Science.gov

Sample records for receptor-like kinase interacts

  1. Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection.

    PubMed

    Liebrand, Thomas W H; van den Berg, Grardy C M; Zhang, Zhao; Smit, Patrick; Cordewener, Jan H G; America, Antoine H P; America, Antione H P; Sklenar, Jan; Jones, Alexandra M E; Tameling, Wladimir I L; Robatzek, Silke; Thomma, Bart P H J; Joosten, Matthieu H A J

    2013-06-11

    The plant immune system is activated by microbial patterns that are detected as nonself molecules. Such patterns are recognized by immune receptors that are cytoplasmic or localized at the plasma membrane. Cell surface receptors are represented by receptor-like kinases (RLKs) that frequently contain extracellular leucine-rich repeats and an intracellular kinase domain for activation of downstream signaling, as well as receptor-like proteins (RLPs) that lack this signaling domain. It is therefore hypothesized that RLKs are required for RLPs to activate downstream signaling. The RLPs Cf-4 and Ve1 of tomato (Solanum lycopersicum) mediate resistance to the fungal pathogens Cladosporium fulvum and Verticillium dahliae, respectively. Despite their importance, the mechanism by which these immune receptors mediate downstream signaling upon recognition of their matching ligand, Avr4 and Ave1, remained enigmatic. Here we show that the tomato ortholog of the Arabidopsis thaliana RLK Suppressor Of BIR1-1/Evershed (SOBIR1/EVR) and its close homolog S. lycopersicum (Sl)SOBIR1-like interact in planta with both Cf-4 and Ve1 and are required for the Cf-4- and Ve1-mediated hypersensitive response and immunity. Tomato SOBIR1/EVR interacts with most of the tested RLPs, but not with the RLKs FLS2, SERK1, SERK3a, BAK1, and CLV1. SOBIR1/EVR is required for stability of the Cf-4 and Ve1 receptors, supporting our observation that these RLPs are present in a complex with SOBIR1/EVR in planta. We show that SOBIR1/EVR is essential for RLP-mediated immunity and propose that the protein functions as a regulatory RLK of this type of cell-surface receptors.

  2. Heterotrimeric G proteins interact with defense-related receptor-like kinases in Arabidopsis.

    PubMed

    Aranda-Sicilia, María Nieves; Trusov, Yuri; Maruta, Natsumi; Chakravorty, David; Zhang, Yuelin; Botella, José Ramón

    2015-09-01

    Heterotrimeric G proteins (G-proteins) are versatile signaling elements conserved in Eukaryotes. In animals G-proteins relay signals from 7-transmembrane spanning G protein-coupled receptors (GPCRs) to intracellular downstream effectors; however, the existence of GPCRs in plants is controversial. Contrastingly, a surplus of receptor-like kinases (RLKs) provides signal recognition at the plant cell surface. It is established that G proteins are involved in plant defense and suggested that they relay signals from defense-related RLKs. However, it is unclear how the signaling is conducted, as physical interaction between the RLKs and G proteins has not been demonstrated. Using yeast split-ubiquitin system and Bimolecular Fluorescence Complementation assays, we demonstrate physical interaction between the Gα, Gγ1 and Gγ2 subunits, and the defense-related RD-type receptor like kinases CERK1, BAK1 and BIR1. At the same time, no interaction was detected with the non-RD RLK FLS2. We hypothesize that G-proteins mediate signal transduction immediately downstream of the pathogenesis-related RLKs.

  3. 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. PMID:27446127

  4. 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

  5. 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.

  6. 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

  7. 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. PMID:27447945

  8. Expression of a gibberellin-induced leucine-rich repeat receptor-like protein kinase in deepwater rice and its interaction with kinase-associated protein phosphatase

    SciTech Connect

    Knaap, E. van der; Sauter, M.; Kende, H. . DOE Plant Research Lab.); Song, W.Y.; Ruan, D.L.; Ronald, P.C. . Dept. of Plant Pathology)

    1999-06-01

    The authors identified in deepwater rice (Oryza sativa L.) a gene encoding a leucine-rich repeat receptor-like transmembrane protein kinase, OsTMK (O. sativa transmembrane kinase). The transcript levels of OsTMK increased in the rice internode in response to gibberellin. Expression of OsTMK was especially high in regions undergoing cell division and elongation. The kinase domain of OsTMK was enzymatically active autophosphorylating on serine and threonine residues. A cDNA encoding a rice ortholog of a kinase-associated type 2C protein phosphatase (OsKAPP) was cloned. KAPPs are putative downstream components in kinase-mediated signal transduction pathways. The kinase interaction domain of OsKAPP was phosphorylated in vitro by the kinase domain of OsTMK. RNA gel-blot analysis indicated that the expression of OsTMK and OsKAPP was similar in different tissues of the rice plant. In protein-binding assays, OsKAPP interacted with a receptor-like protein kinase, RLK5 of Arabidopsis, but not with the protein kinase domains of the rice and maize receptor-like protein kinases Xa21 and ZmPK1, respectively.

  9. SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE Directly Interacts with the Cytoplasmic Domain of SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE and Negatively Regulates Leaf Senescence in Arabidopsis.

    PubMed

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

    2015-10-01

    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.

  10. 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

  11. Isolation and characterization of NgRLK1, a receptor-like kinase of Nicotiana glutinosa that interacts with the elicitin of Phytophthora capsici.

