogen Metabolism in Pinctada fucata

    PubMed Central

    Shi, Yu; He, Mao-xian

    2016-01-01

    The insulin-induced mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways are major intracellular signaling modules and conserved among eukaryotes that are known to regulate diverse cellular processes. However, they have not been investigated in the mollusk species Pinctada fucata. Here, we demonstrate that insulin-related peptide receptor of P. fucata (pfIRR) interacts with human recombinant insulin-like growth factor I (hrIGF-I), and stimulates the MAPK and PI3K signaling pathways in P. fucata oocytes. We also show that inhibition of pfIRR by the inhibitor PQ401 significantly attenuates the basal and hrIGF-I-induced phosphorylation of MAPK and PI3K/Akt at amino acid residues threonine 308 and serine 473. Furthermore, our experiments show that there is cross-talk between the MAPK and PI3K/Akt pathways, in which MAPK kinase positively regulates the PI3K pathway, and PI3K positively regulates the MAPK cascade. Intramuscular injection of hrIGF-I stimulates the PI3K and MAPK pathways to increase the expression of pfirr, protein phosphatase 1, glucokinase, and the phosphorylation of glycogen synthase, decreases the mRNA expression of glycogen synthase kinase-3 beta, decreases glucose levels in hemocytes, and increases glycogen levels in digestive glands. These results suggest that the MAPK and PI3K pathways in P. fucata transmit the hrIGF-I signal to regulate glycogen metabolism. PMID:26911653

  4. Diacylglycerol kinase-zeta localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins.

    PubMed

    Abramovici, Hanan; Hogan, Angela B; Obagi, Christopher; Topham, Matthew K; Gee, Stephen H

    2003-11-01

    Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-zeta (DGK-zeta), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-zeta form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-zeta MARCKS domain. DGK-zeta mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-zeta and regulating its activity. Consistent with a role in actin organization, DGK-zeta and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-zeta regulated its association with the cytoskeleton. In adult muscle, DGK-zeta was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-zeta was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-zeta. Together, our findings suggest syntrophins localize DGK-zeta signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-zeta may cause abnormal cytoskeletal changes that contribute to disease pathogenesis.

  5. Aurora-A kinase phosphorylation of Aurora-A kinase interacting protein (AIP) and stabilization of the enzyme-substrate complex.

    PubMed

    Katayama, Hiroshi; Sasai, Kaori; Czerniak, Bogdan A; Carter, Jennifer L; Sen, Subrata

    2007-12-01

    Aurora-A is an oncogenic kinase that plays essential roles in mitosis as well as cell survival. Aurora-A interacting protein (AIP) was identified as a negative regulator of Aurora-A with its ectopic over expression inducing destabilization of Aurora-A protein. Here we present evidence that in human cells, contrary to the earlier report, AIP functions in stabilizing rather than destabilizing Aurora-A. Furthermore, AIP is phosphorylated on Serine 70 by Aurora-A but not Aurora-B and expression of phosphorylation mimic mutant of AIP results in prolonged protein stability compared to unphosphorylatable mutant. We observed that when co-expressed with AIP, protein levels of both Aurora-A and Aurora-B are markedly elevated regardless of their kinase activities and phosphorylation state of AIP. Interaction of Aurora kinases with AIP is necessary for this elevated stability. This phenomenon is commonly detected in several human cancer cell lines used in this study. Depletion of AIP by RNA interference decreased Aurora-A but not Aurora-B in two of the three cell lines analyzed, indicating that under physiological condition, AIP functions in stabilization of Aurora-A but not Aurora-B, though this regulation may be dependent on additional factors as well. Further, AIP siRNA induced cell cycle arrest at G2/M, which is consistent with anticipated loss of function of Aurora-A in these cells. Thus, our study provides the first evidence of a role for AIP in G2/M cell cycle progression by cooperatively regulating protein stabilization of its up-stream regulator, Aurora-A kinase through protein-protein interaction as well as protein phosphorylation.

  6. Key Structures and Interactions for Binding of Mycobacterium tuberculosis Protein Kinase B Inhibitors from Molecular Dynamics Simulation.

    PubMed

    Punkvang, Auradee; Kamsri, Pharit; Saparpakorn, Patchreenart; Hannongbua, Supa; Wolschann, Peter; Irle, Stephan; Pungpo, Pornpan

    2015-07-01

    Substituted aminopyrimidine inhibitors have recently been introduced as antituberculosis agents. These inhibitors show impressive activity against protein kinase B, a Ser/Thr protein kinase that is essential for cell growth of M. tuberculosis. However, up to now, X-ray structures of the protein kinase B enzyme complexes with the substituted aminopyrimidine inhibitors are currently unavailable. Consequently, structural details of their binding modes are questionable, prohibiting the structural-based design of more potent protein kinase B inhibitors in the future. Here, molecular dynamics simulations, in conjunction with molecular mechanics/Poisson-Boltzmann surface area binding free-energy analysis, were employed to gain insight into the complex structures of the protein kinase B inhibitors and their binding energetics. The complex structures obtained by the molecular dynamics simulations show binding free energies in good agreement with experiment. The detailed analysis of molecular dynamics results shows that Glu93, Val95, and Leu17 are key residues responsible to the binding of the protein kinase B inhibitors. The aminopyrazole group and the pyrimidine core are the crucial moieties of substituted aminopyrimidine inhibitors for interaction with the key residues. Our results provide a structural concept that can be used as a guide for the future design of protein kinase B inhibitors with highly increased antagonistic activity.

  7. Homeodomain-interacting protein kinase 2 plays an important role in normal terminal erythroid differentiation.

    PubMed

    Hattangadi, Shilpa M; Burke, Karly A; Lodish, Harvey F

    2010-06-10

    Gene-targeting experiments report that the homeodomain-interacting protein kinases 1 and 2, Hipk1 and Hipk2, are essential but redundant in hematopoietic development because Hipk1/Hipk2 double-deficient animals exhibit severe defects in hematopoiesis and vasculogenesis, whereas the single knockouts do not. These serine-threonine kinases phosphorylate and consequently modify the functions of several important hematopoietic transcription factors and cofactors. Here we show that Hipk2 knockdown alone plays a significant role in terminal fetal liver erythroid differentiation. Hipk1 and Hipk2 are highly induced during primary mouse fetal liver erythropoiesis. Specific knockdown of Hipk2 inhibits terminal erythroid cell proliferation (explained in part by impaired cell-cycle progression as well as increased apoptosis) and terminal enucleation as well as the accumulation of hemoglobin. Hipk2 knockdown also reduces the transcription of many genes involved in proliferation and apoptosis as well as important, erythroid-specific genes involved in hemoglobin biosynthesis, such as alpha-globin and mitoferrin 1, demonstrating that Hipk2 plays an important role in some but not all aspects of normal terminal erythroid differentiation.

  8. Polo kinase interacts with RacGAP50C and is required to localize the cytokinesis initiation complex.

    PubMed

    Ebrahimi, Saman; Fraval, Hamilton; Murray, Michael; Saint, Robert; Gregory, Stephen L

    2010-09-10

    The assembly and constriction of an actomyosin contractile ring in cytokinesis is dependent on the activation of Rho at the equatorial cortex by a complex, here termed the cytokinesis initiation complex, between a microtubule-associated kinesin-like protein (KLP), a member of the RacGAP family, and the RhoGEF Pebble. Recently, the activity of the mammalian Polo kinase ortholog Plk1 has been implicated in the formation of this complex. We show here that Polo kinase interacts directly with the cytokinesis initiation complex by binding RacGAP50C. We find that a new domain of Polo kinase, termed the intermediate domain, interacts directly with RacGAP50C and that Polo kinase is essential for localization of the KLP-RacGAP centralspindlin complex to the cell equator and spindle midzone. In the absence of Polo kinase, RacGAP50C and Pav-KLP fail to localize normally, instead decorating microtubules along their length. Our results indicate that Polo kinase directly binds the conserved cytokinesis initiation complex and is required to trigger centralspindlin localization as a first step in cytokinesis.

  9. LRRK2 kinase activity mediates toxic interactions between genetic mutation and oxidative stress in a Drosophila model: suppression by curcumin.

    PubMed

    Yang, Dejun; Li, Tianxia; Liu, Zhaohui; Arbez, Nicolas; Yan, Jianqun; Moran, Timothy H; Ross, Christopher A; Smith, Wanli W

    2012-09-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. The pathogenesis of PD is believed to involve both genetic susceptibility and environmental factors. Mutations in Leucine-rich repeat kinase 2 (LRRK2) cause genetic forms of PD, and the LRRK2 locus contributes to sporadic PD. Environmental toxins are believed to act in part by causing oxidative stress. Here we employed cell and Drosophila models to investigate the interaction between LRRK2 genetic mutations and oxidative stress. We found that H(2)O(2) increased LRRK2 kinase activity and enhanced LRRK2 cell toxicity in cultured cells and mouse primary cortical neurons. Furthermore, a sub-toxic dose of H(2)O(2) significantly shortened the survival of LRRK2 transgenic flies and augmented LRRK2-induced locomotor defects and dopamine neuron loss. Treatment with a LRRK2 kinase inhibitor (GW5074) or an anti-oxidant (curcumin) significantly suppressed these PD-like phenotypes in flies. Moreover, curcumin significantly reduced LRRK2 kinase activity and the levels of oxidized proteins, and thus acted as not only an antioxidant but also a LRRK2 kinase inhibitor. These results indicate that LRRK2 genetic alterations can interact with oxidative stress, converging on a pathogenic pathway that may be related to PD. These studies also identified curcumin as a LRRK2 kinase inhibitor that may be a useful candidate for LRRK2-linked PD intervention.

  10. Homeodomain-interacting protein kinase 2 regulates DNA damage response through interacting with heterochromatin protein 1γ.

    PubMed

    Akaike, Y; Kuwano, Y; Nishida, K; Kurokawa, K; Kajita, K; Kano, S; Masuda, K; Rokutan, K

    2015-06-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is a potential tumor suppressor that has a crucial role in the DNA damage response (DDR) by regulating cell-cycle checkpoint activation and apoptosis. However, it is unclear whether HIPK2 exerts distinct roles in DNA damage repair. The aim of this study was to identify novel target molecule(s) of HIPK2, which mediates HIPK2-dependent DNA damage repair. HIPK2-knockdown human colon cancer cells (HCT116) or hipk1/hipk2 double-deficient mouse embryonic fibroblasts could not remove histone H2A.X phosphorylated at Ser139 (γH2A.X) after irradiation with a sublethal dose (10 J/m(2)) of ultraviolet (UV)-C, resulting in apoptosis. Knockdown of HIPK2 in p53-null HCT116 cells similarly promoted the UV-C-induced γH2A.X accumulation and apoptosis. Proteomic analysis of HIPK2-associated proteins using liquid chromatography-tandem mass spectrometry identified heterochromatin protein 1γ (HP1γ) as a novel target for HIPK2. Immunoprecipitation experiments with HCT116 cells expressing FLAG-tagged HIPK2 and one of the HA-tagged HP1 family members demonstrated that HIPK2 specifically associated with HP1γ, but not with HP1α or HP1β, through its chromo-shadow domain. Mutation of the HP1box motif (883-PTVSV-887) within HIPK2 abolished the association. HP1γ knockdown also enhanced accumulation of γH2A.X and apoptosis after sublethal UV-C irradiation. In vitro kinase assay demonstrated an HP1γ-phosphorylating activity of HIPK2. Sublethal UV-C irradiation phosphorylated HP1γ. This phosphorylation was absent in endogenous HIPK2-silenced cells with HIPK2 3'UTR siRNA. Overexpression of FLAG-HIPK2, but not the HP1box-mutated or kinase-dead HIPK2 mutant, in the HIPK2-silenced cells increased HP1γ binding to trimethylated (Lys9) histone H3 (H3K9me3), rescued the UV-C-induced phosphorylation of HP1γ, triggered release of HP1γ from histone H3K9me3 and suppressed γH2A.X accumulation. Our results suggest that HIPK2-dependent

  11. Interaction between protein kinase Cmu and the vanilloid receptor type 1.

    PubMed

    Wang, Yun; Kedei, Noemi; Wang, Min; Wang, Q Jane; Huppler, Anna R; Toth, Attila; Tran, Richard; Blumberg, Peter M

    2004-12-17

    The capsaicin receptor VR1 is a polymodal nociceptor activated by multiple stimuli. It has been reported that protein kinase C plays a role in the sensitization of VR1. Protein kinase D/PKCmu is a member of the protein kinase D serine/threonine kinase family that exhibits structural, enzymological, and regulatory features distinct from those of the PKCs, with which they are related. As part of our effort to optimize conditions for evaluating VR1 pharmacology, we found that treatment of Chinese hamster ovary (CHO) cells heterologously expressing rat VR1 (CHO/rVR1) with butyrate enhanced rVR1 expression and activity. The expression of PKCmu and PKCbeta1, but not of other PKC isoforms, was also enhanced by butyrate treatment, suggesting the possibility that these two isoforms might contribute to the enhanced activity of rVR1. In support of this hypothesis, we found the following. 1) Overexpression of PKCmu enhanced the response of rVR1 to capsaicin and low pH, and expression of a dominant negative variant of PKCmu reduced the response of rVR1. 2) Reduction of endogenous PKCmu using antisense oligonucleotides decreased the response of exogenous rVR1 expressed in CHO cells as well as of endogenous rVR1 in dorsal root ganglion neurons. 3) PKCmu localized to the plasma membrane when overexpressed in CHO/rVR1 cells. 4) PKCmu directly bound to rVR1 expressed in CHO cells as well as to endogenous rVR1 in dorsal root ganglia or to an N-terminal fragment of rVR1, indicating a direct interaction between PKCmu and rVR1. 5) PKCmu directly phosphorylated rVR1 or a longer N-terminal fragment (amino acids 1-118) of rVR1 but not a shorter one (amino acids 1-99). 6) Mutation of S116A in rVR1 blocked both the phosphorylation of rVR1 by PKCmu and the enhancement by PKCmu of the rVR1 response to capsaicin. We conclude that PKCmu functions as a direct modulator of rVR1.

  12. Arabidopsis CBL interacting protein kinase 3 interacts with ABR1, an APETALA2 domain transcription factor, to regulate ABA responses.

    PubMed

    Sanyal, Sibaji K; Kanwar, Poonam; Yadav, Akhilesh K; Sharma, Cheshta; Kumar, Ashish; Pandey, Girdhar K

    2017-01-01

    Calcium (Ca(2+)) plays a vital role as a second messenger in several signaling pathways in plants. The calcineurin B-like proteins (CBLs) represent a family of plant calcium-binding proteins that function in propagating Ca(2+) signals by interacting with CBL interacting protein kinases (CIPKs). Phosphorylation of CBL by CIPK is essential for the module to display full activity towards its target protein. Previous genetic analysis showed that the function of CBL9-CIPK3 module was implicated in negatively regulating seed germination and early development. In the present study, we have biochemically investigated the interaction of CBL9-CIPK3 module and our findings show that CBL9 is phosphorylated by CIPK3. Moreover, Abscisic acid repressor 1 (ABR1) is identified as the downstream target of CIPK3 and CIPK3-ABR1 function to regulate ABA responses during seed germination. Our study also indicates that the role of ABR1 is not limited to seed germination but it also regulates the ABA dependent processes in the adult stage of plant development. Combining our results, we conclude that the CBL9-CIPK3-ABR1 pathway functions to regulate seed germination and ABA dependent physiological processes in Arabidopsis.

  13. Aurora kinase A interacts with H-Ras and potentiates Ras-MAPK signaling | Office of Cancer Genomics

    Cancer.gov

    In cancer, upregulated Ras promotes cellular transformation and proliferation in part through activation of oncogenic Ras-MAPK signaling. While directly inhibiting Ras has proven challenging, new insights into Ras regulation through protein-protein interactions may offer unique opportunities for therapeutic intervention. Here we report the identification and validation of Aurora kinase A (Aurora A) as a novel Ras binding protein. We demonstrate that the kinase domain of Aurora A mediates the interaction with the N-terminal domain of H-Ras.

  14. Characterization of the interactions between the active site of a protein tyrosine kinase and a divalent metal activator

    PubMed Central

    Lin, Xiaofeng; Ayrapetov, Marina K; Sun, Gongqin

    2005-01-01

    Background Protein tyrosine kinases are important enzymes for cell signalling and key targets for anticancer drug discovery. The catalytic mechanisms of protein tyrosine kinase-catalysed phosphorylation are not fully understood. Protein tyrosine kinase Csk requires two Mg2+ cations for activity: one (M1) binds to ATP, and the other (M2) acts as an essential activator. Results Experiments in this communication characterize the interaction between M2 and Csk. Csk activity is sensitive to pH in the range of 6 to 7. Kinetic characterization indicates that the sensitivity is not due to altered substrate binding, but caused by the sensitivity of M2 binding to pH. Several residues in the active site with potential of binding M2 are mutated and the effect on metal activation studied. An active mutant of Asn319 is generated, and this mutation does not alter the metal binding characteristics. Mutations of Glu236 or Asp332 abolish the kinase activity, precluding a positive or negative conclusion on their role in M2 coordination. Finally, the ability of divalent metal cations to activate Csk correlates to a combination of ionic radius and the coordination number. Conclusion These studies demonstrate that M2 binding to Csk is sensitive to pH, which is mainly responsible for Csk activity change in the acidic arm of the pH response curve. They also demonstrate critical differences in the metal activator coordination sphere in protein tyrosine kinase Csk and a protein Ser/Thr kinase, the cAMP-dependent protein kinase. They shed light on the physical interactions between a protein tyrosine kinase and a divalent metal activator. PMID:16305747

  15. Implications of promiscuous Pim-1 kinase fragment inhibitor hydrophobic interactions for fragment-based drug design.

    PubMed

    Good, Andrew C; Liu, Jinyu; Hirth, Bradford; Asmussen, Gary; Xiang, Yibin; Biemann, Hans-Peter; Bishop, Kimberly A; Fremgen, Trisha; Fitzgerald, Maria; Gladysheva, Tatiana; Jain, Annuradha; Jancsics, Katherine; Metz, Markus; Papoulis, Andrew; Skerlj, Renato; Stepp, J David; Wei, Ronnie R

    2012-03-22

    We have studied the subtleties of fragment docking and binding using data generated in a Pim-1 kinase inhibitor program. Crystallographic and docking data analyses have been undertaken using inhibitor complexes derived from an in-house surface plasmon resonance (SPR) fragment screen, a virtual needle screen, and a de novo designed fragment inhibitor hybrid. These investigations highlight that fragments that do not fill their binding pocket can exhibit promiscuous hydrophobic interactions due to the lack of steric constraints imposed on them by the boundaries of said pocket. As a result, docking modes that disagree with an observed crystal structure but maintain key crystallographically observed hydrogen bonds still have potential value in ligand design and optimization. This observation runs counter to the lore in fragment-based drug design that all fragment elaboration must be based on the parent crystal structure alone.

  16. Disruptions in asymmetric centrosome inheritance and WDR62-Aurora kinase B interactions in primary microcephaly

    PubMed Central

    Sgourdou, Paraskevi; Mishra-Gorur, Ketu; Saotome, Ichiko; Henagariu, Octavian; Tuysuz, Beyhan; Campos, Cynthia; Ishigame, Keiko; Giannikou, Krinio; Quon, Jennifer L.; Sestan, Nenad; Caglayan, Ahmet O.; Gunel, Murat; Louvi, Angeliki

    2017-01-01

    Recessive mutations in WD repeat domain 62 (WDR62) cause microcephaly and a wide spectrum of severe brain malformations. Disruption of the mouse ortholog results in microcephaly underlain by reduced proliferation of neocortical progenitors during late neurogenesis, abnormalities in asymmetric centrosome inheritance leading to neuronal migration delays, and altered neuronal differentiation. Spindle pole localization of WDR62 and mitotic progression are defective in patient-derived fibroblasts, which, similar to mouse neocortical progenitors, transiently arrest at prometaphase. Expression of WDR62 is closely correlated with components of the chromosome passenger complex (CPC), a key regulator of mitosis. Wild type WDR62, but not disease-associated mutant forms, interacts with the CPC core enzyme Aurora kinase B and staining of CPC components at centromeres is altered in patient-derived fibroblasts. Our findings demonstrate critical and diverse functions of WDR62 in neocortical development and provide insight into the mechanisms by which its disruption leads to a plethora of structural abnormalities. PMID:28272472

  17. Receptor protein kinase FERONIA controls leaf starch accumulation by interacting with glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Yang, Tao; Wang, Long; Li, Chiyu; Liu, Ying; Zhu, Sirui; Qi, Yinyao; Liu, Xuanming; Lin, Qinglu; Luan, Sheng; Yu, Feng

    2015-09-11

    Cell expansion is coordinated by several cues, but available energy is the major factor determining growth. Receptor protein kinase FERONIA (FER) is a master regulator of cell expansion, but the details of its control mechanisms are not clear. Here we show that FER interacts with cytosolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GAPC1 and GAPC2), that catalyzes a key reaction in glycolysis, which contributes to energy production. When there is an FER deficiency, there are corresponding decreases in the enzyme activity of GAPDH and increased amounts of starch. More importantly, gapc1/2 mutants mimic fer4 mutants. These data indicate that FER regulated starch content is an evolutionarily conserved function in plants that connects the cell expansion and energy metabolism pathways.

  18. Heart failure biomarkers: focus on interleukin-1 receptor-like 1-based blood tests.

    PubMed

    Broch, K; Ueland, T; Yndestad, A; Aukrust, P; Gullestad, L

    2012-07-01

    Heart failure is a leading cause of morbidity and mortality in the Western world. It is often a progressive disease, and the pathophysiology behind this adverse development is not completely understood. Biomarkers are of increasing importance in heart failure research. Despite a growing number of candidate markers, only a select few have made it into clinical practice. Interleukin-1 receptor-like 1 (IL1RL1), also known as protein ST2, is the receptor for interleukin-33 (IL-33), a cytokine involved in T-cell-mediated immune responses. IL1RL1 expression is induced by cardiomyocyte stretch, and IL1RL1 may thus reflect the activity of two interacting processes in heart failure: inflammation and hemodynamic stress. In recent years, the soluble, truncated IL1RL1 isoform B has been shown to provide prognostic information in heart failure. Although ILRL1 isoform B does not seem to aid in the diagnosis of the disease, an elevated plasma/serum concentration of this marker is firmly associated with adverse outcome in patients with heart failure. This association has been established in different heart failure cohorts and is independent of age, etiology of heart failure and left ventricular function. Ultimately, the IL-33/IL1RL1 pathway may become a therapeutic target in heart failure.

  19. Conformational instability of the MARK3 UBA domain compromises ubiquitin recognition and promotes interaction with the adjacent kinase domain

    SciTech Connect

    Murphy, James M.; Korzhnev, Dmitry M.; Ceccarelli, Derek F.; Briant, Douglas J.; Zarrine-Afsar, Arash; Sicheri, Frank; Kay, Lewis E.; Pawson, Tony

    2012-10-23

    The Par-1/MARK protein kinases play a pivotal role in establishing cellular polarity. This family of kinases contains a unique domain architecture, in which a ubiquitin-associated (UBA) domain is located C-terminal to the kinase domain. We have used a combination of x-ray crystallography and NMR dynamics experiments to understand the interaction of the human (h) MARK3 UBA domain with the adjacent kinase domain as compared with ubiquitin. The x-ray crystal structure of the linked hMARK3 kinase and UBA domains establishes that the UBA domain forms a stable intramolecular interaction with the N-terminal lobe of the kinase domain. However, solution-state NMR studies of the isolated UBA domain indicate that it is highly dynamic, undergoing conformational transitions that can be explained by a folding-unfolding equilibrium. NMR titration experiments indicated that the hMARK3 UBA domain has a detectable but extremely weak affinity for mono ubiquitin, which suggests that conformational instability of the isolated hMARK3 UBA domain attenuates binding to ubiquitin despite the presence of residues typically involved in ubiquitin recognition. Our data identify a molecular mechanism through which the hMARK3 UBA domain has evolved to bind the kinase domain, in a fashion that stabilizes an open conformation of the N- and C-terminal lobes, at the expense of its capacity to engage ubiquitin. These results may be relevant more generally to the 30% of UBA domains that lack significant ubiquitin-binding activity, and they suggest a unique mechanism by which interaction domains may evolve new binding properties.

  20. Activation of the Stt7/STN7 Kinase through Dynamic Interactions with the Cytochrome b6f Complex1[OPEN

    PubMed Central

    Shapiguzov, Alexey; Chai, Xin; Fucile, Geoffrey; Longoni, Paolo; Zhang, Lixin

    2016-01-01

    Photosynthetic organisms have the ability to adapt to changes in light quality by readjusting the cross sections of the light-harvesting systems of photosystem II (PSII) and photosystem I (PSI). This process, called state transitions, maintains the redox poise of the photosynthetic electron transfer chain and ensures a high photosynthetic yield when light is limiting. It is mediated by the Stt7/STN7 protein kinase, which is activated through the cytochrome b6f complex upon reduction of the plastoquinone pool. Its probable major substrate, the light-harvesting complex of PSII, once phosphorylated, dissociates from PSII and docks to PSI, thereby restoring the balance of absorbed light excitation energy between the two photosystems. Although the kinase is known to be inactivated under high-light intensities, the molecular mechanisms governing its regulation remain unknown. In this study we monitored the redox state of a conserved and essential Cys pair of the Stt7/STN7 kinase and show that it forms a disulfide bridge. We could not detect any change in the redox state of these Cys during state transitions and high-light treatment. It is only after prolonged anaerobiosis that this disulfide bridge is reduced. It is likely to be mainly intramolecular, although kinase activation may involve a transient covalently linked kinase dimer with two intermolecular disulfide bonds. Using the yeast two-hybrid system, we have mapped one interaction site of the kinase on the Rieske protein of the cytochrome b6f complex. PMID:26941194

  1. Multiple orphan histidine kinases interact directly with Spo0A to control the initiation of endospore formation in Clostridium acetobutylicum

    PubMed Central

    Steiner, Elisabeth; Dago, Angel E.; Young, Danielle I.; Heap, John T.; Minton, Nigel P.; Hoch, James A.

    2011-01-01

    The phosphorylated Spo0A transcription factor controls the initiation of endospore formation in Clostridium acetobutylicum, but genes encoding key phosphorelay components, Spo0F and Spo0B, are missing in the genome. We hypothesized that the five orphan histidine kinases of C. acetobutylicum interact directly with Spo0A to control its phosphorylation state. Sequential targeted gene disruption and gene expression profiling provided evidence for two pathways for Spo0A activation, one dependent on a histidine kinase encoded by cac0323, the other on both histidine kinases encoded by cac0903 and cac3319. Purified Cac0903 and Cac3319 kinases autophosphorylated and transferred phosphoryl groups to Spo0A in vitro, confirming their role in Spo0A activation in vivo. A cac0437 mutant hyper-sporulated, suggesting that Cac0437 is a modulator that prevents sporulation and maintains cellular Spo0A~P homeostasis during growth. Accordingly, Cac0437 has apparently lost the ability to autophosphorylate in vitro; instead it catalyses the ATP-dependent dephosphorylation of Spo0A~P releasing inorganic phosphate. Direct phosphorylation of Spo0A by histidine kinases and dephosphorylation by kinase-like proteins may be a common feature of the clostridia that may represent the ancestral state before the great oxygen event some 2.4 billion years ago, after which additional phosphorelay proteins were recruited in the evolutionary lineage that led to the bacilli. PMID:21401736

  2. Actin interaction and regulation of cyclin-dependent kinase 5/p35 complex activity.

    PubMed

    Xu, Jiqing; Tsutsumi, Koji; Tokuraku, Kiyotaka; Estes, Katherine A; Hisanaga, Shin-ichi; Ikezu, Tsuneya

    2011-01-01

    Cyclin-dependent kinase 5 (Cdk5) plays a critical role during neurodevelopment, synaptic plasticity, and neurodegeneration. Cdk5 activity depends on association with neuronal proteins p35 and p25, a proteolytic product of p35. Cdk5 regulates the actin cytoskeletal dynamics that are essential for neuronal migration, neuritic growth, and synaptogenesis. However, little is known about the interaction of actin and Cdk5 and its effect on neuronal Cdk5 activity. In a previous study, we observed that Cdk5/p35 activity is negatively correlated with co-immunoprecipitated F-actin (filamentous actin) amounts in the mouse brain, and suggested that F-actin inhibits the formation of the Cdk5/p35 complex [Journal of Neuroscience (2008) vol. 28, p. 14511]. The experiments reported here were undertaken to elucidate the relationship between actin and the formation of the Cdk5/p35 complex and its activity. Instead of an F-actin-mediated inhibition, we propose that G-actin (globular actin) in the F-actin preparations is responsible for inhibiting Cdk5/p35 and Cdk5/p25 kinase activity. We found that F-actin binds to p35 but not p25 or Cdk5. We have shown that G-actin binds directly to Cdk5 without disrupting the formation of the Cdk5/p35 or Cdk5/p25 complexes. G-actin potently suppressed Cdk5/p35 and Cdk5/p25 activity when either histone H1 or purified human tau protein were used as substrates, indicating a substrate-independent inhibitory effect of G-actin on Cdk5 activity. Finally, G-actin suppressed the activity of Cdk5 immunoprecipitated from wild type and p35-deficient mouse brain, suggesting that G-actin suppresses endogenous Cdk5 activity in a p35-independent manner. Together, these results suggest a novel mechanism of actin cytoskeletal regulation of Cdk5/p35 activity.

  3. Regulation of glycine receptor diffusion properties and gephyrin interactions by protein kinase C

    PubMed Central

    Specht, Christian G; Grünewald, Nora; Pascual, Olivier; Rostgaard, Nina; Schwarz, Günter; Triller, Antoine

    2011-01-01

    Glycine receptors (GlyRs) can dynamically exchange between synaptic and extrasynaptic locations through lateral diffusion within the plasma membrane. Their accumulation at inhibitory synapses depends on the interaction of the β-subunit of the GlyR with the synaptic scaffold protein gephyrin. An alteration of receptor–gephyrin binding could thus shift the equilibrium between synaptic and extrasynaptic GlyRs and modulate the strength of inhibitory neurotransmission. Using a combination of dynamic imaging and biochemical approaches, we have characterised the molecular mechanism that links the GlyR–gephyrin interaction with GlyR diffusion and synaptic localisation. We have identified a protein kinase C (PKC) phosphorylation site within the cytoplasmic domain of the β-subunit of the GlyR (residue S403) that causes a reduction of the binding affinity between the receptor and gephyrin. In consequence, the receptor's diffusion in the plasma membrane is accelerated and GlyRs accumulate less strongly at synapses. We propose that the regulation of GlyR dynamics by PKC thus contributes to the plasticity of inhibitory synapses and may be involved in maladaptive forms of synaptic plasticity. PMID:21829170

  4. Wheat CBL-interacting protein kinase 25 negatively regulates salt tolerance in transgenic wheat

    PubMed Central

    Jin, Xia; Sun, Tao; Wang, Xiatian; Su, Peipei; Ma, Jingfei; He, Guangyuan; Yang, Guangxiao

    2016-01-01

    CBL-interacting protein kinases are involved in plant responses to abiotic stresses, including salt stress. However, the negative regulating mechanism of this gene family in response to salinity is less reported. In this study, we evaluated the role of TaCIPK25 in regulating salt response in wheat. Under conditions of high salinity, TaCIPK25 expression was markedly down-regulated in roots. Overexpression of TaCIPK25 resulted in hypersensitivity to Na+ and superfluous accumulation of Na+ in transgenic wheat lines. TaCIPK25 expression did not decline in transgenic wheat and remained at an even higher level than that in wild-type wheat controls under high-salinity treatment. Furthermore, transmembrane Na+/H+ exchange was impaired in the root cells of transgenic wheat. These results suggested that TaCIPK25 negatively regulated salt response in wheat. Additionally, yeast-one-hybrid, β-glucuronidase activity and DNA-protein-interaction-enzyme-linked-immunosorbent assays showed that the transcription factor TaWRKY9 bound W-box in the TaCIPK25 promoter region. Quantitative real-time polymerase chain reaction assays showed concomitantly inverted expression patterns of TaCIPK25 and TaWRKY9 in wheat roots under salt treatment, ABA application and inhibition of endogenous ABA condition. Overall, based on our results, in a salt stress condition, the negative salt response in wheat involved TaCIPK25 with the expression regulated by TaWRKY9. PMID:27358166

  5. Receptor-interacting protein kinase 3 promotes platelet activation and thrombosis.

    PubMed

    Zhang, Yiwen; Zhang, Jian; Yan, Rong; Tian, Jingluan; Zhang, Yang; Zhang, Jie; Chen, Mengxing; Cui, Qingya; Zhao, Lili; Hu, Renping; Jiang, Miao; Li, Zhenyu; Ruan, Changgeng; He, Sudan; Dai, Kesheng

    2017-03-14

    Previous studies have shown that receptor-interacting protein kinase 3 (RIP3) is involved in many important biological processes, including necroptosis, apoptosis, and inflammation. Here we show that RIP3 plays a critical role in regulating platelet functions and in vivo thrombosis and hemostasis. Tail bleeding times were significantly longer in RIP3-knockout (RIP3(-/-)) mice compared with their wild-type (WT) littermates. In an in vivo model of arteriole thrombosis, mice lacking RIP3 exhibited prolonged occlusion times. WT mice repopulated with RIP3(-/-) bone marrow-derived cells had longer occlusion times than RIP3(-/-) mice repopulated with WT bone marrow-derived cells, suggesting a role for RIP3-deficient platelets in arterial thrombosis. Consistent with these findings, we observed that RIP3 was expressed in both human and mice platelets. Deletion of RIP3 in mouse platelets caused a marked defect in aggregation and attenuated dense granule secretion in response to low doses of thrombin or a thromboxane A2 analog, U46619. Phosphorylation of Akt induced by U46619 or thrombin was diminished in RIP3(-/-) platelets. Moreover, RIP3 interacted with Gα13 Platelet spreading on fibrinogen and clot retraction were impaired in the absence of RIP3. RIP3 inhibitor dose-dependently inhibited platelet aggregation in vitro and prevented arterial thrombus formation in vivo. These data demonstrate a role for RIP3 in promoting in vivo thrombosis and hemostasis by amplifying platelet activation. RIP3 may represent a novel promising therapeutic target for thrombotic diseases.

  6. Interactions between double-stranded RNA regulators and the protein kinase DAI.

    PubMed Central

    Manche, L; Green, S R; Schmedt, C; Mathews, M B

    1992-01-01

    The interferon-induced protein kinase DAI, the double-stranded RNA (dsRNA)-activated inhibitor of translation, plays a key role in regulating protein synthesis in higher cells. Once activated, in a process that involves autophosphorylation, it phosphorylates the initiation factor eIF-2, leading to inhibition of polypeptide chain initiation. The activity of DAI is controlled by RNA regulators, including dsRNA activators and highly structured single-stranded RNAs which block activation by dsRNA. To elucidate the mechanism of activation, we studied the interaction of DAI with RNA duplexes of discrete sizes. Molecules shorter than 30 bp fail to bind stably and do not activate the enzyme, but at high concentrations they prevent activation by long dsRNA. Molecules longer than 30 bp bind and activate the enzyme, with an efficiency that increases with increasing chain length, reaching a maximum at about 85 bp. These dsRNAs fail to activate at high concentrations and also prevent activation by long dsRNA. Analysis of complexes between dsRNA and DAI suggests that at maximal packing the enzyme interacts with as little as a single helical turn of dsRNA (11 bp) but under conditions that allow activation the binding site protects about 80 bp of duplex. When the RNA-binding site is fully occupied with an RNA activator, the complex appears to undergo a conformational change. Images PMID:1357546

  7. Specific interactions among transmembrane 4 superfamily (TM4SF) proteins and phosphoinositide 4-kinase.

    PubMed Central

    Yauch, R L; Hemler, M E

    2000-01-01

    In earlier work we established that phosphoinositide 4-kinase (PI 4-kinase) may associate with transmembrane 4 superfamily (TM4SF, tetraspanin) proteins, but critical specificity issues were not addressed. Here we demonstrate that at least five different TM4SF proteins (CD9, CD63, CD81, CD151 and A15/TALLA1) can associate with a similar or identical 55 kDa type II PI 4-kinase. These associations were specific, since we found no evidence for other phosphoinositide kinases (e.g. phosphoinositide 3-kinase and phosphoinositide-4-phosphate 5-kinase) associating with TM4SF proteins, and many other TM4SF proteins (including CD82 and CD53) did not associate with PI 4-kinase. CD63-PI 4-kinase complexes were almost entirely intracellular, and thus are distinct from other TM4SF-PI 4-kinase complexes (e.g. involving CD9), which are largely located in the plasma membrane. These results suggest that a specific subset of TM4SF proteins may recruit PI 4-kinase to specific membrane locations, and thereby influence phosphoinositide-dependent signalling. PMID:11042117

  8. The Arabidopsis SERK1 protein interacts with the AAA-ATPase AtCDC48, the 14-3-3 protein GF14lambda and the PP2C phosphatase KAPP.

    PubMed

    Rienties, Ingrid M; Vink, Josefien; Borst, Jan Willem; Russinova, Eugenia; de Vries, Sacco C

    2005-06-01

    Leucine-rich repeat (LRR)-containing transmembrane receptor-like kinases (RLKs) are important components of plant signal transduction. The Arabidopsis thaliana somatic embryogenesis receptor-like kinase 1 (AtSERK1) is an LRR-RLK proposed to participate in a signal transduction cascade involved in embryo development. By yeast two-hybrid screening we identified AtCDC48, a homologue of the mammalian AAA-ATPase p97 and GF14lambda, a member of the Arabidopsis family of 14-3-3 proteins as AtSERK1 interactors. In vitro, the AtSERK1 kinase domain is able to transphosphorylate and bind both AtCDC48 and GF14lambda. In yeast, AtCDC48 interacts with GF14lambda and with the PP2C phosphatase KAPP. In plant protoplasts AtSERK1 interacts with GF14lambda.

  9. Identification of an interaction between EI and a histidine kinase-response regulator hybrid protein in Gluconobacter oxydans.

    PubMed

    Li, Shan; Ma, Yushu; Wei, Dongzhi

    2016-02-05

    Gluconobacter oxydans may contain an incomplete phosphoenolpyruvate: carbohydrate phosphotransferase system consisting of three components--EI, HPr and EIIA, while the function of individual members of the system remains unknown. In this research, a specific interaction between EI and a histidine kinase-response regulator hybrid protein was screened by yeast two-hybrid assay, and the interaction was further identified with GST pull-down assay and bimolecular fluorescence complementation assay in vitro and in vivo, respectively. As the histidine kinase-response regulator hybrid protein serves as a member of two-component system in G. oxydans, its interaction with EI implied that PTS may play certain roles in bacteria under stress.

  10. Cross-interactions of two p38 mitogen-activated protein (MAP) kinase inhibitors and two cholecystokinin (CCK) receptor antagonists with the CCK1 receptor and p38 MAP kinase.

    PubMed

    Morel, Caroline; Ibarz, Géraldine; Oiry, Catherine; Carnazzi, Eric; Bergé, Gilbert; Gagne, Didier; Galleyrand, Jean-Claude; Martinez, Jean

    2005-06-03

    Although SB202190 and SB203580 are described as specific p38 MAP kinase inhibitors, several reports have indicated that other enzymes are also sensitive to SB203580. Using a pharmacological approach, we report for the first time that compounds SB202190 and SB203580 were able to directly and selectively interact with a G-protein-coupled receptor, namely the cholecystokinin receptor subtype CCK1, but not with the CCK2 receptor. We demonstrated that these compounds were non-competitive antagonists of the CCK1 receptor at concentrations typically used to inhibit protein kinases. By chimeric construction of the CCK2 receptor, we determined the involvement of two CCK1 receptor intracellular loops in the binding of SB202190 and SB203580. We also showed that two CCK antagonists, L364,718 and L365,260, were able to regulate p38 mitogen-activated protein (MAP) kinase activity. Using a reporter gene strategy and immunoblotting experiments, we demonstrated that both CCK antagonists inhibited selectively the enzymatic activity of p38 MAP kinase. Kinase assays suggested that this inhibition resulted from a direct interaction with both CCK antagonists. Molecular modeling simulations suggested that this interaction occurs in the ATP binding pocket of p38 MAP kinase. These results suggest that SB202190 and SB203580 bind to the CCK1 receptor and, as such, these compounds should be used with caution in models that express this receptor. We also found that L364,718 and L365,260, two CCK receptor antagonists, directly interacted with p38 MAP kinase and inhibited its activity. These findings suggest that the CCK1 receptor shares structural analogies with the p38 MAP kinase ATP binding site. They open the way to potential design of either a new family of MAP kinase inhibitors from CCK1 receptor ligand structures or new CCK1 receptor ligands based on p38 MAP kinase inhibitor structures.

  11. High-affinity AKAP7δ–protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides

    PubMed Central

    Hundsrucker, Christian; Krause, Gerd; Beyermann, Michael; Prinz, Anke; Zimmermann, Bastian; Diekmann, Oliver; Lorenz, Dorothea; Stefan, Eduard; Nedvetsky, Pavel; Dathe, Margitta; Christian, Frank; Mcsorley, Theresa; Krause, Eberhard; Mcconnachie, George; Herberg, Friedrich W.; Scott, John D.; Rosenthal, Walter; Klussmann, Enno

    2006-01-01

    PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIα subunits with high affinity is AKAP7δ [AKAP18δ; Kd (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7δ mutant binds RIIα subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654–26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7δ bind RIIα subunits with higher affinity (Kd=0.4±0.3 nM) than either full-length or N-terminally truncated AKAP7δ, or peptides derived from other RII binding domains. The AKAP7δ-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP–RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7δ, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction. PMID:16483255

  12. Receptor-Like Function of Heparin in the Binding and Uptake of Neutral Lipids

    NASA Astrophysics Data System (ADS)

    Bosner, Matthew S.; Gulick, Tod; Riley, D. J. S.; Spilburg, Curtis A.; Lange, Louis G.

    1988-10-01

    Molecular mechanisms regulating the binding, amphipathic stabilization, and metabolism of the major neutral lipids (e.g., cholesteryl esters, triglycerides, and fatty acids) are well studied, but the details of their movement from a binding compartment to a metabolic compartment deserve further attention. Since all neutral lipids must cross hydrophilic segments of plasma membranes during such movement, we postulate that a critical receptor-like site exists on the plasma membrane to mediate a step between binding and metabolism and that membrane-associated heparin is a key part of this mediator. For example, intestinal brush border membranes containing heparin bind homogeneous human pancreatic 125I-labeled cholesterol esterase (100 kDa) and 125I-labeled triglyceride lipase (52 kDa). This interaction is enzyme concentration-dependent, specific, and saturable and is reversed upon addition of soluble heparin. Scatchard analysis demonstrates a single class of receptors with a Kd of 100 nM and a Bmax of approximately 50-60 pmol per mg of vesicle protein. In contrast, enzymes associated with the hydrolysis of hydrophilic compounds such as amylase, phospholipase A2, and deoxyribonuclease do not bind to intestinal membranes in this manner. Human pancreatic cholesterol esterase also binds specifically and saturably to cultured intestinal epithelial cells (CaCo-2), and soluble heparin significantly diminishes the cellular uptake of the resultant hydrophobic reaction products (cholesterol and free fatty acids). We conclude that a physiological role for intestinal heparin is that of a mediator to bind neutral lipolytic enzymes at the brush border and thus promote absorption of the subsequent hydrolyzed nutrients in the intestine. This mechanism may be a generalizable pathway for transport of neutral lipids into endothelial and other cells.

  13. T-cell receptor-like antibodies: novel reagents for clinical cancer immunology and immunotherapy.

    PubMed

    Noy, Roy; Eppel, Malka; Haus-Cohen, Maya; Klechevsky, Einav; Mekler, Orian; Michaeli, Yaeil; Denkberg, Galit; Reiter, Yoram

    2005-06-01

    Major histocompatibility complex class I molecules play a central role in the immune response against a variety of cells that have undergone malignant transformation by shaping the T-cell repertoire and presenting peptide antigens from endogeneous antigens to CD8+ cytotoxic T-cells. Diseased tumor or virus-infected cells are present on class I major histocompatibility complex molecule peptides that are derived from tumor-associated antigens or viral-derived proteins. Due to their unique specificity, such major histocompatibility complex-peptide complexes are a desirable target for novel approaches in immunotherapy. Targeted delivery of toxins or other cytotoxic drugs to cells which express specific major histocompatibility complex-peptide complexes that are involved in the immune response against cancer or viral infections would allow for a specific immunotherapeutic treatment of these diseases. It has recently been demonstrated that antibodies with the antigen-specific, major histocompatibility complex-restricted specificity of T-cells can be generated by taking advantage of the selection power of phage display technology. In addition to their tumor targeting capabilities, antibodies that mimic the fine specificity of T-cell receptors can serve as valuable research reagents that enable study of human class I peptide-major histocompatibility complex ligand presentation, as well as T-cell receptor peptide-major histocompatibility complex interactions. T-cell receptor-like antibody molecules may prove to be useful tools for studying major histocompatibility complex class I antigen presentation in health and disease as well as for therapeutic purposes in cancer, infectious diseases and autoimmune disorders.

  14. Signaling between two interacting sensor kinases promotes biofilms and colonization by a bacterial symbiont

    PubMed Central

    Norsworthy, Allison N.; Visick, Karen L.