    PubMed

    Kim, Yeong-Tae; Oh, Jonghee; Kim, Kyung-Hwan; Uhm, Jae-Youl; Lee, Byoung-Moo

    2010-02-01

    Elicitins, extracellular proteins from Phytophthora fungi, elicit a hypersensitivity response (HR), including systemic acquired resistance, in some plants. The elicitin capsicein (approximately 10 kDa) was purified by FPLC from culture filtrates of P. capsici. Purified native and recombinant capsicein induced a hypersensitive response in leaves of the non-host plants Nicotiana glutinosa and Brassica rapa subsp. pekinensis. To search for candidate capsicein-interacting proteins from N. glutinosa, a yeast two-hybrid assay was used. We identified a protein interactor that is homologous to a serine/threonine kinase of the plant receptor-like kinase (RLK) group and designated it NgRLK1. The ORF of NgRLK1 encodes a polypeptide of 832 amino acids (93,490 Da). A conserved domain analysis revealed that NgRLK1 has structural features typical of a plant RLK. NgRLK1 was autophosphorylated, with higher activity in the presence of Mn2+ than Mg2+.

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

    PubMed

    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

  13. 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

  14. 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.

  15. 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.

  16. Tackling Drought Stress: RECEPTOR-LIKE KINASES Present New Approaches

    PubMed Central

    Marshall, Alex; Aalen, Reidunn B.; Audenaert, Dominique; Beeckman, Tom; Broadley, Martin R.; Butenko, Melinka A.; Caño-Delgado, Ana I.; de Vries, Sacco; Dresselhaus, Thomas; Felix, Georg; Graham, Neil S.; Foulkes, John; Granier, Christine; Greb, Thomas; Grossniklaus, Ueli; Hammond, John P.; Heidstra, Renze; Hodgman, Charlie; Hothorn, Michael; Inzé, Dirk; Østergaard, Lars; Russinova, Eugenia; Simon, Rüdiger; Skirycz, Aleksandra; Stahl, Yvonne; Zipfel, Cyril; De Smet, Ive

    2012-01-01

    Global climate change and a growing population require tackling the reduction in arable land and improving biomass production and seed yield per area under varying conditions. One of these conditions is suboptimal water availability. Here, we review some of the classical approaches to dealing with plant response to drought stress and we evaluate how research on RECEPTOR-LIKE KINASES (RLKs) can contribute to improving plant performance under drought stress. RLKs are considered as key regulators of plant architecture and growth behavior, but they also function in defense and stress responses. The available literature and analyses of available transcript profiling data indeed suggest that RLKs can play an important role in optimizing plant responses to drought stress. In addition, RLK pathways are ideal targets for nontransgenic approaches, such as synthetic molecules, providing a novel strategy to manipulate their activity and supporting translational studies from model species, such as Arabidopsis thaliana, to economically useful crops. PMID:22693282

  17. 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.

  18. 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.

  19. 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. PMID:26577786

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

    DOE PAGESBeta

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

    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.

  1. 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 Central

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

    2015-01-01

    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. PMID:25694549

  2. 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

  3. Pepper suppressor of the G2 allele of skp1 interacts with the receptor-like cytoplasmic kinase1 and type III effector AvrBsT and promotes the hypersensitive cell death response in a phosphorylation-dependent manner.

    PubMed

    Kim, Nak Hyun; Kim, Dae Sung; Chung, Eui Hwan; Hwang, Byung Kook

    2014-05-01

    Xanthomonas campestris pv vesicatoria type III effector protein, AvrBsT, triggers hypersensitive cell death in pepper (Capsicum annuum). Here, we have identified the pepper SGT1 (for suppressor of the G2 allele of skp1) as a host interactor of AvrBsT and also the pepper PIK1 (for receptor-like cytoplasmic kinase1). PIK1 specifically phosphorylates SGT1 and AvrBsT in vitro. AvrBsT specifically binds to the CHORD-containing protein and SGT1 domain of SGT1, resulting in the inhibition of PIK1-mediated SGT1 phosphorylation and subsequent nuclear transport of the SGT1-PIK1 complex. Liquid chromatography-tandem mass spectrometry of the proteolytic peptides of SGT1 identified the residues serine-98 and serine-279 of SGT1 as the major PIK1-mediated phosphorylation sites. Site-directed mutagenesis of SGT1 revealed that the identified SGT1 phosphorylation sites are responsible for the activation of AvrBsT-triggered cell death in planta. SGT1 forms a heterotrimeric complex with both AvrBsT and PIK1 exclusively in the cytoplasm. Agrobacterium tumefaciens-mediated coexpression of SGT1 and PIK1 with avrBsT promotes avrBsT-triggered cell death in Nicotiana benthamiana, dependent on PIK1. Virus-induced silencing of SGT1 and/or PIK1 compromises avrBsT-triggered cell death, hydrogen peroxide production, defense gene induction, and salicylic acid accumulation, leading to the enhanced bacterial pathogen growth in pepper. Together, these results suggest that SGT1 interacts with PIK1 and the bacterial effector protein AvrBsT and promotes the hypersensitive cell death associated with PIK1-mediated phosphorylation in plants.

  4. Ligand-induced receptor-like kinase complex regulates floral organ abscission in Arabidopsis

    PubMed Central

    Meng, Xiangzong; Zhou, Jinggeng; Tang, Jiao; Li, Bo; de Oliveira, Marcos V. V.; Chai, Jijie; He, Ping; Shan, Libo

    2016-01-01

    SUMMARY Abscission is a developmental process that enables plants to shed unwanted organs. In Arabidopsis, the floral organ abscission is regulated by a signaling pathway consisting of the peptide ligand IDA, the receptor-like kinases (RLKs) HAE and HSL2, and a downstream MAP kinase (MAPK) cascade. However, little is known about the molecular link between ligand-receptor pairs and intracellular signaling. Here, we report that the SERK family RLKs function redundantly in regulating floral organ abscission downstream of IDA and upstream of the MAPK cascade. IDA induces heterodimerization of HAE/HSL2 and SERKs, which transphosphorylate each other. The SERK3 residues mediating its interaction with the immune receptor FLS2 and the brassinosteroid receptor BRI1 are also required for IDA-induced HAE/HSL2-SERK3 interaction, suggesting SERKs serve as co-receptors of HAE/HSL2 in perceiving IDA. Thus, our study reveals the signaling activation mechanism in floral organ abscission by IDA-induced HAE/HSL2-SERK complex formation accompanied by transphosphorylation. PMID:26854226

  5. 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

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

    DOEpatents

    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.

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

    DOEpatents

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

    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.

  8. Regulatory Role of a Receptor-Like Kinase in Specifying Anther Cell Identity.

    PubMed

    Yang, Li; Qian, Xiaoling; Chen, Mingjiao; Fei, Qili; Meyers, Blake C; Liang, Wanqi; Zhang, Dabing

    2016-07-01

    In flowering plants, sequential formation of anther cell types is a highly ordered process that is essential for successful meiosis and sexual reproduction. Differentiation of meristematic cells and cell-cell communication are proposed to coordinate anther development. Among the proposed mechanisms of cell fate specification are cell surface-localized Leu-rich repeat receptor-like kinases (LRR-RLKs) and their putative ligands. Here, we present the genetic and biochemical evidence that a rice (Oryza sativa) LRR-RLK, MSP1 (MULTIPLE SPOROCYTE1), interacts with its ligand OsTDL1A (TPD1-like 1A), specifying the cell identity of anther wall layers and microsporocytes. An in vitro assay indicates that the 21-amino acid peptide of OsTDL1A has a physical interaction with the LRR domain of MSP1. The ostdl1a msp1 double mutant showed the defect in lacking middle layers and tapetal cells and having an increased number of microsporocytes similar to the ostdl1a or msp1 single mutant, indicating the same pathway of OsTDL1A-MSP1 in regulating anther development. Genome-wide expression profiles showed the altered expression of genes encoding transcription factors, particularly basic helix-loop-helix and basic leucine zipper domain transcription factors in ostdl1a and msp1 Among these reduced expressed genes, one putatively encodes a TGA (TGACGTCA cis-element-binding protein) factor OsTGA10, and another one encodes a plant-specific CC-type glutaredoxin OsGrx_I1. OsTGA10 was shown to interact with OsGrx_I1, suggesting that OsTDL1A-MSP1 signaling specifies anther cell fate directly or indirectly affecting redox status. Collectively, these data point to a central role of the OsTDL1A-MSP1 signaling pathway in specifying somatic cell identity and suppressing overproliferation of archesporial cells in rice. PMID:27208278

  9. Regulatory Role of a Receptor-Like Kinase in Specifying Anther Cell Identity1[OPEN

    PubMed Central

    Yang, Li; Qian, Xiaoling; Chen, Mingjiao

    2016-01-01

    In flowering plants, sequential formation of anther cell types is a highly ordered process that is essential for successful meiosis and sexual reproduction. Differentiation of meristematic cells and cell-cell communication are proposed to coordinate anther development. Among the proposed mechanisms of cell fate specification are cell surface-localized Leu-rich repeat receptor-like kinases (LRR-RLKs) and their putative ligands. Here, we present the genetic and biochemical evidence that a rice (Oryza sativa) LRR-RLK, MSP1 (MULTIPLE SPOROCYTE1), interacts with its ligand OsTDL1A (TPD1-like 1A), specifying the cell identity of anther wall layers and microsporocytes. An in vitro assay indicates that the 21-amino acid peptide of OsTDL1A has a physical interaction with the LRR domain of MSP1. The ostdl1a msp1 double mutant showed the defect in lacking middle layers and tapetal cells and having an increased number of microsporocytes similar to the ostdl1a or msp1 single mutant, indicating the same pathway of OsTDL1A-MSP1 in regulating anther development. Genome-wide expression profiles showed the altered expression of genes encoding transcription factors, particularly basic helix-loop-helix and basic leucine zipper domain transcription factors in ostdl1a and msp1. Among these reduced expressed genes, one putatively encodes a TGA (TGACGTCA cis-element-binding protein) factor OsTGA10, and another one encodes a plant-specific CC-type glutaredoxin OsGrx_I1. OsTGA10 was shown to interact with OsGrx_I1, suggesting that OsTDL1A-MSP1 signaling specifies anther cell fate directly or indirectly affecting redox status. Collectively, these data point to a central role of the OsTDL1A-MSP1 signaling pathway in specifying somatic cell identity and suppressing overproliferation of archesporial cells in rice. PMID:27208278