    2015-01-01

    Summary Cells acclimate to fluctuating environments by utilizing sensory circuits. One common sensory pathway used by bacteria is two-component signaling (TCS), composed of an environmental sensor (the sensor kinase, SK) and a cognate, intracellular effector (the response regulator, RR). The squid symbiont Vibrio fischeri uses an elaborate TCS phosphorelay containing a hybrid SK, RscS, and two RRs, SypE and SypG, to control biofilm formation and host colonization. Here, we found that another hybrid SK, SypF, was essential for biofilms by functioning downstream of RscS to directly control SypE and SypG. Surprisingly, although wild-type SypF functioned as a SK in vitro, this activity was dispensable for colonization. In fact, only a single non-enzymatic domain within SypF, the HPt domain, was critical in vivo. Remarkably, this domain within SypF interacted with RscS to permit a bypass of RscS’s own HPt domain and SypF’s enzymatic function. This represents the first in vivo example of a functional SK that exploits the enzymatic activity of another SK, an adaptation that demonstrates the elegant plasticity in the arrangement of TCS regulators. PMID:25586643

  15. Inhibition of Receptor Interacting Protein Kinases Attenuates Cardiomyocyte Hypertrophy Induced by Palmitic Acid.

    PubMed

    Zhao, Mingyue; Lu, Lihui; Lei, Song; Chai, Hua; Wu, Siyuan; Tang, Xiaoju; Bao, Qinxue; Chen, Li; Wu, Wenchao; Liu, Xiaojing

    2016-01-01

    Palmitic acid (PA) is known to cause cardiomyocyte dysfunction. Cardiac hypertrophy is one of the important pathological features of PA-induced lipotoxicity, but the mechanism by which PA induces cardiomyocyte hypertrophy is still unclear. Therefore, our study was to test whether necroptosis, a receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3-) dependent programmed necrosis, was involved in the PA-induced cardiomyocyte hypertrophy. We used the PA-treated primary neonatal rat cardiac myocytes (NCMs) or H9c2 cells to study lipotoxicity. Our results demonstrated that cardiomyocyte hypertrophy was induced by PA treatment, determined by upregulation of hypertrophic marker genes and cell surface area enlargement. Upon PA treatment, the expression of RIPK1 and RIPK3 was increased. Pretreatment with the RIPK1 inhibitor necrostatin-1 (Nec-1), the PA-induced cardiomyocyte hypertrophy, was attenuated. Knockdown of RIPK1 or RIPK3 by siRNA suppressed the PA-induced myocardial hypertrophy. Moreover, a crosstalk between necroptosis and endoplasmic reticulum (ER) stress was observed in PA-treated cardiomyocytes. Inhibition of RIPK1 with Nec-1, phosphorylation level of AKT (Ser473), and mTOR (Ser2481) was significantly reduced in PA-treated cardiomyocytes. In conclusion, RIPKs-dependent necroptosis might be crucial in PA-induced myocardial hypertrophy. Activation of mTOR may mediate the effect of necroptosis in cardiomyocyte hypertrophy induced by PA.

  16. Homeodomain-Interacting Protein Kinase (HPK-1) regulates stress responses and ageing in C. elegans

    PubMed Central

    Berber, Slavica; Wood, Mallory; Llamosas, Estelle; Thaivalappil, Priya; Lee, Karen; Liao, Bing Mana; Chew, Yee Lian; Rhodes, Aaron; Yucel, Duygu; Crossley, Merlin; Nicholas, Hannah R

    2016-01-01

    Proteins of the Homeodomain-Interacting Protein Kinase (HIPK) family regulate an array of processes in mammalian systems, such as the DNA damage response, cellular proliferation and apoptosis. The nematode Caenorhabditis elegans has a single HIPK homologue called HPK-1. Previous studies have implicated HPK-1 in longevity control and suggested that this protein may be regulated in a stress-dependent manner. Here we set out to expand these observations by investigating the role of HPK-1 in longevity and in the response to heat and oxidative stress. We find that levels of HPK-1 are regulated by heat stress, and that HPK-1 contributes to survival following heat or oxidative stress. Additionally, we show that HPK-1 is required for normal longevity, with loss of HPK-1 function leading to a faster decline of physiological processes that reflect premature ageing. Through microarray analysis, we have found that HPK-1-regulated genes include those encoding proteins that serve important functions in stress responses such as Phase I and Phase II detoxification enzymes. Consistent with a role in longevity assurance, HPK-1 also regulates the expression of age-regulated genes. Lastly, we show that HPK-1 functions in the same pathway as DAF-16 to regulate longevity and reveal a new role for HPK-1 in development. PMID:26791749

  17. Tamoxifen Dependent Interaction Between in Estrogen Receptor and a Novel p21 Activated Kinase

    DTIC Science & Technology

    2004-06-01

    phosphorylation of Tyr 566 by MKK6, a dual-specificity phosphatase, MKP-1 (Lmitogen kinase phosphotase -1), which can dephosphorylate both threonine and tyrosine... phosphotase -1; WT, wild type; HA, hemagglutinin; MAP, mitogen-activated protein 23 Kaur et al. - PAK6 activation via P38 MAP kinase/MKK6 pathway Figure

  18. Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36

    PubMed Central

    Avey, Denis; Tepper, Sarah; Pifer, Benjamin; Bahga, Amritpal; Williams, Hunter; Gillen, Joseph; Li, Wenwei; Ogden, Sarah

    2016-01-01

    ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies. KSHV ORF36 encodes a serine/threonine viral protein kinase, which is conserved throughout all herpesviruses. Although several studies have identified the viral and cellular substrates of conserved herpesvirus protein kinases (CHPKs), the precise functions of KSHV ORF36 during lytic replication remain elusive. Here, we report that ORF36 interacts with another lytic protein, ORF45, in a manner dependent on ORF36 kinase activity. We mapped the regions of ORF36 and ORF45 involved in the binding. Their association appears to be mediated by electrostatic interactions, since deletion of either the highly basic N terminus of ORF36 or an acidic patch of ORF45 abolished the binding. In addition, the dephosphorylation of ORF45 protein dramatically reduced its association with ORF36. Importantly, ORF45 enhances both the stability and kinase activity of ORF36. Consistent with previous studies of CHPK homologs, we detected ORF36 protein in extracellular virions. To investigate the roles of ORF36 in the context of KSHV lytic replication, we used bacterial artificial chromosome mutagenesis to engineer both ORF36-null and kinase-dead mutants. We found that ORF36-null/mutant virions are moderately defective in viral particle production and are further deficient in primary infection. In summary, our results uncover a functionally important interaction between ORF36 and ORF45 and indicate a significant role of ORF36 in the production of infectious progeny virions. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus with a significant public health burden. KSHV ORF36 encodes a serine/threonine viral protein kinase, whose functions throughout the viral life cycle have not been elucidated. Here, we report that ORF36 interacts with another KSHV protein, ORF45. We mapped the regions of ORF36 and ORF45 involved in their association and further

  19. An intramolecular interaction within the lipid kinase Fab1 regulates cellular phosphatidylinositol 3,5-bisphosphate lipid levels.

    PubMed

    Lang, Michael J; Strunk, Bethany S; Azad, Nadia; Petersen, Jason L; Weisman, Lois S

    2017-04-01

    Phosphorylated phosphoinositide lipids (PPIs) are low-abundance signaling molecules that control signal transduction pathways and are necessary for cellular homeostasis. The PPI phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P2) is essential in multiple organ systems. PI(3,5)P2 is generated from PI3P by the conserved lipid kinase Fab1/PIKfyve. Defects in the dynamic regulation of PI(3,5)P2 are linked to human diseases. However, few mechanisms that regulate PI(3,5)P2 have been identified. Here we report an intramolecular interaction between the yeast Fab1 kinase region and an upstream conserved cysteine-rich (CCR) domain. We identify mutations in the kinase domain that lead to elevated levels of PI(3,5)P2 and impair the interaction between the kinase and CCR domain. We also identify mutations in the CCR domain that lead to elevated levels of PI(3,5)P2 Together these findings reveal a regulatory mechanism that involves the CCR domain of Fab1 and contributes to dynamic control of cellular PI(3,5)P2 synthesis.

  20. A complex network of interactions between mitotic kinases, phosphatases and ESCRT proteins regulates septation and membrane trafficking in S. pombe.

    PubMed

    Bhutta, Musab S; Roy, Brinta; Gould, Gwyn W; McInerny, Christopher J

    2014-01-01

    Cytokinesis and cell separation are critical events in the cell cycle. We show that Endosomal Sorting Complex Required for Transport (ESCRT) genes are required for cell separation in Schizosaccharomyces pombe. We identify genetic interactions between ESCRT proteins and polo and aurora kinases and Cdc14 phosphatase that manifest as impaired growth and exacerbated defects in septation, suggesting that the encoded proteins function together to control these processes. Furthermore, we observed defective endosomal sorting in mutants of plo1, ark1 and clp1, as has been reported for ESCRT mutants, consistent with a role for these kinases in the control of ESCRT function in membrane traffic. Multiple observations indicate functional interplay between polo and ESCRT components: firstly, two-hybrid in vivo interactions are reported between Plo1p and Sst4p, Vps28p, Vps25p, Vps20p and Vps32p; secondly, co-immunoprecipitation of human homologues of Vps20p, Vps32p, Vps24p and Vps2p by human Plk1; and thirdly, in vitro phosphorylation of budding yeast Vps32p and Vps20p by polo kinase. Two-hybrid analyses also identified interactions between Ark1p and Vps20p and Vps32p, and Clp1p and Vps28p. These experiments indicate a network of interactions between ESCRT proteins, plo1, ark1 and clp1 that coordinate membrane trafficking and cell separation in fission yeast.

  1. Prediction of Protein Kinase-Ligand Interactions through 2.5D Kinochemometrics.

    PubMed

    Bosc, Nicolas; Wroblowski, Berthold; Meyer, Christophe; Bonnet, Pascal

    2017-01-23

    So far, 518 protein kinases have been identified in the human genome. They share a common mechanism of protein phosphorylation and are involved in many critical biological processes of eukaryotic cells. Deregulation of the kinase phosphorylation function induces severe illnesses such as cancer, diabetes, or inflammatory diseases. Many actors in the pharmaceutical domain have made significant efforts to design potent and selective protein kinase inhibitors as new potential drugs. Because the ATP binding site is highly conserved in the protein kinase family, the design of selective inhibitors remains a challenge and has negatively impacted the progression of drug candidates to late-stage clinical development. The work presented here adopts a 2.5D kinochemometrics (KCM) approach, derived from proteochemometrics (PCM), in which protein kinases are depicted by a novel 3D descriptor and the ligands by 2D fingerprints. We demonstrate in two examples that the protein descriptor successfully classified protein kinases based on their group membership and their Asp-Phe-Gly (DFG) conformation. We also compared the performance of our models with those obtained from a full 2D KCM model and QSAR models. In both cases, the internal validation of the models demonstrated good capabilities to distinguish "active" from "inactive" protein kinase-ligand pairs. However, the external validation performed on two independent data sets showed that the two statistical models tended to overestimate the number of "inactive" pairs.

  2. Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1.

    PubMed

    Child, Matthew A; Garland, Megan; Foe, Ian; Madzelan, Peter; Treeck, Moritz; van der Linden, Wouter A; Oresic Bender, Kristina; Weerapana, Eranthie; Wilson, Mark A; Boothroyd, John C; Reese, Michael L; Bogyo, Matthew

    2017-02-28

    Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson's disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondiiIMPORTANCE Apicomplexan parasites such as Toxoplasma and Plasmodium are obligate intracellular parasites that require the protective environment of a host cell in order to replicate and survive within a host organism. These parasites secrete effector proteins from specialized apical organelles to select and invade a chosen host cell. The secretion of these organelles is a tightly regulated process coordinated by endogenous small molecules and calcium-dependent protein kinases. We previously identified the Toxoplasma orthologue of the highly conserved protein DJ-1 as a regulator of microneme secretion, but the molecular basis for this was not known. We have now identified the molecular mechanism for how TgDJ-1 regulates microneme secretion. TgDJ-1 interacts with the kinase responsible for the secretion of these

  3. Interactions between the S-Domain Receptor Kinases and AtPUB-ARM E3 Ubiquitin Ligases Suggest a Conserved Signaling Pathway in Arabidopsis1[W][OA

    PubMed Central

    Samuel, Marcus A.; Mudgil, Yashwanti; Salt, Jennifer N.; Delmas, Frédéric; Ramachandran, Shaliny; Chilelli, Andrea; Goring, Daphne R.

    2008-01-01

    The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses. PMID:18552232

  4. Hsp90 interaction with Cdc2 and Plo1 kinases contributes to actomyosin ring condensation in fission yeast.

    PubMed

    Santino, Andrea; Tallada, Victor A; Jimenez, Juan; Garzón, Andrés

    2012-08-01

    In Schizosaccharomyces pombe, cytokinesis occurs by ordered recruitment of actomyosin components at the division site, followed by lateral condensation to produce a ring-like structure early in anaphase, which eventually matures and contracts at the end of mitosis. We found that in temperature-sensitive hsp90-w1 mutant cells, encoding an Hsp90 mutant protein, ring components were recruited to form a cortical network at the division site, but this network failed to condense into a compact ring, suggesting a role for Hsp90 in this particular step. hsp90-w1 mutant shows strong genetic interaction with specific mutant alleles of the fission yeast cdc2, such as cdc2-33. Interestingly, actomyosin ring defects in hsp90-w1 cdc2-33 mutant cells resembled that of hsp90-w1 single mutant at restrictive temperature. Noteworthy, similar genetic interaction was found with a mutant allele of polo-like kinase, plo1-ts4, suggesting that Hsp90 collaborates with Cdc2 and Plo1 cell cycle kinases to condense medial ring components. In vitro analyses suggested that Cdc2 and Plo1 physically interact with Hsp90. Association of Cdc2 to Hsp90 was ATP independent, while Plo1 binds to this chaperone in an ATP-dependent manner, indicating that these two kinases interact with different Hsp90 complexes. Overall, our analyses of hsp90-w1 reveal a possible role for this chaperone in medial ring condensation in association with Cdc2 and Plo1 kinases.

  5. Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila.

    PubMed

    Jeon, Mili; Scott, Matthew P; Zinn, Kai

    2012-06-15

    The respiratory (tracheal) system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. We previously described a unique embryonic tracheal tubulogenesis phenotype caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr) tyrosine kinase (TK). Ptp10D physically interacts with Egfr. Here we demonstrate that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, we show that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. We also show that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways.

  6. Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila

    PubMed Central

    Jeon, Mili; Scott, Matthew P.; Zinn, Kai

    2012-01-01

    Summary The respiratory (tracheal) system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. We previously described a unique embryonic tracheal tubulogenesis phenotype caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr) tyrosine kinase (TK). Ptp10D physically interacts with Egfr. Here we demonstrate that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, we show that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. We also show that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways. PMID:23213447

  7. LIM kinase 1 (LIMK1) interacts with tropomyosin-related kinase B (TrkB) and Mediates brain-derived neurotrophic factor (BDNF)-induced axonal elongation.

    PubMed

    Dong, Qing; Ji, Yun-Song; Cai, Chang; Chen, Zhe-Yu

    2012-12-07

    BDNF/TrkB signaling plays critical roles in axonal outgrowth of neurons, the process of which requires the remodeling of the cytoskeleton structure, including microtubules and filamentous actin. However, the mechanism by which BDNF/TrkB signaling regulates cytoskeleton reorganization is still unclear. Here, we identified a novel interaction between LIMK1 and TrkB, which is required for the BDNF-induced axonal elongation. We demonstrated that BDNF-induced TrkB dimerization led to LIMK1 dimerization and transphosphorylation independent of TrkB kinase activity, which could further enhance the activation and stabilization of LIMK1. Moreover, activated LIMK1 translocated to the membrane fraction and phosphorylated its substrate cofilin, thus promoting actin polymerization and axonal elongation. Our findings provided evidence of a novel mechanism for the BDNF-mediated signal transduction leading to axonal elongation.

  8. Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis.

    PubMed

    Pandey, Girdhar K; Kanwar, Poonam; Singh, Amarjeet; Steinhorst, Leonie; Pandey, Amita; Yadav, Akhlilesh K; Tokas, Indu; Sanyal, Sibaji K; Kim, Beom-Gi; Lee, Sung-Chul; Cheong, Yong-Hwa; Kudla, Jörg; Luan, Sheng

    2015-09-01

    The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.

  9. Ligation of human Fc receptor like-2 by monoclonal antibodies down-regulates B-cell receptor-mediated signalling

    PubMed Central

    Shabani, Mahdi; Bayat, Ali Ahmad; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Hojjat-Farsangi, Mohammad; Ulivieri, Cristina; Amirghofran, Zahra; Baldari, Cosima Tatiana; Shokri, Fazel

    2014-01-01

    B-cell antigen receptor (BCR) signalling and its regulation through negative and positive regulators are critical for balancing B-cell response and function. Human Fc receptor like-2 (FCRL2), a member of the newly identified FCRL family, could influence B-cell signalling due to possession of both immunoreceptor tyrosine-based activation and inhibitory motifs (ITAM and ITIM). Since the natural ligand of FCRL2 has not been identified, we generated FCRL2-specific monoclonal antibodies (mAbs) and employed them to investigate the influence of FCRL2 stimulation on BCR signalling in an FCRL2-expressing B-cell line. Two anti-FCRL2 mAb-producing hybridoma clones (5A7-E7 and 3D8-G8) were selected. None of the mAbs displayed any cross-reactivity with the other members of the FCRL family including recombinant FCRL1, -3, -4 and -5, as tested by FACS and ELISA techniques. Engagement of the FCRL2 by these mAbs resulted in significant inhibition of BCR signalling mediators such as calcium mobilization and phosphorylation of the mitogen-activated protein kinases Erk, p38 and Jnk. These findings indicate that the FCRL2 ITIM motifs are functional and the anti-FCRL2 mAbs may mimic the natural ligand of FCRL2 by induction of inhibitory signals in B cells. PMID:24797767

  10. Analysis of A-kinase anchoring protein (AKAP) interaction with protein kinase A (PKA) regulatory subunits: PKA isoform specificity in AKAP binding.

    PubMed

    Herberg, F W; Maleszka, A; Eide, T; Vossebein, L; Tasken, K

    2000-04-28

    Compartmentalization of cAMP-dependent protein kinase (PKA) is in part mediated by specialized protein motifs in the dimerization domain of the regulatory (R)-subunits of PKA that participate in protein-protein interactions with an amphipathic helix region in A-kinase anchoring proteins (AKAPs). In order to develop a molecular understanding of the subcellular distribution and specific functions of PKA isozymes mediated by association with AKAPs, it is of importance to determine the apparent binding constants of the R-subunit-AKAP interactions. Here, we present a novel approach using surface plasmon resonance (SPR) to examine directly the association and dissociation of AKAPs with all four R-subunit isoforms immobilized on a modified cAMP surface with a high level of accuracy. We show that both AKAP79 and S-AKAP84/D-AKAP1 bind RIIalpha very well (apparent K(D) values of 0.5 and 2 nM, respectively). Both proteins also bind RIIbeta quite well, but with three- to fourfold lower affinities than those observed versus RIIalpha. However, only S-AKAP84/D-AKAP1 interacts with RIalpha at a nanomolar affinity (apparent K(D) of 185 nM). In comparison, AKAP95 binds RIIalpha (apparent K(D) of 5.9 nM) with a tenfold higher affinity than RIIbeta and has no detectable binding to RIalpha. Surface competition assays with increasing concentrations of a competitor peptide covering amino acid residues 493 to 515 of the thyroid anchoring protein Ht31, demonstrated that Ht31, but not a proline-substituted peptide, Ht31-P, competed binding of RIIalpha and RIIbeta to all the AKAPs examined (EC(50)-values from 6 to 360 nM). Furthermore, RIalpha interaction with S-AKAP84/D-AKAP1 was competed (EC(50) 355 nM) with the same peptide. Here we report for the first time an approach to determine apparent rate- and equilibria binding constants for the interaction of all PKA isoforms with any AKAP as well as a novel approach for characterizing peptide competitors that disrupt PKA-AKAP anchoring.

  11. Epstein-Barr virus protein kinase BGLF4 targets the nucleus through interaction with nucleoporins.

    PubMed

    Chang, Chou-Wei; Lee, Chung-Pei; Huang, Yu-Hao; Yang, Pei-Wen; Wang, Jiin-Tarng; Chen, Mei-Ru

    2012-08-01

    BGLF4 of Epstein-Barr virus (EBV) encodes a serine/threonine protein kinase that phosphorylates multiple viral and cellular substrates to optimize the cellular environment for viral DNA replication and the nuclear egress of viral nucleocapsids. BGLF4 is expressed predominantly in the nucleus at early and late stages of virus replication, while a small portion of BGLF4 is distributed in the cytoplasm at the late stage of virus replication and packaged into the virion. Here, we analyzed systematically the functional domains crucial for nuclear localization of BGLF4 and found that both the N and C termini play important modulating roles. Analysis of amino acid substitution mutants revealed that the C terminus of BGLF4 does not contain a conventional nuclear localization signal (NLS). Additionally, deletion of the C-terminal putative helical regions at amino acids 386 to 393 and 410 to 419 diminished the nuclear translocation of BGLF4, indicating that the secondary structure of the C terminus is important for the localization of BGLF4. The green fluorescent protein-fused wild-type or C-terminal helical regions of BGLF4 associate with phenylalanine/glycine repeat-containing nucleoporins (Nups) in nuclear envelope fractionation. Both coimmunoprecipitation and in vitro pull-down assays further demonstrated that BGLF4 binds to Nup62 and Nup153. Remarkably, nuclear import assay with permeabilized HeLa cells demonstrated that BGLF4 translocated into nucleus independent of cytosolic factors. Data presented here suggest that BGLF4 employs a novel mechanism through direct interactions with nucleoporins for its nuclear targeting.

  12. The composition and function of the striatin-interacting phosphatases and kinases (STRIPAK) complex in fungi.

    PubMed

    Kück, Ulrich; Beier, Anna M; Teichert, Ines

    2016-05-01

    The striatin-interacting phosphatases and kinases (STRIPAK) complex is a highly conserved eukaryotic protein complex that was recently described for diverse animal and fungal species. Here, we summarize our current knowledge about the composition and function of the STRIPAK complex from the ascomycete Sordaria macrospora, which we discovered by investigating sexually sterile mutants (pro), having a defect in fruiting body development. Mass spectrometry and yeast two-hybrid analysis defined core subunits of the STRIPAK complex, which have structural homologs in animal and other fungal organisms. These subunits (and their mammalian homologs) are PRO11 (striatin), PRO22 (STRIP1/2), SmMOB3 (Mob3), PRO45 (SLMAP), and PP2AA, the structural, and PP2Ac, the catalytic subunits of protein phosphatase 2A (PP2A). Beside fruiting body formation, the STRIPAK complex controls vegetative growth and hyphal fusion in S. macrospora. Although the contribution of single subunits to diverse cellular and developmental processes is not yet fully understood, functional analysis has already shown that mammalian homologs are able to substitute the function of distinct fungal STRIPAK subunits. This underscores the view that fungal model organisms serve as useful tools to get a molecular insight into cellular and developmental processes of eukaryotes in general. Future work will unravel the precise localization of single subunits within the cell and decipher their STRIPAK-related and STRIPAK-independent functions. Finally, evidence is accumulating that there is a crosstalk between STRIPAK and various signaling pathways, suggesting that eukaryotic development is dependent on STRIPAK signaling.

  13. Drug-Drug Interaction Potentials of Tyrosine Kinase Inhibitors via Inhibition of UDP-Glucuronosyltransferases

    PubMed Central

    Zhang, Nan; Liu, Yong; Jeong, Hyunyoung

    2015-01-01

    Tyrosine kinase inhibitors (TKIs) are anticancer drugs that may be co-administered with other drugs. The aims of this study are to investigate the inhibitory effects of TKIs on UDP-glucuronosyltransferase (UGT) activities, and to quantitatively evaluate their potential to cause drug-drug interactions (DDIs). Inhibition kinetic profiles of a panel of UGT enzymes (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17) by four TKIs (axitinib, imatinib, lapatinib and vandetanib) were characterized by using hepatic microsomes and recombinant proteins. Lapatinib exhibited potent competitive inhibition against UGT1A1 activity with a Ki of 0.5 μM. Imatinib was found to exhibit broad inhibition on several UGTs, particularly potent competitive inhibition against UGT2B17 with a Ki of 0.4 μM. The TKIs also exerted intermediate inhibition against several UGTs (i.e., UGT1A7 by lapatinib; UGT1A1 by imatinib; UGT1A4, 1A7 and 1A9 by axitinib; and UGT1A9 by vandetanib). Results from modeling for the quantitative prediction of DDI risk indicated that the coadministration of lapatinib or imatinib at clinical doses could result in a significant increase in AUC of drugs primarily cleared by UGT1A1 or 2B17. Lapatinib and imatinib may cause clinically significant DDIs when co-administered UGT1A1 or 2B17 substrates. PMID:26642944

  14. Structure-based design of small peptide inhibitors of protein kinase CK2 subunit interaction

    PubMed Central

    Laudet, Béatrice; Barette, Caroline; Dulery, Vincent; Renaudet, Olivier; Dumy, Pascal; Metz, Alexandra; Prudent, Renaud; Deshiere, Alexandre; Dideberg, Otto; Filhol, Odile; Cochet, Claude

    2007-01-01

    X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged the traditional view of protein kinase CK2. Unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumours. Thus the potential intersubunit flexibility suggested by these studies raises the likely prospect that the CK2 holoenzyme complex is subject to disassembly and reassembly. In the present paper, we show evidence for the reversible multimeric organization of the CK2 holoenzyme complex in vitro. We used a combination of site-directed mutagenesis, binding experiments and functional assays to show that, both in vitro and in vivo, only a small set of primary hydrophobic residues of CK2β which contacts at the centre of the CK2α/CK2β interface dominates affinity. The results indicate that a double mutation in CK2β of amino acids Tyr188 and Phe190, which are complementary and fill up a hydrophobic pocket of CK2α, is the most disruptive to CK2α binding both in vitro and in living cells. Further characterization of hotspots in a cluster of hydrophobic amino acids centred around Tyr188–Phe190 led us to the structure-based design of small-peptide inhibitors. One conformationally constrained 11-mer peptide (Pc) represents a unique CK2β-based small molecule that was particularly efficient (i) to antagonize the interaction between the CK2 subunits, (ii) to inhibit the assembly of the CK2 holoenzyme complex, and (iii) to strongly affect its substrate preference. PMID:17714077

  15. Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation.

    PubMed

    Geering, Barbara; Zokouri, Zina; Hürlemann, Samuel; Gerrits, Bertran; Ausländer, David; Britschgi, Adrian; Tschan, Mario P; Simon, Hans-Uwe; Fussenegger, Martin

    2016-01-01

    Death-associated protein kinase 2 (DAPK2) is a Ca(2+)/calmodulin-dependent Ser/Thr kinase that possesses tumor-suppressive functions and regulates programmed cell death, autophagy, oxidative stress, hematopoiesis, and motility. As only few binding partners of DAPK2 have been determined, the molecular mechanisms governing these biological functions are largely unknown. We report the identification of 180 potential DAPK2 interaction partners by affinity purification-coupled mass spectrometry, 12 of which are known DAPK binding proteins. A small subset of established and potential binding proteins detected in this screen was further investigated by bimolecular fluorescence complementation (BiFC) assays, a method to visualize protein interactions in living cells. These experiments revealed that α-actinin-1 and 14-3-3-β are novel DAPK2 binding partners. The interaction of DAPK2 with α-actinin-1 was localized at the plasma membrane, resulting in massive membrane blebbing and reduced cellular motility, whereas the interaction of DAPK2 with 14-3-3-β was localized to the cytoplasm, with no impact on blebbing, motility, or viability. Our results therefore suggest that DAPK2 effector functions are influenced by the protein's subcellular localization and highlight the utility of combining mass spectrometry screening with bimolecular fluorescence complementation to identify and characterize novel protein-protein interactions.

  16. Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation

    PubMed Central

    Zokouri, Zina; Hürlemann, Samuel; Gerrits, Bertran; Ausländer, David; Britschgi, Adrian; Tschan, Mario P.; Simon, Hans-Uwe; Fussenegger, Martin

    2015-01-01

    Death-associated protein kinase 2 (DAPK2) is a Ca2+/calmodulin-dependent Ser/Thr kinase that possesses tumor-suppressive functions and regulates programmed cell death, autophagy, oxidative stress, hematopoiesis, and motility. As only few binding partners of DAPK2 have been determined, the molecular mechanisms governing these biological functions are largely unknown. We report the identification of 180 potential DAPK2 interaction partners by affinity purification-coupled mass spectrometry, 12 of which are known DAPK binding proteins. A small subset of established and potential binding proteins detected in this screen was further investigated by bimolecular fluorescence complementation (BiFC) assays, a method to visualize protein interactions in living cells. These experiments revealed that α-actinin-1 and 14-3-3-β are novel DAPK2 binding partners. The interaction of DAPK2 with α-actinin-1 was localized at the plasma membrane, resulting in massive membrane blebbing and reduced cellular motility, whereas the interaction of DAPK2 with 14-3-3-β was localized to the cytoplasm, with no impact on blebbing, motility, or viability. Our results therefore suggest that DAPK2 effector functions are influenced by the protein's subcellular localization and highlight the utility of combining mass spectrometry screening with bimolecular fluorescence complementation to identify and characterize novel protein-protein interactions. PMID:26483415

  17. Protein kinase A RII-like (R2D2) proteins exhibit differential localization and AKAP interaction.

    PubMed

    Newell, Amy E Hanlon; Fiedler, Sarah E; Ruan, Jenny M; Pan, Jieyan; Wang, P Jeremy; Deininger, Jutta; Corless, Christopher L; Carr, Daniel W

    2008-07-01

    A-kinase anchoring proteins (AKAPs) bind to protein kinase A (PKA) via an amphipathic helix domain that interacts with a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins (ROPN1, ASP, SP17, and CABYR) also contain a highly conserved RII dimerization/docking (R2D2) domain, suggesting all four proteins may interact with all AKAPs in a manner similar to RII. All four of these proteins were originally detected in the flagellum of mammalian sperm. In this report, we demonstrate that all four R2D2 proteins are expressed in a wide variety of tissues and three of the proteins SP17, CABYR, and ASP are located in motile cilia of human bronchus and fallopian tubes. In addition, we detect SP17 in primary cilia. We also provide evidence that ROPN1 and ASP bind to a variety of AKAPs and this interaction can be disrupted with anchoring inhibitor peptides. The interaction of SP17 and CABYR with AKAPs appears to be much more limited. None of the R2D2 proteins appears to bind cAMP, a fundamental characteristic of the regulatory subunits of PKA. These observations suggest that R2D2 proteins utilize docking interactions with AKAPs to accomplish their function of regulating cilia and flagella. Based on location, affinity for AKAPs and lack of affinity for cAMP, it appears that each R2D2 protein has a unique role in this process.

  18. A frequent kinase domain mutation that changes the interaction between PI3K[alpha] and the membrane

    SciTech Connect

    Mandelker, Diana; Gabelli, Sandra B.; Schmidt-Kittler, Oleg; Zhu, Jiuxiang; Cheong, Ian; Huang, Chuan-Hsiang; Kinzler, Kenneth W.; Vogelstein, Bert; Amzel, L. Mario

    2009-12-01

    Mutations in oncogenes often promote tumorigenesis by changing the conformation of the encoded proteins, thereby altering enzymatic activity. The PIK3CA oncogene, which encodes p110{alpha}, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3K{alpha}), is one of the two most frequently mutated oncogenes in human cancers. We report the structure of the most common mutant of p110{alpha} in complex with two interacting domains of its regulatory partner (p85{alpha}), both free and bound to an inhibitor (wortmannin). The N-terminal SH2 (nSH2) domain of p85{alpha} is shown to form a scaffold for the entire enzyme complex, strategically positioned to communicate extrinsic signals from phosphopeptides to three distinct regions of p110{alpha}. Moreover, we found that Arg-1047 points toward the cell membrane, perpendicular to the orientation of His-1047 in the WT enzyme. Surprisingly, two loops of the kinase domain that contact the cell membrane shift conformation in the oncogenic mutant. Biochemical assays revealed that the enzymatic activity of the p110{alpha} His1047Arg mutant is differentially regulated by lipid membrane composition. These structural and biochemical data suggest a previously undescribed mechanism for mutational activation of a kinase that involves perturbation of its interaction with the cellular membrane.

  19. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A

    PubMed Central

    Borthwick, Lee A.; Kerbiriou, Mathieu; Taylor, Christopher J.; Cozza, Giorgio; Lascu, Ioan; Postel, Edith H.; Cassidy, Diane; Trouvé, Pascal; Mehta, Anil; Robson, Louise; Muimo, Richmond

    2016-01-01

    Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36–54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351–727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia. PMID:26950439

  20. Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5'-Terminal Cap and Hairpin.

    PubMed

    Korneeva, Nadejda L; Song, Anren; Gram, Hermann; Edens, Mary Ann; Rhoads, Robert E

    2016-02-12

    The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F.

  1. Overexpression of Rice Wall-Associated Kinase 25 (OsWAK25) Alters Resistance to Bacterial and Fungal Pathogens

    PubMed Central

    Harkenrider, Mitch; Sharma, Rita; De Vleesschauwer, David; Tsao, Li; Zhang, Xuting; Chern, Mawsheng; Canlas, Patrick; Zuo, Shimin; Ronald, Pamela C.

    2016-01-01

    Wall-associated kinases comprise a sub-family of receptor-like kinases that function in plant growth and stress responses. Previous studies have shown that the rice wall-associated kinase, OsWAK25, interacts with a diverse set of proteins associated with both biotic and abiotic stress responses. Here, we show that wounding and BTH treatments induce OsWAK25 transcript expression in rice. We generated OsWAK25 overexpression lines and show that these lines exhibit a lesion mimic phenotype and enhanced expression of rice NH1 (NPR1 homolog 1), OsPAL2, PBZ1 and PR10. Furthermore, these lines show resistance to the hemibiotrophic pathogens, Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae, yet display increased susceptibility to necrotrophic fungal pathogens, Rhizoctonia solani and Cochliobolus miyabeanus. PMID:26795719

  2. CHARACTERIZATION OF A NOVEL INTERACTION BETWEEN TRANSCRIPTION FACTOR TFII-I AND THE TYROSINE KINASE ITK IN T CELLS

    PubMed Central

    Sacristán, Catarina; Schattgen, Stefan A.; Berg, Leslie J.; Bunnell, Stephen C.; Roy, Ananda L.; Rosenstein, Yvonne

    2010-01-01

    Summary TCR signaling leads to the activation of kinases such as Itk, a key regulatory protein in T lymphocyte activation and function. The homolog of Itk in B cells is Btk, previously shown to bind and phosphorylate the transcription factor TFII-I. TFII-I plays major roles in transcription and signaling. Our purpose herein was two-fold: first, to identify some of the molecular determinants involved in TFII-I activation downstream of receptor crosslinking in T cells; and second, to uncover the existence of Itk-TFII-I signaling in T lymphocytes. We report for the first time that TFII-I is tyrosine phosphorylated upon TCR, TCR/CD43, and TCR/CD28 co-receptor engagement in human and/or murine T cells. We show that Itk physically interacts with TFII-I and potentiates TFII-I-driven c-fos transcription. We demonstrate that TFII-I is phosphorylated upon co-expression of wild type, but not kinase-dead, or kinase-dead/R29C mutant Itk, suggesting these residues are important for TFII-I phosphorylation, presumably via an Itk-dependent mechanism. Structural analysis of TFII-I-Itk interactions revealed that the first 90 residues of TFII-I are dispensable for Itk binding. Mutations within Itk’s kinase, pleckstrin-homology and proline-rich regions did not abolish TFII-I-Itk binding. Our results provide an initial step in understanding the biological role of Itk-TFII-I signaling in T cell function. PMID:19701889

  3. Hepatitis C Virus RNA-Dependent RNA Polymerase Interacts with the Akt/PKB Kinase and Induces Its Subcellular Relocalization

    PubMed Central

    Valero, María Llanos; Sabariegos, Rosario; Cimas, Francisco J.; Perales, Celia; Domingo, Esteban; Sánchez-Prieto, Ricardo

    2016-01-01

    Hepatitis C virus (HCV) interacts with cellular components and modulates their activities for its own benefit. These interactions have been postulated as a target for antiviral treatment, and some candidate molecules are currently in clinical trials. The multifunctional cellular kinase Akt/protein kinase B (PKB) must be activated to increase the efficacy of HCV entry but is rapidly inactivated as the viral replication cycle progresses. Viral components have been postulated to be responsible for Akt/PKB inactivation, but the underlying mechanism remained elusive. In this study, we show that HCV polymerase NS5B interacts with Akt/PKB. In the presence of transiently expressed NS5B or in replicon- or virus-infected cells, NS5B changes the cellular localization of Akt/PKB from the cytoplasm to the perinuclear region. Sequestration of Akt/PKB by NS5B could explain its exclusion from its participation in early Akt/PKB inactivation. The NS5B-Akt/PKB interaction represents a new regulatory step in the HCV infection cycle, opening possibilities for new therapeutic options. PMID:27021315

  4. Regulation of polar auxin transport by protein and lipid kinases

    PubMed Central

    Jaillais, Yvon

    2016-01-01

    The directional transport of auxin, known as polar auxin transport, allows asymmetric distribution of this hormone in different cells and tissues. This system creates local auxin maxima, minima and gradients that are instrumental in both organ initiation and shape determination. As such, polar auxin transport is crucial for all aspects of plant development but also for environmental interaction, notably in shaping plant architecture to its environment. Cell-to-cell auxin transport is mediated by a network of auxin carriers that are regulated at the transcriptional and post-translational levels. Here we review our current knowledge on some aspects of the ‘non-genomic’ regulation of auxin transport, putting an emphasis on how phosphorylation by protein and lipid kinases controls the polarity, intracellular trafficking, stability and activity of auxin carriers. We describe the role of several AGC kinases, including PINOID, D6PK and the blue light photoreceptor phot1, in phosphorylating auxin carriers from the PIN and ABCB families. We also highlight the function of some Receptor-Like Kinases (RLK) and two-component histidine kinase receptors in polar auxin transport, noticing that there are likely RLKs involved in coordinating auxin distribution yet to be discovered. In addition, we describe the emerging role of phospholipid phosphorylation in polarity establishment and intracellular trafficking of PIN proteins. We outline these various phosphorylation mechanisms in the context of primary and lateral root development, leaf cell shape acquisition as well as root gravitropism and shoot phototropism. PMID:27242371

  5. Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity*

    PubMed Central

    Hughes, Siobhan; Jenkins, Victoria; Dar, Mohd Jamal; Engelman, Alan; Cherepanov, Peter

    2010-01-01

    Lens epithelium-derived growth factor (LEDGF) is an important co-factor of human immunodeficiency virus DNA integration; however, its cellular functions are poorly characterized. We now report identification of the Cdc7-activator of S-phase kinase (ASK) heterodimer as a novel interactor of LEDGF. Both kinase subunits co-immunoprecipitated with endogenous LEDGF from human cell extracts. Truncation analyses identified the integrase-binding domain of LEDGF as essential and minimally sufficient for the interaction with Cdc7-ASK. Reciprocally, the interaction required autophosphorylation of the kinase and the presence of 50 C-terminal residues of ASK. The kinase phosphorylated LEDGF in vitro, with Ser-206 being the major target, and LEDGF phosphorylated at this residue could be detected during S phase of the cell cycle. LEDGF potently stimulated the enzymatic activity of Cdc7-ASK, increasing phosphorylation of MCM2 in vitro by more than 10-fold. This enzymatic stimulation as well as phosphorylation of LEDGF depended on the protein-protein interaction. Intriguingly, removing the C-terminal region of ASK, involved in the interaction with LEDGF, resulted in a hyperactive kinase. Our results indicate that the interaction with LEDGF relieves autoinhibition of Cdc7-ASK kinase, imposed by the C terminus of ASK. PMID:19864417

  6. Ca2+-independent contraction of longitudinal ileal smooth muscle is potentiated by a zipper-interacting protein kinase pseudosubstrate peptide.

    PubMed

    Ihara, Eikichi; Moffat, Lori; Borman, Meredith A; Amon, Jennifer E; Walsh, Michael P; MacDonald, Justin A

    2009-08-01

    As a regulator of smooth muscle contraction, zipper-interacting protein kinase (ZIPK) can directly phosphorylate the myosin regulatory light chains (LC20) and produce contractile force. Synthetic peptides (SM-1 and AV25) derived from the autoinhibitory region of smooth muscle myosin light chain kinase can inhibit ZIPK activity in vitro. Paradoxically, treatment of Triton-skinned ileal smooth muscle strips with AV25, but not SM-1, potentiated Ca2+-independent, microcystin- and ZIPK-induced contractions. The AV25-induced potentiation was limited to ileal and colonic smooth muscles and was not observed in rat caudal artery. Thus the potentiation of Ca2+-independent contractions by AV25 appeared to be mediated by a mechanism unique to intestinal smooth muscle. AV25 treatment elicited increased phosphorylation of LC20 (both Ser-19 and Thr-18) and myosin phosphatase-targeting subunit (MYPT1, inhibitory Thr-697 site), suggesting involvement of a Ca2+-independent LC20 kinase with coincident inhibition of myosin phosphatase. The phosphorylation of the inhibitor of myosin phosphatase, CPI-17, was not affected. The AV25-induced potentiation was abolished by pretreatment with staurosporine, a broad-specificity kinase inhibitor, but specific inhibitors of Rho-associated kinase, PKC, and MAPK pathways had no effect. When a dominant-negative ZIPK [kinase-dead ZIPK((1-320))-D161A] was added to skinned ileal smooth muscle, the potentiation of microcystin-induced contraction by AV25 was blocked. Furthermore, pretreatment of skinned ileal muscle with SM-1 abolished AV25-induced potentiation. We conclude, therefore, that, even though AV25 is an in vitro inhibitor of ZIPK, activation of the ZIPK pathway occurs following application of AV25 to permeabilized ileal smooth muscle. Finally, we propose a mechanism whereby conformational changes in the pseudosubstrate region of ZIPK permit augmentation of ZIPK activity toward LC(20) and MYPT1 in situ. AV25 or molecules based on its structure

  7. Therapeutic potential of a synthetic lethal interaction between the MYC proto-oncogene and inhibition of aurora-B kinase.

    PubMed

    Yang, Dun; Liu, Hong; Goga, Andrei; Kim, Suwon; Yuneva, Mariia; Bishop, J Michael

    2010-08-03

    The Myc protein and proteins that participate in mitosis represent attractive targets for cancer therapy. However, their potential is presently compromised by the threat of side effects and by a lack of pharmacological inhibitors of Myc. Here we report that a circumscribed exposure to the aurora kinase inhibitor, VX-680, selectively kills cells that overexpress Myc. This synthetic lethal interaction is attributable to inhibition of aurora-B kinase, with consequent disabling of the chromosomal passenger protein complex (CPPC) and ensuing DNA replication in the absence of cell division; executed by sequential apoptosis and autophagy; not reliant on the tumor suppressor protein p53; and effective against mouse models for B-cell and T-cell lymphomas initiated by transgenes of MYC. Our findings cast light on how inhibitors of aurora-B kinase may kill tumor cells, implicate Myc in the induction of a lethal form of autophagy, indicate that expression of Myc be a useful biomarker for sensitivity of tumor cells to inhibition of the CPPC, dramatize the virtue of bimodal killing by a single therapeutic agent, and suggest a therapeutic strategy for killing tumor cells that overexpress Myc while sparing normal cells.

  8. Physical and functional interaction of CARMA1 and CARMA3 with Ikappa kinase gamma-NFkappaB essential modulator.

    PubMed

    Stilo, Romania; Liguoro, Domenico; Di Jeso, Bruno; Formisano, Silvestro; Consiglio, Eduardo; Leonardi, Antonio; Vito, Pasquale

    2004-08-13

    CARMA proteins are scaffold molecules that contain a caspase recruitment domain and a membrane-associated guanylate kinase-like domain. CARMA1 plays a critical role in mediating activation of the NFkappaB transcription factor following antigen receptor stimulation of both B and T lymphocytes. However, the biochemical mechanism by which CARMA1 regulates activation of NFkappaB remains to be determined. Here we have shown that CARMA1 and CARMA3 physically associate with Ikappa kinase gamma/NFkappaB essential modulator (IkappaKgamma-NEMO) in lymphoid and non-lymphoid cells. CARMA1 participates to an inducible large molecular complex that contains IkappaKgamma/NEMO, Bcl10, and IkappaKalpha/beta kinases. Expression of the NEMO-binding region of CARMA3 exerts a dominant negative effect on Bcl10-mediated activation of NFkappaB. Thus, our results provide direct evidence for physical and functional interaction between CARMA and the IkappaK complex and offer a biochemical framework to understand the molecular activities controlled by CARMA-1, -2, and -3 and Bcl10.

  9. Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1

    PubMed Central

    Child, Matthew A.; Garland, Megan; Foe, Ian; Madzelan, Peter; Treeck, Moritz; van der Linden, Wouter A.; Oresic Bender, Kristina; Weerapana, Eranthie; Wilson, Mark A.; Boothroyd, John C.; Reese, Michael L.