  10. Uterine activin receptor-like kinase 5 is crucial for blastocyst implantation and placental development.

    PubMed

    Peng, Jia; Monsivais, Diana; You, Ran; Zhong, Hua; Pangas, Stephanie A; Matzuk, Martin M

    2015-09-01

    Members of the transforming growth factor β (TGF-β) superfamily are key regulators in most developmental and physiological processes. However, the in vivo roles of TGF-β signaling in female reproduction remain uncertain. Activin receptor-like kinase 5 (ALK5) is the major type 1 receptor for the TGF-β subfamily. Absence of ALK5 leads to early embryonic lethality because of severe defects in vascular development. In this study, we conditionally ablated uterine ALK5 using progesterone receptor-cre mice to define the physiological roles of ALK5 in female reproduction. Despite normal ovarian functions and artificial decidualization in conditional knockout (cKO) mice, absence of uterine ALK5 resulted in substantially reduced female reproduction due to abnormalities observed at different stages of pregnancy, including implantation defects, disorganization of trophoblast cells, fewer uterine natural killer (uNK) cells, and impairment of spiral artery remodeling. In our microarray analysis, genes encoding proteins involved in cytokine-cytokine receptor interactions and NK cell-mediated cytotoxicity were down-regulated in cKO decidua compared with control decidua. Flow cytometry confirmed a 10-fold decrease in uNK cells in cKO versus control decidua. According to these data, we hypothesize that TGF-β acts on decidual cells via ALK5 to induce expression of other growth factors and cytokines, which are key regulators in luminal epithelium proliferation, trophoblast development, and uNK maturation during pregnancy. Our findings not only generate a mouse model to study TGF-β signaling in female reproduction but also shed light on the pathogenesis of many pregnancy complications in human, such as recurrent spontaneous abortion, preeclampsia, and intrauterine growth restriction. PMID:26305969

  11. Positional signaling mediated by a receptor-like kinase in Arabidopsis.

    PubMed

    Kwak, Su-Hwan; Shen, Ronglai; Schiefelbein, John

    2005-02-18

    The position-dependent specification of root epidermal cells in Arabidopsis provides an elegant paradigm for cell patterning during development. Here, we describe a new gene, SCRAMBLED (SCM), required for cells to appropriately interpret their location within the developing root epidermis. SCM encodes a receptor-like kinase protein with a predicted extracellular domain of six leucine-rich repeats and an intracellular serine-threonine kinase domain. SCM regulates the expression of the GLABRA2, CAPRICE, WEREWOLF, and ENHANCER OF GLABRA3 transcription factor genes that define the cell fates. Further, the SCM gene is expressed throughout the developing root. Therefore, SCM likely enables developing epidermal cells to detect positional cues and establish an appropriate cell-type pattern.

  12. 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.

  13. Diverse Transcriptional Programs Associated with Environmental Stress and Hormones in the Arabidopsis Receptor-Like Kinase Gene Family

    PubMed Central

    Chae, Lee; Sudat, Sylvia; Dudoit, Sandrine; Zhu, Tong; Luan, Sheng

    2009-01-01

    The genome of Arabidopsis thaliana encodes more than 600 receptor-like kinase (RLK) genes, by far the dominant class of receptors found in land plants. Although similar to the mammalian receptor tyrosine kinases, plant RLKs are serine/threonine kinases that represent a novel signaling innovation unique to plants and, consequently, an excellent opportunity to understand how extracellular signaling evolved and functions in plants as opposed to animals. RLKs are predicted to be major components of the signaling pathways that allow plants to respond to environmental and developmental conditions. However, breakthroughs in identifying these processes have been limited to only a handful of individual RLKs. Here, we used a Syngenta custom Arabidopsis GeneChip array to compile a detailed profile of the transcriptional activity of 604 receptor-like kinase genes after exposure to a cross-section of known signaling factors in plants, including abiotic stresses, biotic stresses, and hormones. In the 68 experiments comprising the study, we found that 582 of the 604 RLK genes displayed a two-fold or greater change in expression to at least one of 12 types of treatments, thereby providing a large body of experimental evidence for targeted functional screens of individual RLK genes. We investigated whether particular subfamilies of RLK genes are responsive to specific types of signals and found that each subfamily displayed broad ranges of expression, as opposed to being targeted towards particular signal classes. Finally, by analyzing the divergence of sequence and gene expression among the RLK subfamilies, we present evidence as to the functional basis for the expansion of the RLKs and how this expansion may have affected conservation and divergences in their function. Taken as a whole, our study represents a preliminary, working model of processes and interactions in which the members of the RLK gene family may be involved, where such information has remained elusive for so many

  14. Overexpression of receptor-like kinase ERECTA improves thermotolerance in rice and tomato.

    PubMed

    Shen, Hui; Zhong, Xiangbin; Zhao, Fangfang; Wang, Yanmei; Yan, Bingxiao; Li, Qun; Chen, Genyun; Mao, Bizeng; Wang, Jianjun; Li, Yangsheng; Xiao, Guoying; He, Yuke; Xiao, Han; Li, Jianming; He, Zuhua