    2017-01-01

    ABSTRACT Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson’s disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondii. PMID:28246362

  10. Competition between members of the tribbles pseudokinase protein family shapes their interactions with mitogen activated protein kinase pathways

    PubMed Central

    Guan, Hongtao; Shuaib, Aban; Leon, David Davila De; Angyal, Adrienn; Salazar, Maria; Velasco, Guillermo; Holcombe, Mike; Dower, Steven K.; Kiss-Toth, Endre

    2016-01-01

    Spatio-temporal regulation of intracellular signalling networks is key to normal cellular physiology; dysregulation of which leads to disease. The family of three mammalian tribbles proteins has emerged as an important controller of signalling via regulating the activity of mitogen activated protein kinases (MAPK), the PI3-kinase induced signalling network and E3 ubiquitin ligases. However, the importance of potential redundancy in the action of tribbles and how the differences in affinities for the various binding partners may influence signalling control is currently unclear. We report that tribbles proteins can bind to an overlapping set of MAPK-kinases (MAPKK) in live cells and dictate the localisation of the complexes. Binding studies in transfected cells reveal common regulatory mechanisms and suggest that tribbles and MAPKs may interact with MAPKKs in a competitive manner. Computational modelling of the impact of tribbles on MAPK activation suggests a high sensitivity of this system to changes in tribbles levels, highlighting that these proteins are ideally placed to control the dynamics and balance of activation of concurrent signalling pathways. PMID:27600771

  11. The cytoplasmic PASC domain of the sensor kinase DcuS of Escherichia coli: role in signal transduction, dimer formation, and DctA interaction

    PubMed Central

    Monzel, Christian; Degreif-Dünnwald, Pia; Gröpper, Christina; Griesinger, Christian; Unden, Gottfried

    2013-01-01

    The cytoplasmic PASC domain of the fumarate responsive sensor kinase DcuS of Escherichia coli links the transmembrane to the kinase domain. PASC is also required for interaction with the transporter DctA serving as a cosensor of DcuS. Earlier studies suggested that PASC functions as a hinge and transmits the signal to the kinase. Reorganizing the PASC dimer interaction and, independently, removal of DctA, converts DcuS to the constitutive ON state (active without fumarate stimulation). ON mutants were categorized with respect to these two biophysical interactions and the functional state of DcuS: type I-ON mutations grossly reorganize the homodimer, and decrease interaction with DctA. Type IIA-ON mutations create the ON state without grossly reorganizing the homodimer, whereas interaction with DctA is decreased. The type IIB-ON mutations were neither in PASC/PASC, nor in DctA/DcuS interaction affected, similar to fumarate activated wild-typic DcuS. OFF mutations never affected dimer stability. The ON mutations provide novel mechanistic insight: PASC dimerization is essential to silence the kinase. Reorganizing the homodimer and its interaction with DctA activate the kinase. The study suggests a novel ON homo-dimer conformation (type IIB) and an OFF conformation for PASC. Type IIB-ON corresponds to the fumarate induced wild-type conformation, representing an interesting target for structural biology. PMID:24039243

  12. Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha with actin filaments and focal adhesion kinase

    PubMed Central

    1995-01-01

    Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti- phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas

  13. Protein kinase A modulates transforming growth factor-β signaling through a direct interaction with Smad4 protein.

    PubMed

    Yang, Huibin; Li, Gangyong; Wu, Jing-Jiang; Wang, Lidong; Uhler, Michael; Simeone, Diane M

    2013-03-22

    Transforming growth factor β (TGFβ) signaling normally functions to regulate embryonic development and cellular homeostasis. It is increasingly recognized that TGFβ signaling is regulated by cross-talk with other signaling pathways. We previously reported that TGFβ activates protein kinase A (PKA) independent of cAMP through an interaction of an activated Smad3-Smad4 complex and the regulatory subunit of the PKA holoenzyme (PKA-R). Here we define the interaction domains of Smad4 and PKA-R and the functional consequences of this interaction. Using a series of Smad4 and PKA-R truncation mutants, we identified amino acids 290-300 of the Smad4 linker region as critical for the specific interaction of Smad4 and PKA-R. Co-immunoprecipitation assays showed that the B cAMP binding domain of PKA-R was sufficient for interaction with Smad4. Targeting of B domain regions conserved among all PKA-R isoforms and exposed on the molecular surface demonstrated that amino acids 281-285 and 320-329 were required for complex formation with Smad4. Interactions of these specific regions of Smad4 and PKA-R were necessary for TGFβ-mediated increases in PKA activity, CREB (cAMP-response element-binding protein) phosphorylation, induction of p21, and growth inhibition. Moreover, this Smad4-PKA interaction was required for TGFβ-induced epithelial mesenchymal transition, invasion of pancreatic tumor cells, and regulation of tumor growth in vivo.

  14. Interaction Between a Novel p21 Activated Kinase (PAK6) and Androgen Receptor in Prostate Cancer

    DTIC Science & Technology

    2005-02-01

    not altered by . 2o treatment with DHT (data not shown). Overexpressed 3-cate- nin protein with AR vector, in the absence of DHT, showed the same...Taken together, we due primarily to loss or decreased expression E-cadherin, is conclude that overexpression of E-cadherin in TSU.pr-1 in- frequently...such as glycogen synthase of AR activity by LY294002 is mediated through phos- kinase (GSK3), Bad, and caspase9 and the forkhead transcrip

  15. Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration

    PubMed Central

    Zurnic, Irena; Hütter, Sylvia; Rzeha, Ute; Stanke, Nicole; Reh, Juliane; Müllers, Erik; Hamann, Martin V.; Kern, Tobias; Gerresheim, Gesche K.; Serrao, Erik; Lesbats, Paul; Engelman, Alan N.; Cherepanov, Peter; Lindemann, Dirk

    2016-01-01

    Unlike for other retroviruses, only a few host cell factors that aid the replication of foamy viruses (FVs) via interaction with viral structural components are known. Using a yeast-two-hybrid (Y2H) screen with prototype FV (PFV) Gag protein as bait we identified human polo-like kinase 2 (hPLK2), a member of cell cycle regulatory kinases, as a new interactor of PFV capsids. Further Y2H studies confirmed interaction of PFV Gag with several PLKs of both human and rat origin. A consensus Ser-Thr/Ser-Pro (S-T/S-P) motif in Gag, which is conserved among primate FVs and phosphorylated in PFV virions, was essential for recognition by PLKs. In the case of rat PLK2, functional kinase and polo-box domains were required for interaction with PFV Gag. Fluorescently-tagged PFV Gag, through its chromatin tethering function, selectively relocalized ectopically expressed eGFP-tagged PLK proteins to mitotic chromosomes in a Gag STP motif-dependent manner, confirming a specific and dominant nature of the Gag-PLK interaction in mammalian cells. The functional relevance of the Gag-PLK interaction was examined in the context of replication-competent FVs and single-round PFV vectors. Although STP motif mutated viruses displayed wild type (wt) particle release, RNA packaging and intra-particle reverse transcription, their replication capacity was decreased 3-fold in single-cycle infections, and up to 20-fold in spreading infections over an extended time period. Strikingly similar defects were observed when cells infected with single-round wt Gag PFV vectors were treated with a pan PLK inhibitor. Analysis of entry kinetics of the mutant viruses indicated a post-fusion defect resulting in delayed and reduced integration, which was accompanied with an enhanced preference to integrate into heterochromatin. We conclude that interaction between PFV Gag and cellular PLK proteins is important for early replication steps of PFV within host cells. PMID:27579920

  16. Mitogen-activated protein kinase cascades in signaling plant growth and development.

    PubMed

    Xu, Juan; Zhang, Shuqun

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are ubiquitous signaling modules in eukaryotes. Early research of plant MAPKs has been focused on their functions in immunity and stress responses. Recent studies reveal that they also play essential roles in plant growth and development downstream of receptor-like protein kinases (RLKs). With only a limited number of MAPK components, multiple functional pathways initiated from different receptors often share the same MAPK components or even a complete MAPK cascade. In this review, we discuss how MAPK cascades function as molecular switches in response to spatiotemporal-specific ligand-receptor interactions and the availability of downstream substrates. In addition, we discuss other possible mechanisms governing the functional specificity of plant MAPK cascades, a question central to our understanding of MAPK functions.

  17. Carboxyl Group Footprinting Mass Spectrometry and Molecular Dynamics Identify Key Interactions in the HER2-HER3 Receptor Tyrosine Kinase Interface* ♦

    PubMed Central

    Collier, Timothy S.; Diraviyam, Karthikeyan; Monsey, John; Shen, Wei; Sept, David; Bose, Ron

    2013-01-01

    The HER2 receptor tyrosine kinase is a driver oncogene in many human cancers, including breast and gastric cancer. Under physiologic levels of expression, HER2 heterodimerizes with other members of the EGF receptor/HER/ErbB family, and the HER2-HER3 dimer forms one of the most potent oncogenic receptor pairs. Previous structural biology studies have individually crystallized the kinase domains of HER2 and HER3, but the HER2-HER3 kinase domain heterodimer structure has yet to be solved. Using a reconstituted membrane system to form HER2-HER3 kinase domain heterodimers and carboxyl group footprinting mass spectrometry, we observed that HER2 and HER3 kinase domains preferentially form asymmetric heterodimers with HER3 and HER2 monomers occupying the donor and acceptor kinase positions, respectively. Conformational changes in the HER2 activation loop, as measured by changes in carboxyl group labeling, required both dimerization and nucleotide binding but did not require activation loop phosphorylation at Tyr-877. Molecular dynamics simulations on HER2-HER3 kinase dimers identify specific inter- and intramolecular interactions and were in good agreement with MS measurements. Specifically, several intermolecular ionic interactions between HER2 Lys-716-HER3 Glu-909, HER2 Glu-717-HER3 Lys-907, and HER2 Asp-871-HER3 Arg-948 were identified by molecular dynamics. We also evaluated the effect of the cancer-associated mutations HER2 D769H/D769Y, HER3 E909G, and HER3 R948K (also numbered HER3 E928G and R967K) on kinase activity in the context of this new structural model. This study provides valuable insights into the EGF receptor/HER/ErbB kinase structure and interactions, which can guide the design of future therapies. PMID:23843458

  18. Influenza Virus Infection Induces Host Pyruvate Kinase M Which Interacts with Viral RNA-Dependent RNA Polymerase

    PubMed Central

    Miyake, Yukari; Ishii, Kosuke; Honda, Ayae

    2017-01-01

    Influenza virus RNA-dependent RNA polymerase (RdRp) is a heterotrimer of three viral proteins, PB1, PB2, and PA and is involved in both transcription and replication of the negative strand of the viral RNA (vRNA) genome. RdRp is multifunctional, possessing RNA polymerase, cap binding, and endonuclease activities. The enzyme synthesizes three different RNAs, complementary RNA (cRNA) and messenger RNA (mRNA) from vRNA, and vRNA from cRNA. To synthesize these three RNAs, RdRp requires conversion of its function by host factor. Here, we performed yeast two-hybrid screening to identify the relevant host factor, revealing that pyruvate kinase M2 (PKM2) interacted with the PA subunit of influenza virus RdRp. PKM2 is one of two enzymes (PKM1 and PKM2) produced by alternative splicing of the pyruvate kinase M (PKM) pre-mRNA. We determined the interacting regions in both PKM2 and PA, the expression level of PKM by western blotting at different time points after viral infection, and the effects of transfection of siRNA targeting PKM on influenza virus replication. The results demonstrated that the C-terminal region of PKM2 interacted with the C-terminus of the PA subunit, that the expression level of PKM2 increased with influenza virus infection time, and that this enzyme is essential for influenza virus multiplication. Moreover, isoelectric focusing of uninfected and influenza virus infected cell extracts, followed by gradient gel electrophoresis to separate the PKM1 and PKM2 isoforms and western blotting indicated that PKM2 became more acidic after influenza infection. The decreased pH of PKM2 may have been due to phosphorylation, and phosphorylated PKM2 is active as a pyruvate kinase and protein kinase; therefore, it is possible that PKM2 may transfer a phosphate group to PA and consequently transform the function of RdRp from transcriptase to replicase. PMID:28232820

  19. Influenza Virus Infection Induces Host Pyruvate Kinase M Which Interacts with Viral RNA-Dependent RNA Polymerase.

    PubMed

    Miyake, Yukari; Ishii, Kosuke; Honda, Ayae

    2017-01-01

    Influenza virus RNA-dependent RNA polymerase (RdRp) is a heterotrimer of three viral proteins, PB1, PB2, and PA and is involved in both transcription and replication of the negative strand of the viral RNA (vRNA) genome. RdRp is multifunctional, possessing RNA polymerase, cap binding, and endonuclease activities. The enzyme synthesizes three different RNAs, complementary RNA (cRNA) and messenger RNA (mRNA) from vRNA, and vRNA from cRNA. To synthesize these three RNAs, RdRp requires conversion of its function by host factor. Here, we performed yeast two-hybrid screening to identify the relevant host factor, revealing that pyruvate kinase M2 (PKM2) interacted with the PA subunit of influenza virus RdRp. PKM2 is one of two enzymes (PKM1 and PKM2) produced by alternative splicing of the pyruvate kinase M (PKM) pre-mRNA. We determined the interacting regions in both PKM2 and PA, the expression level of PKM by western blotting at different time points after viral infection, and the effects of transfection of siRNA targeting PKM on influenza virus replication. The results demonstrated that the C-terminal region of PKM2 interacted with the C-terminus of the PA subunit, that the expression level of PKM2 increased with influenza virus infection time, and that this enzyme is essential for influenza virus multiplication. Moreover, isoelectric focusing of uninfected and influenza virus infected cell extracts, followed by gradient gel electrophoresis to separate the PKM1 and PKM2 isoforms and western blotting indicated that PKM2 became more acidic after influenza infection. The decreased pH of PKM2 may have been due to phosphorylation, and phosphorylated PKM2 is active as a pyruvate kinase and protein kinase; therefore, it is possible that PKM2 may transfer a phosphate group to PA and consequently transform the function of RdRp from transcriptase to replicase.

  20. A Conserved Streptococcal Membrane Protein, LsrS, Exhibits a Receptor-Like Function for Lantibiotics

    PubMed Central

    Biswas, Saswati

    2014-01-01

    Streptococcus mutans strain GS-5 produces a two-peptide lantibiotic, Smb, which displays inhibitory activity against a broad spectrum of bacteria, including other streptococci. For inhibition, lantibiotics must recognize specific receptor molecules present on the sensitive bacterial cells. However, so far no such receptor proteins have been identified for any lantibiotics. In this study, using a powerful transposon mutagenesis approach, we have identified in Streptococcus pyogenes a gene that exhibits a receptor-like function for Smb. The protein encoded by that gene, which we named LsrS, is a membrane protein belonging to the CAAX protease family. We also found that nisin, a monopeptide lantibiotic, requires LsrS for its optimum inhibitory activity. However, we found that LsrS is not required for inhibition by haloduracin and galolacticin, both of which are two-peptide lantibiotics closely related to Smb. LsrS appears to be a well-conserved protein that is present in many streptococci, including S. mutans. Inactivation of SMU.662, an LsrS homolog, in S. mutans strains UA159 and V403 rendered the cells refractory to Smb-mediated killing. Furthermore, overexpression of LsrS in S. mutans created cells more susceptible to Smb. Although LsrS and its homolog contain the CAAX protease domain, we demonstrate that inactivation of the putative active sites on the LsrS protein has no effect on its receptor-like function. This is the first report describing a highly conserved membrane protein that displays a receptor-like function for lantibiotics. PMID:24509319

  1. Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

    NASA Technical Reports Server (NTRS)

    Wang, W.; Poovaiah, B. W.

    1999-01-01

    A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

  2. Cell-cycle-specific interaction of nuclear DNA-binding proteins with a CCAAT sequence from the human thymidine kinase gene.

    PubMed Central

    Knight, G B; Gudas, J M; Pardee, A B

    1987-01-01

    Induction of thymidine kinase parallels the onset of DNA synthesis. To investigate the transcriptional regulation of the thymidine kinase gene, we have examined whether specific nuclear factors interact in a cell-cycle-dependent manner with sequences upstream of this gene. Two inverted CCAAT boxes near the transcriptional initiation sites were observed to form complexes with nuclear DNA-binding proteins. The nature of the complexes changes dramatically as the cells approach DNA synthesis and correlates well with the previously reported transcriptional increase of the thymidine kinase gene. Images PMID:3479796

  3. Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution

    PubMed Central

    Tse, Amanda; Verkhivker, Gennady M.

    2015-01-01

    Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib) and promiscuous (Bosutinib, Dasatinib) kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations of key mediating

  4. Signalling-Dependent Interactions Between the Kinase-Coupling Protein CheW and Chemoreceptors in Living Cells

    PubMed Central

    Pedetta, Andrea; Parkinson, John S.; Studdert, Claudia A.

    2014-01-01

    Summary Chemical signals sensed on the periplasmic side of bacterial cells by transmembrane chemoreceptors are transmitted to the flagellar motors via the histidine kinase CheA, which controls the phosphorylation level of the effector protein CheY. Chemoreceptor arrays comprise remarkably stable supramolecular structures in which thousands of chemoreceptors are networked through interactions between their cytoplasmic tips, CheA, and the small coupling protein CheW. To explore the conformational changes that occur within this protein assembly during signalling, we used in vivo crosslinking methods to detect close interactions between the coupling protein CheW and the serine receptor Tsr in intact E. coli cells. We identified two signal-sensitive contacts between CheW and the cytoplasmic tip of Tsr. Our results suggest that ligand binding triggers changes in the receptor that alter its signalling contacts with CheW (and/or CheA). PMID:25060668

  5. Dickkopf-3 Upregulates VEGF in Cultured Human Endothelial Cells by Activating Activin Receptor-Like Kinase 1 (ALK1) Pathway

    PubMed Central

    Busceti, Carla L.; Marchitti, Simona; Bianchi, Franca; Di Pietro, Paola; Riozzi, Barbara; Stanzione, Rosita; Cannella, Milena; Battaglia, Giuseppe; Bruno, Valeria; Volpe, Massimo; Fornai, Francesco; Nicoletti, Ferdinando; Rubattu, Speranza

    2017-01-01

    Dkk-3 is a member of the dickkopf protein family of secreted inhibitors of the Wnt pathway, which has been shown to enhance angiogenesis. The mechanism underlying this effect is currently unknown. Here, we used cultured HUVECs to study the involvement of the TGF-β and VEGF on the angiogenic effect of Dkk-3. Addition of hrDkk-3 peptide (1 or 10 ng/ml) to HUVECs for 6 or 12 h enhanced the intracellular and extracellular VEGF protein levels, as assessed by RTPCR, immunoblotting, immunocytochemistry and ELISA. The increase in the extracellular VEGF levels was associated to the VEGFR2 activation. Pharmacological blockade of VEGFR2 abrogated Dkk-3-induced endothelial cell tubes formation, indicating that VEGF is a molecular player of the angiogenic effects of Dkk-3. Moreover, Dkk-3 enhanced Smad1/5/8 phosphorylation and recruited Smad4 to the VEGF gene promoter, suggesting that Dkk-3 activated ALK1 receptor leading to a transcriptional activation of VEGF. This mechanism was instrumental to the increased VEGF expression and endothelial cell tubes formation mediated by Dkk-3, because both effects were abolished by siRNA-mediated ALK1 knockdown. In summary, we have found that Dkk-3 activates ALK1 to stimulate VEGF production and induce angiogenesis in HUVECs. PMID:28352232

  6. Interaction of the Small GTPase Cdc42 with Arginine Kinase Restricts White Spot Syndrome Virus in Shrimp.

    PubMed

    Xu, Ji-Dong; Jiang, Hai-Shan; Wei, Tian-Di; Zhang, Ke-Yi; Wang, Xian-Wei; Zhao, Xiao-Fan; Wang, Jin-Xing

    2017-03-01

    Many types of small GTPases are widely expressed in eukaryotes and have different functions. As a crucial member of the Rho GTPase family, Cdc42 serves a number of functions, such as regulating cell growth, migration, and cell movement. Several RNA viruses employ Cdc42-hijacking tactics in their target cell entry processes. However, the function of Cdc42 in shrimp antiviral immunity is not clear. In this study, we identified a Cdc42 protein in the kuruma shrimp (Marsupenaeus japonicus) and named it MjCdc42. MjCdc42 was upregulated in shrimp challenged by white spot syndrome virus (WSSV). The knockdown of MjCdc42 and injection of Cdc42 inhibitors increased the proliferation of WSSV. Further experiments determined that MjCdc42 interacted with an arginine kinase (MjAK). By analyzing the binding activity and enzyme activity of MjAK and its mutant, ΔMjAK, we found that MjAK could enhance the replication of WSSV in shrimp. MjAK interacted with the envelope protein VP26 of WSSV. An inhibitor of AK activity, quercetin, could impair the function of MjAK in WSSV replication. Further study demonstrated that the binding of MjCdc42 and MjAK depends on Cys(271) of MjAK and suppresses the WSSV replication-promoting effect of MjAK. By interacting with the active site of MjAK and suppressing its enzyme activity, MjCdc42 inhibits WSSV replication in shrimp. Our results demonstrate a new function of Cdc42 in the cellular defense against viral infection in addition to the regulation of actin and phagocytosis, which has been reported in previous studies. IMPORTANCE The interaction of Cdc42 with arginine kinase plays a crucial role in the host defense against WSSV infection. This study identifies a new mechanism of Cdc42 in innate immunity and enriches the knowledge of the antiviral innate immunity of invertebrates.

  7. Death-associated protein kinase-mediated cell death modulated by interaction with DANGER.

    PubMed

    Kang, Bingnan N; Ahmad, Abdullah S; Saleem, Sofiyan; Patterson, Randen L; Hester, Lynda; Doré, Sylvain; Snyder, Solomon H

    2010-01-06

    Death-associated protein kinase (DAPK) is a key player in multiple cell death signaling pathways. We report that DAPK is regulated by DANGER, a partial MAB-21 domain-containing protein. DANGER binds directly to DAPK and inhibits DAPK catalytic activity. DANGER-deficient mouse embryonic fibroblasts and neurons exhibit greater DAPK activity and increased sensitivity to cell death stimuli than do wild-type control cells. In addition, DANGER-deficient mice manifest more severe brain damage after acute excitotoxicity and transient cerebral ischemia than do control mice. Accordingly, DANGER may physiologically regulate the viability of neurons and represent a potential therapeutic target for stroke and neurodegenerative diseases.

  8. ErbB3 (HER3) interaction with the p85 regulatory subunit of phosphoinositide 3-kinase.

    PubMed Central

    Hellyer, N J; Cheng, K; Koland, J G

    1998-01-01

    ErbB3 (HER3), a unique member of the ErbB receptor family, lacks intrinsic protein tyrosine kinase activity and contains six Tyr-Xaa-Xaa-Met (YXXM) consensus binding sites for the SH2 domains of the p85 regulatory subunit of phosphoinositide 3-kinase. ErbB3 also has a proline-rich sequence that forms a consensus binding site for the SH3 domain of p85. Here we have investigated the interacting domains of ErbB3 and p85 by a unique application of the yeast two-hybrid system. A chimaeric ErbB3 molecule containing the epidermal growth factor receptor protein tyrosine kinase domain was developed so that the C-terminal domain of ErbB3 could become phosphorylated in the yeast system. We also generated several ErbB3 deletion and Tyr-->Phe site-specific mutants, and observed that a single ErbB3 YXXM motif was necessary and sufficient for the association of ErbB3 with p85. The incorporation of multiple YXXM motifs into the ErbB3 C-terminus enabled a stronger ErbB3/p85 interaction. The proline-rich region of ErbB3 was not necessary for interaction with p85. However, either deletion or mutation of the p85 SH3 domain decreased the observed ErbB3/p85 association. Additionally an ErbB3/p85 SH3 domain interaction was detected by an assay in vitro. These results were consistent with a model in which pairs of phosphorylated ErbB3 YXXM motifs co-operate in binding to the tandem SH2 domains of p85. Although a contributing role for the p85 SH3 domain was suggested, the N- and C-terminal SH2 domains seemed to be primarily responsible for the high-affinity association of p85 and ErbB3. PMID:9677338

  9. ErbB3 (HER3) interaction with the p85 regulatory subunit of phosphoinositide 3-kinase.

    PubMed

    Hellyer, N J; Cheng, K; Koland, J G

    1998-08-01

    ErbB3 (HER3), a unique member of the ErbB receptor family, lacks intrinsic protein tyrosine kinase activity and contains six Tyr-Xaa-Xaa-Met (YXXM) consensus binding sites for the SH2 domains of the p85 regulatory subunit of phosphoinositide 3-kinase. ErbB3 also has a proline-rich sequence that forms a consensus binding site for the SH3 domain of p85. Here we have investigated the interacting domains of ErbB3 and p85 by a unique application of the yeast two-hybrid system. A chimaeric ErbB3 molecule containing the epidermal growth factor receptor protein tyrosine kinase domain was developed so that the C-terminal domain of ErbB3 could become phosphorylated in the yeast system. We also generated several ErbB3 deletion and Tyr-->Phe site-specific mutants, and observed that a single ErbB3 YXXM motif was necessary and sufficient for the association of ErbB3 with p85. The incorporation of multiple YXXM motifs into the ErbB3 C-terminus enabled a stronger ErbB3/p85 interaction. The proline-rich region of ErbB3 was not necessary for interaction with p85. However, either deletion or mutation of the p85 SH3 domain decreased the observed ErbB3/p85 association. Additionally an ErbB3/p85 SH3 domain interaction was detected by an assay in vitro. These results were consistent with a model in which pairs of phosphorylated ErbB3 YXXM motifs co-operate in binding to the tandem SH2 domains of p85. Although a contributing role for the p85 SH3 domain was suggested, the N- and C-terminal SH2 domains seemed to be primarily responsible for the high-affinity association of p85 and ErbB3.

  10. Phosphorylation Regulates Interaction of 210-kDa Myosin Light Chain Kinase N-terminal Domain with Actin Cytoskeleton.

    PubMed

    Vilitkevich, E L; Khapchaev, A Y; Kudryashov, D S; Nikashin, A V; Schavocky, J P; Lukas, T J; Watterson, D M; Shirinsky, V P

    2015-10-01

    High molecular weight myosin light chain kinase (MLCK210) is a multifunctional protein involved in myosin II activation and integration of cytoskeletal components in cells. MLCK210 possesses actin-binding regions both in the central part of the molecule and in its N-terminal tail domain. In HeLa cells, mitotic protein kinase Aurora B was suggested to phosphorylate MLCK210 N-terminal tail at serine residues (Dulyaninova, N. G., and Bresnick, A. R. (2004) Exp. Cell Res., 299, 303-314), but the functional significance of the phosphorylation was not established. We report here that in vitro, the N-terminal actin-binding domain of MLCK210 is located within residues 27-157 (N27-157, avian MLCK210 sequence) and is phosphorylated by cAMP-dependent protein kinase (PKA) and Aurora B at serine residues 140/149 leading to a decrease in N27-157 binding to actin. The same residues are phosphorylated in a PKA-dependent manner in transfected HeLa cells. Further, in transfected cells, phosphomimetic mutants of N27-157 showed reduced association with the detergent-stable cytoskeleton, whereas in vitro, the single S149D mutation reduced N27-157 association with F-actin to a similar extent as that achieved by N27-157 phosphorylation. Altogether, our results indicate that phosphorylation of MLCK210 at distinct serine residues, mainly at S149, attenuates the interaction of MLCK210 N-terminus with the actin cytoskeleton and might serve to regulate MLCK210 microfilament cross-linking activity in cells.

  11. The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL).

    PubMed

    West, K A; Zhang, H; Brown, M C; Nikolopoulos, S N; Riedy, M C; Horwitz, A F; Turner, C E

    2001-07-09

    The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinDeltaLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin-PKL interaction. In cells overexpressing paxillinDeltaLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLDeltaPBS2) induced phenotypic changes reminiscent of paxillinDeltaLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

  12. Ligand-protein interactions of selective casein kinase 1δ inhibitors.

    PubMed

    Mente, Scot; Arnold, Eric; Butler, Todd; Chakrapani, Subramanyam; Chandrasekaran, Ramalakshmi; Cherry, Kevin; DiRico, Ken; Doran, Angela; Fisher, Katherine; Galatsis, Paul; Green, Michael; Hayward, Matthew; Humphrey, John; Knafels, John; Li, Jianke; Liu, Shenping; Marconi, Michael; McDonald, Scott; Ohren, Jeff; Paradis, Vanessa; Sneed, Blossom; Walton, Kevin; Wager, Travis

    2013-09-12

    Casein kinase 1δ (CK1δ) and 1ε (CK1ε) are believed to be necessary enzymes for the regulation of circadian rhythms in all mammals. On the basis of our previously published work demonstrating a CK1ε-preferring compound to be an ineffective circadian clock modulator, we have synthesized a series of pyrazole-substitued pyridine inhibitors, selective for the CK1δ isoform. Additionally, using structure-based drug design, we have been able to exploit differences in the hinge region between CK1δ and p38 to find selective inhibitors that have minimal p38 activity. The SAR, brain exposure, and the effect of these inhibitors on mouse circadian rhythms are described. The in vivo evaluation of these inhibitors demonstrates that selective inhibition of CK1δ at sufficient central exposure levels is capable of modulating circadian rhythms.

  13. Thioredoxin-interacting protein regulates haematopoietic stem cell ageing and rejuvenation by inhibiting p38 kinase activity

    PubMed Central

    Jung, Haiyoung; Kim, Dong Oh; Byun, Jae-Eun; Kim, Won Sam; Kim, Mi Jeong; Song, Hae Young; Kim, Young Kwan; Kang, Du-Kyeong; Park, Young-Jun; Kim, Tae-Don; Yoon, Suk Ran; Lee, Hee Gu; Choi, Eun-Ji; Min, Sang-Hyun; Choi, Inpyo

    2016-01-01

    Ageing is a natural process in living organisms throughout their lifetime, and most elderly people suffer from ageing-associated diseases. One suggested way to tackle such diseases is to rejuvenate stem cells, which also undergo ageing. Here we report that the thioredoxin-interacting protein (TXNIP)-p38 mitogen-activated protein kinase (p38) axis regulates the ageing of haematopoietic stem cells (HSCs), by causing a higher frequency of long-term HSCs, lineage skewing, a decrease in engraftment, an increase in reactive oxygen species and loss of Cdc42 polarity. TXNIP inhibits p38 activity via direct interaction in HSCs. Furthermore, cell-penetrating peptide (CPP)-conjugated peptide derived from the TXNIP-p38 interaction motif inhibits p38 activity via this docking interaction. This peptide dramatically rejuvenates aged HSCs in vitro and in vivo. Our findings suggest that the TXNIP-p38 axis acts as a regulatory mechanism in HSC ageing and indicate the potent therapeutic potential of using CPP-conjugated peptide to rejuvenate aged HSCs. PMID:27929088

  14. Tamoxifen inhibits CDK5 kinase activity by interacting with p35/p25 and modulates the pattern of tau phosphorylation.

    PubMed

    Corbel, Caroline; Zhang, Bing; Le Parc, Annabelle; Baratte, Blandine; Colas, Pierre; Couturier, Cyril; Kosik, Kenneth S; Landrieu, Isabelle; Le Tilly, Véronique; Bach, Stéphane

    2015-04-23

    Cyclin-dependent kinase 5 (CDK5) is a multifunctional enzyme that plays numerous roles, notably in brain development. CDK5 is activated through its association with the activators, p35 and p39, rather than by cyclins. Proteolytic procession of the N-terminal part of its activators has been linked to Alzheimer's disease and various other neuropathies. The interaction with the proteolytic product p25 prolongs CDK5 activation and modifies the substrate specificity. In order to discover small-molecule inhibitors of the interaction between CDK5 and p25, we have used a bioluminescence resonance energy transfer (BRET)-based screening assay. Among the 1,760 compounds screened, the generic drug tamoxifen has been identified. The inhibition of the CDK5 activity by tamoxifen was notably validated by monitoring the phosphorylation state of tau protein. The study of the molecular mechanism of inhibition indicates that tamoxifen interacts with p25 to block the CDK5/p25 interaction and pave the way for new treatments of tauopathies.

  15. Comprehensive Modeling and Discovery of Mebendazole as a Novel TRAF2- and NCK-interacting Kinase Inhibitor

    PubMed Central

    Tan, Zhi; Chen, Lu; Zhang, Shuxing

    2016-01-01

    TRAF2- and NCK-interacting kinase (TNIK) represents one of the crucial targets for Wnt-activated colorectal cancer. In this study, we curated two datasets and conducted a comprehensive modeling study to explore novel TNIK inhibitors with desirable biopharmaceutical properties. With Dataset I, we derived Comparative Molecular Similarity Indices Analysis (CoMSIA) and variable-selection k-nearest neighbor models, from which 3D-molecular fields and 2D-descriptors critical for the TNIK inhibitor activity were revealed. Based on Dataset II, predictive CoMSIA-SIMCA (Soft Independent Modelling by Class Analogy) models were obtained and employed to screen 1,448 FDA-approved small molecule drugs. Upon experimental evaluations, we discovered that mebendazole, an approved anthelmintic drug, could selectively inhibit TNIK kinase activity with a dissociation constant Kd = ~1 μM. The subsequent CoMSIA and kNN analyses indicated that mebendazole bears the favorable molecular features that are needed to bind and inhibit TNIK. PMID:27650168

  16. Interaction of Rio1 Kinase with Toyocamycin Reveals a Conformational Switch That Controls Oligomeric State and Catalytic Activity

    SciTech Connect

    Kiburu, Irene N.; LaRonde-LeBlanc, Nicole

    2012-10-10

    Rio1 kinase is an essential ribosome-processing factor required for proper maturation of 40 S ribosomal subunit. Although its structure is known, several questions regarding its functional remain to be addressed. We report that both Archaeoglobus fulgidus and human Rio1 bind more tightly to an adenosine analog, toyocamycin, than to ATP. Toyocamycin has antibiotic, antiviral and cytotoxic properties, and is known to inhibit ribosome biogenesis, specifically the maturation of 40 S. We determined the X-ray crystal structure of toyocamycin bound to Rio1 at 2.0 {angstrom} and demonstrated that toyocamycin binds in the ATP binding pocket of the protein. Despite this, measured steady state kinetics were inconsistent with strict competitive inhibition by toyocamycin. In analyzing this interaction, we discovered that Rio1 is capable of accessing multiple distinct oligomeric states and that toyocamycin may inhibit Rio1 by stabilizing a less catalytically active oligomer. We also present evidence of substrate inhibition by high concentrations of ATP for both archaeal and human Rio1. Oligomeric state studies show both proteins access a higher order oligomeric state in the presence of ATP. The study revealed that autophosphorylation by Rio1 reduces oligomer formation and promotes monomerization, resulting in the most active species. Taken together, these results suggest the activity of Rio1 may be modulated by regulating its oligomerization properties in a conserved mechanism, identifies the first ribosome processing target of toyocamycin and presents the first small molecule inhibitor of Rio1 kinase activity.

  17. Mitogen-Activated Protein Kinase Kinase 2, a Novel E2-Interacting Protein, Promotes the Growth of Classical Swine Fever Virus via Attenuation of the JAK-STAT Signaling Pathway

    PubMed Central

    Wang, Jinghan; Chen, Shucheng; Liao, Yajin; Zhang, Enyu; Feng, Shuo; Yu, Shaoxiong; Li, Lian-Feng; He, Wen-Rui; Li, Yongfeng; Luo, Yuzi; Sun, Yuan; Zhou, Mo; Wang, Xiao; Munir, Muhammad

    2016-01-01

    ABSTRACT The mitogen-activated protein kinase kinase/extracellular regulated kinase (MEK1/2/ERK1/2) cascade is involved in the replication of several members of the Flaviviridae family, including hepatitis C virus and dengue virus. The effects of the cascade on the replication of classical swine fever virus (CSFV), a fatal pestivirus of pigs, remain unknown. In this study, MEK2 was identified as a novel binding partner of the E2 protein of CSFV using yeast two-hybrid screening. The E2-MEK2 interaction was confirmed by glutathione S-transferase pulldown, coimmunoprecipitation, and laser confocal microscopy assays. The C termini of E2 (amino acids [aa] 890 to 1053) and MEK2 (aa 266 to 400) were mapped to be crucial for the interaction. Overexpression of MEK2 significantly promoted the replication of CSFV, whereas knockdown of MEK2 by lentivirus-mediated small hairpin RNAs dramatically inhibited CSFV replication. In addition, CSFV infection induced a biphasic activation of ERK1/2, the downstream signaling molecules of MEK2. Furthermore, the replication of CSFV was markedly inhibited in PK-15 cells treated with U0126, a specific inhibitor for MEK1/2/ERK1/2, whereas MEK2 did not affect CSFV replication after blocking the interferon-induced Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway by ruxolitinib, a JAK-STAT-specific inhibitor. Taken together, our results indicate that MEK2 positively regulates the replication of CSFV through inhibiting the JAK-STAT signaling pathway. IMPORTANCE Mitogen-activated protein kinase kinase 2 (MEK2) is a kinase that operates immediately upstream of extracellular regulated kinase 1/2 (ERK1/2) and links to Raf and ERK via phosphorylation. Currently, little is known about the role of MEK2 in the replication of classical swine fever virus (CSFV), a devastating porcine pestivirus. Here, we investigated the roles of MEK2 and the MEK2/ERK1/2 cascade in the growth of CSFV for the first time. We show

  18. A subnanomolar fluorescent probe for protein kinase CK2 interaction studies.

    PubMed

    Enkvist, Erki; Viht, Kaido; Bischoff, Nils; Vahter, Jürgen; Saaver, Siiri; Raidaru, Gerda; Issinger, Olaf-Georg; Niefind, Karsten; Uri, Asko

    2012-11-21

    Up-regulation of an acidophilic protein kinase, CK2, has been established in several types of cancer. This cognition has made CK2 an important target for drug development for cancer chemotherapy. The characterization of potential drug candidates, determination of the structure and clarification of the functions of CK2 could be facilitated by the application of small-molecule fluorescent probes that bind to the active site of the enzyme with high affinity and selectivity. We have used a bisubstrate approach for the development of a highly potent inhibitor of CK2. 4,5,6,7-Tetrabromo-1H-benzimidazole was conjugated with peptides containing multiple aspartate residues via different linkers. The design of the inhibitors was by crystallographic analysis of the complex of an inhibitor with the catalytic subunit of the enzyme (CK2α). The inhibitory potency of the synthesized compounds was established in a kinetic assay that used thin layer chromatography for the measurement of the rate of phosphorylation of fluorescently labelled peptide 5-TAMRA-RADDSDDDDD. The most potent inhibitor, ARC-1502 (K(i) = 0.5 nM), revealed high selectivity for CK2α in a panel of 140 protein kinases. Labelling of ARC-1502 with PromoFluor-647 gave the fluorescent probe ARC-1504 that possessed subnanomolar affinity towards both CK2α and the holoenzyme. The probe was used in a fluorescence anisotropy-based binding assay to measure the concentration of CK2α and characterize non-labelled ligands binding to the active site of CK2α.

  19. Microglia activation and interaction with neuronal cells in a biochemical model of mevalonate kinase deficiency.

    PubMed

    Tricarico, Paola Maura; Piscianz, Elisa; Monasta, Lorenzo; Kleiner, Giulio; Crovella, Sergio; Marcuzzi, Annalisa

    2015-08-01

    Mevalonate kinase deficiency is a rare disease whose worst manifestation, characterised by severe neurologic impairment, is called mevalonic aciduria. The progressive neuronal loss associated to cell death can be studied in vitro with a simplified model based on a biochemical block of the mevalonate pathway and a subsequent inflammatory trigger. The aim of this study was to evaluate the effect of the mevalonate blocking on glial cells (BV-2) and the following effects on neuronal cells (SH-SY5Y) when the two populations were cultured together. To better understand the cross-talk between glial and neuronal cells, as it happens in vivo, BV-2 and SH-SY5Y were co-cultured in different experimental settings (alone, transwell, direct contact); the effect of mevalonate pathway biochemical block by Lovastatin, followed by LPS inflammatory trigger, were evaluated by analysing programmed cell death and mitochondrial membrane potential, cytokines' release and cells' morphology modifications. In this experimental condition, glial cells underwent an evident activation, confirmed by elevated pro-inflammatory cytokines release, typical of these disorders, and a modification in morphology. Moreover, the activation induced an increase in apoptosis. When glial cells were co-cultured with neurons, their activation caused an increase of programmed cell death also in neuronal cells, but only if the two populations were cultured in direct contact. Our findings, being aware of the limitations related to the cell models used, represent a preliminary step towards understanding the pathological and neuroinflammatory mechanisms occurring in mevalonate kinase diseases. Contact co-culture between neuronal and microglial cells seems to be a good model to study mevalonic aciduria in vitro, and to contribute to the identification of potential drugs able to block microglial activation for this orphan disease. In fact, in such a pathological condition, we demonstrated that microglial cells are

  20. Protein kinase CK2 interacts at the neuromuscular synapse with Rapsyn, Rac1, 14-3-3γ, and Dok-7 proteins and phosphorylates the latter two.

    PubMed

    Herrmann, Dustin; Straubinger, Marion; Hashemolhosseini, Said

    2015-09-11

    Previously, we demonstrated that the protein kinase CK2 associates with and phosphorylates the receptor tyrosine kinase MuSK (muscle specific receptor tyrosine kinase) at the neuromuscular junction (NMJ), thereby preventing fragmentation of the NMJs (Cheusova, T., Khan, M. A., Schubert, S. W., Gavin, A. C., Buchou, T., Jacob, G., Sticht, H., Allende, J., Boldyreff, B., Brenner, H. R., and Hashemolhosseini, S. (2006) Genes Dev. 20, 1800-1816). Here, we asked whether CK2 interacts with other proteins involved in processes at the NMJ, which would be consistent with the previous observation that CK2 appears enriched at the NMJ. We identified the following proteins to interact with protein kinase CK2: (a) the α and β subunits of the nicotinic acetylcholine receptors with weak interaction, (b) dishevelled (Dsh), and (c) another four proteins, Rapsyn, Rac1, 14-3-3γ, and Dok-7, with strong interaction. CK2 phosphorylated 14-3-3γ at serine residue 235 and Dok-7 at several serine residues but does not phosphorylate Rapsyn or Rac1. Furthermore, phosphomimetic Dok-7 mutants aggregated nicotinic acetylcholine receptors in C2C12 myotubes with significantly higher frequency than wild type Dok-7. Additionally, we mapped the interacting epitopes of all four binding partners to CK2 and thereby gained insights into the potential role of the CK2/Rapsyn interaction.

  1. Ca2+-dependent hydrophobic-interaction chromatography. Isolation of a novel Ca2+-binding protein and protein kinase C from bovine brain.

    PubMed

    Walsh, M P; Valentine, K A; Ngai, P K; Carruthers, C A; Hollenberg, M D

    1984-11-15

    Several bovine brain proteins have been found to interact with a hydrophobic chromatography resin (phenyl-Sepharose CL-4B) in a Ca2+-dependent manner. These include calmodulin, the Ca2+/phospholipid-dependent protein kinase (protein kinase C) and a novel Ca2+-binding protein that has now been purified to electrophoretic homogeneity. This latter protein is acidic (pI 5.1) and, like calmodulin and some other high-affinity Ca2+-binding proteins, exhibits a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, with an apparent Mr of 22 000 in the absence of Ca2+ and Mr 21 000 in the presence of Ca2+. This novel calciprotein is distinct from known Ca2+-binding proteins on the basis of Mr under denaturing conditions, Cleveland peptide mapping and amino acid composition analysis. It may be a member of the calmodulin superfamily of Ca2+-binding proteins. This calciprotein does not activate two calmodulin-dependent enzymes, namely cyclic nucleotide phosphodiesterase and myosin light-chain kinase, nor does it have any effect on protein kinase C. It may be a Ca2+-dependent regulatory protein of an as-yet-undefined enzymic activity. The Ca2+/phospholipid-dependent protein kinase is also readily purified by Ca2+-dependent hydrophobic-interaction chromatography followed by ion-exchange chromatography, during which it is easily separated from calmodulin. A preparation of protein kinase C that lacks contaminating kinase or phosphatase activities is thereby obtained rapidly and simply. Such a preparation is ideal for the study of phosphorylation reactions catalysed in vitro by protein kinase C.

  2. Interaction of hexa-His tag with acidic amino acids results in facilitated refolding of halophilic nucleoside diphosphate kinase.

    PubMed

    Ishibashi, Matsujiro; Ida, Keiko; Tatsuda, Shuhei; Arakawa, Tsutomu; Tokunaga, Masao

    2011-11-01

    We have previously reported that amino-terminal extension sequence containing hexa-His facilitated refolding and assembly of hexameric nucleoside diphosphate kinase from extremely halophilic archaeon Halobacterium salinarum (NDK). In this study, we made various mutations in both the tag sequence and within NDK molecule. SerNDK, in which hexa-His was replaced with hexa-Ser, showed no facilitated folding. In addition, HisD58GD63G, in which both Asp58 and Asp63 in NDK were replaced with Gly, also showed no refolding enhancement. These results suggest that hexa-His in His-tag interact cooperatively with either Asp58 or Asp63 or both. Furthermore, G114D mutant, which formed a dimer in low salt solution, was strongly stabilized by His-tag to form a stable hexamer.

  3. Identification of extracellular signal-regulated kinase 3 as a new interaction partner of cyclin D3

    SciTech Connect

    Sun Maoyun; Wei Yuanyan; Yao Luyang; Xie Jianhui; Chen Xiaoning; Wang Hanzhou; Jiang Jianhai; Gu Jianxin . E-mail: jxgu@shmu.edu.cn

    2006-02-03

    Cyclin D3, like cyclin D1 and D2 isoforms, is a crucial component of the core cell cycle machinery in mammalian cells. It also exhibits its unique properties in many other physiological processes. In the present study, using yeast two-hybrid screening, we identified ERK3, an atypical mitogen-activated protein kinase (MAPK), as a cyclin D3 binding partner. GST pull-down assays showed that cyclin D3 interacts directly and specifically with ERK3 in vitro. The binding of cyclin D3 and ERK3 was further confirmed in vivo by co-immunoprecipitation assay and confocal microscopic analysis. Moreover, carboxy-terminal extension of ERK3 was responsible for its association with intact cyclin D3. These findings further expand distinct roles of cyclin D3 and suggest the potential activity of ERK3 in cell proliferation.

  4. Feedback regulation on PTEN/AKT pathway by the ER stress kinase PERK mediated by interaction with the Vault complex.

    PubMed

    Zhang, Wei; Neo, Suat Peng; Gunaratne, Jayantha; Poulsen, Anders; Boping, Liu; Ong, Esther Hongqian; Sangthongpitag, Kanda; Pendharkar, Vishal; Hill, Jeffrey; Cohen, Stephen M

    2015-03-01

    The high proliferation rate of cancer cells, together with environmental factors such as hypoxia and nutrient deprivation can cause Endoplasmic Reticulum (ER) stress. The protein kinase PERK is an essential mediator in one of the three ER stress response pathways. Genetic and pharmacological inhibition of PERK has been reported to limit tumor growth in xenograft models. Here we provide evidence that inactive PERK interacts with the nuclear pore-associated Vault complex protein and that this compromises Vault-mediated nuclear transport of PTEN. Pharmacological inhibition of PERK under ER stress results is abnormal sequestration of the Vault complex, leading to increased cytoplasmic PTEN activity and lower AKT activation. As the PI3K/PTEN/AKT pathway is crucial for many aspects of cell growth and survival, this unexpected effect of PERK inhibitors on AKT activity may have implications for their potential use as therapeutic agents.

  5. Mitogen-stimulated TIS21 protein interacts with a protein-kinase-Calpha-binding protein rPICK1.

    PubMed Central

    Lin, W J; Chang, Y F; Wang, W L; Huang, C Y

    2001-01-01

    TIS21 is induced transiently by PMA and a number of extracellular stimuli. Yeast two-hybrid screening has identified three TIS21 interacting clones from a rat cDNA library [Lin, Gary, Yang, Clarke and Herschman (1996) J. Biol. Chem 271, 15034-15044]. The amino acid sequence deduced from clone 5A shows 96.9% identity with the murine PICK1, a protein kinase Calpha (PKCalpha)-binding protein postulated to act as an intracellular receptor for PKC. A fusion protein of glutathione S-transferase and rPICK1 associates with the TIS21 translated in vitro, suggesting a direct physical interaction between these two proteins. TIS21 and rPICK1 are co-immunoprecipitated from NIH 3T3 cells overexpressing these two proteins. This indicates that the interaction also occurs in mammalian cells. Deletion of the PDZ domain at the N-terminus of rPICK1 abolishes its interaction with TIS21. A putative carboxylate-binding loop required for PICK1 to bind PKCalpha [Staudinger, Lu and Olson (1997) J. Biol. Chem 272, 32019-32024] is within this deleted region. Our results suggest a potential competition between TIS21 and PKC for binding to PICK1. We show that recombinant TIS21 is phosphorylated by PKC in vitro. The catalytic activity of PKC towards TIS21 is significantly decreased in the presence of rPICK1, whereas phosphorylation of histone by PKC is not affected. rPICK1 seems to modulate the phosphorylation of TIS21 through specific interactions between these two proteins. TIS21 might have a role in PKC-mediated extracellular signal transduction through its interaction with rPICK1. PMID:11237868

  6. A Direct Interaction between Leucine-rich Repeat Kinase 2 and Specific β-Tubulin Isoforms Regulates Tubulin Acetylation*

    PubMed Central

    Law, Bernard M. H.; Spain, Victoria A.; Leinster, Veronica H. L.; Chia, Ruth; Beilina, Alexandra; Cho, Hyun J.; Taymans, Jean-Marc; Urban, Mary K.; Sancho, Rosa M.; Ramírez, Marian Blanca; Biskup, Saskia; Baekelandt, Veerle; Cai, Huaibin; Cookson, Mark R.; Berwick, Daniel C.; Harvey, Kirsten

    2014-01-01

    Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease. PMID:24275654

  7. A model for the chemical interactions of adenosine 3':5'-monophosphate with the R subunit of protein kinase type I. Refinement of the cyclic phosphate binding moiety of protein kinase type I.

    PubMed

    Jastorff, B; Hoppe, J; Morr, M

    1979-11-01

    The cAMP receptor site in the regulatory subunit of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase type I was mapped using analogues of cAMP in which the ribose phosphate moiety was systematically modified. Electronical alteration of the cyclophosphate ring at the 3' and 5' positions by sulfur and nitrogen decreased the affinity of these analogues towards the kinase. Substituents at these positions are not tolerated. Testing the separated diastereomers of derivatives in which one of the exocyclic oxygens at the phosphorus has been substituted by sulfur, it was found that one diastereoisomer is preferentially recognized. Based on these results it is proposed that the hydrophylic cyclic phosphate-ribose moiety of cAMP is bound to the kinase via its 3' and 5'-oxygens, the 2'-hydroxy group and the negative charge in a fixed position. Based on our and other published results it is further proposed, that the adenine moiety is bound in a hydrophobic cleft without any hydrogen bond interactions. The chemical interactions between cAMP and the R subunit of protein kinase type I differ from those found for the binding of cAMP to the chemoreceptor of Dictyostelium discoideum [18].

  8. Structural analysis reveals features of the spindle checkpoint kinase Bub1–kinetochore subunit Knl1 interaction

    PubMed Central

    Krenn, Veronica; Wehenkel, Annemarie; Santaguida, Stefano

    2012-01-01

    The function of the essential checkpoint kinases Bub1 and BubR1 requires their recruitment to mitotic kinetochores. Kinetochore recruitment of Bub1 and BubR1 is proposed to rely on the interaction of the tetratricopeptide repeats (TPRs) of Bub1 and BubR1 with two KI motifs in the outer kinetochore protein Knl1. We determined the crystal structure of the Bub1 TPRs in complex with the cognate Knl1 KI motif and compared it with the structure of the equivalent BubR1TPR–KI motif complex. The interaction developed along the convex surface of the TPR assembly. Point mutations on this surface impaired the interaction of Bub1 and BubR1 with Knl1 in vitro and in vivo but did not cause significant displacement of Bub1 and BubR1 from kinetochores. Conversely, a 62-residue segment of Bub1 that includes a binding domain for the checkpoint protein Bub3 and is C terminal to the TPRs was necessary and largely sufficient for kinetochore recruitment of Bub1. These results shed light on the determinants of kinetochore recruitment of Bub1. PMID:22331848

  9. The Ciliopathy-Associated Cep104 Protein Interacts with Tubulin and Nek1 Kinase.

    PubMed

    Al-Jassar, Caezar; Andreeva, Antonina; Barnabas, Deepak D; McLaughlin, Stephen H; Johnson, Christopher M; Yu, Minmin; van Breugel, Mark

    2017-01-03

    Cilia are thin cell projections with essential roles in cell motility, fluid movement, sensing, and signaling. They are templated from centrioles that dock against the plasma membrane and subsequently extend their peripheral microtubule array. The molecular mechanisms underpinning cilia assembly are incompletely understood. Cep104 is a key factor involved in cilia formation and length regulation that rides on the ends of elongating and shrinking cilia. It is mutated in Joubert syndrome, a genetically heterogeneous ciliopathy. Here we provide structural and biochemical data that Cep104 contains a tubulin-binding TOG (tumor overexpressed gene) domain and a novel C2HC zinc finger array. Furthermore, we identify the kinase Nek1, another ciliopathy-associated protein, as a potential binding partner of this array. Finally, we show that Nek1 competes for binding to Cep104 with the distal centriole-capping protein CP110. Our data suggest a model for Cep104 activity during ciliogenesis and provide a novel link between Cep104 and Nek1.

  10. Aluminum interaction with human brain tau protein phosphorylation by various kinases

    SciTech Connect

    El-Sebae; Abou Zeid, M.M.; Saleh, M.A. . Environmental Chemistry and Toxicology Lab.); Abdel-Ghany, M.E.; Shalloway, D. . Section of Biochemistry, Mol, and Cell Biology); Blancato, J. . Environmental Monit. Systems Lab.)

    1993-01-01

    Phosphorylation is an indispensable process for energy and signal transduction in biological systems. AlCl[sub 3] at 10 nM to 10 [mu]M range activated in-vitro [[gamma][sup [minus]32]P]ATP phosphorylation of the brain ([tau]) [Gamma] protein in both normal human or E.coli expressed [Gamma] forms; in the presence of the kinases P34,PKP, and PKC. However, higher concentrations of AlCl[sub 3] inhibited the [Gamma] phosphorylation with P34, PKP, and PKC to a maximum at 1 mM level. AlCl[sub 3] at 100 [mu]M to 500 [mu]M range induced non-enzymatic phosphorylation of [Gamma] with [gamma]-ATP, [gamma]-GTP, and [alpha]-GRP. AlCl[sub 3] activated histone phosphorylation by P34 in a similar pattern. The hyperphosphorylation of [Gamma] by Al[sup 3+] was accompanied in molecular shift and mobility retardation in SDS-PAGE. This may demonstrate the mechanism of the long term neurological effect of Al[sub 3+] in human brain leading to the formation of the neutrofibrillary tangles related to Alzeheimer's disease.

  11. An interaction map of small-molecule kinase inhibitors with anaplastic lymphoma kinase (ALK) mutants in ALK-positive non-small cell lung cancer.

    PubMed

    Ai, Xinghao; Shen, Shengping; Shen, Lan; Lu, Shun

    2015-05-01

    Human anaplastic lymphoma kinase (ALK) has become a well-established target for the treatment of ALK-positive non-small cell lung cancer (NSCLC). Here, we have profiled seven small-molecule inhibitors, including 2 that are approved drugs, against a panel of clinically relevant mutations in ALK tyrosine kinase (TK) domain, aiming at a comprehensive understanding of molecular mechanism and biological implication underlying inhibitor response to ALK TK mutation. We find that (i) the gatekeeper mutation L1196M causes crizotinib resistance by simultaneously increasing and decreasing the binding affinities of, respectively, ATP and inhibitor to ALK, whereas the secondary mutation C1156Y, which is located far away from the ATP-binding site of ALK TK domain, causes the resistance by inducing marked allosteric effect on the site, (ii) the 2nd and 3rd generation kinase inhibitors exhibit relatively high sensitivity towards ALK mutants as compared to 1st generation inhibitors, (iii) the pan-kinase inhibitor staurosporine is insensitive for most mutations due to its high structural compatibility, and (iv) ATP affinity to ALK is generally reduced upon most clinically relevant mutations. Furthermore, we also identify six novel mutation-inhibitor pairs that are potentially associated with drug resistance. In addition, the G1202R and C1156Y mutations are expected to generally cause resistance for many existing inhibitors, since they can address significant effect on the geometric shape and physicochemical property of ALK active pocket.

  12. Type IV pilins regulate their own expression via direct intramembrane interactions with the sensor kinase PilS

    PubMed Central

    Kilmury, Sara L. N.

    2016-01-01

    Type IV pili are important virulence factors for many pathogens, including Pseudomonas aeruginosa. Transcription of the major pilin gene—pilA—is controlled by the PilS-PilR two-component system in response to unknown signals. The absence of a periplasmic sensing domain suggested that PilS may sense an intramembrane signal, possibly PilA. We suggest that direct interactions between PilA and PilS in the inner membrane reduce pilA transcription when PilA levels are high. Overexpression in trans of PilA proteins with diverse and/or truncated C termini decreased native pilA transcription, suggesting that the highly conserved N terminus of PilA was the regulatory signal. Point mutations in PilA or PilS that disrupted their interaction prevented autoregulation of pilA transcription. A subset of PilA point mutants retained the ability to interact with PilS but could no longer decrease pilA transcription, suggesting that interaction between the pilin and sensor kinase is necessary but not sufficient for pilA autoregulation. Furthermore, PilS’s phosphatase motif was required for the autoregulation of pilA transcription, suggesting that under conditions where PilA is abundant, the PilA–PilS interaction promotes PilR dephosphorylation and thus down-regulation of further pilA transcription. These data reveal a clever bacterial inventory control strategy in which the major subunit of an important P. aeruginosa virulence factor controls its own expression. PMID:27162347

  13. OsBRI1 Activates BR Signaling by Preventing Binding between the TPR and Kinase Domains of OsBSK3 via Phosphorylation1

    PubMed Central

    Wang, Xiaolong; Zhao, Zhiying; Wang, Ruiju; Huang, Xiahe; Zhu, Yali; Yuan, Li; Wang, Yingchun; Burlingame, Alma L.; Gao, Yingjie

    2016-01-01

    Many plant receptor kinases transduce signals through receptor-like cytoplasmic kinases (RLCKs); however, the molecular mechanisms that create an effective on-off switch are unknown. The receptor kinase BR INSENSITIVE1 (BRI1) transduces brassinosteroid (BR) signal by phosphorylating members of the BR-signaling kinase (BSK) family of RLCKs, which contain a kinase domain and a C-terminal tetratricopeptide repeat (TPR) domain. Here, we show that the BR signaling function of BSKs is conserved in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) and that the TPR domain of BSKs functions as a “phospho-switchable” autoregulatory domain to control BSKs’ activity. Genetic studies revealed that OsBSK3 is a positive regulator of BR signaling in rice, while in vivo and in vitro assays demonstrated that OsBRI1 interacts directly with and phosphorylates OsBSK3. The TPR domain of OsBSK3, which interacts directly with the protein’s kinase domain, serves as an autoinhibitory domain to prevent OsBSK3 from interacting with bri1-SUPPRESSOR1 (BSU1). Phosphorylation of OsBSK3 by OsBRI1 disrupts the interaction between its TPR and kinase domains, thereby increasing the binding between OsBSK3’s kinase domain and BSU1. Our results not only demonstrate that OsBSK3 plays a conserved role in regulating BR signaling in rice, but also provide insight into the molecular mechanism by which BSK family proteins are inhibited under basal conditions but switched on by the upstream receptor kinase BRI1. PMID:26697897

  14. Three wall-associated kinases required for rice basal immunity form protein complexes in the plasma membrane.

    PubMed

    Cayrol, Bastien; Delteil, Amandine; Gobbato, Enrico; Kroj, Thomas; Morel, Jean-Benoit

    2016-01-01

    Receptor-like kinases (RLKs) play key roles in disease resistance, in particular basal immunity. They recognize patterns produced by the pathogen invasion and often work as complexes in the plasma membrane. Among these RLKs, there is increasing evidence in several plant species of the key role of Wall-associated kinases (WAKs) in disease resistance. We recently showed using rice (Oryza sativa) loss-of-function mutants of three transcriptionally co-regulated OsWAK genes that individual OsWAKs are positively required for quantitative resistance to the rice blast fungus, Magnaporthe oryzae. This finding was unexpected since WAK genes belong to large gene families where functional redundancy is expected. Here we provide evidence that this may be due to complex physical interaction between OsWAK proteins.

  15. Synthesis and characterization of N-parinaroyl analogs of ganglioside GM3 and de-N-acetyl GM3. Interactions with the EGF receptor kinase

    NASA Technical Reports Server (NTRS)

    Song, W.; Welti, R.; Hafner-Strauss, S.; Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    A specific plasma membrane glycosphingolipid, known as ganglioside GM3, can regulate the intrinsic tyrosyl kinase activity of the epidermal growth factor (EGF) receptor; this modulation is not associated with alterations in hormone binding to the receptor. GM3 inhibits EGF receptor tyrosyl kinase activity in detergent micelles, in plasma membrane vesicles, and in whole cells. In addition, immunoaffinity-purified EGF receptor preparations contain ganglioside GM3 (Hanai et al. (1988) J. Biol. Chem. 263, 10915-10921), implying that the glycosphingolipid is intimately associated with the receptor kinase in cell membranes. Both the nature of this association and the molecular mechanism of kinase inhibition remain to be elucidated. In this report, we describe the synthesis of a fluorescent analog of ganglioside GM3, in which the native fatty acid was replaced with trans-parinaric acid. This glycosphingolipid inhibited the receptor kinase activity in a manner similar to that of the native ganglioside. A modified fluorescent glycosphingolipid, N-trans-parinaroyl de-N-acetyl ganglioside GM3, was also prepared. This analog, like the nonfluorescent de-N-acetyl ganglioside GM3, had no effect on receptor kinase activity. Results from tryptophan fluorescence quenching and steady-state anisotropy measurements in membranes containing these fluorescent probes and the human EGF receptor were consistent with the notion that GM3, but not de-N-acetyl GM3, interacts specifically with the receptor in intact membranes.

  16. An Amino-Terminal Polo Kinase Interaction Motif Acts in the Regulation of Centrosome Formation and Reveals a Novel Function for centrosomin (cnn) in Drosophila

    PubMed Central

    Eisman, Robert C.; Phelps, Melissa A. S.; Kaufman, Thomas

    2015-01-01

    The formation of the pericentriolar matrix (PCM) and a fully functional centrosome in syncytial Drosophila melanogaster embryos requires the rapid transport of Cnn during initiation of the centrosome replication cycle. We show a Cnn and Polo kinase interaction is apparently required during embryogenesis and involves the exon 1A-initiating coding exon, suggesting a subset of Cnn splice variants is regulated by Polo kinase. During PCM formation exon 1A Cnn-Long Form proteins likely bind Polo kinase before phosphorylation by Polo for Cnn transport to the centrosome. Loss of either of these interactions in a portion of the total Cnn protein pool is sufficient to remove native Cnn from the pool, thereby altering the normal localization dynamics of Cnn to the PCM. Additionally, Cnn-Short Form proteins are required for polar body formation, a process known to require Polo kinase after the completion of meiosis. Exon 1A Cnn-LF and Cnn-SF proteins, in conjunction with Polo kinase, are required at the completion of meiosis and for the formation of functional centrosomes during early embryogenesis. PMID:26447129

  17. An Amino-Terminal Polo Kinase Interaction Motif Acts in the Regulation of Centrosome Formation and Reveals a Novel Function for centrosomin (cnn) in Drosophila.

    PubMed

    Eisman, Robert C; Phelps, Melissa A S; Kaufman, Thomas

    2015-10-01

    The formation of the pericentriolar matrix (PCM) and a fully functional centrosome in syncytial Drosophila melanogaster embryos requires the rapid transport of Cnn during initiation of the centrosome replication cycle. We show a Cnn and Polo kinase interaction is apparently required during embryogenesis and involves the exon 1A-initiating coding exon, suggesting a subset of Cnn splice variants is regulated by Polo kinase. During PCM formation exon 1A Cnn-Long Form proteins likely bind Polo kinase before phosphorylation by Polo for Cnn transport to the centrosome. Loss of either of these interactions in a portion of the total Cnn protein pool is sufficient to remove native Cnn from the pool, thereby altering the normal localization dynamics of Cnn to the PCM. Additionally, Cnn-Short Form proteins are required for polar body formation, a process known to require Polo kinase after the completion of meiosis. Exon 1A Cnn-LF and Cnn-SF proteins, in conjunction with Polo kinase, are required at the completion of meiosis and for the formation of functional centrosomes during early embryogenesis.

  18. SUMO-interacting motifs (SIMs) in Polo-like kinase 1-interacting checkpoint helicase (PICH) ensure proper chromosome segregation during mitosis.

    PubMed

    Sridharan, Vinidhra; Azuma, Yoshiaki

    2016-08-17

    Polo-like kinase 1 (Plk1)-interacting checkpoint helicase (PICH) localizes at the centromere and is critical for proper chromosome segregation during mitosis. However, the precise molecular mechanism of PICH's centromeric localization and function at the centromere is not yet fully understood. Recently, using Xenopus egg extract assays, we showed that PICH is a promiscuous SUMO binding protein. To further determine the molecular consequence of PICH/SUMO interaction on PICH function, we identified 3 SUMO-interacting motifs (SIMs) on PICH and generated a SIM-deficient PICH mutant. Using the conditional expression of PICH in cells, we found distinct roles of PICH SIMs during mitosis. Although all SIMs are dispensable for PICH's localization on ultrafine anaphase DNA bridges, only SIM3 (third SIM, close to the C-terminus end of PICH) is critical for its centromeric localization. Intriguingly, the other 2 SIMs function in chromatin bridge prevention. With these results, we propose a novel SUMO-dependent regulation of PICH's function on mitotic centromeres.

  19. Peroxisome proliferator activated receptor-γ-Rho-kinase interactions contribute to vascular remodeling after chronic intrauterine pulmonary hypertension

    PubMed Central

    Tseng, Nancy; Seedorf, Gregory; Roe, Gates; Abman, Steven H.

    2013-01-01

    Peroxisome proliferator-activated receptor-γ (PPARγ) and Rho-kinase (ROCK) regulate smooth muscle cell (SMC) proliferation and contribute to vascular remodeling in adult pulmonary hypertension. Whether these pathways interact to contribute to the development of vascular remodeling in persistent pulmonary hypertension of the newborn (PPHN) remains unknown. We hypothesized that ROCK-PPARγ interactions increase SMC proliferation resulting in vascular remodeling in experimental PPHN. Pulmonary artery SMCs (PASMCs) were harvested from fetal sheep after partial ligation of the ductus arteriosus in utero (PPHN) and controls. Cell counts were performed daily for 5 days with or without PPARγ agonists and ROCK inhibition. PPARγ and ROCK protein expression/activity were measured by Western blot in normal and PPHN PASMCs. We assessed PPARγ-ROCK interactions by studying the effect of ROCK activation on PPARγ activity and PPARγ inhibition (siRNA) on ROCK activity and PASMC proliferation. At baseline, PPHN PASMC cell number was increased by 38% above controls on day 5. ROCK protein expression/activity were increased by 25 and 34% and PPARγ protein/activity decreased by 40 and 50% in PPHN PASMC. ROCK inhibition and PPARγ activation restored PPHN PASMC growth to normal values. ROCK inhibition increased PPARγ activity by 50% in PPHN PASMC, restoring PPARγ activity to normal. In normal PASMCs, ROCK activation decreased PPARγ activity and PPARγ inhibition increased ROCK activity and cell proliferation, resulting in a PPHN hyperproliferative PASMC phenotype. PPARγ-ROCK interactions regulate SMC proliferation and contribute to increased PPHN PASMC proliferation and vascular remodeling in PPHN. Restoring normal PPARγ-ROCK signaling may prevent vascular remodeling and improve outcomes in PPHN. PMID:24375792

  20. Gene-Environment Interactions Target Mitogen-activated Protein 3 Kinase 1 (MAP3K1) Signaling in Eyelid Morphogenesis*

    PubMed Central

    Mongan, Maureen; Meng, Qinghang; Wang, Jingjing; Kao, Winston W.-Y.; Puga, Alvaro; Xia, Ying

    2015-01-01

    Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1+/− embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure. PMID:26109068

  1. Heat Stress Response in Pea Involves Interaction of Mitochondrial Nucleoside Diphosphate Kinase with a Novel 86-Kilodalton Protein1

    PubMed Central

    Escobar Galvis, Martha L.; Marttila, Salla; Håkansson, Gunilla; Forsberg, Jens; Knorpp, Carina

    2001-01-01

    In this work we have further characterized the first mitochondrial nucleoside diphosphate kinase (mtNDPK) isolated from plants. The mitochondrial isoform was found to be especially abundant in reproductive and young tissues. Expression of the pea (Pisum sativum L. cv Oregon sugarpod) mtNDPK was not affected by different stress conditions. However, the pea mtNDPK was found to interact with a novel 86-kD protein, which is de novo synthesized in pea leaves upon exposure to heat. Thus, we have evidence for the involvement of mtNDPK in mitochondrial heat response in pea in vivo. Studies on oligomerization revealed that mtNDPK was found in complexes of various sizes, corresponding to the sizes of e.g. hexamers, tetramers, and dimers, indicating flexibility in oligomerization. This flexibility, also found for other NDPK isoforms, has been correlated with the ability of this enzyme to interact with other proteins. We believe that the mtNDPK is involved in heat stress response in pea, possibly as a modulator of the 86-kD protein. PMID:11351071

  2. Selective Inhibition of Mitochondrial JNK Signaling Achieved Using Peptide Mimicry of the Sab Kinase Interacting Motif-1 (KIM1)

    PubMed Central

    Chambers, Jeremy W.; Cherry, Lisa; Laughlin, John D.; Figuera-Losada, Mariana; LoGrasso, Philip V.

    2011-01-01

    The c-jun N-terminal kinases (JNKs) are responsive to stress stimuli leading to activation of proapoptotic proteins and transcription. Additionally, JNK mitochondrial localization has been reported. To selectively target mitochondrial JNK signaling, we exploited JNKs interaction with its mitochondrial scaffold, Sab, using small interfering RNAs (siRNAs) and a cell permeable peptide corresponding to the KIM1 domain of Sab. Gene silencing and peptide interference of this interaction disrupted JNK translocation to the mitochondria and reduced phosphorylation of Bcl-2 without significant impact on c-Jun phosphorylation or AP-1 transcription. In contrast, the JNK inhibitory peptide (TI-JIP1) prevented these three functions. Tat-SabKIM1 selectivity was also demonstrated in anisomycin-stressed HeLa cells where Tat-SabKIM1 prevented Bcl-2 phosphorylation, cell death, loss of mitochondrial membrane potential, and superoxide generation, but not c-Jun phosphorylation. Conversely, TI-JIP1 prevented all aforementioned stress-induced events. This probe introduces a means to evaluate JNK-mediated events on the mitochondria without intervening in nuclear functions of JNK. PMID:21563797

  3. Selective inhibition of mitochondrial JNK signaling achieved using peptide mimicry of the Sab kinase interacting motif-1 (KIM1).

    PubMed

    Chambers, Jeremy W; Cherry, Lisa; Laughlin, John D; Figuera-Losada, Mariana; Lograsso, Philip V

    2011-08-19

    The c-jun N-terminal kinases (JNKs) are responsive to stress stimuli leading to activation of proapoptotic proteins and transcription. Additionally, JNK mitochondrial localization has been reported. To selectively target mitochondrial JNK signaling, we exploited JNK interaction with its mitochondrial scaffold, Sab, using small interfering RNAs (siRNAs) and a cell-permeable peptide corresponding to the KIM1 domain of Sab. Gene silencing and peptide interference of this interaction disrupted JNK translocation to the mitochondria and reduced phosphorylation of Bcl-2 without significant impact on c-Jun phosphorylation or AP-1 transcription. In contrast, the JNK inhibitory peptide (TI-JIP1) prevented these three functions. Tat-Sab(KIM1) selectivity was also demonstrated in anisomycin-stressed HeLa cells where Tat-Sab(KIM1) prevented Bcl-2 phosphorylation, cell death, loss of mitochondrial membrane potential, and superoxide generation but not c-Jun phosphorylation. Conversely, TI-JIP1 prevented all aforementioned stress-induced events. This probe introduces a means to evaluate JNK-mediated events on the mitochondria without intervening in nuclear functions of JNK.

  4. 5-HT2A SEROTONIN RECEPTOR BIOLOGY: Interacting proteins, kinases and paradoxical regulation

    PubMed Central

    Roth, Bryan L

    2011-01-01

    5-hydroxytryptamine2A (5-HT2A) serotonin receptors are important pharmacological targets for a large number of central nervous system and peripheral serotonergic medications. In this review article I summarize work mainly from my lab regarding serotonin receptor anatomy, pharmacology, signaling and regulation. I highlight the role of serotonin receptor interacting proteins and the emerging paradigm of G-protein coupled receptor functional selectivity. PMID:21288474

  5. MUC13 Interaction with Receptor Tyrosine Kinase HER2 Drives Pancreatic Ductal Adenocarcinoma Progression

    PubMed Central

    Khan, Sheema; Sikander, Mohammed; Ebeling, Mara C.; Ganju, Aditya; Kumari, Sonam; Yallapu, Murali M.; Hafeez, Bilal Bin; Ise, Tomoko; Nagata, Satoshi; Zafar, Nadeem; Behrman, Stephen W.; Wan, Jim Y.; Ghimire, Hemendra M.; Sahay, Peeyush; Pradhan, Prabhakar; Chauhan, Subhash C.; Jaggi, Meena

    2016-01-01

    Although MUC13, a transmembrane mucin, is aberrantly expressed in pancreatic ductal adenocarcinoma (PDAC) and generally correlates with increased expression of HER2, the underlying mechanism remains poorly understood. Herein, we found that MUC13 co-localizes and interacts with HER2 in PDAC cells (reciprocal co-immunoprecipitation, immunofluorescence, proximity ligation, co-capping assays) and tissues (immunohistofluorescence). The results from this study demonstrate that MUC13 functionally interacts and activates HER2 at p1248 in PDAC cells, leading to stimulation of HER2 signaling cascade including, ERK1/2, FAK, AKT and PAK1 as well as regulation of the growth, cytoskeleton remodeling and motility and invasion of PDAC cells - all collectively contributing to PDAC progression. Interestingly, all of these phenotypic effects of MUC13-HER2 co-localization could be effectively compromised by depleting MUC13 and mediated by the first and second EGF-like domains of MUC13. Further, MUC13-HER2 co-localization also holds true in PDAC tissues with a strong functional correlation with events contributing to increased degree of disorder and cancer aggressiveness. In brief, findings presented here provide compelling evidence of a functional ramification of MUC13-HER2: this interaction could be potentially exploited for targeted therapeutics in a subset of patients harboring an aggressive form of PDAC. PMID:27321183

  6. The LIM-Only Protein PINCH Directly Interacts with Integrin-Linked Kinase and Is Recruited to Integrin-Rich Sites in Spreading Cells

    PubMed Central

    Tu, Yizeng; Li, Fugang; Goicoechea, Silvia; Wu, Chuanyue

    1999-01-01

    PINCH is a widely expressed and evolutionarily conserved protein comprising primarily five LIM domains, which are cysteine-rich consensus sequences implicated in mediating protein-protein interactions. We report here that PINCH is a binding protein for integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays important roles in the cell adhesion, growth factor, and Wnt signaling pathways. The interaction between ILK and PINCH has been consistently observed under a variety of experimental conditions. They have interacted in yeast two-hybrid assays, in solution, and in solid-phase-based binding assays. Furthermore, ILK, but not vinculin or focal adhesion kinase, has been coisolated with PINCH from mammalian cells by immunoaffinity chromatography, indicating that PINCH and ILK associate with each other in vivo. The PINCH-ILK interaction is mediated by the N-terminal-most LIM domain (LIM1, residues 1 to 70) of PINCH and multiple ankyrin (ANK) repeats located within the N-terminal domain (residues 1 to 163) of ILK. Additionally, biochemical studies indicate that ILK, through the interaction with PINCH, is capable of forming a ternary complex with Nck-2, an SH2/SH3-containing adapter protein implicated in growth factor receptor kinase and small GTPase signaling pathways. Finally, we have found that PINCH is concentrated in peripheral ruffles of cells spreading on fibronectin and have detected clusters of PINCH that are colocalized with the α5β1 integrins. These results demonstrate a specific protein recognition mechanism utilizing a specific LIM domain and multiple ANK repeats and suggest that PINCH functions as an adapter protein connecting ILK and the integrins with components of growth factor receptor kinase and small GTPase signaling pathways. PMID:10022929

  7. Inhibition of the focal adhesion kinase and vascular endothelial growth factor receptor-3 interaction leads to decreased survival in human neuroblastoma cell lines.

    PubMed

    Beierle, Elizabeth A; Ma, Xiaojie; Stewart, Jerry E; Megison, Michael; Cance, William G; Kurenova, Elena V

    2014-03-01

    Neuroblastoma continues to be a devastating childhood solid tumor and is responsible for over 15% of all childhood cancer-related deaths. Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor-3 (VEGFR-3) are protein tyrosine kinases that are overexpressed in a number of human cancers, including neuroblastoma. These two kinases can directly interact and provide survival signals to cancer cells. In this study, we utilized siRNA to VEGFR-3 to demonstrate the biologic importance of this kinase in neuroblastoma cell survival. We also used confocal microscopy and immunoprecipitation to show that FAK and VEGFR-3 bind in neuroblastoma. Finally, employing a 12-amino-acid peptide (AV3) specific to VEGFR-3, we showed that the colocalization between FAK and VEGFR-3 could be disrupted, and that disruption resulted in decreased neuroblastoma cell survival. These studies provide insight to the FAK-VEGFR-3 interaction in neuroblastoma and demonstrate its importance in this tumor type. Focusing upon the FAK-VEGFR-3 interaction may provide a novel therapeutic target for the development of new strategies for treatment of neuroblastoma.

  8. Selective disruption of rb-raf-1 kinase interaction inhibits pancreatic adenocarcinoma growth irrespective of gemcitabine sensitivity.

    PubMed

    Treviño, José G; Verma, Monika; Singh, Sandeep; Pillai, Smitha; Zhang, Dongyu; Pernazza, Daniele; Sebti, Said M; Lawrence, Nicholas J; Centeno, Barbara A; Chellappan, Srikumar P

    2013-12-01

    Inactivation of the retinoblastoma (Rb) tumor suppressor protein is widespread in human cancers. Inactivation of Rb is thought to be initiated by association with Raf-1 (C-Raf) kinase, and here we determined how RRD-251, a disruptor of the Rb-Raf-1 interaction, affects pancreatic tumor progression. Assessment of phospho-Rb levels in resected human pancreatic tumor specimens by immunohistochemistry (n = 95) showed that increased Rb phosphorylation correlated with increasing grade of resected human pancreatic adenocarcinomas (P = 0.0272), which correlated with reduced overall patient survival (P = 0.0186). To define the antitumor effects of RRD-251 (50 μmol/L), cell-cycle analyses, senescence, cell viability, cell migration, anchorage-independent growth, angiogenic tubule formation and invasion assays were conducted on gemcitabine-sensitive and -resistant pancreatic cancer cells. RRD-251 prevented S-phase entry, induced senescence and apoptosis, and inhibited anchorage-independent growth and invasion (P < 0.01). Drug efficacy on subcutaneous and orthotopic xenograft models was tested by intraperitoneal injections of RRD-251 (50 mg/kg) alone or in combination with gemcitabine (250 mg/kg). RRD-251 significantly reduced tumor growth in vivo accompanied by reduced Rb phosphorylation and lymph node and liver metastasis (P < 0.01). Combination of RRD-251 with gemcitabine showed cooperative effect on tumor growth (P < 0.01). In conclusion, disruption of the Rb-Raf-1 interaction significantly reduces the malignant properties of pancreatic cancer cells irrespective of their gemcitabine sensitivity. Selective targeting of Rb-Raf-1 interaction might be a promising strategy targeting pancreatic cancer.

  9. IκB kinase-induced interaction of TPL-2 kinase with 14-3-3 is essential for Toll-like receptor activation of ERK-1 and -2 MAP kinases

    PubMed Central

    Ben-Addi, Abduelhakem; Mambole-Dema, Agnes; Brender, Christine; Martin, Stephen R.; Janzen, Julia; Kjaer, Sven; Smerdon, Stephen J.; Ley, Steven C.

    2014-01-01

    The MEK-1/2 kinase TPL-2 is critical for Toll-like receptor activation of the ERK-1/2 MAP kinase pathway during inflammatory responses, but it can transform cells following C-terminal truncation. IκB kinase (IKK) complex phosphorylation of the TPL-2 C terminus regulates full-length TPL-2 activation of ERK-1/2 by a mechanism that has remained obscure. Here, we show that TPL-2 Ser-400 phosphorylation by IKK and TPL-2 Ser-443 autophosphorylation cooperated to trigger TPL-2 association with 14-3-3. Recruitment of 14-3-3 to the phosphorylated C terminus stimulated TPL-2 MEK-1 kinase activity, which was essential for TPL-2 activation of ERK-1/2. The binding of 14-3-3 to TPL-2 was also indispensible for lipopolysaccharide-induced production of tumor necrosis factor by macrophages, which is regulated by TPL-2 independently of ERK-1/2 activation. Our data identify a key step in the activation of TPL-2 signaling and provide a mechanistic insight into how C-terminal deletion triggers the oncogenic potential of TPL-2 by rendering its kinase activity independent of 14-3-3 binding. PMID:24912162

  10. Glutamate receptor-like channels in plants: a role as amino acid sensors in plant defence?

    PubMed Central

    Roberts, Michael R.

    2014-01-01

    Plant glutamate receptor-like genes (GLRs) are homologous to the genes for mammalian ionotropic glutamate receptors (iGluRs), after which they were named, but in the 16 years since their existence was first revealed, progress in elucidating their biological role has been disappointingly slow. Recently, however, studies from a number of laboratories focusing on the model plant species Arabidopsis thaliana (L.) have thrown new light on the functional properties of some members of the GLR gene family. One important finding has been that plant GLR receptors have a much broader ligand specificity than their mammalian iGluR counterparts, with evidence that some individual GLR receptors can be gated by as many as seven amino acids. These results, together with the ubiquity of their expression throughout the plant, open up the possibility that GLR receptors could have a pervasive role in plants as non-specific amino acid sensors in diverse biological processes. Addressing what one of these roles could be, recent studies examining the wound response and disease susceptibility in GLR knockout mutants have provided evidence that some members of clade 3 of the GLR gene family encode important components of the plant's defence response. Ways in which this family of amino acid receptors might contribute to the plant's ability to respond to an attack from pests and pathogens are discussed. PMID:24991414

  11. Ontogeny of cells containing estrogen receptor-like immunoreactivity in the Brazilian opossum brain.

    PubMed

    Fox, C A; Ross, L R; Jacobson, C D

    1991-11-19

    In this study, we have used the Brazilian short-tailed opossum (Monodelphis domestica) as a model to study the ontogeny of estrogen receptors in the mammalian brain. Monodelphis is a small, pouchless marsupial which breeds well under laboratory conditions and whose young are born in an immature sexually undifferentiated state. The Abbott H222 monoclonal rat estrogen receptor antibody (gift of Abbott Laboratories) was utilized in an indirect immunohistochemical procedure to detect estrogen receptors in developing opossum brains. Estrogen receptors were first expressed in the dorsomedial and ventromedial hypothalamus of the opossum 10 days after birth (10PN). Most regions that contained estrogen receptor-like immunoreactivity (ER LI) in the adult opossum contained ER LI at 15 PN. These areas include the lateral septum, medial preoptic area, bed nucleus of the stria terminalis, periventricular preoptic area and hypothalamus, amygdala, dorsomedial and ventromedial hypothalamic nuclei, arcuate nucleus, ventral premammillary nucleus, and the midbrain central grey. The number of cells that contain ER LI increased through 60PN in all regions that will contain ER LI in the adult opossum. These results indicate that estrogen receptors are present in early development of the Monodelphis brain and may mark the beginning of a critical period for sexual differentiation of the opossum brain.

  12. A nuclear receptor-like pathway regulating multidrug resistance in fungi.

    PubMed

    Thakur, Jitendra K; Arthanari, Haribabu; Yang, Fajun; Pan, Shih-Jung; Fan, Xiaochun; Breger, Julia; Frueh, Dominique P; Gulshan, Kailash; Li, Darrick K; Mylonakis, Eleftherios; Struhl, Kevin; Moye-Rowley, W Scott; Cormack, Brendan P; Wagner, Gerhard; Näär, Anders M

    2008-04-03

    Multidrug resistance (MDR) is a serious complication during treatment of opportunistic fungal infections that frequently afflict immunocompromised individuals, such as transplant recipients and cancer patients undergoing cytotoxic chemotherapy. Improved knowledge of the molecular pathways controlling MDR in pathogenic fungi should facilitate the development of novel therapies to combat these intransigent infections. MDR is often caused by upregulation of drug efflux pumps by members of the fungal zinc-cluster transcription-factor family (for example Pdr1p orthologues). However, the molecular mechanisms are poorly understood. Here we show that Pdr1p family members in Saccharomyces cerevisiae and the human pathogen Candida glabrata directly bind to structurally diverse drugs and xenobiotics, resulting in stimulated expression of drug efflux pumps and induction of MDR. Notably, this is mechanistically similar to regulation of MDR in vertebrates by the PXR nuclear receptor, revealing an unexpected functional analogy of fungal and metazoan regulators of MDR. We have also uncovered a critical and specific role of the Gal11p/MED15 subunit of the Mediator co-activator and its activator-targeted KIX domain in antifungal/xenobiotic-dependent regulation of MDR. This detailed mechanistic understanding of a fungal nuclear receptor-like gene regulatory pathway provides novel therapeutic targets for the treatment of multidrug-resistant fungal infections.

  13. Interactions between glycogen synthase kinase 3beta, protein kinase B, and protein phosphatase 2A in tau phosphorylation in mouse N2a neuroblastoma cells.

    PubMed

    Zhou, Xin-Wen; Winblad, Bengt; Guan, Zhizhong; Pei, Jin-Jing

    2009-01-01

    In this study, we investigated how tau phosphorylation is regulated by protein kinase glycogen synthase kinase 3beta (GSK3 beta), protein kinase B (PKB), and protein phosphatase 2A (PP2A) in mouse N2a neuroblastoma cells. Results showed that GSK3 beta overexpression significantly increased PKB phosphorylation at the S473 site but not the T308 site. Neither GSK3 beta nor PKB overexpression could reduce the PP2AC phosphorylation at the Y307 site. In contrast, either PKB or GSK3 beta knockdown could increase PP2A phosphorylation at the Y307 site. PP2AC knockdown increased GSK3 beta phosphorylation at the S9 site but not at the Y216 site, and PKB phosphorylation at the T308 site but not at the S473 site. Tau phosphorylation at the S396 site was increased by GSK3 beta or PKB overexpression. Tau phosphorylation at the S214 site was only induced by PKB overexpression in the study. While GSK3 beta knockdown decreased tau phosphorylation at the S396 site, PKB knockdown increased tau phosphorylation at both the S396 and S214 sites. PP2AC knockdown decreased tau phosphorylation at the S396 and S214 sites. These findings suggest that tau phosphorylation at the S396 and S214 sites is differentially regulated by GSK3 beta, PKB, and PP2A in N2a cells. The final phosphorylation state of tau is possibly caused by the synergic action of the three enzymes.

  14. Comparison of FDA Approved Kinase Targets to Clinical Trial Ones: Insights from Their System Profiles and Drug-Target Interaction Networks

    PubMed Central

    Xu, Jingyu; Wang, Panpan; Yang, Hong; Li, Yinghong; Yu, Chunyan; Tian, Yubin

    2016-01-01

    Kinase is one of the most productive classes of established targets, but the majority of approved drugs against kinase were developed only for cancer. Intensive efforts were therefore exerted for releasing its therapeutic potential by discovering new therapeutic area. Kinases in clinical trial could provide great opportunities for treating various diseases. However, no systematic comparison between system profiles of established targets and those of clinical trial ones was conducted. The reveal of probable difference or shift of trend would help to identify key factors defining druggability of established targets. In this study, a comparative analysis of system profiles of both types of targets was conducted. Consequently, the systems profiles of the majority of clinical trial kinases were identified to be very similar to those of established ones, but percentages of established targets obeying the system profiles appeared to be slightly but consistently higher than those of clinical trial targets. Moreover, a shift of trend in the system profiles from the clinical trial to the established targets was identified, and popular kinase targets were discovered. In sum, this comparative study may help to facilitate the identification of the druggability of established drug targets by their system profiles and drug-target interaction networks. PMID:27547755

  15. Molecular and biochemical analysis of symbiotic plant receptor kinase complexes

    SciTech Connect

    Cook, Douglas R; Riely, Brendan K

    2010-09-01

    DE-FG02-01ER15200 was a 36-month project, initiated on Sept 1, 2005 and extended with a one-year no cost extension to August 31, 2009. During the project period we published seven manuscripts (2 in review). Including the prior project period (2002-2005) we published 12 manuscripts in journals that include Science, PNAS, The Plant Cell, Plant Journal, Plant Physiology, and MPMI. The primary focus of this work was to further elucidate the function of the Nod factor signaling pathway that is involved in initiation of the legume-rhizobium symbiosis and in particular to explore the relationship between receptor kinase-like proteins and downstream effectors of symbiotic development. During the project period we have map-base cloned two additional players in symbiotic development, including an ERF transcription factor and an ethylene pathway gene (EIN2) that negatively regulates symbiotic signaling; we have also further characterized the subcellular distribution and function of a nuclear-localized symbiosis-specific ion channel, DMI1. The major outcome of the work has been the development of systems for exploring and validating protein-protein interactions that connect symbiotic receptor-like proteins to downstream responses. In this regard, we have developed both homologous (i.e., in planta) and heterologous (i.e., in yeast) systems to test protein interactions. Using yeast 2-hybrid screens we isolated the only known interactor of the nuclear-localized calcium-responsive kinase DMI3. We have also used yeast 2-hybrid methodology to identify interactions between symbiotic signaling proteins and certain RopGTPase/RopGEF proteins that regulate root hair polar growth. More important to the long-term goals of our work, we have established a TAP tagging system that identifies in planta interactions based on co-immuno precipitation and mass spectrometry. The validity of this approach has been shown using known interactors that either co-iummnoprecipate (i.e., remorin) or co

  16. Protein interacting with C kinase 1 suppresses invasion and anchorage-independent growth of astrocytic tumor cells

    PubMed Central

    Cockbill, Louisa M. R.; Murk, Kai; Love, Seth; Hanley, Jonathan G.