    2015-09-01

    The detrimental effects of global warming on crop productivity threaten to reduce the world's food supply. Although plant responses to changes in temperature have been studied, genetic modification of crops to improve thermotolerance has had little success to date. Here we demonstrate that overexpression of the Arabidopsis thaliana receptor-like kinase ERECTA (ER) in Arabidopsis, rice and tomato confers thermotolerance independent of water loss and that Arabidopsis er mutants are hypersensitive to heat. A loss-of-function mutation of a rice ER homolog and reduced expression of a tomato ER allele decreased thermotolerance of both species. Transgenic tomato and rice lines overexpressing Arabidopsis ER showed improved heat tolerance in the greenhouse and in field tests at multiple locations in China during several seasons. Moreover, ER-overexpressing transgenic Arabidopsis, tomato and rice plants had increased biomass. Our findings could contribute to engineering or breeding thermotolerant crops with no growth penalty. PMID:26280413

  15. Overexpression of receptor-like kinase ERECTA improves thermotolerance in rice and tomato.

    PubMed

    Shen, Hui; Zhong, Xiangbin; Zhao, Fangfang; Wang, Yanmei; Yan, Bingxiao; Li, Qun; Chen, Genyun; Mao, Bizeng; Wang, Jianjun; Li, Yangsheng; Xiao, Guoying; He, Yuke; Xiao, Han; Li, Jianming; He, Zuhua

    2015-09-01

    The detrimental effects of global warming on crop productivity threaten to reduce the world's food supply. Although plant responses to changes in temperature have been studied, genetic modification of crops to improve thermotolerance has had little success to date. Here we demonstrate that overexpression of the Arabidopsis thaliana receptor-like kinase ERECTA (ER) in Arabidopsis, rice and tomato confers thermotolerance independent of water loss and that Arabidopsis er mutants are hypersensitive to heat. A loss-of-function mutation of a rice ER homolog and reduced expression of a tomato ER allele decreased thermotolerance of both species. Transgenic tomato and rice lines overexpressing Arabidopsis ER showed improved heat tolerance in the greenhouse and in field tests at multiple locations in China during several seasons. Moreover, ER-overexpressing transgenic Arabidopsis, tomato and rice plants had increased biomass. Our findings could contribute to engineering or breeding thermotolerant crops with no growth penalty.

  16. 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.

  17. 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

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

    PubMed Central

    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. PMID:25490905

  19. Plasmodesmal receptor-like kinases identified through analysis of rice cell wall extracted proteins.

    PubMed

    Jo, Yeonhwa; Cho, Won Kyong; Rim, Yeonggil; Moon, Juyeon; Chen, Xiong-Yan; Chu, Hyosub; Kim, Cha Young; Park, Zee-Yong; Lucas, William J; Kim, Jae-Yean

    2011-01-01

    In plants, plasmodesmata (PD) are intercellular channels that function in both metabolite exchange and the transport of proteins and RNAs. Currently, many of the PD structural and regulatory components remain to be elucidated. Receptor-like kinases (RLKs) belonging to a notably expanded protein family in plants compared to the animal kingdom have been shown to play important roles in plant growth, development, pathogen resistance, and cell death. In this study, cell biological approaches were used to identify potential PD-associated RLK proteins among proteins contained within cell walls isolated from rice callus cultured cells. A total of 15 rice RLKs were investigated to determine their subcellular localization, using an Agrobacterium-mediated transient expression system. Of these six PD-associated RLKs were identified based on their co-localization with a viral movement protein that served as a PD marker, plasmolysis experiments, and subcellular localization at points of wall contact between spongy mesophyll cells. These findings suggest potential PD functions in apoplasmic signaling in response to environmental stimuli and developmental inputs. PMID:21161304

  20. A receptor-like kinase mutant with absent endodermal diffusion barrier displays selective nutrient homeostasis defects

    PubMed Central

    Pfister, Alexandre; Barberon, Marie; Alassimone, Julien; Kalmbach, Lothar; Lee, Yuree; Vermeer, Joop EM; Yamazaki, Misako; Li, Guowei; Maurel, Christophe; Takano, Junpei; Kamiya, Takehiro; Salt, David E; Roppolo, Daniele; Geldner, Niko

    2014-01-01

    The endodermis represents the main barrier to extracellular diffusion in plant roots, and it is central to current models of plant nutrient uptake. Despite this, little is known about the genes setting up this endodermal barrier. In this study, we report the identification and characterization of a strong barrier mutant, schengen3 (sgn3). We observe a surprising ability of the mutant to maintain nutrient homeostasis, but demonstrate a major defect in maintaining sufficient levels of the macronutrient potassium. We show that SGN3/GASSHO1 is a receptor-like kinase that is necessary for localizing CASPARIAN STRIP DOMAIN PROTEINS (CASPs)—major players of endodermal differentiation—into an uninterrupted, ring-like domain. SGN3 appears to localize into a broader band, embedding growing CASP microdomains. The discovery of SGN3 strongly advances our ability to interrogate mechanisms of plant nutrient homeostasis and provides a novel actor for localized microdomain formation at the endodermal plasma membrane. DOI: http://dx.doi.org/10.7554/eLife.03115.001 PMID:25233277