    2015-01-01

    Astrocytic tumors are the most common form of primary brain tumor. Astrocytic tumor cells infiltrate the surrounding CNS tissue, allowing them to evade removal upon surgical resection of the primary tumor. Dynamic changes to the actin cytoskeleton are crucial to cancer cell invasion, but the specific mechanisms that underlie the particularly invasive phenotype of astrocytic tumor cells are unclear. Protein interacting with C kinase 1 (PICK1) is a PDZ and BAR domain–containing protein that inhibits actin-related protein 2/3 (Arp2/3)-dependent actin polymerization and is involved in regulating the trafficking of a number of cell-surface receptors. Here we report that, in contrast to other cancers, PICK1 expression is down-regulated in grade IV astrocytic tumor cell lines and also in clinical cases of the disease in which grade IV tumors have progressed from lower-grade tumors. Exogenous expression of PICK1 in the grade IV astrocytic cell line U251 reduces their capacity for anchorage-independent growth, two-dimensional migration, and invasion through a three-dimensional matrix, strongly suggesting that low PICK1 expression plays an important role in astrocytic tumorigenesis. We propose that PICK1 negatively regulates neoplastic infiltration of astrocytic tumors and that manipulation of PICK1 is an attractive possibility for therapeutic intervention. PMID:26466675

  17. Receptor-interacting protein kinase 3-mediated programmed cell necrosis in rats subjected to focal cerebral ischemia-reperfusion injury

    PubMed Central

    DONG, YANRU; BAO, CUIFEN; YU, JINGWEI; LIU, XIA

    2016-01-01

    In the current study, the activation of tumor necrosis factor-α receptor 1 (TNFR1) and receptor-interacting protein kinase 3 (RIP3) were investigated following cerebral ischemia-reperfusion injury (CIRI). Healthy SD rats were randomly divided into 3 groups: Sham operation group, model group and inhibitor group. The model group and inhibitor group were further divided into 4 subgroups of 6, 12, 24 and 72 h following CIRI. Using right middle cerebral artery embolization, the CIRI model was generated. To confirm that the CIRI model was established, neurological scores, TTC staining and brain water content measurements were conducted. Immunohistochemistry and western blotting were conducted to investigate the expression of TNFR1 and RIP3 in the cerebral cortex. It was observed that nerve cell necrosis occurred following 6 h of CIRI. The appearance of necrotic cells was gradually increased with increasing CIRI duration. TNFR1 and RIP3 were positively expressed following 6 h of CIRI. With increasing durations of CIRI, the protein expression levels of TNFR1 and RIP3 were significantly increased. Pre-administration with Z-VAD-FMK (zVAD) significantly increased the protein level of RIP3, however, had no effect on the levels of TNFR1, and was accompanied by a reduction in necrosis. In conclusion, RIP3-mediated cell necrosis was enhanced by caspase blockade zVAD and the function of zVAD was independent of TNFR1 signaling following IR. PMID:27220678

  18. The CBL-Interacting Protein Kinase CIPK23 Regulates HAK5-Mediated High-Affinity K+ Uptake in Arabidopsis Roots.

    PubMed

    Ragel, Paula; Ródenas, Reyes; García-Martín, Elena; Andrés, Zaida; Villalta, Irene; Nieves-Cordones, Manuel; Rivero, Rosa M; Martínez, Vicente; Pardo, Jose M; Quintero, Francisco J; Rubio, Francisco

    2015-12-01

    Plant growth and development requires efficient acquisition of essential elements. Potassium (K(+)) is an important macronutrient present in the soil solution at a wide range of concentrations. Regulation of the K(+) uptake systems in the roots is essential to secure K(+) supply. It has been shown in Arabidopsis (Arabidopsis thaliana) that when the external K(+) concentration is very low (<10 µm), K(+) nutrition depends exclusively on the high-affinity K(+) transporter5 (HAK5). Low-K(+)-induced transcriptional activation of the gene encoding HAK5 has been previously reported. Here, we show the posttranscriptional regulation of HAK5 transport activity by phosphorylation. Expression in a heterologous system showed that the Ca(2+) sensors calcineurin B-like (CBL1), CBL8, CBL9, and CBL10, together with CBL-interacting protein kinase23 (CIPK23), activated HAK5 in vivo. This activation produced an increase in the affinity and the Vmax of K(+) transport. In vitro experiments show that the N terminus of HAK5 is phosphorylated by CIPK23. This supports the idea that phosphorylation of HAK5 induces a conformational change that increases its affinity for K(+). Experiments of K(+) (Rb(+)) uptake and growth measurements in low-K(+) medium with Arabidopsis single mutants hak5, akt1, and cipk23, double mutants hak5 akt1, hak5 cipk23, and akt1 cipk23, and the triple mutant hak5 akt1 cipk23 confirmed the regulatory role of CIPK23 in planta.

  19. Design of novel ligands of CDP-methylerythritol kinase by mimicking direct protein-protein and solvent-mediated interactions.

    PubMed

    Giménez-Oya, Victor; Villacañas, Oscar; Obiol-Pardo, Cristian; Antolin-Llovera, Meritxell; Rubio-Martinez, Jaime; Imperial, Santiago

    2011-01-01

    The methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of the isoprenoid universal building blocks (isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP)) is present in most of human pathogens and is absent in animals, turning it into a promising therapeutic druggable pathway. Two different strategies, a pharmacophore-directed virtual screening and a protein-protein interaction (PPI)-mimicking cyclic peptide were used to search for compounds that bind to the PPI surface of the 4-(cytidine 5-diphospho)-2C-methyl-D-erythritol kinase (CMK), which catalyzes the fourth step of the MEP pathway. A significant part of the pharmacophore hypothesis used in this study was designed by mimicking water-mediated PPI relevant in the CMK homodimer complex stabilization. After database search and with the aid of docking and molecular dynamics (MD) simulations, a 7H-furo[3,2-g]chromen-7-one derivative and a cyclic peptide were chosen as candidates to be ligands of CMK. Their binding affinities were measured using surface plasmon resonance (SPR) technology.

  20. Dynamics of PLCγ and Src Family Kinase 1 Interactions during Nuclear Envelope Formation Revealed by FRET-FLIM

    PubMed Central

    Byrne, Richard D.; Applebee, Christopher; Poccia, Dominic L.; Larijani, Banafshé

    2012-01-01

    The nuclear envelope (NE) breaks down and reforms during each mitotic cycle. A similar process happens to the sperm NE following fertilisation. The formation of the NE in both these circumstances involves endoplasmic reticulum membranes enveloping the chromatin, but PLCγ-dependent membrane fusion events are also essential. Here we demonstrate the activation of PLCγ by a Src family kinase (SFK1) during NE assembly. We show by time-resolved FRET for the first time the direct in vivo interaction and temporal regulation of PLCγ and SFK1 in sea urchins. As a prerequisite for protein activation, there is a rapid phosphorylation of PLCγ on its Y783 residue in response to GTP in vitro. This phosphorylation is dependent upon SFK activity; thus Y783 phosphorylation and NE assembly are susceptible to SFK inhibition. Y783 phosphorylation is also observed on the surface of the male pronucleus (MPN) in vivo during NE formation. Together the corroborative in vivo and in vitro data demonstrate the phosphorylation and activation of PLCγ by SFK1 during NE assembly. We discuss the potential generality of such a mechanism. PMID:22848394

  1. Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions

    PubMed Central

    Hussain, Rohanah; Harding, Stephen E.; Hughes, Charlotte S.; Ma, Pikyee; Patching, Simon G.; Edara, Shalini; Siligardi, Giuliano; Henderson, Peter J.F.; Phillips-Jones, Mary K.

    2016-01-01

    This article reviews current methods for the reliable heterologous overexpression in Escherichia coli and purification of milligram quantities of bacterial membrane sensor kinase (MSK) proteins belonging to the two-component signal transduction family of integral membrane proteins. Many of these methods were developed at Leeds alongside Professor Steve Baldwin to whom this review is dedicated. It also reviews two biophysical methods that we have adapted successfully for studies of purified MSKs and other membrane proteins–synchrotron radiation circular dichroism (SRCD) spectroscopy and analytical ultracentrifugation (AUC), both of which are non-immobilization and matrix-free methods that require no labelling strategies. Other techniques such as isothermal titration calorimetry (ITC) also share these features but generally require high concentrations of material. In common with many other biophysical techniques, both of these biophysical methods provide information regarding membrane protein conformation, oligomerization state and ligand binding, but they possess the additional advantage of providing direct assessments of whether ligand binding interactions are accompanied by conformational changes. Therefore, both methods provide a powerful means by which to identify and characterize inhibitor binding and any associated protein conformational changes, thereby contributing valuable information for future drug intervention strategies directed towards bacterial MSKs. PMID:27284046

  2. Downregulation of homeodomain-interacting protein kinase-2 contributes to bladder cancer metastasis by regulating Wnt signaling.

    PubMed

    Tan, Mingyue; Gong, Hua; Zeng, Yigang; Tao, Le; Wang, Jun; Jiang, Juntao; Xu, Dongliang; Bao, Erdun; Qiu, Jianxin; Liu, Zhihong

    2014-10-01

    Homeodomain-interacting protein kinase-2 (Hipk2) has been shown to have important regulatory roles in cancer biology, such as cancer cell proliferation, cell cycle, and cell invasion. However, the contributions of Hipk2 to bladder cancer metastasis remain largely unknown. In the current study, we assayed the expression level of Hipk2 in bladder cancer tissues by real-time PCR, and defined its biological functions. We found that Hipk2 levels were downregulated in most bladder cancer tissues compared with adjacent normal tissues, and Hipk2 levels were remarkably decreased in metastasized tumor tissues when compared with primary tumors. SiRNA-mediated Hipk2 silencing increased bladder cancer cell invasion. Hipk2 knockdown resulted in decrease of E-cadherin expression and increase of N-cadherin and fibronectin expression, indicated that epithelial-mesenchymal transition (EMT) was induced. We further demonstrated that Hipk2 knockdown induced Wnt signaling activation and β-catenin nuclear localization. Finally, we confirmed that Hipk2 inhibition promoted EMT and subsequent cell invasion, at least in part by activating Wnt signaling. These data suggest an important role of Hipk2 in regulating metastasis of bladder cancer and implicate the potential application of Hipk2 in bladder cancer therapy.

  3. Tyrosine kinase receptor Axl enhances entry of Zaire ebolavirus without direct interactions with the viral glycoprotein

    SciTech Connect

    Brindley, Melinda A.; Hunt, Catherine L.; Kondratowicz, Andrew S.; Bowman, Jill; Sinn, Patrick L.; McCray, Paul B.; Quinn, Kathrina; Weller, Melodie L.; Chiorini, John A.; Maury, Wendy

    2011-07-05

    In a bioinformatics-based screen for cellular genes that enhance Zaire ebolavirus (ZEBOV) transduction, AXL mRNA expression strongly correlated with ZEBOV infection. A series of cell lines and primary cells were identified that require Axl for optimal ZEBOV entry. Using one of these cell lines, we identified ZEBOV entry events that are Axl-dependent. Interactions between ZEBOV-GP and the Axl ectodomain were not detected in immunoprecipitations and reduction of surface-expressed Axl by RNAi did not alter ZEBOV-GP binding, providing evidence that Axl does not serve as a receptor for the virus. However, RNAi knock down of Axl reduced ZEBOV pseudovirion internalization and {alpha}-Axl antisera inhibited pseudovirion fusion with cellular membranes. Consistent with the importance of Axl for ZEBOV transduction, Axl transiently co-localized on the surface of cells with ZEBOV virus particles and was internalized during virion transduction. In total, these findings indicate that endosomal uptake of filoviruses is facilitated by Axl.

  4. Protein Kinases and Addiction

    PubMed Central

    Lee, Anna M.; Messing, Robert O.

    2011-01-01

    Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharma-cotherapies to treat drug addiction. PMID:18991950

  5. Interaction of a tyrosine kinase inhibitor, vandetanib with human serum albumin as studied by fluorescence quenching and molecular docking.

    PubMed

    Kabir, Md Zahirul; Feroz, Shevin R; Mukarram, Abdul Kadir; Alias, Zazali; Mohamad, Saharuddin B; Tayyab, Saad

    2016-08-01

    Interaction of a tyrosine kinase inhibitor, vandetanib (VDB), with the major transport protein in the human blood circulation, human serum albumin (HSA), was investigated using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking analysis. The binding constant of the VDB-HSA system, as determined by fluorescence quenching titration method was found in the range, 8.92-6.89 × 10(3 )M(-1) at three different temperatures, suggesting moderate binding affinity. Furthermore, decrease in the binding constant with increasing temperature revealed involvement of static quenching mechanism, thus affirming the formation of the VDB-HSA complex. Thermodynamic analysis of the binding reaction between VDB and HSA yielded positive ΔS (52.76 J mol(-1) K(-1)) and negative ΔH (-6.57 kJ mol(-1)) values, which suggested involvement of hydrophobic interactions and hydrogen bonding in stabilizing the VDB-HSA complex. Far-UV and near-UV CD spectral results suggested alterations in both secondary and tertiary structures of HSA upon VDB-binding. Three-dimensional fluorescence spectral results also showed significant microenvironmental changes around the Trp residue of HSA consequent to the complex formation. Use of site-specific marker ligands, such as phenylbutazone (site I marker) and diazepam (site II marker) in competitive ligand displacement experiments indicated location of the VDB binding site on HSA as Sudlow's site I (subdomain IIA), which was further established by molecular docking results. Presence of some common metal ions, such as Ca(2+), Zn(2+), Cu(2+), Ba(2+), Mg(2+), and Mn(2+) in the reaction mixture produced smaller but significant alterations in the binding affinity of VDB to HSA.

  6. Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo

    PubMed Central

    Lageix, Sebastien; Zhang, Jinwei; Rothenburg, Stefan; Hinnebusch, Alan G.

    2015-01-01

    The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd- phenotype), while other substitutions block kinase activation (Gcn- phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction. PMID:25695491

  7. PUB1 Interacts with the Receptor Kinase DMI2 and Negatively Regulates Rhizobial and Arbuscular Mycorrhizal Symbioses through Its Ubiquitination Activity in Medicago truncatula1

    PubMed Central

    Camut, Sylvie; Camps, Céline; Rembliere, Céline; de Carvalho-Niebel, Fernanda; Timmers, Ton; Gasciolli, Virginie; Thompson, Richard; Lefebvre, Benoit; Cullimore, Julie; Hervé, Christine

    2016-01-01

    PUB1, an E3 ubiquitin ligase, which interacts with and is phosphorylated by the LYK3 symbiotic receptor kinase, negatively regulates rhizobial infection and nodulation during the nitrogen-fixing root nodule symbiosis in Medicago truncatula. In this study, we show that PUB1 also interacts with and is phosphorylated by DOES NOT MAKE INFECTIONS 2, the key symbiotic receptor kinase of the common symbiosis signaling pathway, required for both the rhizobial and the arbuscular mycorrhizal (AM) endosymbioses. We also show here that PUB1 expression is activated during successive stages of root colonization by Rhizophagus irregularis that is compatible with its interaction with DOES NOT MAKE INFECTIONS 2. Through characterization of a mutant, pub1-1, affected by the E3 ubiquitin ligase activity of PUB1, we have shown that the ubiquitination activity of PUB1 is required to negatively modulate successive stages of infection and development of rhizobial and AM symbioses. In conclusion, PUB1 represents, to our knowledge, a novel common component of symbiotic signaling integrating signal perception through interaction with and phosphorylation by two key symbiotic receptor kinases, and downstream signaling via its ubiquitination activity to fine-tune both rhizobial and AM root endosymbioses. PMID:26839127

  8. A new c-Jun N-terminal kinase (JNK)-interacting protein, Sab (SH3BP5), associates with mitochondria.

    PubMed

    Wiltshire, Carolyn; Matsushita, Masato; Tsukada, Satoshi; Gillespie, David A F; May, Gerhard H W

    2002-11-01

    We have identified a novel c-Jun N-terminal kinase (JNK)-interacting protein, Sab, by yeast two-hybrid screening. Sab binds to and serves as a substrate for JNK in vitro, and was previously found to interact with the Src homology 3 (SH3) domain of Bruton's tyrosine kinase (Btk). Inspection of the sequence of Sab reveals the presence of two putative mitogen-activated protein kinase interaction motifs (KIMs) similar to that found in the JNK docking domain of the c-Jun transcription factor, and four potential serine-proline JNK phosphorylation sites in the C-terminal half of the molecule. Using deletion and site-directed mutagenesis, we demonstrate that the most N-terminal KIM in Sab is essential for JNK binding, and that, as with c-Jun, physical interaction with JNK is necessary for Sab phosphorylation. Interestingly, confocal immunocytochemistry and cell fractionation studies indicate that Sab is associated with mitochondria, where it co-localizes with a fraction of active JNK. These and previously reported properties of Sab suggest a possible role in targeting JNK to this subcellular compartment and/or mediating cross-talk between the Btk and JNK signal transduction pathways.

  9. A new c-Jun N-terminal kinase (JNK)-interacting protein, Sab (SH3BP5), associates with mitochondria.

    PubMed Central

    Wiltshire, Carolyn; Matsushita, Masato; Tsukada, Satoshi; Gillespie, David A F; May, Gerhard H W

    2002-01-01

    We have identified a novel c-Jun N-terminal kinase (JNK)-interacting protein, Sab, by yeast two-hybrid screening. Sab binds to and serves as a substrate for JNK in vitro, and was previously found to interact with the Src homology 3 (SH3) domain of Bruton's tyrosine kinase (Btk). Inspection of the sequence of Sab reveals the presence of two putative mitogen-activated protein kinase interaction motifs (KIMs) similar to that found in the JNK docking domain of the c-Jun transcription factor, and four potential serine-proline JNK phosphorylation sites in the C-terminal half of the molecule. Using deletion and site-directed mutagenesis, we demonstrate that the most N-terminal KIM in Sab is essential for JNK binding, and that, as with c-Jun, physical interaction with JNK is necessary for Sab phosphorylation. Interestingly, confocal immunocytochemistry and cell fractionation studies indicate that Sab is associated with mitochondria, where it co-localizes with a fraction of active JNK. These and previously reported properties of Sab suggest a possible role in targeting JNK to this subcellular compartment and/or mediating cross-talk between the Btk and JNK signal transduction pathways. PMID:12167088

  10. Fc Receptor-Like Proteins in Pathophysiology of B-cell Disorder

    PubMed Central

    Capone, Mollie; Bryant, John Matthew; Sutkowski, Natalie; Haque, Azizul

    2016-01-01

    Members of the family of Fc receptor-like (FcRL) proteins, homologous to FcγRI, have been identified by multiple research groups. Consequently, they have been described using multiple nomenclatures including Fc receptor homologs (FcRH), immunoglobulin superfamily receptor translocation-associated genes (IRTA), immunoglobulin-Fc-gp42-related genes (IFGP), Src homology 2 domain-containing phosphatase anchor proteins (SPAP), and B cell cross-linked by anti-immunoglobulin M-activating sequences (BXMAS). They are now referred to under a unified nomenclature as FCRL. Eight different human FCRL genes have been identified, all of which appear to be related to the genes of the immunoglobulin superfamily (IgSF) of cellular adhesion molecules. These type 1 transmembrane glycoproteins are composed of different combinations of 5 types of immunoglobulin-like domains, with each protein consisting of 3 to 9 domains, and no individual domain type conserved throughout all of the FCRL proteins. Ligands for the majority of the FCRLs remain unknown. In general, FCRL expression is restricted to lymphocytes and is primarily expressed in B-lymphocytes, supporting FCRL’s involvement in a variety of immune disorders. Most FCRLs functionally repress B-cell activation; however, they might have dual roles in lymphocyte functions as these proteins often possess immunoreceptor tyrosine activation (ITAM) and inhibitory (ITIM) motif elements. The biological functions of these newly recognized FCRL proteins are just beginning to emerge, and might provide the insight necessary for understanding pathophysiology of lymphocyte disorders and treating different immune diseases. PMID:27446638

  11. The role of effectors and host immunity in plant-necrotrophic fungal interactions.

    PubMed

    Wang, Xuli; Jiang, Nan; Liu, Jinling; Liu, Wende; Wang, Guo-Liang

    2014-01-01

    Fungal diseases pose constant threats to the global economy and food safety. As the largest group of plant fungal pathogens, necrotrophic fungi cause heavy crop losses worldwide. The molecular mechanisms of the interaction between necrotrophic fungi and plants are complex and involve sophisticated recognition and signaling networks. Here, we review recent findings on the roles of phytotoxin and proteinaceous effectors, pathogen-associated molecular patterns (PAMPs), and small RNAs from necrotrophic fungi. We also consider the functions of damage-associated molecular patterns (DAMPs), the receptor-like protein kinase BIK1, and epigenetic regulation in plant immunity to necrotrophic fungi.

  12. Interaction with receptor for activated C-kinase 1 (RACK1) sensitizes the phosphodiesterase PDE4D5 towards hydrolysis of cAMP and activation by protein kinase C

    PubMed Central

    Bird, Rebecca J.; Baillie, George S.; Yarwood, Stephen J.

    2010-01-01

    We have previously identified the PKC (protein kinase C)-anchoring protein RACK1 (receptor for activated C-kinase 1), as a specific binding partner for the cAMP-specific phosphodiesterase PDE4D5, suggesting a potential site for cross-talk between the PKC and cAMP signalling pathways. In the present study we found that elevation of intracellular cAMP, with the β2-adrenoceptor agonist isoproterenol (isoprenaline), led to activation of PDE4 enzymes in the particulate and soluble fractions of HEK (human embryonic kidney)-293 cells. In contrast activation of PDE4D5, with isoproterenol and the PKC activator PMA, was restricted to the particulate fraction, where it interacts with RACK1; however, RACK1 is dispensable for anchoring PDE4D5 to the particulate fraction. Kinetic studies demonstrated that RACK1 alters the conformation of particulate-associated PDE4D5 so that it more readily interacts with its substrate cAMP and with rolipram, a PDE4 inhibitor that specifically targets the active site of the enzyme. Interaction with RACK1 was also essential for PKC-dependent and ERK (extracellular-signal-regulated kinase)-independent phosphorylation (on Ser126), and activation of PDE4D5 in response to PMA and isoproterenol, both of which trigger the recruitment of PKCα to RACK1. Together these results reveal novel signalling cross-talk, whereby RACK1 mediates PKC-dependent activation of PDE4D5 in the particulate fraction of HEK-293 cells in response to elevations in intracellular cAMP. PMID:20819076

  13. Function of the cytoplasmic tail of human calcitonin receptor-like receptor in complex with receptor activity-modifying protein 2

    SciTech Connect

    Kuwasako, Kenji; Kitamura, Kazuo; Nagata, Sayaka; Hikosaka, Tomomi; Kato, Johji

    2010-02-12

    Receptor activity-modifying protein 2 (RAMP2) enables calcitonin receptor-like receptor (CRLR) to form an adrenomedullin (AM)-specific receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or receptor internalization, but did not affect [{sup 125}I]AM binding. We found that CRLR residues 428-439 are required for AM-evoked cAMP production, though deleting this region had little effect on receptor internalization. Moreover, pretreatment with pertussis toxin (100 ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2 complexes. This effect was canceled by deleting CRLR residues 454-457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser{sup 449} to Ser{sup 467} were unable to undergo AM-induced receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449-467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.

  14. Protein interacting with C kinase 1 (PICK1) reduces reinsertion rates of interaction partners sorted to Rab11-dependent slow recycling pathway.

    PubMed

    Madsen, Kenneth L; Thorsen, Thor S; Rahbek-Clemmensen, Troels; Eriksen, Jacob; Gether, Ulrik

    2012-04-06

    The scaffolding protein PICK1 (protein interacting with C kinase 1) contains an N-terminal PSD-95/Discs large/ZO-1 (PDZ) domain and a central lipid-binding Bin/amphiphysin/Rvs (BAR) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which primarily sorts to degradation upon internalization, did not form perinuclear clusters with PICK1, and PICK1 did not affect DAT internalization/recycling. However, transfer of the PICK1-binding DAT C terminus to the β(2)-adrenergic receptor, which sorts to recycling upon internalization, led to formation of PICK1 co-clusters in Rab11-positive compartments. Furthermore, PICK1 inhibited Rab11-mediated recycling of the receptor in a BAR and PDZ domain-dependent manner. In contrast, transfer of the DAT C terminus to the δ-opioid receptor, which sorts to degradation, did not result in PICK1 co-clusters or any change in internalization/recycling. Further support for a role of PICK1 determined by its PDZ cargo was obtained for the PICK1 interaction partner prolactin-releasing peptide receptor (GPR10). GPR10 co-localized with Rab11 and clustered with PICK1 upon constitutive internalization but co-localized with the late endosomal marker Rab7 and did not cluster with PICK1 upon agonist-induced internalization. Our data suggest a selective role of PICK1 in clustering and reducing the recycling rates of PDZ domain binding partners sorted to the Rab11-dependent recycling pathway.

  15. Caveolin-1 - A Novel Interacting Partner of Organic Cation/Carnitine Transporter (Octn2): Effect of Protein Kinase C on This Interaction in Rat Astrocytes

    PubMed Central

    Czeredys, Magdalena; Samluk, Łukasz; Michalec, Katarzyna; Tułodziecka, Karolina; Skowronek, Krzysztof; Nałęcz, Katarzyna A.

    2013-01-01

    OCTN2 - the Organic Cation Transporter Novel family member 2 (SLC22A5) is known to be a xenobiotic/drug transporter. It transports as well carnitine - a compound necessary for oxidation of fatty acids and mutations of its gene cause primary carnitine deficiency. Octn2 regulation by protein kinase C (PKC) was studied in rat astrocytes - cells in which β-oxidation takes place in the brain. Activation of PKC with phorbol ester stimulated L-carnitine transport and increased cell surface presence of the transporter, although no PKC-specific phosphorylation of Octn2 could be detected. PKC activation resulted in an augmented Octn2 presence in cholesterol/sphingolipid-rich microdomains of plasma membrane (rafts) and increased co-precipitation of Octn2 with raft-proteins, caveolin-1 and flotillin-1. Deletion of potential caveolin-1 binding motifs pointed to amino acids 14–22 and 447–454 as the caveolin-1 binding sites within Octn2 sequence. A direct interaction of Octn2 with caveolin-1 in astrocytes upon PKC activation was detected by proximity ligation assay, while such an interaction was excluded in case of flotillin-1. Functioning of a multi-protein complex regulated by PKC has been postulated in rOctn2 trafficking to the cell surface, a process which could be important both under physiological conditions, when carnitine facilitates fatty acids catabolism and controls free Coenzyme A pool as well as in pathology, when transport of several drugs can induce secondary carnitine deficiency. PMID:24349196

  16. Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits.

    PubMed Central

    Holt, K H; Olson, L; Moye-Rowley, W S; Pessin, J E

    1994-01-01

    Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110. Images PMID:8264609

  17. SRPK2: A Differentially Expressed SR Protein-specific Kinase Involved in Mediating the Interaction and Localization of Pre-mRNA Splicing Factors in Mammalian Cells

    PubMed Central

    Wang, Huan-You; Lin, Wen; Dyck, Jacqueline A.; Yeakley, Joanne M.; Songyang, Zhou; Cantley, Lewis C.; Fu, Xiang-Dong

    1998-01-01

    Abstract. Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells. Two kinases, SR protein-specific kinase (SRPK1) and Clk/Sty, have been shown to phosphorylate the SR family of splicing factors. We report here the cloning and characterization of SRPK2, which is highly related to SRPK1 in sequence, kinase activity, and substrate specificity. Random peptide selection for preferred phosphorylation sites revealed a stringent preference of SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites in candidate arginine and serine-rich (RS) domain–containing proteins. Phosphorylation of an SR protein (ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain–containing protein (U1 70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus. These observations likely reflect the function of the SRPK family of kinases in spliceosome assembly and in mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities between SRPK1 and 2, however, are in contrast to their differences in expression. SRPK1 is highly expressed in pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues and in many experimental cell lines. Interestingly, SRPK2 also contains a proline-rich sequence at its NH2 terminus, and a recent study showed that this NH2-terminal sequence has the capacity to interact with a WW domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely regulated and targeted, thereby contributing to splicing regulation in different tissues, during development, or in response to signaling. PMID:9472028

  18. Protein Kinase C Activation Promotes α1B-Adrenoceptor Internalization and Late Endosome Trafficking through Rab9 Interaction. Role in Heterologous Desensitization.

    PubMed

    Alfonzo-Méndez, Marco A; Hernández-Espinosa, David A; Carmona-Rosas, Gabriel; Romero-Ávila, M Teresa; Reyes-Cruz, Guadalupe; García-Sáinz, J Adolfo

    2017-04-01

    Upon agonist stimulation, α1B-adrenergic receptors couple to Gq proteins, calcium signaling and protein kinase C activation; subsequently, the receptors are phosphorylated, desensitized, and internalized. Internalization seems to involve scaffolding proteins, such as β-arrestin and clathrin. However, the fine mechanisms that participate remain unsolved. The roles of protein kinase C and the small GTPase, Rab9, in α1B-AR vesicular traffic were investigated by studying α1B-adrenergic receptor-Rab protein interactions, using Förster resonance energy transfer (FRET), confocal microscopy, and intracellular calcium quantitation. In human embryonic kidney 293 cells overexpressing Discosoma spp. red fluorescent protein (DsRed)-tagged α1B-ARs and enhanced green fluorescent protein--tagged Rab proteins, pharmacological protein kinase C activation mimicked α1B-AR traffic elicited by nonrelated agents, such as sphingosine 1-phosphate (i.e., transient α1B-AR-Rab5 FRET signal followed by a sustained α1B-AR-Rab9 interaction), suggesting brief receptor localization in early endosomes and transfer to late endosomes. This latter interaction was abrogated by blocking protein kinase C activity, resulting in receptor retention at the plasma membrane. Similar effects were observed when a dominant-negative Rab9 mutant (Rab9-GDP) was employed. When α1B-adrenergic receptors that had been mutated at protein kinase C phosphorylation sites (S396A, S402A) were used, phorbol ester-induced desensitization of the calcium response was markedly decreased; however, interaction with Rab9 was only partially decreased and internalization was observed in response to phorbol esters and sphingosine 1-phosphate. Finally, Rab9-GDP expression did not affect adrenergic-mediated calcium response but abolished receptor traffic and altered desensitization. Data suggest that protein kinase C modulates α1B-adrenergic receptor transfer to late endosomes and that Rab9 regulates this process and

  19. Impaired Angiogenesis during Fracture Healing in GPCR Kinase 2 Interacting Protein-1 (GIT1) Knock Out Mice

    PubMed Central

    Menon, Prashanthi; Pang, Jinjiang; Ho, Hsin-Chiu; Shi, Shanshan; Xie, Chao; Smolock, Elaine; Yan, Chen; Zuscik, Michael J.; Berk, Bradford C.

    2014-01-01

    G protein coupled receptor kinase 2 (GRK2) interacting protein-1 (GIT1), is a scaffold protein that plays an important role in angiogenesis and osteoclast activity. We have previously demonstrated that GIT1 knockout (GIT1 KO) mice have impaired angiogenesis and dysregulated osteoclast podosome formation leading to a reduction in the bone resorbing ability of these cells. Since both angiogenesis and osteoclast-mediated bone remodeling are involved in the fracture healing process, we hypothesized that GIT1 participates in the normal progression of repair following bone injury. In the present study, comparison of fracture healing in wild type (WT) and GIT1 KO mice revealed altered healing in mice with loss of GIT1 function. Alcian blue staining of fracture callus indicated a persistence of cartilagenous matrix in day 21 callus samples from GIT1 KO mice which was temporally correlated with increased type 2 collagen immunostaining. GIT1 KO mice also showed a decrease in chondrocyte proliferation and apoptosis at days 7 and 14, as determined by PCNA and TUNEL staining. Vascular microcomputed tomography analysis of callus samples at days 7, 14 and 21 revealed decreased blood vessel volume, number, and connection density in GIT1 KO mice compared to WT controls. Correlating with this, VEGF-A, phospho-VEGFR2 and PECAM1 (CD31) were decreased in GIT1 KO mice, indicating reduced angiogenesis with loss of GIT1. Finally, calluses from GIT1 KO mice displayed a reduced number of tartrate resistant acid phosphatase-positive osteoclasts at days 14 and 21. Collectively, these results indicate that GIT1 is an important signaling participant in fracture healing, with gene ablation leading to reduced callus vascularity and reduced osteoclast number in the healing callus. PMID:24586541

  20. Immune-Mediated Nephropathy and Systemic Autoimmunity in Mice Does Not Require Receptor Interacting Protein Kinase 3 (RIPK3)

    PubMed Central

    Corradetti, Chelsea; Jog, Neelakshi R.; Gallucci, Stefania; Madaio, Michael; Balachandran, Siddharth

    2016-01-01

    Immune mediated nephropathy is one of the most serious manifestations of lupus and is characterized by severe inflammation and necrosis that, if untreated, eventually leads to renal failure. Although lupus has a higher incidence in women, both sexes can develop lupus glomerulonephritis; nephritis in men develops earlier and is more severe than in women. It is therefore important to understand the cellular and molecular mechanisms mediating nephritis in each sex. Previous work by our lab found that the absence or pharmacological inhibition of Poly [ADP-ribose] polymerase 1 (PARP-1), an enzyme involved in DNA repair and necrotic cell death, affects only male mice and results in milder nephritis, with less in situ inflammation, and diminished incidence of necrotic lesions, allowing for higher survival rates. A second pathway mediating necrosis involves Receptor-Interacting Serine-Threonine Kinase 3 (RIPK3); in this study we sought to investigate the impact of RIPK3 on the development of lupus and nephritis in both sexes. To this end, we used two inducible murine models of lupus: chronic graft versus host disease (cGvHD) and pristane-induced lupus; and nephrotoxic serum (NTS)-induced nephritis as a model of immune mediated nephropathy. We found that the absence of RIPK3 has neither positive nor negative impact on the disease development or progression of lupus and nephritis in all three models, and in both male and female mice. We conclude that RIPK3 is dispensable for the pathogenesis of lupus and immune mediated nephropathy as to accelerate, worsen or ameliorate the disease. PMID:27669412

  1. Homeodomain Interacting Protein Kinase 2 Regulates Postnatal Development of Enteric Dopaminergic Neurons and Glia via BMP Signaling

    PubMed Central

    Chalazonitis, Alcmène; Tang, Amy A.; Shang, Yulei; Pham, Tuan D.; Hsieh, Ivy; Setlik, Wanda; Gershon, Michael D.; Huang, Eric J.

    2011-01-01

    Trophic factor signaling is important for the migration, differentiation and survival of enteric neurons during development. The mechanisms that regulate the maturation of enteric neurons in postnatal life, however, are poorly understood. Here, we show that transcriptional cofactor HIPK2 (homeodomain interacting protein kinase 2) is required for the maturation of enteric neurons and for regulating gliogenesis during postnatal development. Mice lacking HIPK2 display a spectrum of gastrointestinal (GI) phenotypes, including distention of colon and slowed GI transit time. Although loss of HIPK2 does not affect enteric neuron in prenatal development, a progressive loss of enteric neurons occurs during postnatal life in Hipk2−/− -mutant mice that preferentially affects the dopaminergic population of neurons in the caudal region of the intestine. The mechanism by which HIPK2 regulates postnatal enteric neuron development appears to involve the response of enteric neurons to bone morphogenetic proteins (BMPs). Specifically, compared to wild type mice, a larger proportion of enteric neurons in Hipk2−/−mutants have abnormally high level of phosphorylated Smad1/5/8. Consistent with the ability of BMP signaling to promote gliogenesis, Hipk2−/− mutants show a significant increase in glia in the ENS. In addition, numbers of autophagosomes are increased in enteric neurons in Hipk2−/−mutants and synaptic maturation is arrested. These results reveal a new role for HIPK2 as an important transcriptional cofactor that regulates the BMP signaling pathway in the maintenance of enteric neurons and glia, and further suggest that HIPK2 and its associated signaling mechanisms may be therapeutically altered to promote postnatal neuronal maturation. PMID:21957238

  2. Myotubularin-related proteins 3 and 4 interact with polo-like kinase 1 and centrosomal protein of 55 kDa to ensure proper abscission.

    PubMed

    St-Denis, Nicole; Gupta, Gagan D; Lin, Zhen Yuan; Gonzalez-Badillo, Beatriz; Pelletier, Laurence; Gingras, Anne-Claude

    2015-04-01

    The myotubularins are a family of phosphatases that dephosphorylate the phosphatidylinositols phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-phosphate. Several family members are mutated in disease, yet the biological functions of the majority of myotubularins remain unknown. To gain insight into the roles of the individual enzymes, we have used affinity purification coupled to mass spectrometry to identify protein-protein interactions for the myotubularins. The myotubularin interactome comprises 66 high confidence (false discovery rate ≤1%) interactions, including 18 pairwise interactions between individual myotubularins. The results reveal a number of potential signaling contexts for this family of enzymes, including an intriguing, novel role for myotubularin-related protein 3 and myotubularin-related protein 4 in the regulation of abscission, the final step of mitosis in which the membrane bridge remaining between two daughter cells is cleaved. Both depletion and overexpression of either myotubularin-related protein 3 or myotubularin-related protein 4 result in abnormal midbody morphology and cytokinesis failure. Interestingly, myotubularin-related protein 3 and myotubularin-related protein 4 do not exert their effects through lipid regulation at the midbody, but regulate abscission during early mitosis, by interacting with the mitotic kinase polo-like kinase 1, and with centrosomal protein of 55 kDa (CEP55), an important regulator of abscission. Structure-function analysis reveals that, consistent with known intramyotubularin interactions, myotubularin-related protein 3 and myotubularin-related protein 4 interact through their respective coiled coil domains. The interaction between myotubularin-related protein 3 and polo-like kinase 1 relies on the divergent, nonlipid binding Fab1, YOTB, Vac1, and EEA1 domain of myotubularin-related protein 3, and myotubularin-related protein 4 interacts with CEP55 through a short GPPXXXY motif, analogous to

  3. The WASH complex, an endosomal Arp2/3 activator, interacts with the Hermansky–Pudlak syndrome complex BLOC-1 and its cargo phosphatidylinositol-4-kinase type IIα

    PubMed Central

    Ryder, P. V.; Vistein, R.; Gokhale, A.; Seaman, M. N.; Puthenveedu, M. A.; Faundez, V.

    2013-01-01

    Vesicle biogenesis machinery components such as coat proteins can interact with the actin cytoskeleton for cargo sorting into multiple pathways. It is unknown, however, whether these interactions are a general requirement for the diverse endosome traffic routes. In this study, we identify actin cytoskeleton regulators as previously unrecognized interactors of complexes associated with the Hermansky–Pudlak syndrome. Two complexes mutated in the Hermansky–Pudlak syndrome, adaptor protein complex-3 and biogenesis of lysosome-related organelles complex-1 (BLOC-1), interact with and are regulated by the lipid kinase phosphatidylinositol-4-kinase type IIα (PI4KIIα). We therefore hypothesized that PI4KIIα interacts with novel regulators of these complexes. To test this hypothesis, we immunoaffinity purified PI4KIIα from isotope-labeled cell lysates to quantitatively identify interactors. Strikingly, PI4KIIα isolation preferentially coenriched proteins that regulate the actin cytoskeleton, including guanine exchange factors for Rho family GTPases such as RhoGEF1 and several subunits of the WASH complex. We biochemically confirmed several of these PI4KIIα interactions. Of importance, BLOC-1 complex, WASH complex, RhoGEF1, or PI4KIIα depletions altered the content and/or subcellular distribution of the BLOC-1–sensitive cargoes PI4KIIα, ATP7A, and VAMP7. We conclude that the Hermansky–Pudlak syndrome complex BLOC-1 and its cargo PI4KIIα interact with regulators of the actin cytoskeleton. PMID:23676666

  4. Programmed cell death 4 protein (Pdcd4) and homeodomain-interacting protein kinase 2 (Hipk2) antagonistically control translation of Hipk2 mRNA.

    PubMed

    Ohnheiser, Johanna; Ferlemann, Eva; Haas, Astrid; Müller, Jan P; Werwein, Eugen; Fehler, Olesja; Biyanee, Abhiruchi; Klempnauer, Karl-Heinz

    2015-07-01

    The tumor suppressor protein programmed cell death 4 (Pdcd4) is a highly conserved RNA-binding protein that inhibits the translation of specific mRNAs. Here, we have identified the homeobox-interacting protein kinase-2 (Hipk2) mRNA as a novel translational target of Pdcd4. Unlike most other protein kinases Hipk2 is constitutively active after being synthesized by the ribosome and its expression and activity are thought to be mainly controlled by modulation of the half-life of the kinase. Our work provides the first evidence that Hipk2 expression is also controlled on the level of translation. We show that Hipk2 stimulates the translation of its own mRNA and that Pdcd4 suppresses the translation of Hipk2 mRNA by interfering with this auto-regulatory feedback mechanism. We also show that the translation of the related kinase Hipk1 is controlled by a similar feedback loop and that Hipk2 also stimulates the translation of Hipk1 mRNA. Taken together, our work describes a novel mechanism of translational suppression by Pdcd4 and shows for the first time that Hipk2 controls its own synthesis by an auto-regulatory feedback mechanism. Furthermore, the effect of Hipk2 on the translation of Hipk1 RNA suggests that Hipk2 and Pdcd4 can act in similar manner to control the translation of other mRNAs.

  5. STRUBBELIG defines a receptor kinase-mediated signaling pathway regulating organ development in Arabidopsis

    PubMed Central

    Chevalier, David; Batoux, Martine; Fulton, Lynette; Pfister, Karen; Yadav, Ram Kishor; Schellenberg, Maja; Schneitz, Kay

    2005-01-01

    An open question remains as to what coordinates cell behavior during organogenesis, permitting organs to reach their appropriate size and shape. The Arabidopsis gene STRUBBELIG (SUB) defines a receptor-mediated signaling pathway in plants. SUB encodes a putative leucine-rich repeat transmembrane receptor-like kinase. The mutant sub phenotype suggests that SUB affects the formation and shape of several organs by influencing cell morphogenesis, the orientation of the division plane, and cell proliferation. Mutational analysis suggests that the kinase domain is important for SUB function. Biochemical assays using bacterially expressed fusion proteins indicate that the SUB kinase domain lacks enzymatic phosphotransfer activity. Furthermore, transgenes encoding WT and different mutant variants of SUB were tested for their ability to rescue the mutant sub phenotype. These genetic data also indicate that SUB carries a catalytically inactive kinase domain. The SUB receptor-like kinase may therefore signal in an atypical fashion. PMID:15951420

  6. The Brassica napus blackleg resistance gene LepR3 encodes a receptor-like protein triggered by the Leptosphaeria maculans effector AVRLM1.

    PubMed

    Larkan, N J; Lydiate, D J; Parkin, I A P; Nelson, M N; Epp, D J; Cowling, W A; Rimmer, S R; Borhan, M H

    2013-01-01

    LepR3, found in the Brassica napus cv 'Surpass 400', provides race-specific resistance to the fungal pathogen Leptosphaeria maculans, which was overcome after great devastation in Australia in 2004. We investigated the LepR3 locus to identify the genetic basis of this resistance interaction. We employed a map-based cloning strategy, exploiting collinearity with the Arabidopsis thaliana and Brassica rapa genomes to enrich the map and locate a candidate gene. We also investigated the interaction of LepR3 with the L. maculans avirulence gene AvrLm1 using transgenics. LepR3 was found to encode a receptor-like protein (RLP). We also demonstrated that avirulence towards LepR3 is conferred by AvrLm1, which is responsible for both the Rlm1 and LepR3-dependent resistance responses in B. napus. LepR3 is the first functional B. napus disease resistance gene to be cloned. AvrLm1's interaction with two independent resistance loci, Rlm1 and LepR3, highlights the need to consider redundant phenotypes in 'gene-for-gene' interactions and offers an explanation as to why LepR3 was overcome so rapidly in parts of Australia.

  7. Receptor-interacting protein kinase 2 promotes triple-negative breast cancer cell migration and invasion via activation of nuclear factor-kappaB and c-Jun N-terminal kinase pathways

    PubMed Central

    2014-01-01

    Introduction Metastasis is the main cause of breast cancer morbidity and mortality. Processes that allow for tumor cell migration and invasion are important therapeutic targets. Here we demonstrate that receptor-interacting protein kinase 2 (RIP2), a kinase known to be involved in inflammatory processes, also has novel roles in cancer cell migration and invasion. Methods A total of six breast cancer expression databases, including The Cancer Genome Atlas, were assessed for RIP2 expression among various clinical subtypes and its role as a prognostic biomarker. mRNA fluorescence in situ hybridization (FISH) for RIP2 was performed on 17 stage III breast cancers to determine if there was a correlation between RIP2 expression and lymph node involvement. RNA-interference was used to knock-down RIP2 expression in MDA-MB-231, Htb126, SUM149PT, MCF7, T47D, and HCC1428 cells. Cell migration and invasion were measured in vitro by scratch/wound healing and transwell migration assays. A xenograft mouse model was used to assess tumor growth and chemosensitivity to docetaxel in vivo in MDA-MB-231 cells with and without RIP2 small hairpin RNA knockdown. Western blot and immunofluorescence imaging were used to evaluate protein expressions. Results Interrogation of expression databases showed that RIP2 expression is significantly over-expressed in triple-negative breast cancers (TNBC: estrogen-receptor (ER) negative, progesterone-receptor (PR) negative, Her2/neu- (Her2) negative), compared to other clinical subtypes. High RIP2 expression correlates with worse progression-free survival using a combined breast cancer expression array dataset consisting of 946 patients. Multivariate analysis shows RIP2 as an independent prognostic biomarker. Knock-down of RIP2 significantly decreases migration in both scratch/wound healing and transwell migration assays in MDA-MB-231, Htb126, SUM149PT, MCF7, and T47D cells and is correlated with decreased Nuclear Factor-kappaB and c-Jun N

  8. Interaction of SOS2 with nucleoside diphosphate kinase 2 and catalases reveals a point of connection between salt stress and H2O2 signaling in Arabidopsis thaliana.

    PubMed

    Verslues, Paul E; Batelli, Giorgia; Grillo, Stefania; Agius, Fernanda; Kim, Yong-Sig; Zhu, Jianhua; Agarwal, Manu; Katiyar-Agarwal, Surekha; Zhu, Jian-Kang

    2007-11-01

    SOS2, a class 3 sucrose-nonfermenting 1-related kinase, has emerged as an important mediator of salt stress response and stress signaling through its interactions with proteins involved in membrane transport and in regulation of stress responses. We have identified additional SOS2-interacting proteins that suggest a connection between SOS2 and reactive oxygen signaling. SOS2 was found to interact with the H2O2 signaling protein nucleoside diphosphate kinase 2 (NDPK2) and to inhibit its autophosphorylation activity. A sos2-2 ndpk2 double mutant was more salt sensitive than a sos2-2 single mutant, suggesting that NDPK2 and H2O2 are involved in salt resistance. However, the double mutant did not hyperaccumulate H2O2 in response to salt stress, suggesting that it is altered signaling rather than H2O2 toxicity alone that is responsible for the increased salt sensitivity of the sos2-2 ndpk2 double mutant. SOS2 was also found to interact with catalase 2 (CAT2) and CAT3, further connecting SOS2 to H2O2 metabolism and signaling. The interaction of SOS2 with both NDPK2 and CATs reveals a point of cross talk between salt stress response and other signaling factors including H2O2.

  9. A kinase anchoring protein (AKAP) interaction and dimerization of the RIalpha and RIbeta regulatory subunits of protein kinase a in vivo by the yeast two hybrid system.

    PubMed

    Carlson, Cathrine R; Ruppelt, Anja; Taskén, Kjetil

    2003-03-28

    Protein kinase A (PKA) regulatory (R) subunits dimerize through an N-terminal motif. Such dimerization is necessary for binding to PKA anchoring proteins (AKAPs) and targeting of PKA to its site of action. In the present study, we used the yeast two-hybrid system as an in vivo bio-reporter assay and analyzed the formation of homo- and heterodimeric complexes of RIalpha and RIbeta as well as AKAP binding of RI dimers. Native polyacrylamide gel electrophoresis (PAGE) of yeast extracts confirmed the two-hybrid data. Both RIalpha- and RIbeta homodimers as well as an RIalpha:RIbeta heterodimer were observed. Single, double and one triple mutation were introduced into the RIalpha and RIbeta subunits and dimerization properties of the mutants were analyzed. Consistent with previous reports, RIalpha(C37H) dimerized, although the disulfide bridges were disrupted, whereas the additional mutation of F47 or F52 abolished the dimerization. Corresponding mutations (C38H, F48A, F53A) in RIbeta were not sufficient to abolish the RIbeta dimerization, indicating that additional or other amino acids are important. RIalpha:RIbeta heterodimers of the mutants were formed at intermediate stringency. Analysis of ternary complexes by the yeast two-hybrid system revealed that RIalpha and RIbeta homodimers as well as an RIalpha:RIbeta heterodimer and several of the mutants were able to bind to the R-binding domain of AKAP149/D-AKAP1. Furthermore, an RIbeta:AKAP149 complex was identified following introduction of RIbeta into HEK293 cells. Importantly, RIbeta revealed AKAP binding properties similar to those of RIalpha, indicating that RIbeta holoenzymes may be anchored.

  10. Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein

    PubMed Central

    Jagadish, Nirmala; Rana, Ritu; Selvi, Ramasamy; Mishra, Deepshikha; Garg, Manoj; Yadav, Shikha; Herr, John C.; Okumura, Katsuzumi; Hasegawa, Akiko; Koyama, Koji; Suri, Anil

    2005-01-01

    We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561–565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an α-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38α or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa–egg interaction. PMID:15693750

  11. Acetyl-lysine Binding Site of Bromodomain-Containing Protein 4 (BRD4) Interacts with Diverse Kinase Inhibitors

    PubMed Central

    2014-01-01

    Members of the bromodomain and extra terminal (BET) family of proteins are essential for the recognition of acetylated lysine (KAc) residues in histones and have emerged as promising drug targets in cancer, inflammation, and contraception research. In co-crystallization screening campaigns using the first bromodomain of BRD4 (BRD4-1) against kinase inhibitor libraries, we identified and characterized 14 kinase inhibitors (10 distinct chemical scaffolds) as ligands of the KAc binding site. Among these, the PLK1 inhibitor BI2536 and the JAK2 inhibitor TG101209 displayed strongest inhibitory potential against BRD4 (IC50 = 25 nM and 130 nM, respectively) and high selectivity for BET bromodomains. Comparative structural analysis revealed markedly different binding modes of kinase hinge-binding scaffolds in the KAc binding site, suggesting that BET proteins are potential off-targets of diverse kinase inhibitors. Combined, these findings provide a new structural framework for the rational design of next-generation BET-selective and dual-activity BET-kinase inhibitors. PMID:24568369

  12. Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with the SNF4 protein.

    PubMed Central

    Celenza, J L; Carlson, M

    1989-01-01

    The SNF1 gene of Saccharomyces cerevisiae encodes a protein-serine/threonine kinase that is required for derepression of gene expression in response to glucose limitation. We present evidence that the protein kinase activity is essential for SNF1 function: substitution of Arg for Lys in the putative ATP-binding site results in a mutant phenotype. A polyhistidine tract near the N terminus was found to be dispensable. Deletion of the large region C terminal to the kinase domain only partially impaired SNF1 function, causing expression of invertase to be somewhat reduced but still glucose repressible. The function of the SNF4 gene, another component of the regulatory system, was required for maximal in vitro activity of the SNF1 protein kinase. Increased SNF1 gene dosage partially alleviated the requirement for SNF4. C-terminal deletions of SNF1 also reduced dependence on SNF4. Our findings suggest that SNF4 acts as a positive effector of the kinase but does not serve a regulatory function in signaling glucose availability. Images PMID:2557546

  13. The phosphotransferase protein EIIA(Ntr) modulates the phosphate starvation response through interaction with histidine kinase PhoR in Escherichia coli.

    PubMed

    Lüttmann, Denise; Göpel, Yvonne; Görke, Boris

    2012-10-01

    Many Proteobacteria possess the paralogous PTS(Ntr), in addition to the sugar transport phosphotransferase system (PTS). In the PTS(Ntr) phosphoryl-groups are transferred from phosphoenolpyruvate to protein EIIA(Ntr) via the phosphotransferases EI(Ntr) and NPr. The PTS(Ntr) has been implicated in regulation of diverse physiological processes. In Escherichia coli, the PTS(Ntr) plays a role in potassium homeostasis. In particular, EIIA(Ntr) binds to and stimulates activity of a two-component histidine kinase (KdpD) resulting in increased expression of the genes encoding the high-affinity K(+) transporter KdpFABC. Here, we show that the phosphate (pho) regulon is likewise modulated by PTS(Ntr). The pho regulon, which comprises more than 30 genes, is activated by the two-component system PhoR/PhoB under conditions of phosphate starvation. Mutants lacking EIIA(Ntr) are unable to fully activate the pho genes and exhibit a growth delay upon adaptation to phosphate limitation. In contrast, pho expression is increased above the wild-type level in mutants deficient for EIIA(Ntr) phosphorylation suggesting that non-phosphorylated EIIA(Ntr) modulates pho. Protein interaction analyses reveal binding of EIIA(Ntr) to histidine kinase PhoR. This interaction increases the amount of phosphorylated response regulator PhoB. Thus, EIIA(Ntr) is an accessory protein that modulates the activities of two distinct sensor kinases, KdpD and PhoR, in E. coli.

  14. Promotion of DNA strand breaks in cocultured mononuclear leukocytes by protein kinase C-dependent prooxidative interactions of benoxaprofen, human polymorphonuclear leukocytes, and ultraviolet radiation

    SciTech Connect

    Schwalb, G.; Beyers, A.D.; Anderson, R.; Nel, A.E.

    1988-06-01

    At concentrations of 5 micrograms/ml and greater the nonsteroidal antiinflammatory drug benoxaprofen caused dose-related activation of lucigenin-enhanced chemiluminescence in human polymorphonuclear leukocytes (PMNL). Benoxaprofen-mediated activation of lucigenin-enhanced chemiluminescence by PMNL was increased by UV radiation and was particularly sensitive to inhibition by the selective protein kinase C inhibitor H-7. To identify the molecular mechanism of the prooxidative activity of benoxaprofen, the effects of the nonsteroidal antiinflammatory drug on the activity of purified protein kinase C in a cell-free system were investigated. Benoxaprofen caused a dose-related activation of protein kinase C by interaction with the binding site for the physiological activator phosphatidylserine, but could not replace diacylglycerol. When autologous mononuclear leukocytes (MNL) were cocultured with PMNL and benoxaprofen in combination, but not individually, the frequency of DNA strand breaks in MNL was markedly increased. UV radiation significantly potentiated damage to DNA mediated by benoxaprofen and PMNL. Inclusion of superoxide dismutase, H-7, and, to a much lesser extent, catalase during exposure of MNL to benoxaprofen-activated PMNL prevented oxidant damage to DNA. These results clearly demonstrate that potentially carcinogenic prooxidative interactions, which are unlikely to be detected by conventional assays of mutagenicity, may occur between phagocytes, UV radiation, and certain pharmacological agents.

  15. Direct Association of Sprouty-related Protein with an EVH1 Domain (SPRED) 1 or SPRED2 with DYRK1A Modifies Substrate/Kinase Interactions*

    PubMed Central

    Li, Dan; Jackson, Rebecca A.; Yusoff, Permeen; Guy, Graeme R.

    2010-01-01

    The mammalian SPRED (Sprouty-related protein with an EVH1 domain) proteins include a family of three members, SPRED1–3. Currently, little is known about their biochemistry. The best described, SPRED1, has been shown to inhibit the Ras/ERK pathway downstream of Ras. All three SPREDs have a cysteine-rich domain (CRD) that has high homology to the CRD of the Sprouty family of proteins, several of which are also Ras/ERK inhibitors. In the belief that binding partners would clarify SPRED function, we assayed for their associated proteins. Here, we describe the direct and endogenous interaction of SPRED1 and SPRED2 with the novel kinase, DYRK1A. DYRK1A has become the subject of recent research focus as it plays a central role in Caenorhabditis elegans oocyte maturation and egg activation, and there is strong evidence that it could be involved in Down syndrome in humans. Both SPRED1 and SPRED2 inhibit the ability of DYRK1A to phosphorylate its substrates, Tau and STAT3. This inhibition occurs via an interaction of the CRD of the SPREDs with the kinase domain of DYRK1A. DYRK1A substrates must bind to the kinase to enable phosphorylation, and SPRED proteins compete for the same binding site to modify this process. Our accumulated evidence indicates that the SPRED proteins are likely physiological modifiers of DYRK1A. PMID:20736167

  16. PDCD10 interacts with Ste20-related kinase MST4 to promote cell growth and transformation via modulation of the ERK pathway.

    PubMed

    Ma, Xi; Zhao, Hongshan; Shan, Jingxuan; Long, Feng; Chen, Yaoyao; Chen, Yingyu; Zhang, Yingmei; Han, Xiao; Ma, Dalong

    2007-06-01

    PDCD10 (programmed cell death 10, TFAR15), a novel protein associated with cell apoptosis has been recently implicated in mutations associated with Cerebral Cavernous Malformations (CCM). Yeast two-hybrid screening revealed that PDCD10 interacts with MST4, a member of Ste20-related kinases. This interaction was confirmed by coimmunoprecipitation and colocalization assays in mammalian cells. Furthermore, the co-overexpression of PDCD10 and MST4 promoted cell proliferation and transformation via modulation of the extracellular signal-regulated kinase (ERK) pathway. Potent short interfering RNAs (siRNAs) against PDCD10 (siPDCD10) and MST4 (siMST4) were designed to specifically inhibit the expression of PDCD10 and MST4 mRNA, respectively. The induction of siPDCD10 or siMST4 resulted in decreased expression of endogenous PDCD10 or MST4, which was accompanied by reduced ERK activity and attenuated cell growth and anchorage-independent growth. On the other hand, siMST4 had similar effects in PDCD10-overexpressed cells. And more importantly, we confirmed that either overexpressing or endogenous PDCD10 can increase the MST4 kinase activity in vitro. Our results demonstrated that PDCD10 modulation of ERK signaling was mediated by MST4, and PDCD10 could be a regulatory adaptor necessary for MST4 function, suggesting a link between cerebral cavernous malformation pathogenesis and the ERK-MAPK cascade via PDCD10/MST4.

  17. rugose (rg), a Drosophila A kinase anchor protein, is required for retinal pattern formation and interacts genetically with multiple signaling pathways.

    PubMed Central

    Shamloula, Hoda K; Mbogho, Mkajuma P; Pimentel, Angel C; Chrzanowska-Lightowlers, Zosia M A; Hyatt, Vanneta; Okano, Hideyuki; Venkatesh, Tadmiri R

    2002-01-01

    In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the EGFR- and Notch-mediated signaling pathways. Our results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the EGFR and Notch signaling pathways. PMID:12072466

  18. Interaction of Omega, Sigma, and Theta Glutathione Transferases with p38b Mitogen-Activated Protein Kinase from the Fruit Fly, Drosophila melanogaster

    PubMed Central

    Wongtrakul, J.; Janphen, K.; Saisawang, C.; Ketterman, A.J.

    2014-01-01

    Glutathione S-transferases (GSTs) are a diverse family of phase II detoxification enzymes found in almost all organisms. Besides playing a major role in the detoxification of xenobiotic and toxic compounds, GSTs are also involved in the regulation of mitogen activated protein (MAP) kinase signal transduction by interaction with proteins in the pathway. An in vitro study was performed for Theta, Omega, Sigma GSTs and their interaction with MAP kinase p38b protein from the fruit fly Drosophila melanogaster Meigen (Diptera: Drosophilidae). The study included the effects of all five Omega class GSTs (DmGSTO1, DmGSTO2a, DmGSTO2b, DmGSTO3, DmGSTO4), all five Theta class GSTs (DmGSTT1, DmGSTT2, DmGSTT3a, DmGSTT3b, DmGSTT4), and one Sigma class glutathione transferase on the activity of Drosophila p38b, including the reciprocal effect of this kinase protein on glutathione transferase activity. It was found that DmGSTT2, DmGSTT3b, DmGSTO1, and DmGSTO3 activated p38b significantly. Substrate specificities of GSTs were also altered after co-incubation with p38b. Although p38b activated DmGSTO1, DmGSTO2a, and DmGSTT2, it inhibited DmGSTT3b and DmGSTO3 activity toward xenobiotic and physiological substrates tested. These results suggest a novel link between Omega and Theta GSTs with the p38b MAP kinase pathway. PMID:25373207

  19. The cyclin-dependent kinase 5 activators p35 and p39 interact with the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II and alpha-actinin-1 in a calcium-dependent manner.

    PubMed

    Dhavan, Rani; Greer, Paul L; Morabito, Maria A; Orlando, Lianna R; Tsai, Li-Huei

    2002-09-15

    Cyclin-dependent kinase 5 (Cdk5) is a critical regulator of neuronal migration in the developing CNS, and recent studies have revealed a role for Cdk5 in synaptogenesis and regulation of synaptic transmission. Deregulation of Cdk5 has been linked to the pathology of neurodegenerative diseases such as Alzheimer's disease. Activation of Cdk5 requires its association with a regulatory subunit, and two Cdk5 activators, p35 and p39, have been identified. To gain further insight into the functions of Cdk5, we identified proteins that interact with p39 in a yeast two-hybrid screen. In this study we report that alpha-actinin-1 and the alpha-subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKIIalpha), two proteins localized at the postsynaptic density, interact with Cdk5 via their association with p35 and p39. CaMKIIalpha and alpha-actinin-1 bind to distinct regions of p35 and p39 and also can interact with each other. The association of CaMKIIalpha and alpha-actinin-1 to the Cdk5 activators, as well as to each other, is stimulated by calcium. Further, the activation of glutamate receptors increases the association of p35 and p39 with CaMKIIalpha, and the inhibition of CaMKII activation diminishes this effect. The glutamate-mediated increase in association of p35 and CaMKIIalpha is mediated in large part by NMDA receptors, suggesting that cross talk between the Cdk5 and CaMKII signal transduction pathways may be a component of the complex molecular mechanisms contributing to synaptic plasticity, memory, and learning.

  20. Phosphatidylinositol 3'-kinase associates with an insulin receptor substrate-1 serine kinase distinct from its intrinsic serine kinase.

    PubMed Central

    Cengel, K A; Kason, R E; Freund, G G

    1998-01-01

    Serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been proposed as a counter-regulatory mechanism in insulin and cytokine signalling. Here we report that IRS-1 is phosphorylated by a wortmannin insensitive phosphatidylinositol 3'-kinase (PI 3-kinase)-associated serine kinase (PAS kinase) distinct from PI 3-kinase serine kinase. We found that PI 3-kinase immune complexes contain 5-fold more wortmannin-insensitive serine kinase activity than SH2-containing protein tyrosine phosphatase-2 (SHP2) and IRS-1 immune complexes. Affinity chromatography of cell lysates with a glutathione S-transferase fusion protein for the p85 subunit of PI 3-kinase showed that PAS kinase associated with the p85 subunit of PI 3-kinase. This interaction required unoccupied SH2 domain(s) but did not require the PI 3-kinase p110 subunit binding domain. In terms of function, PAS kinase phosphorylated IRS-1 and, after insulin stimulation, PAS kinase phosphorylated IRS-1 in PI 3-kinase-IRS-1 complexes. Phosphopeptide mapping showed that insulin-dependent in vivo sites of IRS-1 serine phosphorylation were comparable to those of PAS kinase phosphorylated IRS-1. More importantly, PAS kinase-dependent phosphorylation of IRS-1 reduced by 4-fold the ability of IRS-1 to act as an insulin receptor substrate. Taken together, these findings indicate that: (a) PAS kinase is distinct from the intrinsic serine kinase activity of PI 3-kinase, (b) PAS kinase associates with the p85 subunit of PI 3-kinase through SH2 domain interactions, and (c) PAS kinase is an IRS-1 serine kinase that can reduce the ability of IRS-1 to serve as an insulin receptor substrate. PMID:9761740

  1. Gankyrin is an ankyrin-repeat oncoprotein that interacts with CDK4 kinase and the S6 ATPase of the 26 S proteasome.

    PubMed

    Dawson, Simon; Apcher, Sebastien; Mee, Maureen; Higashitsuji, Hiroaki; Baker, Rohan; Uhle, Stefan; Dubiel, Wolfgang; Fujita, Jun; Mayer, R John

    2002-03-29

    A yeast two-hybrid screen with the human S6 (TBP7, RPT3) ATPase of the 26 S proteasome has identified gankyrin, a liver oncoprotein, as an interacting protein. Gankyrin interacts with both free and regulatory complex-associated S6 ATPase and is not stably associated with the 26 S particle. Deletional mutagenesis shows that the C-terminal 78 amino acids of the S6 ATPase are necessary and sufficient to mediate the interaction with gankyrin. Deletion of an orthologous gene in Saccharomyces cerevisiae suggests that it is dispensable for cell growth and viability. Overexpression and precipitation of tagged gankyrin from cultured cells detects a complex containing co-transfected tagged S6 ATPase (or endogenous S6) and endogenous cyclin D-dependent kinase CDK4. The proteasomal ATPases are part of the AAA (ATPases associated with diverse cellular activities) family, members of which are molecular chaperones; gankyrin complexes may therefore influence CDK4 function during oncogenesis.

  2. DNA-Encoded Library Screening Identifies Benzo[b][1,4]oxazepin-4-ones as Highly Potent and Monoselective Receptor Interacting Protein 1 Kinase Inhibitors.

    PubMed

    Harris, Philip A; King, Bryan W; Bandyopadhyay, Deepak; Berger, Scott B; Campobasso, Nino; Capriotti, Carol A; Cox, Julie A; Dare, Lauren; Dong, Xiaoyang; Finger, Joshua N; Grady, LaShadric C; Hoffman, Sandra J; Jeong, Jae U; Kang, James; Kasparcova, Viera; Lakdawala, Ami S; Lehr, Ruth; McNulty, Dean E; Nagilla, Rakesh; Ouellette, Michael T; Pao, Christina S; Rendina, Alan R; Schaeffer, Michelle C; Summerfield, Jennifer D; Swift, Barbara A; Totoritis, Rachel D; Ward, Paris; Zhang, Aming; Zhang, Daohua; Marquis, Robert W; Bertin, John; Gough, Peter J

    2016-03-10

    The recent discovery of the role of receptor interacting protein 1 (RIP1) kinase in tumor necrosis factor (TNF)-mediated inflammation has led to its emergence as a highly promising target for the treatment of multiple inflammatory diseases. We screened RIP1 against GSK's DNA-encoded small-molecule libraries and identified a novel highly potent benzoxazepinone inhibitor series. We demonstrate that this template possesses complete monokinase selectivity for RIP1 plus unique species selectivity for primate versus nonprimate RIP1. We elucidate the conformation of RIP1 bound to this benzoxazepinone inhibitor driving its high kinase selectivity and design specific mutations in murine RIP1 to restore potency to levels similar to primate RIP1. This series differentiates itself from known RIP1 inhibitors in combining high potency and kinase selectivity with good pharmacokinetic profiles in rodents. The favorable developability profile of this benzoxazepinone template, as exemplified by compound 14 (GSK'481), makes it an excellent starting point for further optimization into a RIP1 clinical candidate.

  3. Attenuation of Helicobacter pylori CagA x SHP-2 signaling by interaction between CagA and C-terminal Src kinase.

    PubMed

    Tsutsumi, Ryouhei; Higashi, Hideaki; Higuchi, Megumi; Okada, Masato; Hatakeyama, Masanori

    2003-02-07

    Helicobacter pylori (H. pylori) is a causative agent of gastric diseases ranging from gastritis to cancer. The CagA protein is the product of the cagA gene carried among virulent H. pylori strains and is associated with severe disease outcomes, most notably gastric carcinoma. CagA is injected from the attached H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation. The phosphorylated CagA binds and activates SHP-2 phosphatase and thereby induces a growth factor-like morphological change termed the "hummingbird phenotype." In this work, we demonstrate that CagA is also capable of interacting with C-terminal Src kinase (Csk). As is the case with SHP-2, Csk selectively binds tyrosine-phosphorylated CagA via its SH2 domain. Upon complex formation, CagA stimulates Csk, which in turn inactivates the Src family of protein-tyrosine kinases. Because Src family kinases are responsible for CagA phosphorylation, an essential prerequisite of CagA.SHP-2 complex formation and subsequent induction of the hummingbird phenotype, our results indicate that CagA-Csk interaction down-regulates CagA.SHP-2 signaling by both competitively inhibiting CagA.SHP-2 complex formation and reducing levels of CagA phosphorylation. We further demonstrate that CagA.SHP-2 signaling eventually induces apoptosis in AGS cells. Our results thus indicate that CagA-Csk interaction prevents excess cell damage caused by deregulated activation of SHP-2. Attenuation of CagA activity by Csk may enable cagA-positive H. pylori to persistently infect the human stomach for decades while avoiding excess CagA toxicity to the host.

  4. Small Molecule AKAP-Protein Kinase A (PKA) Interaction Disruptors That Activate PKA Interfere with Compartmentalized cAMP Signaling in Cardiac Myocytes*

    PubMed Central

    Christian, Frank; Szaszák, Márta; Friedl, Sabine; Drewianka, Stephan; Lorenz, Dorothea; Goncalves, Andrey; Furkert, Jens; Vargas, Carolyn; Schmieder, Peter; Götz, Frank; Zühlke, Kerstin; Moutty, Marie; Göttert, Hendrikje; Joshi, Mangesh; Reif, Bernd; Haase, Hannelore; Morano, Ingo; Grossmann, Solveig; Klukovits, Anna; Verli, Judit; Gáspár, Róbert; Noack, Claudia; Bergmann, Martin; Kass, Robert; Hampel, Kornelia; Kashin, Dmitry; Genieser, Hans-Gottfried; Herberg, Friedrich W.; Willoughby, Debbie; Cooper, Dermot M. F.; Baillie, George S.; Houslay, Miles D.; von Kries, Jens Peter; Zimmermann, Bastian; Rosenthal, Walter; Klussmann, Enno

    2011-01-01

    A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3′-diamino-4,4′-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating β-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure. PMID:21177871

  5. Tobacco Translationally Controlled Tumor Protein Interacts with Ethylene Receptor Tobacco Histidine Kinase1 and Enhances Plant Growth through Promotion of Cell Proliferation1[OPEN

    PubMed Central

    Tao, Jian-Jun; Cao, Yang-Rong; Chen, Hao-Wei; Wei, Wei; Li, Qing-Tian; Ma, Biao; Zhang, Wan-Ke; Chen, Shou-Yi; Zhang, Jin-Song

    2015-01-01

    Ethylene is an important phytohormone in the regulation of plant growth, development, and stress response throughout the lifecycle. Previously, we discovered that a subfamily II ethylene receptor tobacco (Nicotiana tabacum) Histidine Kinase1 (NTHK1) promotes seedling growth. Here, we identified an NTHK1-interacting protein translationally controlled tumor protein (NtTCTP) by the yeast (Saccharomyces cerevisiae) two-hybrid assay and further characterized its roles in plant growth. The interaction was further confirmed by in vitro glutathione S-transferase pull down and in vivo coimmunoprecipitation and bimolecular fluorescence complementation assays, and the kinase domain of NTHK1 mediates the interaction with NtTCTP. The NtTCTP protein is induced by ethylene treatment and colocalizes with NTHK1 at the endoplasmic reticulum. Overexpression of NtTCTP or NTHK1 reduces plant response to ethylene and promotes seedling growth, mainly through acceleration of cell proliferation. Genetic analysis suggests that NtTCTP is required for the function of NTHK1. Furthermore, association of NtTCTP prevents NTHK1 from proteasome-mediated protein degradation. Our data suggest that plant growth inhibition triggered by ethylene is regulated by a unique feedback mechanism, in which ethylene-induced NtTCTP associates with and stabilizes ethylene receptor NTHK1 to reduce plant response to ethylene and promote plant growth through acceleration of cell proliferation. PMID:25941315

  6. Identification of cofilin and LIM-domain-containing protein kinase 1 as novel interaction partners of 14-3-3 zeta.

    PubMed Central

    Birkenfeld, Jörg; Betz, Heinrich; Roth, Dagmar

    2003-01-01

    Proteins of the 14-3-3 family have been implicated in various physiological processes, and are thought to function as adaptors in various signal transduction pathways. In addition, 14-3-3 proteins may contribute to the reorganization of the actin cytoskeleton by interacting with as yet unidentified actin-binding proteins. Here we show that the 14-3-3 zeta isoform interacts with both the actin-depolymerizing factor cofilin and its regulatory kinase, LIM (Lin-11/Isl-1/Mec-3)-domain-containing protein kinase 1 (LIMK1). In both yeast two-hybrid assays and glutathione S-transferase pull-down experiments, these proteins bound efficiently to 14-3-3 zeta. Deletion analysis revealed consensus 14-3-3 binding sites on both cofilin and LIMK1. Furthermore, the C-terminal region of 14-3-3 zeta inhibited the binding of cofilin to actin in co-sedimentation experiments. Upon co-transfection into COS-7 cells, 14-3-3 zeta-specific immunoreactivity was redistributed into characteristic LIMK1-induced actin aggregations. Our data are consistent with 14-3-3-protein-induced changes to the actin cytoskeleton resulting from interactions with cofilin and/or LIMK1. PMID:12323073

  7. Small molecule AKAP-protein kinase A (PKA) interaction disruptors that activate PKA interfere with compartmentalized cAMP signaling in cardiac myocytes.

    PubMed

    Christian, Frank; Szaszák, Márta; Friedl, Sabine; Drewianka, Stephan; Lorenz, Dorothea; Goncalves, Andrey; Furkert, Jens; Vargas, Carolyn; Schmieder, Peter; Götz, Frank; Zühlke, Kerstin; Moutty, Marie; Göttert, Hendrikje; Joshi, Mangesh; Reif, Bernd; Haase, Hannelore; Morano, Ingo; Grossmann, Solveig; Klukovits, Anna; Verli, Judit; Gáspár, Róbert; Noack, Claudia; Bergmann, Martin; Kass, Robert; Hampel, Kornelia; Kashin, Dmitry; Genieser, Hans-Gottfried; Herberg, Friedrich W; Willoughby, Debbie; Cooper, Dermot M F; Baillie, George S; Houslay, Miles D; von Kries, Jens Peter; Zimmermann, Bastian; Rosenthal, Walter; Klussmann, Enno

    2011-03-18

    A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating β-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.

  8. Protein interaction module-assisted function X (PIMAX) approach to producing challenging proteins including hyperphosphorylated tau and active CDK5/p25 kinase complex.

    PubMed

    Sui, Dexin; Xu, Xinjing; Ye, Xuemei; Liu, Mengyu; Mianecki, Maxwell; Rattanasinchai, Chotirat; Buehl, Christopher; Deng, Xiexiong; Kuo, Min-Hao

    2015-01-01

    Many biomedically critical proteins are underrepresented in proteomics and biochemical studies because of the difficulty of their production in Escherichia coli. These proteins might possess posttranslational modifications vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific "facilitator," such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex. Here we report the design and application of a protein interaction module-assisted function X (PIMAX) system that effectively overcomes these hurdles. By fusing two proteins of interest to a pair of well-studied protein-protein interaction modules, we were able to potentiate the association of these two proteins, resulting in successful production of an enzymatically active cyclin-dependent kinase complex and hyperphosphorylated tau protein, which is intimately linked to Alzheimer disease. Furthermore, using tau isoforms quantitatively phosphorylated by GSK-3β and CDK5 kinases via PIMAX, we demonstrated the hyperphosphorylation-stimulated tau oligomerization in vitro, paving the way for new Alzheimer disease drug discoveries. Vectors for PIMAX can be easily modified to meet the needs of different applications. This approach thus provides a convenient and modular suite with broad implications for proteomics and biomedical research.

  9. Tobacco Translationally Controlled Tumor Protein Interacts with Ethylene Receptor Tobacco Histidine Kinase1 and Enhances Plant Growth through Promotion of Cell Proliferation.

    PubMed

    Tao, Jian-Jun; Cao, Yang-Rong; Chen, Hao-Wei; Wei, Wei; Li, Qing-Tian; Ma, Biao; Zhang, Wan-Ke; Chen, Shou-Yi; Zhang, Jin-Song

    2015-09-01

    Ethylene is an important phytohormone in the regulation of plant growth, development, and stress response throughout the lifecycle. Previously, we discovered that a subfamily II ethylene receptor tobacco (Nicotiana tabacum) Histidine Kinase1 (NTHK1) promotes seedling growth. Here, we identified an NTHK1-interacting protein translationally controlled tumor protein (NtTCTP) by the yeast (Saccharomyces cerevisiae) two-hybrid assay and further characterized its roles in plant growth. The interaction was further confirmed by in vitro glutathione S-transferase pull down and in vivo coimmunoprecipitation and bimolecular fluorescence complementation assays, and the kinase domain of NTHK1 mediates the interaction with NtTCTP. The NtTCTP protein is induced by ethylene treatment and colocalizes with NTHK1 at the endoplasmic reticulum. Overexpression of NtTCTP or NTHK1 reduces plant response to ethylene and promotes seedling growth, mainly through acceleration of cell proliferation. Genetic analysis suggests that NtTCTP is required for the function of NTHK1. Furthermore, association of NtTCTP prevents NTHK1 from proteasome-mediated protein degradation. Our data suggest that plant growth inhibition triggered by ethylene is regulated by a unique feedback mechanism, in which ethylene-induced NtTCTP associates with and stabilizes ethylene receptor NTHK1 to reduce plant response to ethylene and promote plant growth through acceleration of cell proliferation.

  10. LYR3, a high-affinity LCO-binding protein of Medicago truncatula, interacts with LYK3, a key symbiotic receptor.

    PubMed

    Fliegmann, Judith; Jauneau, Alain; Pichereaux, Carole; Rosenberg, Charles; Gasciolli, Virginie; Timmers, Antonius C J; Burlet-Schiltz, Odile; Cullimore, Julie; Bono, Jean-Jacques

    2016-05-01

    LYR3, LYK3, and NFP are lysin motif-containing receptor-like kinases (LysM-RLKs) from Medicago truncatula, involved in perception of symbiotic lipo-chitooligosaccharide (LCO) signals. Here, we show that LYR3, a high-affinity LCO-binding protein, physically interacts with LYK3, a key player regulating symbiotic interactions. In vitro, LYR3 is phosphorylated by the active kinase domain of LYK3. Fluorescence lifetime imaging/Förster resonance energy transfer (FLIM/FRET) experiments in tobacco protoplasts show that the interaction between LYR3 and LYK3 at the plasma membrane is disrupted or inhibited by addition of LCOs. Moreover, LYR3 attenuates the cell death response, provoked by coexpression of NFP and LYK3 in tobacco leaves.

  11. Human biliverdin reductase-based peptides activate and inhibit glucose uptake through direct interaction with the kinase domain of insulin receptor

    PubMed Central

    Gibbs, Peter E. M.; Lerner-Marmarosh, Nicole; Poulin, Amelia; Farah, Elie; Maines, Mahin D.

    2014-01-01

    Insulin binding changes conformation of the insulin receptor kinase (IRK) domain and initiates glucose uptake through the insulin, IGF-1, phosphatidyl inositol 3-kinase (PI3K), and MAPK pathways; human biliverdin reductase (hBVR) is an IRK substrate and pathway effector. This is the first report on hBVR peptide-mediated IRK activation and conformational change. 290KYCCSRK, which increased IRK Vmax without changing Km, stimulated glucose uptake and potentiated insulin and IGF-1 stimulation in 4 cell lines. KYCCSRK in native hBVR was necessary for the hBVR and IRK cross-activation. Peptide treatment also activated PI3K downstream effectors, Akt and ERK, phosphorylation, and Elk transcriptional activity. In cells transfected with CMV-regulated EGFP-VP-peptide plasmid, C292→A mutant did not stimulate glucose uptake; K296→A decreased uptake and kinase activity. KEDQYMKMTV, corresponding to hBVR's SH2-binding domain, was a potent inhibitor of glucose uptake and IRK. The mechanism of action of peptides was examined using cells expressing IRK (aa 988–1263) activated by coexpressed KYCCSRK. Three active cys-mutants of IRK, with fluorophore coupled to cysteines, C1056, C1138, or C1234, were examined for changes in fluorescence emission spectra in the presence of peptides. KYCCSRK and KEDQYMKMTV bound to different sites in IRK. The findings identify novel agents for activating or inhibiting insulin signaling and offer a new approach for treatment of type 2 diabetes and hypoglycemia.—Gibbs, P. E. M., Lerner-Marmarosh, N., Poulin, A., Farah, E., Maines, M. D. Human biliverdin reductase-based peptides activate and inhibit glucose uptake through direct interaction with the kinase domain of insulin receptor. PMID:24568842

  12. Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses1[OPEN

    PubMed Central

    Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; Munemasa, Shintaro; Booker, Matthew A.; Brandt, Benjamin; Waadt, Christian; Nusinow, Dmitri A.; Kay, Steve A.; Kunz, Hans-Henning; Schumacher, Karin; DeLong, Alison; Yates, John R.; Schroeder, Julian I.

    2015-01-01

    The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases. PMID:26175513

  13. Oncogenic potential of TAR RNA binding protein TRBP and its regulatory interaction with RNA-dependent protein kinase PKR.

    PubMed Central

    Benkirane, M; Neuveut, C; Chun, R F; Smith, S M; Samuel, C E; Gatignol, A; Jeang, K T

    1997-01-01

    TAR RNA binding protein (TRBP) belongs to an RNA binding protein family that includes the double-stranded RNA-activated protein kinase (PKR), Drosophila Staufen and Xenopus xlrbpa. One member of this family, PKR, is a serine/threonine kinase which has anti-viral and anti-proliferative effects. In this study we show that TRBP is a cellular down-regulator of PKR function. Assaying expression from an infectious HIV-1 molecular clone, we found that PKR inhibited viral protein synthesis and that over-expression of TRBP effectively countered this inhibition. In intracellular and in cell-free assays we show that TRBP directly inhibits PKR autophosphorylation through an RNA binding-independent pathway. Biologically, TRBP serves a growth-promoting role; cells that overexpress TRBP exhibit transformed phenotypes. Our results demonstrate the oncogenic potential of TRBP and are consistent with the notion that intracellular PKR function contributes physiologically towards regulating cellular proliferation. PMID:9034343

  14. Interaction with Pyruvate Kinase M2 Destabilizes Tristetraprolin by Proteasome Degradation and Regulates Cell Proliferation in Breast Cancer

    PubMed Central

    Huang, Liangqian; Yu, Zhenhai; Zhang, Zhenchao; Ma, Wenjing; Song, Shaoli; Huang, Gang

    2016-01-01

    Pyruvate kinase M2 (PKM2), which is predominantly expressed in most cancers, plays a key role in the Warburg effect. However, how PKM2 functions as a tumor supportive protein has not been fully elucidated. Here, we identified tristetraprolin (TTP), an AU-rich, element-binding protein that regulates mRNA stability, as a new binding partner of PKM2. Our data reveal that PKM2 suppresses TTP protein levels by promoting its phosphorylation, ubiquitination, and proteasome degradation, reducing its mRNA turnover ability and ultimately impairing cell viability in breast cancer cells. The p38/mitogen-activated protein kinase (MAPK) pathway might be involved in PKM2-mediated TTP degradation, while treatment with the p38 inhibitor or siRNA abolished PKM2-induced TTP protein degradation. These findings demonstrate that PKM2–TTP association is crucial for regulating breast cancer cell proliferation and is therefore a potential therapeutic target in cancer. PMID:26926077

  15. Calcium-Dependent Protein Kinase in Ginger Binds with Importin-α through Its Junction Domain for Nuclear Localization, and Further Interacts with NAC Transcription Factor

    PubMed Central

    Vivek, Padmanabhan Jayanthi; Resmi, Mohankumar Saraladevi; Sreekumar, Sweda; Sivakumar, K. C.; Tuteja, Narendra; Soniya, Eppurathu Vasudevan

    2017-01-01

    Calcium-dependent protein kinases (CDPKs) are important sensors of Ca2+ elevations in plant cells regulating the gene expression linked with various cellular processes like stress response, growth and development, metabolism, and cytoskeleton dynamics. Ginger is an extensively used spice due to its unique flavor and immense medicinal value. The two major threats that interfere with the large scale production of ginger are the salinity and drought stress. ZoCDPK1 (Zingiber officinale Calcium-dependent protein kinase 1) is a salinity and drought-inducible CDPK gene isolated from ginger and undergoes dynamic subcellular localization during stress conditions. ZoCDPK1, with signature features of a typical Ca2+ regulated kinase, also possesses a bipartite nuclear localization sequence (NLS) in its junction domain (JD). A striking feature in ZoCDPK1 is the rare occurrence of a coupling between the NLS in JD and consensus sequences in regulatory domain. Here, we further identified its nature of nuclear localization and its interaction partners. In the homology model generated for ZoCDPK1, the regulatory domain mimics the crystal structure of the regulatory domain in Arabidopsis CDPK1. Molecular docking simulation of importin (ZoIMPα), an important protein involved in nuclear translocation, into the NLS of ZoCDPK1 was well-visualized. Furthermore, the direct interaction of ZoCDPK1 and ZoIMPα proteins was studied by the yeast 2-hybrid (Y2H) system, which confirmed that junction domain (JD) is an important interaction module required for ZoCDPK1 and ZoIMPα binding. The probable interacting partners of ZoCDPK1 were also identified using Y2H experiment. Of the 10 different stress-related interacting partners identified for ZoCDPK1, NAC transcription factor (TF) needs special mention, especially in the context of ZoCDPK1 function. The interaction between ZoCDPK1 and NAC TF, in fact, corroborate with the results of gene expression and over-expression studies of ZoCDPK1. Hence

  16. Wall-associated kinase-like polypeptide mediates nutritional status perception and response

    DOEpatents

    Yang, Zhenbiao; Karr, Stephen

    2014-02-11

    The disclosure relates to methods for modulating plant growth and organogenesis using dominant-negative receptor-like kinases. The disclosure further provides a method for increasing plant yield relative to corresponding wild type plants comprising modulating the expression in a plant of a nucleic acid encoding a Wall-Associated Kinase-like 14 polypeptide or a homolog thereof, and selecting for plants having increased yield or growth on a nutrient deficient substrate.

  17. Direct interaction of Syk and Lyn protein tyrosine kinases in rat basophilic leukemia cells activated via type I Fc epsilon receptors.

    PubMed

    Amoui, M; Dráberová, L; Tolar, P; Dráber, P

    1997-01-01

    Activation of rat mast cells through the receptor with high affinity for IgE (Fc epsilonRI) requires a complex set of interactions involving transmembrane subunits of the Fc epsilonRI and two classes of nonreceptor protein tyrosine kinase (PTK). the Src family PTK p53/p56(lyn) (Lyn) and the Syk/ZAP-family PTK p72(syk) (Syk). Early activation events involve increased activity of Lyn and Syk kinases and their translocation into membrane domains containing aggregated Fc epsilonRI, but the molecular mechanisms responsible for these changes have remained largely unclear. To determine the role of Fc epsilonRI subunits in this process, we have analyzed Syk- and Lyn-associated proteins in activated rat basophilic leukemia (RBL) cells and their variants deficient in the expression of Fc epsilonRI beta or gamma subunits. Sepharose 4B gel chromatography of postnuclear supernatants from Nonidet-P40-solubilized antigen (Ag)- or pervanadate-activated RBL cells revealed extensive changes in the size of complexes formed by Lyn and Syk kinases and other cellular components. A fusion protein containing Src homology 2 (SH2) and SH3 domains of Lyn bound Syk from lysates of nonactivated RBL cells; an increased binding was observed when lysates from Ag- or pervanadate-activated cells were used. A similar amount of Syk was bound when lysates from pervanadate-activated variant cells deficient in the expression of Fc epsilonRI beta or gamma subunits were used, suggesting that Fc epsilonRI does not function as the only intermediate in the formation of the Syk-Lyn complexes. Further experiments have indicated that Syk-Lyn interactions occur in Ag-activated RBL cells under in vivo conditions and that these interactions could involve direct binding of the Lyn SH2 domain with phosphorylated tyrosine of Syk. The physical association of Lyn and Syk during mast-like cell activation supports the recently proposed functional cooperation of these two tyrosine kinases in Fc epsilonRI signaling.

  18. Identification of Potential Plk1 Targets in a Cell-Cycle Specific Proteome through Structural Dynamics of Kinase and Polo Box-Mediated Interactions

    PubMed Central

    Bibi, Nousheen; Parveen, Zahida; Rashid, Sajid

    2013-01-01

    Polo like kinase 1 (Plk1) is a key player in orchestrating the wide variety of cell-cycle events ranging from centrosome maturation, mitotic entry, checkpoint recovery, transcriptional control, spindle assembly, mitotic progression, cytokinesis and DNA damage checkpoints recovery. Due to its versatile nature, Plk1 is considered an imperative regulator to tightly control the diverse aspects of the cell cycle network. Interactions among Plk1 polo box domain (PBD) and its putative binding proteins are crucial for the activation of Plk1 kinase domain (KD). To date, only a few substrate candidates have been characterized through the inclusion of both polo box and kinase domain-mediated interactions. Thus it became compelling to explore precise and specific Plk1 substrates through reassessment and extension of the structure-function paradigm. To narrow this apparently wide gap in knowledge, here we employed a thorough sequence search of Plk1 phosphorylation signature containing proteins and explored their structure-based features like conceptual PBD-binding capabilities and subsequent recruitment of KD directed phosphorylation to dissect novel targets of Plk1. Collectively, we identified 4,521 phosphodependent proteins sharing similarity to the consensus phosphorylation and PBD recognition motifs. Subsequent application of filters including similarity index, Gene Ontology enrichment and protein localization resulted in stringent pre-filtering of irrelevant candidates and isolated unique targets with well-defined roles in cell-cycle machinery and carcinogenesis. These candidates were further refined structurally using molecular docking and dynamic simulation assays. Overall, our screening approach enables the identification of several undefined cell-cycle associated functions of Plk1 by uncovering novel phosphorylation targets. PMID:23967120

  19. Axl-EGFR receptor tyrosine kinase hetero-interaction provides EGFR with access to pro-invasive signalling in cancer cells

    PubMed Central

    Vouri, M; Croucher, D R; Kennedy, S P; An, Q; Pilkington, G J; Hafizi, S

    2016-01-01

    Acquired resistance to conventional and targeted therapies is becoming a major hindrance in cancer management. It is increasingly clear that cancer cells are able to evolve and rewire canonical signalling pathways to their advantage, thus evading cell death and promoting cell invasion. The Axl receptor tyrosine kinase (RTK) has been shown to modulate acquired resistance to EGFR-targeted therapies in both breast and lung cancers. Glioblastoma multiforme (GBM) is a highly infiltrative and invasive form of brain tumour with little response to therapy. Both Axl and EGFR have been identified as major players in gliomagenesis and invasiveness. However, the mechanisms underlying a potential signalling crosstalk between EGFR and Axl RTKs are unknown. The purpose of this study was to investigate this novel and unconventional interaction among RTKs of different families in human GBM cells. With the use of western blotting, in vitro kinase activity, co-immunoprecipitation and bimolecular fluorescence complementation assays, we show that EGF stimulates activation of Axl kinase and that there is a hetero-interaction between the two RTKs. Through small interfering RNA knockdown and quantitative PCR screening, we identified distinct gene expression patterns in GBM cells that were specifically regulated by signalling from EGFR-EGFR, Axl–Axl and EGFR-Axl RTK parings. These included genes that promote invasion, which were activated only via the EGFR-Axl axis (MMP9), while EGFR-EGFR distinctly regulated the cell cycle and Axl–Axl regulated invasion. Our findings provide critical insights into the role of EGFR-Axl hetero-dimerisation in cancer cells and reveal regulation of cell invasion via Axl as a novel function of EGFR signalling. PMID:27775700

  20. The Interaction between Polynucleotide Kinase Phosphatase and the DNA Repair Protein XRCC1 Is Critical for Repair of DNA Alkylation Damage and Stable Association at DNA Damage Sites*

    PubMed Central

    Della-Maria, Julie; Hegde, Muralidhar L.; McNeill, Daniel R.; Matsumoto, Yoshihiro; Tsai, Miaw-Sheue; Ellenberger, Tom; Wilson, David M.; Mitra, Sankar; Tomkinson, Alan E.