  1. Targeting tumour vasculature by inhibiting activin receptor-like kinase (ALK)1 function.

    PubMed

    de Vinuesa, Amaya García; Bocci, Matteo; Pietras, Kristian; Ten Dijke, Peter

    2016-08-15

    Angiogenesis is a hallmark of cancer and is now a validated therapeutic target in the clinical setting. Despite the initial success, anti-angiogenic compounds impinging on the vascular endothelial growth factor (VEGF) pathway display limited survival benefits in patients and resistance often develops due to activation of alternative pathways. Thus, finding and validating new targets is highly warranted. Activin receptor-like kinase (ALK)1 is a transforming growth factor beta (TGF-β) type I receptor predominantly expressed in actively proliferating endothelial cells (ECs). ALK1 has been shown to play a pivotal role in regulating angiogenesis by binding to bone morphogenetic protein (BMP)9 and 10. Two main pharmacological inhibitors, an ALK1-Fc fusion protein (Dalantercept/ACE-041) and a fully human antibody against the extracellular domain of ALK1 (PF-03446962) are currently under clinical development. Herein, we briefly recapitulate the role of ALK1 in blood vessel formation and the current status of the preclinical and clinical studies on inhibition of ALK1 signalling as an anti-angiogenic strategy. Future directions in terms of new combination regimens will also be presented. PMID:27528762

  2. Cellular pattern formation by SCRAMBLED, a leucine-rich repeat receptor-like kinase in Arabidopsis.

    PubMed

    Kwak, Su-Hwan; Schiefelbein, John

    2008-02-01

    The appropriate specification of distinct cell types is important for generating the proper tissues and bodies of multicellular organisms. In the root epidermis of Arabidopsis, cell fate determination is accomplished by a transcriptional regulatory circuit that is influenced by positional signaling. A leucine-rich repeat receptor-like kinase, SCRAMBLED (SCM), has been shown to be responsible for the position-dependent aspect of this epidermal pattern. In a recent report, we find that SCM affects the transcriptional regulatory network by down-regulating the WEREWOLF (WER) MYB gene expression in a set of epidermal cells located in a specific position. We also find that SCM and the SCM-related SRF1 and SRF3 are not required for embryonic epidermal patterning and that SRF1 and SRF3 do not act redundantly with SCM. This suggests that distinct positional signaling mechanisms exist for embryonic and post-embryonic epidermal patterning. In this addendum, we discuss the implications of our recent findings and extend our working model for epidermal cell pattering.

  3. The role of the SCRAMBLED receptor-like kinase in patterning the Arabidopsis root epidermis.

    PubMed

    Kwak, Su-Hwan; Schiefelbein, John

    2007-02-01

    Cell-type patterning in the Arabidopsis root epidermis is achieved by a network of transcription factors and influenced by a position-dependent mechanism. The SCRAMBLED receptor-like kinase is required for the normal pattern to arise, but its precise role is not understood. Here we describe genetic and molecular studies to define the spatial and temporal role of SCM in epidermal patterning and its relationship to the transcriptional network. Our results suggest that SCM helps unspecified epidermal cells interpret their position in relation to the underlying cortical cells and establish distinct cell identities. Furthermore, SCM loss-of-function and overexpression analyses suggest that SCM influences cell fate through its negative transcriptional regulation of the WEREWOLF MYB gene in epidermal cells at the H position. We also find that SCM function is specifically required for patterning the post-embryonic root epidermis and not for the analogous epidermal cell-type patterning during embryogenesis or hypocotyl development. In addition, we show that two closely related SCM-like genes in Arabidopsis (SRF1 and SRF3) are not required alone or together with SCM for proper epidermal patterning. These findings help define the developmental and mechanistic role of SCM and suggest a new model for its action in root epidermal cell patterning.

  4. Extracellular signals and receptor-like kinases regulating ROP GTPases in plants

    PubMed Central

    Miyawaki, Kaori N.; Yang, Zhenbiao

    2014-01-01

    Rho-like GTPase from plants (ROPs) function as signaling switches that control a wide variety of cellular functions and behaviors including cell morphogenesis, cell division and cell differentiation. The Arabidopsis thaliana genome encodes 11 ROPs that form a distinct single subfamily contrarily to animal or fungal counterparts where multiple subfamilies of Rho GTPases exist. Since Rho proteins bind to their downstream effector proteins only in their GTP-bound “active” state, the activation of ROPs by upstream factor(s) is a critical step in the regulation of ROP signaling. Therefore, it is critical to examine the input signals that lead to the activation of ROPs. Recent findings showed that the plant hormone auxin is an important signal for the activation of ROPs during pavement cell morphogenesis as well as for other developmental processes. In contrast to auxin, another plant hormone, abscisic acid, negatively regulates ROP signaling. Calcium is another emerging signal in the regulation of ROP signaling. Several lines of evidence indicate that plasma membrane localized-receptor like kinases play a critical role in the transmission of the extracellular signals to intracellular ROP signaling pathways. This review focuses on how these signals impinge upon various direct regulators of ROPs to modulate various plant processes. PMID:25295042

  5. The Secreted Peptide PIP1 Amplifies Immunity through Receptor-Like Kinase 7

    PubMed Central

    Hou, Shuguo; Wang, Xin; Chen, Donghua; Yang, Xue; Wang, Mei; Turrà, David; Di Pietro, Antonio; Zhang, Wei