    2012-01-01

    XRCC1 plays a key role in the repair of DNA base damage and single-strand breaks. Although it has no known enzymatic activity, XRCC1 interacts with multiple DNA repair proteins and is a subunit of distinct DNA repair protein complexes. Here we used the yeast two-hybrid genetic assay to identify mutant versions of XRCC1 that are selectively defective in interacting with a single protein partner. One XRCC1 mutant, A482T, that was defective in binding to polynucleotide kinase phosphatase (PNKP) not only retained the ability to interact with partner proteins that bind to different regions of XRCC1 but also with aprataxin and aprataxin-like factor whose binding sites overlap with that of PNKP. Disruption of the interaction between PNKP and XRCC1 did not impact their initial recruitment to localized DNA damage sites but dramatically reduced their retention there. Furthermore, the interaction between PNKP and the DNA ligase IIIα-XRCC1 complex significantly increased the efficiency of reconstituted repair reactions and was required for complementation of the DNA damage sensitivity to DNA alkylation agents of xrcc1 mutant cells. Together our results reveal novel roles for the interaction between PNKP and XRCC1 in the retention of XRCC1 at DNA damage sites and in DNA alkylation damage repair. PMID:22992732

  1. Chickpea transcription factor CaTLP1 interacts with protein kinases, modulates ROS accumulation and promotes ABA-mediated stomatal closure

    PubMed Central

    Wardhan, Vijay; Pandey, Aarti; Chakraborty, Subhra; Chakraborty, Niranjan

    2016-01-01

    Tubby and Tubby-like proteins (TLPs), in mammals, play critical roles in neural development, while its function in plants is largely unknown. We previously demonstrated that the chickpea TLP, CaTLP1, participates in osmotic stress response and might be associated with ABA-dependent network. However, how CaTLP1 is connected to ABA signaling remains unclear. The CaTLP1 was found to be engaged in ABA-mediated gene expression and stomatal closure. Complementation of the yeast yap1 mutant with CaTLP1 revealed its role in ROS scavenging. Furthermore, complementation of Arabidopsis attlp2 mutant displayed enhanced stress tolerance, indicating the functional conservation of TLPs across the species. The presence of ABA-responsive element along with other motifs in the proximal promoter regions of TLPs firmly established their involvement in stress signalling pathways. The CaTLP1 promoter driven GUS expression was restricted to the vegetative organs, especially stem and rosette leaves. Global protein expression profiling of wild-type, attlp2 and complemented Arabidopsis plants revealed 95 differentially expressed proteins, presumably involved in maintaining physiological and biological processes under dehydration. Immunoprecipitation assay revealed that protein kinases are most likely to interact with CaTLP1. This study provides the first demonstration that the TLPs act as module for ABA-mediated stomatal closure possibly via interaction with protein kinase. PMID:27934866

  2. Phospho-Ser383-Elk-1 is localized to the mitotic spindles during cell cycle and interacts with mitotic kinase Aurora-A.

    PubMed

    Demir, Ozlem; Kurnaz, Isil Aksan

    2013-10-01

    Elk-1 is a member of the E-twenty-six (ETS) domain superfamily of transcription factors and has been traditionally associated with mitogen-induced immediate early gene transcription upon phosphorylation by mitogen activated protein kinases (ERK/MAPK). Elk-1 is not only upregulated but also phosphorylated in brain tumour cells. However, in this study, we show for the first time that S383-phosphorylated Elk-1 (P-S383-Elk-1) is associated with mitotic spindle poles from metaphase through telophase and relocates to the spindle midbody during cytokinesis, while Thr417Ala mutation is associated with DNA throughout mitosis. Serine 383 phosphorylation appears to be important for polar localization of Elk-1, since exogenous protein including serine-to-alanine mutation was seen to be distributed throughout the spindle fibres. We further show that Elk-1 interacts with the cell cycle kinase Aurora-A, and when Aurora inhibitors are used, P-S383-Elk-1 fails to localize to the poles and remains associated with DNA. Apart from one transcriptional repressor molecule, Kaiso, this is the first time a transactivator was shown to possess such mitotic localization and interaction. The functional significance and detailed mechanism of this cell cycle-related localization of Elk-1 are yet to be determined.

  3. Interaction between cyclin-dependent kinases and human papillomavirus replication-initiation protein E1 is required for efficient viral replication

    PubMed Central

    Ma, Tianlin; Zou, Nianxiang; Lin, Biing Yuan; Chow, Louise T.; Harper, J. Wade

    1999-01-01

    We have identified the human papillomavirus (HPV) DNA replication initiation protein E1 as a tight-binding substrate of cyclin E/cyclin-dependent kinase (Cdk) complexes by using expression cloning. E1, a DNA helicase, collaborates with the HPV E2 protein in ori-dependent replication. E1 formed complexes with cyclin E in insect and mammalian cells, independent of Cdks and E2. Additional cyclins, including A-, B-, and F-type (but not D-type), interacted with the E1/E2 complex, and A- and E-type cyclin kinases were capable of phosphorylating E1 and E2 in vitro. Association with cyclins and efficient phosphorylation of E1 required the presence of a cyclin interaction motif (the RXL motif). E1 lacking the RXL motif displayed defects in E2-dependent HPV ori replication in vivo. Consistent with a role for Cdk-mediated phosphorylation in E1 function, an E1 protein lacking all four candidate Cdk phosphorylation sites still associated with E2 and cyclin E but was impaired in HPV replication in vitro and in vivo. Our data reveal a link between cyclin/Cdk function and activation of HPV DNA replication through targeting of Cdk complexes to the E1 replication-initiation protein and suggest a functional role for E1 phosphorylation by Cdks. The use of cyclin-binding RXL motifs is now emerging as a major mechanism by which cyclins are targeted to key substrates. PMID:9892642

  4. [Tyrosine kinase inhibitors].

    PubMed

    Robert, Jacques

    2011-11-01

    Membrane receptors with tyrosine kinase activity and cytoplasmic tyrosine kinases have emerged as important potential targets in oncology. Starting from basic structures such as anilino-quinazoline, numerous compounds have been synthesised, with the help of tyrosine kinase crystallography, which has allowed to optimise protein-ligand interactions. The catalytic domains of all kinases present similar three-dimensional structures, which explains that it may be difficult to identify molecules having a high specificity for a given tyrosine kinase. Some tyrosine kinase inhibitors are relatively specific for epidermal growth factor receptor (EGFR) such as géfitinib and erlotinib; other are mainly active against platelet-derived growth factor receptor (PDGFR) and the receptor KIT, such as imatinib or nilotinib, and other against vascular endothelial growth factor (VEGF) receptors involved in angiogenesis, such as sunitinib and sorafenib. The oral formulation of tyrosine kinase inhibitors is well accepted by the patients but may generate sometimes compliance problems requiring pharmacokinetic monitoring. This chemical family is in full expansion and several dozens of compounds have entered clinical trials.

  5. Diacylglycerol kinase α promotes 3D cancer cell growth and limits drug sensitivity through functional interaction with Src

    PubMed Central

    Torres-Ayuso, Pedro; Daza-Martín, Manuel; Martín-Pérez, Jorge; Ávila-Flores, Antonia; Mérida, Isabel

    2014-01-01

    Diacylglycerol kinase (DGK)α converts diacylglycerol to phosphatidic acid. This lipid kinase sustains survival, migration and invasion of tumor cells, with no effect over untransformed cells, suggesting its potential as a cancer-specific target. Nonetheless the mechanisms that underlie DGKα specific contribution to cancer survival have not been elucidated. Using three-dimensional (3D) colon and breast cancer cell cultures, we demonstrate that DGKα upregulation is part of the transcriptional program that results in Src activation in these culture conditions. Pharmacological or genetic DGKα silencing impaired tumor growth in vivo confirming its function in malignant transformation. DGKα-mediated Src regulation contributed to limit the effect of Src inhibitors, and its transcriptional upregulation in response to PI3K/Akt inhibitors resulted in reduced toxicity. Src oncogenic properties and contribution to pharmacological resistance have been linked to its overactivation in cancer. DGKα participation in this central node helps to explain why its pharmacological inhibition or siRNA-mediated targeting specifically alters tumor viability with no effect on untransformed cells. Our results identify DGKα-mediated stabilization of Src activation as an important mechanism in tumor growth, and suggest that targeting this enzyme, alone or in combination with other inhibitors in wide clinical use, could constitute a treatment strategy for aggressive forms of cancer. PMID:25339152

  6. GnRH-II receptor-like antigenicity in human placenta and in cancers of the human reproductive organs.

    PubMed

    Eicke, Nicola; Günthert, Andreas R; Viereck, Volker; Siebold, Doreen; Béhé, Martin; Becker, Tamara; Emons, Günter; Gründker, Carsten

    2005-10-01

    We have recently demonstrated that the antiproliferative activity of GnRH-II on human endometrial and ovarian cancer cell lines is not mediated through the GnRH-I receptor. A functional receptor for human GnRH-II has not yet been identified. In this study, we have generated a polyclonal antiserum to the putative human GnRH-II receptor using a peptide (YSPTMLTEVPPC) corresponding to the third extracellular domain coupled to keyhole limpet haemocyanin via the Cys residue. A database search showed no identical peptide sequences in any other human gene. To avoid cross-reactions against two similar amino acid sequences the antiserum was pre-absorbed using these peptides. Immune histological sections of human placenta and human endometrial, ovarian and prostate cancers using rabbit anti-human GnRH-II receptor antiserum showed GnRH-II receptor-like staining. Western blot analysis of cell membrane preparations of human endometrial and ovarian cancer cell lines yielded a band at approximately 43 kDa whereas Western blot analysis of cell membrane preparations of ovaries obtained from the marmoset monkey (Callithrix jacchus) yielded a band at approximately 54 kDa. To identify the GnRH-II receptor-like antigen we used the photo-affinity labelling technique. Photochemical reaction of (125)I-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[d-Lys(6)]-GnRH-II (10(-9) M) with cell membrane preparations of human endometrial and ovarian cancer cells yielded a band at approximately 43 kDa. In competition experiments, the GnRH-I agonist Triptorelin (10(-7) M) showed a weak decrease of (125)I-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[d-Lys(6)]-GnRH-II binding to its binding site. The GnRH-I antagonist Cetrorelix (10(-7) M) showed a clearly stronger decrease, whereas GnRH-II agonist [d-Lys(6)]-GnRH-II (10(-7) M) was the most potent competitor. Western blot analysis of the same gel using rabbit anti-human GnRH-II receptor antiserum identified this band as GnRH-II receptor-like

  7. Interaction of new kinase inhibitors cabozantinib and tofacitinib with human serum alpha-1 acid glycoprotein. A comprehensive spectroscopic and molecular Docking approach.

    PubMed

    Ajmal, Mohammad Rehan; Abdelhameed, Ali Saber; Alam, Parvez; Khan, Rizwan Hasan

    2016-04-15

    In the current study we have investigated the interaction of newly approved kinase inhibitors namely Cabozantinib (CBZ) and Tofacitinib (TFB) with human Alpha-1 acid glycoprotein (AAG) under simulated physiological conditions using fluorescence quenching measurements, circular dichroism, dynamic light scattering and molecular docking methods. CBZ and TFB binds to AAG with significant affinity and the calculated binding constant for the drugs lie in the order of 10(4). With the increase in temperature the binding constant values decreased for both CBZ and TFB. The fluorescence resonance energy transfer (FRET) from AAG to CBZ and TFB suggested the fluorescence intensity of AAG was quenched by the two studied drugs via the formation of a non-fluorescent complex in the static manner. The molecular distance r value calculated from FRET is around 2 nm for both drugs, fluorescence spectroscopy data was employed for the study of thermodynamic parameters, standard Gibbs free energy change at 300 K was calculated as -5.234 kcal mol(-1) for CBZ-AAG interaction and -6.237 kcal mol(-1) for TFB-AAG interaction, standard enthalpy change and standard entropy change for CBZ-AAG interaction are -9.553 kcal mol(-1) and -14.618 cal mol(-1) K(-1) respectively while for AAG-TFB interaction, standard enthalpy and standard entropy change was calculated as 4.019 kcal mol(-1) and 7.206 cal mol(-1) K(-1) respectively. Protein binding of the two drugs caused the tertiary structure alterations. Dynamic light scattering measurements demonstrated the reduction in the hydrodynamic radii of the protein. Furthermore molecular docking results suggested the Hydrophobic interaction and hydrogen bonding were the interactive forces in the binding process of CBZ to AAG while in case of TFB only hydrophobic interactions were found to be involved, overlap of the binding site for two studied drugs on the AAG molecule was revealed by docking results.

  8. Interaction of new kinase inhibitors cabozantinib and tofacitinib with human serum alpha-1 acid glycoprotein. A comprehensive spectroscopic and molecular Docking approach

    NASA Astrophysics Data System (ADS)

    Ajmal, Mohammad Rehan; Abdelhameed, Ali Saber; Alam, Parvez; Khan, Rizwan Hasan

    2016-04-01

    In the current study we have investigated the interaction of newly approved kinase inhibitors namely Cabozantinib (CBZ) and Tofacitinib (TFB) with human Alpha-1 acid glycoprotein (AAG) under simulated physiological conditions using fluorescence quenching measurements, circular dichroism, dynamic light scattering and molecular docking methods. CBZ and TFB binds to AAG with significant affinity and the calculated binding constant for the drugs lie in the order of 104. With the increase in temperature the binding constant values decreased for both CBZ and TFB. The fluorescence resonance energy transfer (FRET) from AAG to CBZ and TFB suggested the fluorescence intensity of AAG was quenched by the two studied drugs via the formation of a non-fluorescent complex in the static manner. The molecular distance r value calculated from FRET is around 2 nm for both drugs, fluorescence spectroscopy data was employed for the study of thermodynamic parameters, standard Gibbs free energy change at 300K was calculated as - 5.234 kcal mol- 1 for CBZ-AAG interaction and - 6.237 kcal mol- 1 for TFB-AAG interaction, standard enthalpy change and standard entropy change for CBZ-AAG interaction are - 9.553 kcal mol- 1 and - 14.618 cal mol- 1K- 1 respectively while for AAG-TFB interaction, standard enthalpy and standard entropy change was calculated as 4.019 kcal mol- 1 and 7.206 cal mol- 1K- 1 respectively. Protein binding of the two drugs caused the tertiary structure alterations. Dynamic light scattering measurements demonstrated the reduction in the hydrodynamic radii of the protein. Furthermore molecular docking results suggested the Hydrophobic interaction and hydrogen bonding were the interactive forces in the binding process of CBZ to AAG while in case of TFB only hydrophobic interactions were found to be involved, overlap of the binding site for two studied drugs on the AAG molecule was revealed by docking results.

  9. High-Resolution Structure of the Histidine-Containing Phosphocarrier Protein (HPr) from Staphylococcus aureus and Characterization of Its Interaction with the Bifunctional HPr Kinase/Phosphorylase

    PubMed Central

    Maurer, Till; Meier, Sebastian; Kachel, Norman; Munte, Claudia Elisabeth; Hasenbein, Sonja; Koch, Brigitte; Hengstenberg, Wolfgang; Kalbitzer, Hans Robert

    2004-01-01

    A high-resolution structure of the histidine-containing phosphocarrier protein (HPr) from Staphylococcus aureus was obtained by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy on the basis of 1,766 structural restraints. Twenty-three hydrogen bonds in HPr could be directly detected by polarization transfer from the amide nitrogen to the carbonyl carbon involved in the hydrogen bond. Differential line broadening was used to characterize the interaction of HPr with the HPr kinase/phosphorylase (HPrK/P) of Staphylococcus xylosus, which is responsible for phosphorylation-dephosphorylation of the hydroxyl group of the regulatory serine residue at position 46. The dissociation constant Kd was determined to be 0.10 ± 0.02 mM at 303 K from the NMR data, assuming independent binding. The data are consistent with a stoichiometry of 1 HPr molecule per HPrK/P monomer in solution. Using transversal relaxation optimized spectroscopy-heteronuclear single quantum correlation, we mapped the interaction site of the two proteins in the 330-kDa complex. As expected, it covers the region around Ser46 and the small helix b following this residue. In addition, HPrK/P also binds to the second phosphorylation site of HPr at position 15. This interaction may be essential for the recognition of the phosphorylation state of His15 and the phosphorylation-dependent regulation of the kinase/phosphorylase activity. In accordance with this observation, the recently published X-ray structure of the HPr/HPrK core protein complex from Lactobacillus casei shows interactions with the two phosphorylation sites. However, the NMR data also suggest differences for the full-length protein from S. xylosus: there are no indications for an interaction with the residues preceding the regulatory Ser46 residue (Thr41 to Lys45) in the protein of S. xylosus. In contrast, it seems to interact with the C-terminal helix of HPr in solution, an interaction which is not observed for the

  10. S100B Protein Regulates Astrocyte Shape and Migration via Interaction with Src Kinase: IMPLICATIONS FOR ASTROCYTE DEVELOPMENT, ACTIVATION, AND TUMOR GROWTH.

    PubMed

    Brozzi, Flora; Arcuri, Cataldo; Giambanco, Ileana; Donato, Rosario

    2009-03-27

    S100B is a Ca(2+)-binding protein of the EF-hand type that is abundantly expressed in astrocytes and has been implicated in the regulation of several intracellular activities, including proliferation and differentiation. We show here that reducing S100B levels in the astrocytoma cell line GL15 and the Müller cell line MIO-M1 by small interference RNA technique results in a rapid disassembly of stress fibers, collapse of F-actin onto the plasma membrane and reduced migration, and acquisition of a stellate shape. Also, S100B-silenced GL15 and MIO-M1 Müller cells show a higher abundance of glial fibrillary acidic protein filaments, which mark differentiated astrocytes, compared with control cells. These effects are dependent on reduced activation of the phosphatidylinositol 3-kinase (PI3K) downstream effectors, Akt and RhoA, and consequently elevated activity of GSK3beta and Rac1 and decreased activity of the RhoA-associated kinase. Also, rat primary astrocytes transiently down-regulate S100B expression when exposed to the differentiating agent dibutyryl cyclic AMP and re-express S100B at later stages of dibutyryl cyclic AMP-induced differentiation. Moreover, reducing S100B levels results in a remarkably slow resumption of S100B expression, suggesting the S100B might regulate its own expression. Finally, we show that S100B interacts with Src kinase, thereby stimulating the PI3K/Akt and PI3K/RhoA pathways. These results suggest that S100B might contribute to reduce the differentiation potential of cells of the astrocytic lineage and participate in the astrocyte activation process in the case of brain insult and in invasive properties of glioma cells.

  11. Fcgamma receptor-like activity of hepatitis C virus core protein.

    PubMed

    Maillard, Patrick; Lavergne, Jean-Pierre; Sibéril, Sophie; Faure, Grazyna; Roohvand, Farzin; Petres, Stephane; Teillaud, Jean Luc; Budkowska, Agata

    2004-01-23

    We have previously demonstrated that viral particles with the properties of nonenveloped hepatitis C virus (HCV) nucleocapsids occur in the serum of HCV-infected individuals (1). We show here that nucleocapsids purified directly from serum or isolated from HCV virions have FcgammaR-like activity and bind "nonimmune" IgG via its Fcgamma domain. HCV core proteins produced in Escherichia coli and in the baculovirus expression system also bound "nonimmune" IgG and their Fcgamma fragments. Folded conformation was required for IgG binding because the FcgammaR-like site of the core protein was inactive in denaturing conditions. Studies with synthetic core peptides showed that the region spanning amino acids 3-75 was essential for formation of the IgG-binding site. The interaction between the HCV core and human IgG is more efficient in acidic (pH 6.0) than in neutral conditions. The core protein-binding site on the IgG molecule differs from those for C1q, FcgammaRII (CD32), and FcgammaRIII (CD16) but overlaps with that for soluble protein A from Staphylococcus aureus (SpA), which is located in the CH2-CH3 interface of IgG. These characteristics of the core-IgG interaction are very similar to those of the neonatal FcRn. Surface plasmon resonance studies suggested that the binding of an anti-core antibody to HCV core protein might be "bipolar" through its paratope to the corresponding epitope and by its Fcgamma region to the FcgammaR-like motif on this protein. These features of HCV nucleocapsids and HCV core protein may confer an advantage for HCV in terms of survival by interfering with host defense mechanisms mediated by the Fcgamma part of IgG.

  12. Oncogenic mutations weaken the interactions that stabilize the p110α-p85α heterodimer in phosphatidylinositol 3-kinase α.

    PubMed

    Echeverria, Ignacia; Liu, Yunlong; Gabelli, Sandra B; Amzel, L Mario

    2015-09-01

    Phosphatidylinositol 3-kinase (PI3K) α is a heterodimeric lipid kinase that catalyzes the conversion of phosphoinositol-4,5-bisphosphate to phosphoinositol-3,4,5-trisphosphate. The PI3Kα signaling pathway plays an important role in cell growth, proliferation, and survival. This pathway is activated in numerous cancers, where the PI3KCA gene, which encodes for the p110α PI3Kα subunit, is mutated. Its mutation often results in gain of enzymatic activity; however, the mechanism of activation by oncogenic mutations remains unknown. Here, using computational methods, we show that oncogenic mutations that are far from the catalytic site and increase the enzymatic affinity destabilize the p110α-p85α dimer. By affecting the dynamics of the protein, these mutations favor the conformations that reduce the autoinhibitory effect of the p85α nSH2 domain. For example, we determined that, in all of the mutants, the nSH2 domain shows increased positional heterogeneity as compared with the wild-type, as demonstrated by changes in the fluctuation profiles computed by normal mode analysis of coarse-grained elastic network models. Analysis of the interdomain interactions of the wild-type and mutants at the p110α-p85α interface obtained with molecular dynamics simulations suggest that all of the tumor-associated mutations effectively weaken the interactions between p110α and p85α by disrupting key stabilizing interactions. These findings have important implications for understanding how oncogenic mutations change the conformational multiplicity of PI3Kα and lead to increased enzymatic activity. This mechanism may apply to other enzymes and/or macromolecular complexes that play a key role in cell signaling.

  13. The oligomeric assembly of the novel haem-degrading protein HbpS is essential for interaction with its cognate two-component sensor kinase.

    PubMed

    Ortiz de Orué Lucana, Darío; Bogel, Gabriele; Zou, Peijian; Groves, Matthew R

    2009-03-06

    HbpS, a novel protein of previously unknown function from Streptomyces reticuli, is up-regulated in response to haemin- and peroxide-based oxidative stress and interacts with the SenS/SenR two-component signal transduction system. In this study, we report the high-resolution crystal structures (2.2 and 1.6 A) of octomeric HbpS crystallized in the presence and in the absence of haem and demonstrate that iron binds to surface-exposed lysine residues of an octomeric assembly. Based on an analysis of the crystal structures, we propose that the iron atom originates from the haem group and report subsequent biochemical experiments that demonstrate that HbpS possesses haem-degrading activity in vitro. Further examination of the crystal structures has identified amino acids that are essential for assembly of the octomer. The role of these residues is confirmed by biophysical experiments. Additionally, we show that while the octomeric assembly state of HbpS is not essential for haem-degrading activity, the assembly of HbpS is required for its interaction with the cognate sensor kinase, SenS. Homologs of HbpS and SenS/SenR have been identified in a number of medically and ecologically relevant bacterial species (including Vibrio cholerae, Klebsiella pneumoniae, Corynebacterium diphtheriae, Arthrobacter aurescens and Pseudomonas putida), suggesting the existence of a previously undescribed bacterial oxidative stress-response pathway common to Gram-negative and Gram-positive bacteria. Thus, the data presented provide the first insight into the function of a novel protein family and an example of an iron-mediated interaction between an accessory protein and its cognate two-component sensor kinase.

  14. p300/β-Catenin Interactions Regulate Adult Progenitor Cell Differentiation Downstream of WNT5a/Protein Kinase C (PKC)*

    PubMed Central

    Rieger, Megan E.; Zhou, Beiyun; Solomon, Nicola; Sunohara, Mitsuhiro; Li, Changgong; Nguyen, Cu; Liu, Yixin; Pan, Jie-hong; Minoo, Parviz; Crandall, Edward D.; Brody, Steven L.; Kahn, Michael; Borok, Zea

    2016-01-01

    Maintenance of stem/progenitor cell-progeny relationships is required for tissue homeostasis during normal turnover and repair. Wnt signaling is implicated in both maintenance and differentiation of adult stem/progenitor cells, yet how this pathway serves these dichotomous roles remains enigmatic. We previously proposed a model suggesting that specific interaction of β-catenin with either of the homologous Kat3 co-activators, p300 or CREB-binding protein, differentially regulates maintenance versus differentiation of embryonic stem cells. Limited knowledge of endogenous mechanisms driving differential β-catenin/co-activator interactions and their role in adult somatic stem/progenitor cell maintenance versus differentiation led us to explore this process in defined models of adult progenitor cell differentiation. We focused primarily on alveolar epithelial type II (AT2) cells, progenitors of distal lung epithelium, and identified a novel axis whereby WNT5a/protein kinase C (PKC) signaling regulates specific β-catenin/co-activator interactions to promote adult progenitor cell differentiation. p300/β-catenin but not CBP/β-catenin interaction increases as AT2 cells differentiate to a type I (AT1) cell-like phenotype. Additionally, p300 transcriptionally activates AT1 cell-specific gene Aqp-5. IQ-1, a specific inhibitor of p300/β-catenin interaction, prevents differentiation of not only primary AT2 cells, but also tracheal epithelial cells, and C2C12 myoblasts. p300 phosphorylation at Ser-89 enhances p300/β-catenin interaction, concurrent with alveolar epithelial cell differentiation. WNT5a, a traditionally non-canonical WNT ligand regulates Ser-89 phosphorylation and p300/β-catenin interactions in a PKC-dependent manner, likely involving PKCζ. These studies identify a novel intersection of canonical and non-canonical Wnt signaling in adult progenitor cell differentiation that has important implications for targeting β-catenin to modulate adult progenitor cell

  15. Genetic analysis of a Drosophila neural cell adhesion molecule: interaction of fasciclin I and Abelson tyrosine kinase mutations.

    PubMed

    Elkins, T; Zinn, K; McAllister, L; Hoffmann, F M; Goodman, C S

    1990-02-23

    Drosophila fasciclin I is a homophilic cell adhesion molecule expressed in the developing embryo on the surface of a subset of fasciculating CNS axons, all PNS axons, and some nonneuronal cells. We have identified protein-null mutations in the fasciclin I (fas I) gene, and show that these mutants are viable and do not display gross defects in nervous system morphogenesis. The Drosophila Abelson (abl) proto-oncogene homolog encodes a cytoplasmic tyrosine kinase that is expressed during embryogenesis primarily in developing CNS axons; abl mutants show no gross defects in CNS morphogenesis. However, embryos doubly mutant for fas I and abl display major defects in CNS axon pathways, particularly in the commissural tracts where expression of these two proteins normally overlaps. The double mutant shows a clear defect in growth cone guidance; for example, the RP1 growth cone (normally fas I positive) does not follow its normal path across the commissure.

  16. The Gα4 G protein subunit interacts with the MAP kinase ERK2 using a D-motif that regulates developmental morphogenesis in Dictyostelium

    PubMed Central

    Nguyen, Hoai-Nghia; Hadwiger, Jeffrey A.

    2009-01-01

    G protein Gα subunits contribute to the specificity of different signal transduction pathways in Dictyostelium discoideum but Gα subunit-effector interactions have not been previously identified. The requirement of the Dictyostelium Gα4 subunit for MAP kinase (MAPK) activation and the identification of a putative MAPK docking site (D-motif) in this subunit suggested a possible interaction between the Gα4 subunit and MAPKs. In vivo association of the Gα4 subunit and ERK2 was demonstrated by pull-down and co-immunoprecipitation assays. Alteration of the D-motif reduced Gα4 subunit-ERK2 interactions but only slightly altered MAPK activation in response to folate. Expression of the Gα4 subunit with the altered D-motif in gα4− cells allowed for slug formation but not the morphogenesis associated with culmination. Expression of this mutant Gα4 subunit was sufficient to rescue chemotactic movement to folate. Alteration of the D-motif also reduced the aggregation defect associated with constitutively active Gα4 subunits. These results suggest Gα4 subunit-MAPK interactions are necessary for developmental morphogenesis but not for chemotaxis to folate. PMID:19765570

  17. Mitogen-activated protein kinase (MAPK)-regulated interactions between Osterix and Runx2 are critical for the transcriptional osteogenic program.

    PubMed

    Artigas, Natalia; Ureña, Carlos; Rodríguez-Carballo, Edgardo; Rosa, José Luis; Ventura, Francesc

    2014-09-26

    The transcription factors Runx2 and Osx (Osterix) are required for osteoblast differentiation and bone formation. Runx2 expression occurs at early stages of osteochondroprogenitor determination, followed by Osx induction during osteoblast maturation. We demonstrate that coexpression of Osx and Runx2 leads to cooperative induction of expression of the osteogenic genes Col1a1, Fmod, and Ibsp. Functional interaction of Osx and Runx2 in the regulation of these promoters is mediated by enhancer regions with adjacent Sp1 and Runx2 DNA-binding sites. These enhancers allow formation of a cooperative transcriptional complex, mediated by the binding of Osx and Runx2 to their specific DNA promoter sequences and by the protein-protein interactions between them. We also identified the domains involved in the interaction between Osx and Runx2. These regions contain the amino acids in Osx and Runx2 known to be phosphorylated by p38 and ERK MAPKs. Inhibition of p38 and ERK kinase activities or mutation of their known phosphorylation sites in Osx or Runx2 strongly disrupts their physical interaction and cooperative transcriptional effects. Altogether, our results provide a molecular description of a mechanism for Osx and Runx2 transcriptional cooperation that is subject to further regulation by MAPK-activating signals during osteogenesis.

  18. Protein kinase CK2 phosphorylation regulates the interaction of Kaposi's sarcoma-associated herpesvirus regulatory protein ORF57 with its multifunctional partner hnRNP K

    PubMed Central

    Malik, Poonam; Clements, J. Barklie

    2004-01-01

    ORF57 protein of Kaposi's sarcoma-associated herpesvirus has a counterpart in all herpesvirus of mammals and birds and regulates gene expression at transcriptional and post-transcriptional levels. ORF57 was capable of self-interaction and bound a rapidly migrating form of heterogeneous nuclear ribonucleoprotein K (hnRNP K), a multifunctional cellular protein involved in gene expression. In virus infected cell extracts, ORF57 was present in a complex with hnRNP K that had protein kinase CK2 activity, and was phosphorylated by CK2. Different regions of ORF57 bound both catalytic α/α′ and regulatory β subunits of CK2. CK2 modification enhanced the ORF57–hnRNP K interaction, and may regulate the presence and activities of components in the complex. We suggest that ORF57 and hnRNP K interaction may modulate ORF57-mediated regulation of viral gene expression. Herpesviral ORF57 (Rhadinovirus) and ICP27 (Simplexvirus) proteins both interact with hnRNP K and CK2 implying that adaptation of the ancestral hnRNP K and CK2 to associate with viral regulatory ancestor protein likely pre-dates divergence of these Herpesviridae genera that occurred 200 million years ago. PMID:15486205

  19. A non-mitotic role for Aurora kinase A as a direct activator of cell migration upon interaction with PLD, FAK and Src.

    PubMed

    Mahankali, Madhu; Henkels, Karen M; Speranza, Francis; Gomez-Cambronero, Julian

    2015-02-01

    Timely activation of Aurora kinase A (AURA, also known as AURKA) is vital for centrosome formation and the progression of mitosis. Nonetheless, it is still unclear if and when other cellular functions are activated by AURA. We report here that Src phosphorylates and activates AURA at T288, and AURA also activates focal adhesion kinase (FAK, also known as PTK2), leading to initiation of cell movement. An additional and new way by which AURA is regulated, is by phospholipase D2 (PLD2), which causes AURA activation. In addition, AURA phosphorylates PLD, so both proteins engage in a positive reinforcement loop. AURA and PLD2 form a protein–protein complex and colocalize to cytoplasmic regions in cells. The reason why PLD activates AURA is because of the production of phosphatidic acid by the lipase, which binds directly to AURA, with the region E171–E211 projected to be a phosphatidic-acid-binding pocket. Furthermore, this direct interaction with phosphatidic acid enhances tubulin polymerization and cooperates synergistically with AURA, FAK and Src in yielding a fully effectual cellular migration. Thus, Src and FAK, and PLD and phosphatidic acid are new upstream regulators of AURA that mediate its role in the non-mitotic cellular function of cell migration.

  20. Targeted disruption of the murine homeodomain-interacting protein kinase-2 causes growth deficiency in vivo and cell cycle arrest in vitro.

    PubMed

    Trapasso, Francesco; Aqeilan, Rami I; Iuliano, Rodolfo; Visone, Rosa; Gaudio, Eugenio; Ciuffini, Laura; Alder, Hansjuerg; Paduano, Francesco; Pierantoni, Giovanna Maria; Soddu, Silvia; Croce, Carlo M; Fusco, Alfredo

    2009-04-01

    The homeodomain-interacting protein kinase 2 (HIPK2) protein is a member of a recently identified family of nuclear protein kinases that are well conserved in various organisms. HIPK2 can bind to several homeotic factors and to a series of proteins involved in the regulation of cell survival and proliferation in response to morphogenetic and genotoxic signals. Here we report Hipk2-targeted disruption in mouse; Hipk2(-/-) mice are viable and fertile but significantly smaller than their wild-type littermates. This feature is present at birth and retained throughout the mouse adulthood. Mouse embryo fibroblasts from Hipk2(-/-) mice show a reduced proliferation rate, compared to the wild-type counterparts, with accumulation in the G0/G1 phase of the cell cycle and altered levels of the cell cycle regulators cyclin D and CDK6. Restoration of wild-type HIPK2 expression in Hipk2(-/-) cells rescues the normal phenotype supporting a role for HIPK2 in the regulation of cell proliferation.

  1. Structure of Ctk3, a subunit of the RNA polymerase II CTD kinase complex, reveals a noncanonical CTD-interacting domain fold.

    PubMed

    Mühlbacher, Wolfgang; Mayer, Andreas; Sun, Mai; Remmert, Michael; Cheung, Alan C M; Niesser, Jürgen; Soeding, Johannes; Cramer, Patrick

    2015-10-01

    CTDK-I is a yeast kinase complex that phosphorylates the C-terminal repeat domain (CTD) of RNA polymerase II (Pol II) to promote transcription elongation. CTDK-I contains the cyclin-dependent kinase Ctk1 (homologous to human CDK9/CDK12), the cyclin Ctk2 (human cyclin K), and the yeast-specific subunit Ctk3, which is required for CTDK-I stability and activity. Here we predict that Ctk3 consists of a N-terminal CTD-interacting domain (CID) and a C-terminal three-helix bundle domain. We determine the X-ray crystal structure of the N-terminal domain of the Ctk3 homologue Lsg1 from the fission yeast Schizosaccharomyces pombe at 2.0 Å resolution. The structure reveals eight helices arranged into a right-handed superhelical fold that resembles the CID domain present in transcription termination factors Pcf11, Nrd1, and Rtt103. Ctk3 however shows different surface properties and no binding to CTD peptides. Together with the known structure of Ctk1 and Ctk2 homologues, our results lead to a molecular framework for analyzing the structure and function of the CTDK-I complex.

  2. The Pto kinase conferring resistance to tomato bacterial speck disease interacts with proteins that bind a cis-element of pathogenesis-related genes.

    PubMed Central

    Zhou, J; Tang, X; Martin, G B

    1997-01-01

    In tomato, the Pto kinase confers resistance to bacterial speck disease by recognizing the expression of a corresponding avirulence gene, avrPto, in the pathogen Pseudomonas syringae pv. tomato. Using the yeast two-hybrid system, we have identified three genes, Pti4, Pti5 and Pti6, that encode proteins that physically interact with the Pto kinase. Pti4/5/6 each encode a protein with characteristics that are typical of transcription factors and are similar to the tobacco ethylene-responsive element-binding proteins (EREBPs). Using a gel mobility-shift assay, we demonstrate that, similarly to EREBPs, Pti4/5/6 specifically recognize and bind to a DNA sequence that is present in the promoter region of a large number of genes encoding 'pathogenesis-related' (PR) proteins. Expression of several PR genes and a tobacco EREBP gene is specifically enhanced upon Pto-avrPto recognition in tobacco. These observations establish a direct connection between a disease resistance gene and the specific activation of plant defense genes. PMID:9214637

  3. Homeodomain-interacting Protein Kinase-2 (HIPK2) Phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77 and Modulates Its DNA Binding Affinity

    PubMed Central

    Zhang, Qingchun; Wang, Yinsheng

    2008-01-01

    The chromosomal high-mobility group A (HMGA) proteins, comprising of HMGA1a, HMGA1b and HMGA2, play important roles in the regulation of numerous processes in eukaryotic cells, such as transcriptional regulation, DNA repair, RNA processing, and chromatin remodeling. The biological activities of HMGA1 proteins are highly regulated by their post-translational modifications (PTMs), including acetylation, methylation and phosphorylation. Recently, it was found that the homeodomain-interacting protein kinase-2 (HIPK2), a newly identified serine/threonine kinase, co-immunoprecipitated with, and phosphorylated HMGA1 proteins. However, the sites and the biological significance of the phosphorylation have not been elucidated. Here, we found that HIPK2 phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77, and HMGA1b at Thr-41 and Thr-66. In addition, we demonstrated that cdc2, which is known to phosphorylate HMGA1 proteins, could induce the phosphorylation of HMGA1 proteins at the same Ser/Thr sites. The two kinases, however, exhibited different site preferences for the phosphorylation: The preference for HIPK2 phosphorylation followed the order of Thr-77 > Thr-52 > Ser-35, whereas the order for cdc2 phosphorylation was Thr-52 > Thr-77 > Ser-35. Moreover, we found that the HIPK2-phosphorylated HMGA1a reduced the binding affinity of HMGA1a to human germ line ε promoter, and the drop in binding affinity induced by HIPK2 phosphorylation was lower than that introduced by cdc2 phosphorylation, which is consistent with the notion that the second AT-hook in HMGA1a is more important for DNA binding than the third AT-hook. Synopsis Here we report that both HIPK2 and cdc2 phosphorylate HMGA1a at Ser-35, Thr-52 and Thr-77, but the two kinases exhibit different site preferences. Moreover, we found that HIPK2-induced phosphorylation of HMGA1a reduced the binding affinity of HMGA1a to DNA, and the drop in binding affinity was lower than that introduced by cdc2 phosphorylation, confirming

  4. Rational development of high-affinity T-cell receptor-like antibodies

    PubMed Central

    Stewart-Jones, Guillaume; Wadle, Andreas; Hombach, Anja; Shenderov, Eugene; Held, Gerhard; Fischer, Eliane; Kleber, Sascha; Nuber, Natko; Stenner-Liewen, Frank; Bauer, Stefan; McMichael, Andrew; Knuth, Alexander; Abken, Hinrich; Hombach, Andreas A.; Cerundolo, Vincenzo; Jones, E. Yvonne; Renner, Christoph

    2009-01-01

    T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1157–165 peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW “peg” dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2–4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1157–165 target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes. PMID:19307587

  5. The Brassica napus receptor-like protein RLM2 is encoded by a second allele of the LepR3/Rlm2 blackleg resistance locus.

    PubMed

    Larkan, Nicholas J; Ma, Lisong; Borhan, Mohammad Hossein

    2015-09-01

    Leucine-rich repeat receptor-like proteins (LRR-RLPs) are highly adaptable parts of the signalling apparatus for extracellular detection of plant pathogens. Resistance to blackleg disease of Brassica spp. caused by Leptosphaeria maculans is largely governed by host race-specific R-genes, including the LRR-RLP gene LepR3. The blackleg resistance gene Rlm2 was previously mapped to the same genetic interval as LepR3. In this study, the LepR3 locus of the Rlm2 Brassica napus line 'Glacier DH24287' was cloned, and B. napus transformants were analysed for recovery of the Rlm2 phenotype. Multiple B. napus, B. rapa and B. juncea lines were assessed for sequence variation at the locus. Rlm2 was found to be an allelic variant of the LepR3 LRR-RLP locus, conveying race-specific resistance to L. maculans isolates harbouring AvrLm2. Several defence-related LRR-RLPs have previously been shown to associate with the RLK SOBIR1 to facilitate defence signalling. Bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation of RLM2-SOBIR1 studies revealed that RLM2 interacts with SOBIR1 of Arabidopsis thaliana when co-expressed in Nicotiana benthamiana. The interaction of RLM2 with AtSOBIR1 is suggestive of a conserved defence signalling pathway between B. napus and its close relative A. thaliana.

  6. Role of Protein Kinase C, PI3-kinase and Tyrosine Kinase in Activation of MAP Kinase by Glucose and Agonists of G-protein Coupled Receptors in INS-1 Cells

    PubMed Central

    Böcker, Dietmar

    2001-01-01

    MAP (mitogen-activated protein) kinase (also called Erk 1/2) plays a crucial role in cell proliferation and differentiation. Its impact on secretory events is less well established. The interplay of protein kinase C (PKC), PI3-kinase nd cellular tyrosine kinase with MAP kinase activity using inhibitors and compounds such as glucose, phorbol 12-myristate 13-acetate (PMA) and agonists of G-protein coupled receptors like gastrin releasing peptide (GRP), oxytocin (OT) and glucose-dependent insulinotropic peptide (GIP) was investigated in INS-1 cells, an insulin secreting cell line. MAP kinase activity was determined by using a peptide derived from the EGF receptor as a MAP kinase substrate and [ P 32 ]ATP. Glucose as well as GRP, OT and GIP exhibited a time-dependent increase in MAP kinase activity with a maximum at time point 2.5 min. All further experiments were performed using 2.5 min incubations. The flavone PD 098059 is known to bind to the inactive forms of MEK1 (MAPK/ERK-Kinase) thus preventing activation by upstream activators. 20 μM PD 098059 ( IC 50 =51 μM) inhibited MAP kinase stimulated by either glucose, GRP, OT, GIP or PMA. Inhibiton (“downregulation”) of PKC by a long term (22h) pretreatment with 1 μM PMA did not influence MAP kinase activity when augmented by either of the above mentioned compound. To investigate whether PI3-kinase and cellular tyrosine kinase are involved in G-protein mediated effects on MAP kinase, inhibitors were used: 100 nM wortmannin (PI3-kinase inhibitor) reduced the effects of GRP, OT and GIP but not that of PMA; 100 μM genistein (tyrosine kinase inhibitor) inhibited the stimulatory effect of either above mentioned compound on MAP kinase activation. Inhibition of MAP kinase by 20 μM PD 098059 did not influence insulin secretion modulated by either compound (glucose, GRP, OT or GIP). [ H 3 ]Thymidine incorporation, however, was severely inhibited by PD 098059. Thus MAP kinase is important for INS-1 cell proliferation but

  7. Leucine-Rich Repeat Kinase 2 interacts with Parkin, DJ-1 and PINK-1 in a Drosophila melanogaster model of Parkinson's disease.

    PubMed

    Venderova, Katerina; Kabbach, Ghassan; Abdel-Messih, Elizabeth; Zhang, Yi; Parks, Robin J; Imai, Yuzuru; Gehrke, Stephan; Ngsee, Johnny; Lavoie, Matthew J; Slack, Ruth S; Rao, Yong; Zhang, Zhuohua; Lu, Bingwei; Haque, M Emdadul; Park, David S

    2009-11-15

    Mutations in the LRRK2 gene are the most common genetic cause of familial Parkinson's disease (PD). However, its physiological and pathological functions are unknown. Therefore, we generated several independent Drosophila lines carrying WT or mutant human LRRK2 (mutations in kinase, COR or LRR domains, resp.). Ectopic expression of WT or mutant LRRK2 in dopaminergic neurons caused their significant loss accompanied by complex age-dependent changes in locomotor activity. Overall, the ubiquitous expression of LRRK2 increased lifespan and fertility of the flies. However, these flies were more sensitive to rotenone. LRRK2 expression in the eye exacerbated retinal degeneration. Importantly, in double transgenic flies, various indices of the eye and dopaminergic survival were modified in a complex fashion by a concomitant expression of PINK1, DJ-1 or Parkin. This evidence suggests a genetic interaction between these PD-relevant genes.