    2014-01-01

    In plants, innate immune responses are initiated by plasma membrane-located pattern recognition receptors (PRRs) upon recognition of elicitors, including exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs). Arabidopsis thaliana produces more than 1000 secreted peptide candidates, but it has yet to be established whether any of these act as elicitors. Here we identified an A. thaliana gene family encoding precursors of PAMP-induced secreted peptides (prePIPs) through an in-silico approach. The expression of some members of the family, including prePIP1 and prePIP2, is induced by a variety of pathogens and elicitors. Subcellular localization and proteolytic processing analyses demonstrated that the prePIP1 product is secreted into extracellular spaces where it is cleaved at the C-terminus. Overexpression of prePIP1 and prePIP2, or exogenous application of PIP1 and PIP2 synthetic peptides corresponding to the C-terminal conserved regions in prePIP1 and prePIP2, enhanced immune responses and pathogen resistance in A. thaliana. Genetic and biochemical analyses suggested that the receptor-like kinase 7 (RLK7) functions as a receptor of PIP1. Once perceived by RLK7, PIP1 initiates overlapping and distinct immune signaling responses together with the DAMP PEP1. PIP1 and PEP1 cooperate in amplifying the immune responses triggered by the PAMP flagellin. Collectively, these studies provide significant insights into immune modulation by Arabidopsis endogenous secreted peptides. PMID:25188390

  6. 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.

  7. 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. PMID:25271257

  8. Two Redundant Receptor-Like Cytoplasmic Kinases Function Downstream of Pattern Recognition Receptors to Regulate Activation of SA Biosynthesis.

    PubMed

    Kong, Qing; Sun, Tongjun; Qu, Na; Ma, Junling; Li, Meng; Cheng, Yu-Ti; Zhang, Qian; Wu, Di; Zhang, Zhibin; Zhang, Yuelin

    2016-06-01

    Salicylic acid (SA) serves as a critical signaling molecule in plant defense. Two transcription factors, SARD1 and CBP60g, control SA biosynthesis through regulating pathogen-induced expression of Isochorismate Synthase1, which encodes a key enzyme for SA biosynthesis. Here, we report that Pattern-Triggered Immunity Compromised Receptor-like Cytoplasmic Kinase1 (PCRK1) and PCRK2 function as key regulators of SA biosynthesis. In the pcrk1 pcrk2 double mutant, pathogen-induced expression of SARD1, CBP60g, and ICS1 is greatly reduced. The pcrk1 pcrk2 double mutant, but neither of the single mutants, exhibits reduced accumulation of SA and enhanced disease susceptibility to bacterial pathogens. Both PCRK1 and PCRK2 interact with the pattern recognition receptor FLS2, and treatment with pathogen-associated molecular patterns leads to rapid phosphorylation of PCRK2. Our data suggest that PCRK1 and PCRK2 function downstream of pattern recognition receptor in a signal relay leading to the activation of SA biosynthesis. PMID:27208222

  9. 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.

  10. 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

  11. ANXUR Receptor-Like Kinases Coordinate Cell Wall Integrity with Growth at the Pollen Tube Tip Via NADPH Oxidases

    PubMed Central

    Boisson-Dernier, Aurélien; Lituiev, Dmytro S.; Nestorova, Anna; Franck, Christina Maria; Thirugnanarajah, Sharme; Grossniklaus, Ueli

    2013-01-01

    It has become increasingly apparent that the extracellular matrix (ECM), which in plants corresponds to the cell wall, can influence intracellular activities in ways that go far beyond their supposedly passive mechanical support. In plants, growing cells use mechanisms sensing cell wall integrity to coordinate cell wall performance with the internal growth machinery to avoid growth cessation or loss of integrity. How this coordination precisely works is unknown. Previously, we reported that in the tip-growing pollen tube the ANXUR receptor-like kinases (RLKs) of the CrRLK1L subfamily are essential to sustain growth without loss of cell wall integrity in Arabidopsis. Here, we show that over-expression of the ANXUR RLKs inhibits growth by over-activating exocytosis and the over-accumulation of secreted cell wall material. Moreover, the characterization of mutations in two partially redundant pollen-expressed NADPH oxidases coupled with genetic interaction studies demonstrate that the ANXUR RLKs function upstream of these NADPH oxidases. Using the H2O2-sensitive HyPer and the Ca2+-sensitive YC3.60 sensors in NADPH oxidase-deficient mutants, we reveal that NADPH oxidases generate tip-localized, pulsating H2O2 production that functions, possibly through Ca2+ channel activation, to maintain a steady tip-focused Ca2+ gradient during growth. Our findings support a model where ECM-sensing receptors regulate reactive oxygen species production, Ca2+ homeostasis, and exocytosis to coordinate ECM-performance with the internal growth machinery. PMID:24302886

  12. Molecular Characterization and Comparative Sequence Analysis of Defense-Related Gene, Oryza rufipogon Receptor-Like Protein Kinase 1

    PubMed Central

    Law, Yee-Song; Gudimella, Ranganath; Song, Beng-Kah; Ratnam, Wickneswari; Harikrishna, Jennifer Ann

    2012-01-01

    Many of the plant leucine rich repeat receptor-like kinases (LRR-RLKs) have been found to regulate signaling during plant defense processes. In this study, we selected and sequenced an LRR-RLK gene, designated as Oryza rufipogon receptor-like protein kinase 1 (OrufRPK1), located within yield QTL yld1.1 from the wild rice Oryza rufipogon (accession IRGC105491). A 2055 bp coding region and two exons were identified. Southern blotting determined OrufRPK1 to be a single copy gene. Sequence comparison with cultivated rice orthologs (OsI219RPK1, OsI9311RPK1 and OsJNipponRPK1, respectively derived from O. sativa ssp. indica cv. MR219, O. sativa ssp. indica cv. 9311 and O. sativa ssp. japonica cv. Nipponbare) revealed the presence of 12 single nucleotide polymorphisms (SNPs) with five non-synonymous substitutions, and 23 insertion/deletion sites. The biological role of the OrufRPK1 as a defense related LRR-RLK is proposed on the basis of cDNA sequence characterization, domain subfamily classification, structural prediction of extra cellular domains, cluster analysis and comparative gene expression. PMID:22942769

  13. 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.