  8. Specificity Rendering ‘Hot-Spots’ for Aurora Kinase Inhibitor Design: The Role of Non-Covalent Interactions and Conformational Transitions

    PubMed Central

    Badrinarayan, Preethi; Sastry, G. Narahari

    2014-01-01

    The present study examines the conformational transitions occurring among the major structural motifs of Aurora kinase (AK) concomitant with the DFG-flip and deciphers the role of non-covalent interactions in rendering specificity. Multiple sequence alignment, docking and structural analysis of a repertoire of 56 crystal structures of AK from Protein Data Bank (PDB) has been carried out. The crystal structures were systematically categorized based on the conformational disposition of the DFG-loop [in (DI) 42, out (DO) 5 and out-up (DOU) 9], G-loop [extended (GE) 53 and folded (GF) 3] and αC-helix [in (CI) 42 and out (CO) 14]. The overlapping subsets on categorization show the inter-dependency among structural motifs. Therefore, the four distinct possibilities a) 2W1C (DI, CI, GE) b) 3E5A (DI, CI, GF) c) 3DJ6 (DI, CO, GF) d) 3UNZ (DOU, CO, GF) along with their co-crystals and apo-forms were subjected to molecular dynamics simulations of 40 ns each to evaluate the variations of individual residues and their impact on forming interactions. The non-covalent interactions formed by the 157 AK co-crystals with different regions of the binding site were initially studied with the docked complexes and structure interaction fingerprints. The frequency of the most prominent interactions was gauged in the AK inhibitors from PDB and the four representative conformations during 40 ns. Based on this study, seven major non-covalent interactions and their complementary sites in AK capable of rendering specificity have been prioritized for the design of different classes of inhibitors. PMID:25485544

  9. N-Acetyl-D-Glucosamine Kinase Interacts with Dynein-Lis1-NudE1 Complex and Regulates Cell Division

    PubMed Central

    Sharif, Syeda Ridita; Islam, Ariful; Moon, Il Soo

    2016-01-01

    N-acetyl-D-glucosamine kinase (GlcNAc kinase or NAGK) primarily catalyzes phosphoryl transfer to GlcNAc during amino sugar metabolism. Recently, it was shown NAGK interacts with dynein light chain roadblock type 1 (DYNLRB1) and upregulates axo-dendritic growth, which is an enzyme activity-independent, non-canonical structural role. The authors examined the distributions of NAGK and NAGK-dynein complexes during the cell cycle in HEK293T cells. NAGK was expressed throughout different stages of cell division and immunocytochemistry (ICC) showed NAGK was localized at nuclear envelope, spindle microtubules (MTs), and kinetochores (KTs). A proximity ligation assay (PLA) for NAGK and DYNLRB1 revealed NAGK-dynein complex on nuclear envelopes in prophase cells and on chromosomes in metaphase cells. NAGK-DYNLRB1 PLA followed by Lis1/NudE1 immunostaining showed NAGK-dynein complexes were colocalized with Lis1 and NudE1 signals, and PLA for NAGK-Lis1 showed similar signal patterns, suggesting a functional link between NAGK and dynein-Lis1 complex. Subsequently, NAGK-dynein complexes were found in KTs and on nuclear membranes where KTs were marked with CENP-B ICC and nuclear membrane with lamin ICC. Furthermore, knockdown of NAGK by small hairpin (sh) RNA was found to delay cell division. These results indicate that the NAGK-dynein interaction with the involvements of Lis1 and NudE1 plays an important role in prophase nuclear envelope breakdown (NEB) and metaphase MT-KT attachment during eukaryotic cell division. PMID:27646688

  10. Characterization of the specific interaction between the DNA aptamer sgc8c and protein tyrosine kinase-7 receptors at the surface of T-cells by biosensing AFM.

    PubMed

    Leitner, Michael; Poturnayova, Alexandra; Lamprecht, Constanze; Weich, Sabine; Snejdarkova, Maja; Karpisova, Ivana; Hianik, Tibor; Ebner, Andreas

    2017-04-01

    We studied the interaction of the specific DNA aptamer sgc8c immobilized at the AFM tip with its corresponding receptor, the protein tyrosine kinase-7 (PTK7) embedded in the membrane of acute lymphoblastic leukemia (ALL) cells (Jurkat T-cells). Performing single molecule force spectroscopy (SMFS) experiments, we showed that the aptamer sgc8c bound with high probability (38.3 ± 7.48%) and high specificity to PTK7, as demonstrated by receptor blocking experiments and through comparison with the binding behavior of a nonspecific aptamer. The determined kinetic off-rate (koff = 5.16 s(-1)) indicates low dissociation of the sgc8c-PTK7 complex. In addition to the pulling force experiments, simultaneous topography and recognition imaging (TREC) experiments using AFM tips functionalized with sgc8c aptamers were realized on the outer regions surface of surface-immobilized Jurkat cells for the first time. This allowed determination of the distribution of PTK7 without any labeling and at near physiological conditions. As a result, we could show a homogeneous distribution of PTK7 molecules on the outer regions of ALL cells with a surface density of 325 ± 12 PTK7 receptors (or small receptor clusters) per μm(2). Graphical Abstract The specific interaction of the DNA aptamer sgc8c and protein tyrosine kinase-7 (PTK7) on acute lymphoblastic leukemia (ALL) cells was characterized. AFM based single molecule force spectroscopy (SMFS) yielded a kinetic off-rate of 5.16 s(-1) of the complex. Simultaneous topography and recognition imaging (TREC) revealed a PTK7 density of 325 ± 12 molecules or clusters per μm(2) in the cell membrane.

  11. Insight into the interactions between novel isoquinolin-1,3-dione derivatives and cyclin-dependent kinase 4 combining QSAR and molecular docking.

    PubMed

    Zheng, Junxia; Kong, Hao; Wilson, James M; Guo, Jialiang; Chang, Yiqun; Yang, Mengjia; Xiao, Gaokeng; Sun, Pinghua

    2014-01-01

    Several small-molecule CDK inhibitors have been identified, but none have been approved for clinical use in the past few years. A new series of 4-[(3-hydroxybenzylamino)-methylene]-4H-isoquinoline-1,3-diones were reported as highly potent and selective CDK4 inhibitors. In order to find more potent CDK4 inhibitors, the interactions between these novel isoquinoline-1,3-diones and cyclin-dependent kinase 4 was explored via in silico methodologies such as 3D-QSAR and docking on eighty-one compounds displaying potent selective activities against cyclin-dependent kinase 4. Internal and external cross-validation techniques were investigated as well as region focusing, bootstraping and leave-group-out. A training set of 66 compounds gave the satisfactory CoMFA model (q2 = 0.695, r2 = 0.947) and CoMSIA model (q2 = 0.641, r2 = 0.933). The remaining 15 compounds as a test set also gave good external predictive abilities with r2pred values of 0.875 and 0.769 for CoMFA and CoMSIA, respectively. The 3D-QSAR models generated here predicted that all five parameters are important for activity toward CDK4. Surflex-dock results, coincident with CoMFA/CoMSIA contour maps, gave the path for binding mode exploration between the inhibitors and CDK4 protein. Based on the QSAR and docking models, twenty new potent molecules have been designed and predicted better than the most active compound 12 in the literatures. The QSAR, docking and interactions analysis expand the structure-activity relationships of constrained isoquinoline-1,3-diones and contribute towards the development of more active CDK4 subtype-selective inhibitors.

  12. Distinct interaction modes of an AKAP bound to two regulatory subunit isoforms of protein kinase A revealed by amide hydrogen/deuterium exchange.

    PubMed

    Burns-Hamuro, Lora L; Hamuro, Yoshitomo; Kim, Jack S; Sigala, Paul; Fayos, Rosa; Stranz, David D; Jennings, Patricia A; Taylor, Susan S; Woods, Virgil L

    2005-12-01

    The structure of an AKAP docked to the dimerization/docking (D/D) domain of the type II (RIIalpha) isoform of protein kinase A (PKA) has been well characterized, but there currently is no detailed structural information of an AKAP docked to the type I (RIalpha) isoform. Dual-specific AKAP2 (D-AKAP2) binds in the nanomolar range to both isoforms and provided us with an opportunity to characterize the isoform-selective nature of AKAP binding using a common docked ligand. Hydrogen/deuterium (H/D) exchange combined with mass spectrometry (DXMS) was used to probe backbone structural changes of an alpha-helical A-kinase binding (AKB) motif from D-AKAP2 docked to both RIalpha and RIIalpha D/D domains. The region of protection upon complex formation and the magnitude of protection from H/D exchange were determined for both interacting partners in each complex. The backbone of the AKB ligand was more protected when bound to RIalpha compared to RIIalpha, suggesting an increased helical stabilization of the docked AKB ligand. This combined with a broader region of backbone protection induced by the AKAP on the docking surface of RIalpha indicated that there were more binding constraints for the AKB ligand when bound to RIalpha. This was in contrast to RIIalpha, which has a preformed, localized binding surface. These distinct modes of AKAP binding may contribute to the more discriminating nature of the RIalpha AKAP-docking surface. DXMS provides valuable structural information for understanding binding specificity in the absence of a high-resolution structure, and can readily be applied to other protein-ligand and protein-protein interactions.

  13. Contribution of the interaction between the rabies virus P protein and I-kappa B kinase ϵ to the inhibition of type I IFN induction signalling.

    PubMed

    Masatani, Tatsunori; Ozawa, Makoto; Yamada, Kentaro; Ito, Naoto; Horie, Masayuki; Matsuu, Aya; Okuya, Kosuke; Tsukiyama-Kohara, Kyoko; Sugiyama, Makoto; Nishizono, Akira

    2016-02-01

    The P protein of rabies virus (RABV) is known to interfere with the phosphorylation of the host IFN regulatory factor 3 (IRF-3) and to consequently inhibit type I IFN induction. Previous studies, however, have only tested P proteins from laboratory-adapted fixed virus strains, and to the best of our knowledge there is no report about the effect of P proteins from street RABV strains or other lyssaviruses on the IRF-3-mediated type I IFN induction system. In this study, we evaluated the inhibitory effect of P proteins from several RABV strains, including fixed and street virus strains and other lyssaviruses (Lagos bat, Mokola and Duvenhage viruses), on IRF-3 signalling. All P proteins tested inhibited retinoic acid-inducible gene-1 (RIG-I)- and TANK binding kinase 1 (TBK1)-mediated IRF-3-dependent IFN-β promoter activities. On the other hand, the P proteins from the RABV street strains 1088 and HCM-9, but not from fixed strains Nishigahara (Ni) and CVS-11 and other lyssaviruses tested, significantly inhibited I-kappa B kinase ϵ (IKKϵ)-inducible IRF-3-dependent IFN-β promoter activity. Importantly, we revealed that the P proteins from the 1088 and HCM-9 strains, but not from the remaining viruses, interacted with IKKϵ. By using expression plasmids encoding chimeric P proteins from the 1088 strain and Ni strain, we found that the C-terminal region of the P protein is important for the interaction with IKKϵ. These findings suggest that the P protein of RABV street strains may contribute to efficient evasion of host innate immunity.

  14. Protein Kinase A (PKA) Type I Interacts with P-Rex1, a Rac Guanine Nucleotide Exchange Factor

    PubMed Central

    Chávez-Vargas, Lydia; Adame-García, Sendi Rafael; Cervantes-Villagrana, Rodolfo Daniel; Castillo-Kauil, Alejandro; Bruystens, Jessica G. H.; Fukuhara, Shigetomo; Taylor, Susan S.; Mochizuki, Naoki; Reyes-Cruz, Guadalupe; Vázquez-Prado, José

    2016-01-01

    Morphology of migrating cells is regulated by Rho GTPases and fine-tuned by protein interactions and phosphorylation. PKA affects cell migration potentially through spatiotemporal interactions with regulators of Rho GTPases. Here we show that the endogenous regulatory (R) subunit of type I PKA interacts with P-Rex1, a Rac guanine nucleotide exchange factor that integrates chemotactic signals. Type I PKA holoenzyme interacts with P-Rex1 PDZ domains via the CNB B domain of RIα, which when expressed by itself facilitates endothelial cell migration. P-Rex1 activation localizes PKA to the cell periphery, whereas stimulation of PKA phosphorylates P-Rex1 and prevents its activation in cells responding to SDF-1 (stromal cell-derived factor 1). The P-Rex1 DEP1 domain is phosphorylated at Ser-436, which inhibits the DH-PH catalytic cassette by direct interaction. In addition, the P-Rex1 C terminus is indirectly targeted by PKA, promoting inhibitory interactions independently of the DEP1-PDZ2 region. A P-Rex1 S436A mutant construct shows increased RacGEF activity and prevents the inhibitory effect of forskolin on sphingosine 1-phosphate-dependent endothelial cell migration. Altogether, these results support the idea that P-Rex1 contributes to the spatiotemporal localization of type I PKA, which tightly regulates this guanine exchange factor by a multistep mechanism, initiated by interaction with the PDZ domains of P-Rex1 followed by direct phosphorylation at the first DEP domain and putatively indirect regulation of the C terminus, thus promoting inhibitory intramolecular interactions. This reciprocal regulation between PKA and P-Rex1 might represent a key node of integration by which chemotactic signaling is fine-tuned by PKA. PMID:26797121

  15. Tyrosine Phosphorylation of the BRI1 Receptor Kinase Emerges as a Component of Brassinosteriod Signaling in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brassinosteroids (BRs) are essential growth-promoting hormones that regulate many aspects of plant growth and development. Two leucine-rich repeat receptor-like kinases (LRR-RLKs) are involved in BR perception and signal transduction: BRASSINOSTEROID INSENSITIVE 1 (BRI1), which is the BR receptor, ...

  16. The beneficial effects of AMP kinase activation against oxidative stress are associated with prevention of PPARα-cyclophilin D interaction in cardiomyocytes.

    PubMed

    Barreto-Torres, Giselle; Hernandez, Jessica Soto; Jang, Sehwan; Rodríguez-Muñoz, Adlín R; Torres-Ramos, Carlos A; Basnakian, Alexei G; Javadov, Sabzali

    2015-04-01

    AMP kinase (AMPK) plays an important role in the regulation of energy metabolism in cardiac cells. Furthermore, activation of AMPK protects the heart from myocardial infarction and heart failure. The present study examines whether or not AMPK affects the peroxisome proliferator-activated receptor-α (PPARα)/mitochondria pathway in response to acute oxidative stress in cultured cardiomyocytes. Cultured H9c2 rat embryonic cardioblasts were exposed to H2O2-induced acute oxidative stress in the presence or absence of metformin, compound C (AMPK inhibitor), GW6471 (PPARα inhibitor), or A-769662 (AMPK activator). Results showed that AMPK activation by metformin reverted oxidative stress-induced inactivation of AMPK and prevented oxidative stress-induced cell death. In addition, metformin attenuated reactive oxygen species generation and depolarization of the inner mitochondrial membrane. The antioxidative effects of metformin were associated with the prevention of mitochondrial DNA damage in cardiomyocytes. Coimmunoprecipitation studies revealed that metformin abolished oxidative stress-induced physical interactions between PPARα and cyclophilin D (CypD), and the abolishment of these interactions was associated with inhibition of permeability transition pore formation. The beneficial effects of metformin were not due to acetylation or phosphorylation of PPARα in response to oxidative stress. In conclusion, this study demonstrates that the protective effects of metformin-induced AMPK activation against oxidative stress converge on mitochondria and are mediated, at least in part, through the dissociation of PPARα-CypD interactions, independent of phosphorylation and acetylation of PPARα and CypD.

  17. Solution structure of the focal adhesion adaptor PINCH LIM1 domain and characterization of its interaction with the integrin-linked kinase ankyrin repeat domain.

    PubMed

    Velyvis, A; Yang, Y; Wu, C; Qin, J

    2001-02-16

    PINCH is a recently identified adaptor protein that comprises an array of five LIM domains. PINCH functions through LIM-mediated protein-protein interactions that are involved in cell adhesion, growth, and differentiation. The LIM1 domain of PINCH interacts with integrin-linked kinase (ILK), thereby mediating focal adhesions via a specific integrin/ILK signaling pathway. We have solved the NMR structure of the PINCH LIM1 domain and characterized its binding to ILK. LIM1 contains two contiguous zinc fingers of the CCHC and CCCH types and adopts a global fold similar to that of functionally distinct LIM domains from cysteine-rich protein and cysteine-rich intestinal protein families with CCHC and CCCC zinc finger types. Gel-filtration and NMR experiments demonstrated a 1:1 complex between PINCH LIM1 and the ankyrin repeat domain of ILK. A chemical shift mapping experiment identified regions in PINCH LIM1 that are important for interaction with ILK. Comparison of surface features between PINCH LIM1 and other functionally different LIM domains indicated that the LIM motif might have a highly variable mode in recognizing various target proteins.

  18. Modulation of Pantothenate Kinase 3 Activity by Small Molecules that Interact with the Substrate/Allosteric Regulatory Domain

    SciTech Connect

    Leonardi, Roberta; Zhang, Yong-Mei; Yun, Mi-Kyung; Zhou, Ruobing; Zeng, Fu-Yue; Lin, Wenwei; Cui, Jimmy; Chen, Taosheng; Rock, Charles O.; White, Stephen W.; Jackowski, Suzanne

    2010-09-27

    Pantothenate kinase (PanK) catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis. PanK3 is stringently regulated by acetyl-CoA and uses an ordered kinetic mechanism with ATP as the leading substrate. Biochemical analysis of site-directed mutants indicates that pantothenate binds in a tunnel adjacent to the active site that is occupied by the pantothenate moiety of the acetyl-CoA regulator in the PanK3 acetyl-CoA binary complex. A high-throughput screen for PanK3 inhibitors and activators was applied to a bioactive compound library. Thiazolidinediones, sulfonylureas and steroids were inhibitors, and fatty acyl-amides and tamoxifen were activators. The PanK3 activators and inhibitors either stimulated or repressed CoA biosynthesis in HepG2/C3A cells. The flexible allosteric acetyl-CoA regulatory domain of PanK3 also binds the substrates, pantothenate and pantetheine, and small molecule inhibitors and activators to modulate PanK3 activity.

  19. Differentiating a Ligand's Chemical Requirements for Allosteric Interactions from Those for Protein Binding. Phenylalanine Inhibition of Pyruvate Kinase

    SciTech Connect

    Williams,R.; Holyoak, T.; McDonald, G.; Gui, C.; Fenton, A.

    2006-01-01

    The isoform of pyruvate kinase from brain and muscle of mammals (M1-PYK) is allosterically inhibited by phenylalanine. Initial observations in this model allosteric system indicate that Ala binds competitively with Phe, but elicits a minimal allosteric response. Thus, the allosteric ligand of this system must have requirements for eliciting an allosteric response in addition to the requirements for binding. Phe analogues have been used to dissect what chemical properties of Phe are responsible for eliciting the allosteric response. We first demonstrate that the L-2-aminopropanaldehyde substructure of the amino acid ligand is primarily responsible for binding to M1-PYK. Since the allosteric response to Ala is minimal and linear addition of methyl groups beyond the -carbon increase the magnitude of the allosteric response, we conclude that moieties beyond the -carbon are primarily responsible for allostery. Instead of an all-or-none mechanism of allostery, these findings support the idea that the bulk of the hydrophobic side chain, but not the aromatic nature, is the primary determinant of the magnitude of the observed allosteric inhibition. The use of these results to direct structural studies has resulted in a 1.65 Angstroms structure of M1-PYK with Ala bound. The coordination of Ala in the allosteric amino acid binding site confirms the binding role of the L-2-aminopropanaldehyde substructure of the ligand. Collectively, this study confirms that a ligand can have chemical regions specific for eliciting the allosteric signal in addition to the chemical regions necessary for binding.

  20. Molecular cloning and characterization of two novel genes from hexaploid wheat that encode double PR-1 domains coupled with a receptor-like protein kinase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hexaploid wheat (Triticum aestivum L.) contains at least 23 TaPr-1 genes encoding the group 1 pathogenesis-related (PR-1) proteins as identified in our previous work. Here we report the cloning and characterization of TaPr-1-rk1 and TaPr-1-rk2, two novel genes closely related to the wheat PR-1 famil...

  1. Mass Spectrometry Reveals Differences in Stability and Subunit Interactions between Activated and Nonactivated Conformers of the (αβγδ)4 Phosphorylase Kinase Complex*

    PubMed Central

    Lane, Laura A.; Nadeau, Owen W.; Carlson, Gerald M.; Robinson, Carol V.

    2012-01-01

    Phosphorylase kinase (PhK), a 1.3 MDa enzyme complex that regulates glycogenolysis, is composed of four copies each of four distinct subunits (α, β, γ, and δ). The catalytic protein kinase subunit within this complex is γ, and its activity is regulated by the three remaining subunits, which are targeted by allosteric activators from neuronal, metabolic, and hormonal signaling pathways. The regulation of activity of the PhK complex from skeletal muscle has been studied extensively; however, considerably less is known about the interactions among its subunits, particularly within the non-activated versus activated forms of the complex. Here, nanoelectrospray mass spectrometry and partial denaturation were used to disrupt PhK, and subunit dissociation patterns of non-activated and phospho-activated (autophosphorylation) conformers were compared. In so doing, we have established a network of subunit contacts that complements and extends prior evidence of subunit interactions obtained from chemical crosslinking, and these subunit interactions have been modeled for both conformers within the context of a known three-dimensional structure of PhK solved by cryoelectron microscopy. Our analyses show that the network of contacts among subunits differs significantly between the nonactivated and phospho-activated conformers of PhK, with the latter revealing new interprotomeric contact patterns for the β subunit, the predominant subunit responsible for PhK's activation by phosphorylation. Partial disruption of the phosphorylated conformer yields several novel subcomplexes containing multiple β subunits, arguing for their self-association within the activated complex. Evidence for the theoretical αβγδ protomeric subcomplex, which has been sought but not previously observed, was also derived from the phospho-activated complex. In addition to changes in subunit interaction patterns upon phospho-activation, mass spectrometry revealed a large change in the overall

  2. Dysregulated miR34a/diacylglycerol kinase ζ interaction enhances T-cell activation in acquired aplastic anemia

    PubMed Central

    Sun, Yuan-xin; Li, Hui; Feng, Qi; Li, Xin; Yu, Ying-yi; Zhou, Li-wei; Gao, Yan; Li, Guo-sheng; Ren, Juan; Ma, Chun-hong; Gao, Cheng-jiang; Peng, Jun

    2017-01-01

    Acquired aplastic anemia is an idiopathic paradigm of human bone marrow failure syndrome, which involves active destruction of hematopoietic stem cells and progenitors by cytotoxic T cells in the bone marrow. Aberrant expression of microRNAs in T cells has been shown to lead to development of certain autoimmune diseases. In the present study, we performed a microarray analysis of miRNA expression in bone marrow CD3+ T cells from patients with aplastic anemia and healthy controls. Overexpression of miR34a and underexpression of its target gene diacylglycerol kinase (DGK) ζ in bone marrow mononuclear cells were validated in 41 patients and associated with the severity of aplastic anemia. Further, the level of miR34a was higher in naïve T cells from patients than from controls. The role of miR34a and DGKζ in aplastic anemia was investigated in a murine model of immune-mediated bone marrow failure using miR34a−/− mice. After T-cell receptor stimulation in vitro, lymph node T cells from miR34a−/− mice demonstrated reduced activation and proliferation accompanied with a less profound down-regulation of DGKζ expression and decreased ERK phosphorylation compared to those from wild-type C57BL6 control mice. Infusion of 5 × 106 miR34a−/− lymph node T cells into sublethally irradiated CB6F1 recipients led to increased Lin-Sca1+CD117+ cells and less vigorous expansion of CD8+ T cells than injection of same number of wild-type lymph node cells. Our study demonstrates that the miR34a/DGKζ dysregulation enhances T-cell activation in aplastic anemia and targeting miR34a may represent a novel molecular therapeutic approach for patients with aplastic anemia. PMID:28008152

  3. Hepatocyte Growth Factor Modulates MET Receptor Tyrosine Kinase and β-Catenin Functional Interactions to Enhance Synapse Formation

    PubMed Central

    Xie, Zhihui; Eagleson, Kathie L.

    2016-01-01

    MET, a pleiotropic receptor tyrosine kinase implicated in autism risk, influences multiple neurodevelopmental processes. There is a knowledge gap, however, in the molecular mechanism through which MET mediates developmental events related to disorder risk. In the neocortex, MET is expressed transiently during periods of peak dendritic outgrowth and synaptogenesis, with expression enriched at developing synapses, consistent with demonstrated roles in dendritic morphogenesis, modulation of spine volume, and excitatory synapse development. In a recent coimmunoprecipitation/mass spectrometry screen, β-catenin was identified as part of the MET interactome in developing neocortical synaptosomes. Here, we investigated the influence of the MET/β-catenin complex in mouse neocortical synaptogenesis. Western blot analysis confirms that MET and β-catenin coimmunoprecipitate, but N-cadherin is not associated with the MET complex. Following stimulation with hepatocyte growth factor (HGF), β-catenin is phosphorylated at tyrosine142 (Y142) and dissociates from MET, accompanied by an increase in β-catenin/N-cadherin and MET/synapsin 1 protein complexes. In neocortical neurons in vitro, proximity ligation assays confirmed the close proximity of these proteins. Moreover, in neurons transfected with synaptophysin-GFP, HGF stimulation increases the density of synaptophysin/bassoon (a presynaptic marker) and synaptophysin/PSD-95 (a postsynaptic marker) clusters. Mutation of β-catenin at Y142 disrupts the dissociation of the MET/β-catenin complex and prevents the increase in clusters in response to HGF. The data demonstrate a new mechanism for the modulation of synapse formation, whereby MET activation induces an alignment of presynaptic and postsynaptic elements that are necessary for assembly and formation of functional synapses by subsets of neocortical neurons that express MET/β-catenin complex. PMID:27595133

  4. Dysregulated miR34a/diacylglycerol kinase ζ interaction enhances T-cell activation in acquired aplastic anemia.

    PubMed

    Sun, Yuan-Xin; Li, Hui; Feng, Qi; Li, Xin; Yu, Ying-Yi; Zhou, Li-Wei; Gao, Yan; Li, Guo-Sheng; Ren, Juan; Ma, Chun-Hong; Gao, Cheng-Jiang; Peng, Jun

    2017-01-24

    Acquired aplastic anemia is an idiopathic paradigm of human bone marrow failure syndrome, which involves active destruction of hematopoietic stem cells and progenitors by cytotoxic T cells in the bone marrow. Aberrant expression of microRNAs in T cells has been shown to lead to development of certain autoimmune diseases. In the present study, we performed a microarray analysis of miRNA expression in bone marrow CD3+ T cells from patients with aplastic anemia and healthy controls. Overexpression of miR34a and underexpression of its target gene diacylglycerol kinase (DGK) ζ in bone marrow mononuclear cells were validated in 41 patients and associated with the severity of aplastic anemia. Further, the level of miR34a was higher in naïve T cells from patients than from controls. The role of miR34a and DGKζ in aplastic anemia was investigated in a murine model of immune-mediated bone marrow failure using miR34a-/- mice. After T-cell receptor stimulation in vitro, lymph node T cells from miR34a-/- mice demonstrated reduced activation and proliferation accompanied with a less profound down-regulation of DGKζ expression and decreased ERK phosphorylation compared to those from wild-type C57BL6 control mice. Infusion of 5 × 106 miR34a-/- lymph node T cells into sublethally irradiated CB6F1 recipients led to increased Lin-Sca1+CD117+ cells and less vigorous expansion of CD8+ T cells than injection of same number of wild-type lymph node cells. Our study demonstrates that the miR34a/DGKζ dysregulation enhances T-cell activation in aplastic anemia and targeting miR34a may represent a novel molecular therapeutic approach for patients with aplastic anemia.

  5. The receptor kinase CERK1 has dual functions in symbiosis and immunity signalling.

    PubMed

    Zhang, Xiaowei; Dong, Wentao; Sun, Jongho; Feng, Feng; Deng, Yiwen; He, Zuhua; Oldroyd, Giles E D; Wang, Ertao

    2015-01-01

    The establishment of symbiotic interactions between mycorrhizal fungi, rhizobial bacteria and their legume hosts involves a common symbiosis signalling pathway. This signalling pathway is activated by Nod factors produced by rhizobia and these are recognised by the Nod factor receptors NFR1/LYK3 and NFR5/NFP. Mycorrhizal fungi produce lipochitooligosaccharides (LCOs) similar to Nod factors, as well as short-chain chitin oligomers (CO4/5), implying commonalities in signalling during mycorrhizal and rhizobial associations. Here we show that NFR1/LYK3, but not NFR5/NFP, is required for the establishment of the mycorrhizal interaction in legumes. NFR1/LYK3 is necessary for the recognition of mycorrhizal fungi and the activation of the symbiosis signalling pathway leading to induction of calcium oscillations and gene expression. Chitin oligosaccharides also act as microbe associated molecular patterns that promote plant immunity via similar LysM receptor-like kinases. CERK1 in rice has the highest homology to NFR1 and we show that this gene is also necessary for the establishment of the mycorrhizal interaction as well as for resistance to the rice blast fungus. Our results demonstrate that NFR1/LYK3/OsCERK1 represents a common receptor for chitooligosaccharide-based signals produced by mycorrhizal fungi, rhizobial bacteria (in legumes) and fungal pathogens. It would appear that mycorrhizal recognition has been conserved in multiple receptors across plant species, but additional diversification in certain plant species has defined other signals that this class of receptors can perceive.

  6. The Legionella pneumophila orphan sensor kinase LqsT regulates competence and pathogen-host interactions as a component of the LAI-1 circuit.

    PubMed

    Kessler, Aline; Schell, Ursula; Sahr, Tobias; Tiaden, André; Harrison, Christopher; Buchrieser, Carmen; Hilbi, Hubert

    2013-02-01

    Legionella pneumophila is an amoeba-resistant opportunistic pathogen that performs cell-cell communication through the signalling molecule 3-hydroxypentadecane-4-one (LAI-1, Legionella autoinducer-1). The lqs (Legionella quorum sensing) gene cluster encodes the LAI-1 autoinducer synthase LqsA, the cognate sensor kinase LqsS and the response regulator LqsR. Here we show that the Lqs system includes an 'orphan' homologue of LqsS termed LqsT. Compared with wild-type L. pneumophila, strains lacking lqsT or both lqsS and lqsT show increased salt resistance, greatly enhanced natural competence for DNA acquisition and impaired uptake by phagocytes. Sensitive novel single round growth assays and competition experiments using Acanthamoeba castellanii revealed that ΔlqsT and ΔlqsS-ΔlqsT, as well as ΔlqsA and other lqs mutant strains are impaired for intracellular growth and cannot compete against wild-type bacteria upon co-infection. In contrast to the ΔlqsS strain, ΔlqsT does not produce extracellular filaments. The phenotypes of the ΔlqsS-ΔlqsT strain are partially complemented by either lqsT or lqsS, but are not reversed by overexpression of lqsA, suggesting that LqsT and LqsS are the sole LAI-1-responsive sensor kinases in L. pneumophila. In agreement with the different phenotypes of the ΔlqsT and ΔlqsS strains, lqsT and lqsS are differentially expressed in the post-exponential growth phase, and transcriptome studies indicated that 90% of the genes, which are downregulated in absence of lqsT, are upregulated in absence of lqsS. Reciprocally regulated genes encode components of a 133 kb genomic 'fitness island' or translocated effector proteins implicated in virulence. Together, these results reveal a unique organization of the L. pneumophila Lqs system comprising two partially antagonistic LAI-1-responsive sensor kinases, LqsT and LqsS, which regulate distinct pools of genes implicated in pathogen-host cell interactions, competence, expression of a

  7. Protein kinase C mediates the synergistic interaction between agonists acting at alpha2-adrenergic and delta-opioid receptors in spinal cord.

    PubMed

    Overland, Aaron C; Kitto, Kelley F; Chabot-Doré, Anne-Julie; Rothwell, Patrick E; Fairbanks, Carolyn A; Stone, Laura S; Wilcox, George L

    2009-10-21

    Coactivation of spinal alpha(2)-adrenergic receptors (ARs) and opioid receptors produces antinociceptive synergy. Antinociceptive synergy between intrathecally administered alpha(2)AR and opioid agonists is well documented, but the mechanism underlying this synergy remains unclear. The delta-opioid receptor (DOP) and the alpha(2A)ARs are coexpressed on the terminals of primary afferent fibers in the spinal cord where they may mediate this phenomenon. We evaluated the ability of the DOP-selective agonist deltorphin II (DELT), the alpha(2)AR agonist clonidine (CLON) or their combination to inhibit calcitonin gene-related peptide (CGRP) release from spinal cord slices. We then examined the possible underlying signaling mechanisms involved through coadministration of inhibitors of phospholipase C (PLC), protein kinase C (PKC) or protein kinase A (PKA). Potassium-evoked depolarization of spinal cord slices caused concentration-dependent release of CGRP. Coadministration of DELT and CLON inhibited the release of CGRP in a synergistic manner as confirmed statistically by isobolograpic analysis. Synergy was dependent on the activation of PLC and PKC, but not PKA, whereas the effect of agonist administration alone was only dependent on PLC. The importance of these findings was confirmed in vivo, using a thermal nociceptive test, demonstrating the PKC dependence of CLON-DELT antinociceptive synergy in mice. That inhibition of CGRP release by the combination was maintained in the presence of tetrodotoxin in spinal cord slices suggests that synergy does not rely on interneuronal signaling and may occur within single subcellular compartments. The present study reveals a novel signaling pathway underlying the synergistic analgesic interaction between DOP and alpha(2)AR agonists in the spinal cord.

  8. Protein kinase C-alpha regulates insulin action and degradation by interacting with insulin receptor substrate-1 and 14-3-3 epsilon.

    PubMed

    Oriente, Francesco; Andreozzi, Francesco; Romano, Chiara; Perruolo, Giuseppe; Perfetti, Anna; Fiory, Francesca; Miele, Claudia; Beguinot, Francesco; Formisano, Pietro

    2005-12-09

    Protein kinase C (PKC)-alpha exerts a regulatory function on insulin action. We showed by overlay blot that PKCalpha directly binds a 180-kDa protein, corresponding to IRS-1, and a 30-kDa molecular species, identified as 14-3-3epsilon. In intact NIH-3T3 cells overexpressing insulin receptors (3T3-hIR), insulin selectively increased PKCalpha co-precipitation with IRS-1, but not with IRS-2, and with 14-3-3epsilon, but not with other 14-3-3 isoforms. Overexpression of 14-3-3epsilon in 3T3-hIR cells significantly reduced IRS-1-bound PKCalpha activity, without altering IRS-1/PKCalpha co-precipitation. 14-3-3epsilon overexpression also increased insulin-stimulated insulin receptor and IRS-1 tyrosine phosphorylation, followed by increased activation of Raf1, ERK1/2, and Akt/protein kinase B. Insulin-induced glycogen synthase activity and thymidine incorporation were also augmented. Consistently, selective depletion of 14-3-3epsilon by antisense oligonucleotides caused a 3-fold increase of IRS-1-bound PKCalpha activity and a similarly sized reduction of insulin receptor and IRS-1 tyrosine phosphorylation and signaling. In turn, selective inhibition of PKCalpha expression by antisense oligonucleotides reverted the negative effect of 14-3-3epsilon depletion on insulin signaling. Moreover, PKCalpha inhibition was accompanied by a >2-fold decrease of insulin degradation. Similar results were also obtained by overexpressing 14-3-3epsilon. Thus, in NIH-3T3 cells, insulin induces the formation of multimolecular complexes, including IRS-1, PKCalpha, and 14-3-3epsilon. The presence of 14-3-3epsilon in the complex is not necessary for IRS-1/PKCalpha interaction but modulates PKCalpha activity, thereby regulating insulin signaling and degradation.

  9. Analysis of protein-protein interactions involved in the activation of the Shc/Grb-2 pathway by the ErbB-2 kinase.

    PubMed

    Ricci, A; Lanfrancone, L; Chiari, R; Belardo, G; Pertica, C; Natali, P G; Pelicci, P G; Segatto, O

    1995-10-19

    In murine fibroblasts activation of the Shc/Grb-2 pathway by the ErbB-2 kinase involves tyrosine phosphorylation of Shc products and the formation of Shc/ErbB-2, Shc/Grb-2 and Grb-2/ErbB-2 complexes. Tyr 1139 of ErbB-2 bound to the Grb-2 SH2 domain in vitro as well as in intact cells. Tyr 1221 and 1248 are binding sites of gp185ErbB-2 for Shc SH2 domain in vitro whereas Tyr 1196 and 1248 are major binding sites of ErbB-2 for Shc PTB domain. Inhibition of Shc/ErbB-2 complex formation in intact cells was obtained by simultaneous mutational inactivation of Shc SH2 and Shc PTB binding sites of gp185ErbB-2. Shc/ErbB-2 complexes are formed upon activation of the ErbB-2 kinase and tyrosine phosphorylation of Shc proteins; they are located in both cytosol and cellular membranes. ErbB-2 activation induces also translocation of Grb-2 from cytosol to membranes. This network of protein-protein interactions may reflect the ability of the Shc/Grb-2 pathway to act as a molecular switch controlling different cellular functions regulated by RTK activation. In fact the Ras GDP exchanger mSOS was recruited in Grb-2/ErbB-2 complexes; furthermore besides mSOS, other polypeptides present in either cytosolic or membrane preparations were able to complex in vitro with Grb-2 SH3 domains.

  10. Critical role of X-box binding protein 1 in NADPH oxidase 4-triggered cardiac hypertrophy is mediated by receptor interacting protein kinase 1.

    PubMed

    Chen, Li; Zhao, Mingyue; Li, Junli; Wang, Yu; Bao, Qinxue; Wu, Siyuan; Deng, Xueqin; Tang, Xiaoju; Wu, Wenchao; Liu, Xiaojing

    2017-02-16

    NADPH oxidase 4 (NOX4) and the NOX4-related redox signaling are implicated in cardiac hypertrophy. NOX4 is interrelated with endoplasmic reticulum stress (ERS). Spliced X-box binding protein 1 (Xbp1s) is a key mediator of ERS while its role in cardiac hypertrophy is still poorly understood. Recently, receptor interacting protein kinase 1(RIPK1) has been increasingly reported to be associated with ERS. Therefore, we aimed to test the hypothesis that Xbp1s mediates NOX4-triggered cardiac hypertrophy via RIPK1 signaling. In the heart tissue of transverse aortic constriction (TAC) rats and in primary cultured neonatal cardiomyocytes(NCMs) treated with angiotensinII(AngII) or isoproterenol (ISO), NOX4 expression and reactive oxygen species (ROS) generation, and expression of Xbp1s as well as RIPK1-related phosphorylation of P65 subunit of NF-κB were elevated. Gene silencing of NOX4 by specific small interfering RNA (siRNA) significantly blocked the upregulation of NOX4, generation of ROS, splicing of Xbp1 and activation of the RIPK1-related NF-κB signaling, meanwhile attenuated cardiomyocyte hypertrophy. In addition, ROS scavenger (N-acetyl-L-cysteine, NAC) and NOX4 inhibitor GKT137831 reduced ROS generation and alleviated activation of Xbp1 and RIPK1-related NF-κB signaling. Furthermore, splicing of Xbp1 was responsible for the increase in RIPK1 expression in AngII or ISO-treated NCMs. Upregulated RIPK1 in turn activates NF-κB signaling in a kinase activity-independent manner. These findings suggest that Xbp1s plays an important role in NOX4-triggered cardiomyocyte hypertrophy via activating its downstream effector RIPK1, which may prove significant for the development of future therapeutic strategies.

  11. Protein Kinase C Mediates the Synergistic Interaction Between Agonists Acting at Alpha-2-Adrenergic and Delta-Opioid Receptors in Spinal Cord

    PubMed Central

    Overland, Aaron C.; Kitto, Kelley F.; Chabot-Doré, Anne-Julie; Rothwell, Patrick E.; Fairbanks, Carolyn A.; Stone, Laura S.; Wilcox, George L.

    2009-01-01

    Co-activation of spinal α2-adrenergic receptors (AR) and opioid receptors (OR) produces antinociceptive synergy. Antinociceptive synergy between intrathecally (i.t.) administered α2AR and OR agonists is well documented, but the mechanism underlying this synergy remains unclear. The delta-opioid receptor (DOP) and the α2AAR are co-expressed on the terminals of primary afferent fibers in the spinal cord where they may mediate this phenomenon. We evaluated the ability of the DOP-selective agonist deltorphin II (DELT), the α2AR agonist clonidine (CLON) or their combination to inhibit calcitonin gene-related peptide (CGRP) release from spinal cord slices. We then examined the possible underlying signaling mechanisms involved through co-administration of inhibitors of phospholipase C (PLC), protein kinase C (PKC) or protein kinase A (PKA). Potassium-evoked depolarization of spinal cord slices caused concentration-dependent release of CGRP. Co-administration of DELT and CLON inhibited the release of CGRP in a synergistic manner as confirmed statistically by isobolograpic analysis. Synergy was dependent on the activation of PLC and PKC, but not PKA, while the effect of agonist administration alone was only dependent on PLC. The importance of these findings was confirmed in vivo, demonstrating the PKC-dependence on CLON-DELT antinociceptive synergy in mice. That inhibition of CGRP release by the combination was maintained in the presence of tetrodotoxin in spinal cord slices suggests that synergy does not rely on interneuronal signaling and may occur within single subcellular compartments. The present study reveals a novel signaling pathway underlying the synergistic analgesic interaction between DOP and α2AR agonists in the spinal cord. PMID:19846714

  12. MZF-1/Elk-1 interaction domain as therapeutic target for protein kinase Cα-based triple-negative breast cancer cells

    PubMed Central

    Lee, Chia-Jen; Hsu, Li-Sung; Yue, Chia-Herng; Lin, Ho; Chiu, Yung-Wei; Lin, Yu-Yu; Huang, Chih-Yang; Hung, Mien-Chie; Liu, Jer-Yuh

    2016-01-01

    Recent reports demonstrate that the expression of protein kinase C alpha (PKCα) in triple-negative breast cancer (TNBC) correlates with decreased survival outcomes. However, off-target effects of targeting PKCα and limited understanding of the signaling mechanisms upstream of PKCα have hampered previous efforts to manipulate this ubiquitous gene. This study shows that the expression of both myeloid zinc finger 1 (MZF-1) and Ets-like protein-1 (Elk-1) correlates with PKCα expression in TNBC. We found that the acidic domain of MZF-1 and the heparin-binding domain of Elk-1 facilitate the heterodimeric interaction between the two genes before the complex formation binds to the PKCα promoter. Blocking the formation of the heterodimer by transfection of MZF-160–72 or Elk-1145–157 peptide fragments at the MZF-1 / Elk-1 interface decreases DNA-binding activity of the MZF-1 / Elk-1 complex at the PKCα promoter. Subsequently, PKCα expression, migration, tumorigenicity, and the epithelial–mesenchymal transition potential of TNBC cells decrease. These subsequent effects are reversed by transfection with full-length PKCα, confirming that the MZF-1/Elk-1 heterodimer is a mediator of PKCα in TNBC cells. These data suggest that the next therapeutic strategy in treating PKCα-related cancer will be developed from blocking MZF-1/Elk-1 interaction through their binding domain. PMID:27542222

  13. Decreased interactions in protein kinase A-glucocorticoid receptor signaling in the hippocampus after selective removal of the basal forebrain cholinergic input.

    PubMed

    Lim, Chol Seung; Kim, Youn Jung; Hwang, Yoo Kyeong; Bañuelos, Christina; Bizon, Jennifer L; Han, Jung-Soo

    2012-03-01

    Removal of the cholinergic innervation to the hippocampus via selective immunolesions of septohippocampal cholinergic neurons induces dysfunction of the hypothalamic-pituitary-adrenocortical (HPA) axis and decreases glucocorticoid receptor (GR) mRNA. This study examined whether removal of the cholinergic innervation decreased GR protein levels and induced changes in the interaction between GR and the cytoplasmic catalytic subunit of protein kinase A (PKAc) in the hippocampus. In lesioned animals, GR protein levels were markedly decreased in the nucleus, but not in the cytosol of hippocampal neurons, whereas mineralocorticoid receptor (MR) levels remained unchanged in both the nucleus and cytosol. PKAc levels did not differ between lesioned and control groups, but PKAc activity was reduced in lesion tissue compared with the controls. The interaction between GR and PKAc was also decreased in the hippocampus without cholinergic input. These results indicate that degeneration of septohippocampal cholinergic neurons leads to reduced PKAc activity in the hippocampus which, in turn, alters GR signaling. The altered GR signaling induced by the degeneration of basal forebrain cholinergic neurons may contribute to dysfunction of the HPA axis in aged animals and patients with Alzheimer's disease (AD) and lead to neuropsychiatric symptoms that occur throughout the course of AD.

  14. SPRi-MALDI MS: characterization and identification of a kinase from cell lysate by specific interaction with different designed ankyrin repeat proteins.

    PubMed

    Anders, Ulrike; Schaefer, Jonas V; Hibti, Fatima-Ezzahra; Frydman, Chiraz; Suckau, Detlev; Plückthun, Andreas; Zenobi, Renato

    2017-03-01

    We report on the direct coupling of surface plasmon resonance imaging (SPRi) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) for the investigation of specific, non-covalent interactions, using the example of designed ankyrin repeat proteins (DARPins) and ribosomal protein S6 kinase 2 (RPS6KA2) directly from lysate of SH-SY5Y cells, derived from human bone marrow. Due to an array format, tracing of binding kinetics of numerous DARPins simultaneously and in real time becomes possible. By optimizing both the proteolytic digest directly on the SPRi chip (amount of trypsin, incubation time, and temperature) as well as the MALDI matrix application (concentration of matrix and number of spray cycles), we are able to identify the specific interaction with RPS6KA2 directly from the cell lysate at a surface coverage of only 0.8 fmol/mm(2). Graphical Abstract Workflow of the direct coupling of SPRi with MALDI mass spectrometry.

  15. LEAFY COTYLEDON1-CASEIN KINASE I-TCP15-PHYTOCHROME INTERACTING FACTOR4 Network Regulates Somatic Embryogenesis by Regulating Auxin Homeostasis1[OPEN

    PubMed Central

    Min, Ling; Hu, Qin; Li, Yaoyao; Xu, Jiao; Ma, Yizan; Zhu, Longfu; Yang, Xiyan; Zhang, Xianlong

    2015-01-01

    Somatic embryogenesis (SE) is an efficient tool for the propagation of plant species and also, a useful model for studying the regulatory networks in embryo development. However, the regulatory networks underlying the transition from nonembryogenic callus to somatic embryos during SE remain poorly understood. Here, we describe an upland cotton (Gossypium hirsutum) CASEIN KINASE I gene, GhCKI, which is a unique key regulatory factor that strongly affects SE. Overexpressing GhCKI halted the formation of embryoids and plant regeneration because of a block in the transition from nonembryogenic callus to somatic embryos. In contrast, defective GhCKI in plants facilitated SE. To better understand the mechanism by which