  14. 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

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

    PubMed

    Rameneni, Jana Jeevan; Lee, Yeon; 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.

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

    PubMed

    Rameneni, Jana Jeevan; Lee, Yeon; 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

  17. A MAPK cascade downstream of ERECTA receptor-like protein kinase regulates Arabidopsis inflorescence architecture by promoting localized cell proliferation.

    PubMed

    Meng, Xiangzong; Wang, Huachun; He, Yunxia; Liu, Yidong; Walker, John C; Torii, Keiko U; Zhang, Shuqun

    2012-12-01

    Spatiotemporal-specific cell proliferation and cell differentiation are critical to the formation of normal tissues, organs, and organisms. The highly coordinated cell differentiation and proliferation events illustrate the importance of cell-cell communication during growth and development. In Arabidopsis thaliana, ERECTA (ER), a receptor-like protein kinase, plays important roles in promoting localized cell proliferation, which determines inflorescence architecture, organ shape, and size. However, the downstream signaling components remain unidentified. Here, we report a mitogen-activated protein kinase (MAPK; or MPK) cascade that functions downstream of ER in regulating localized cell proliferation. Similar to an er mutant, loss of function of MPK3/MPK6 or their upstream MAPK kinases (MAPKKs; or MKKs), MKK4/MKK5, resulted in shortened pedicels and clustered inflorescences. Epistasis analysis demonstrated that the gain of function of MKK4 and MKK5 transgenes could rescue the loss-of-function er mutant phenotype at both morphological and cellular levels, suggesting that the MPK3/MPK6 cascade functions downstream of the ER receptor. Furthermore, YODA (YDA), a MAPKK kinase, was shown to be upstream of MKK4/MKK5 and downstream of ER in regulating inflorescence architecture based on both gain- and loss-of-function data. Taken together, these results suggest that the YDA-MKK4/MKK5-MPK3/MPK6 cascade functions downstream of the ER receptor in regulating localized cell proliferation, which further shapes the morphology of plant organs. PMID:23263767

  18. 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

  19. Receptor-like cytoplasmic kinase MARIS functions downstream of CrRLK1L-dependent signaling during tip growth

    PubMed Central

    Boisson-Dernier, Aurélien; Franck, Christina Maria; Lituiev, Dmytro S.; Grossniklaus, Ueli

    2015-01-01

    Growing plant cells need to rigorously coordinate external signals with internal processes. For instance, the maintenance of cell wall (CW) integrity requires the coordination of CW sensing with CW remodeling and biosynthesis to avoid growth arrest or integrity loss. Despite the involvement of receptor-like kinases (RLKs) of the Catharanthus roseus RLK1-like (CrRLK1L) subfamily and the reactive oxygen species-producing NADPH oxidases, it remains largely unknown how this coordination is achieved. ANXUR1 (ANX1) and ANX2, two redundant members of the CrRLK1L subfamily, are required for tip growth of the pollen tube (PT), and their closest homolog, FERONIA, controls root-hair tip growth. Previously, we showed that ANX1 overexpression mildly inhibits PT growth by oversecretion of CW material, whereas pollen tubes of anx1 anx2 double mutants burst spontaneously after germination. Here, we report the identification of suppressor mutants with improved fertility caused by the rescue of anx1 anx2 pollen tube bursting. Mapping of one these mutants revealed an R240C nonsynonymous substitution in the activation loop of a receptor-like cytoplasmic kinase (RLCK), which we named MARIS (MRI). We show that MRI is a plasma membrane-localized member of the RLCK-VIII subfamily and is preferentially expressed in both PTs and root hairs. Interestingly, mri-knockout mutants display spontaneous PT and root-hair bursting. Moreover, expression of the MRIR240C mutant, but not its wild-type form, partially rescues the bursting phenotypes of anx1 anx2 PTs and fer root hairs but strongly inhibits wild-type tip growth. Thus, our findings identify a novel positive component of the CrRLK1L-dependent signaling cascade that coordinates CW integrity and tip growth. PMID:26378127

  20. Isolation and characterization of a novel wheat cysteine-rich receptor-like kinase gene induced by Rhizoctonia cerealis

    NASA Astrophysics Data System (ADS)

    Yang, Kun; Rong, Wei; Qi, Lin; Li, Jiarui; Wei, Xuening; Zhang, Zengyan

    2013-10-01

    Cysteine-rich receptor kinases (CRKs) belong to the receptor-like kinase family. Little is known about CRK genes in wheat. We isolated a wheat CRK gene TaCRK1 from Rhizoctonia cerealis-resistant wheat CI12633 based on a differentially expressed sequence identified by RNA-Sequencing (RNA-Seq) analysis. TaCRK1 was more highly expressed in CI12633 than in susceptible Wenmai 6. Transcription of TaCRK1 in wheat was induced in CI12633 after R. cerealis infection and exogenous abscisic acid (ABA) treatment. The deduced TaCRK1 protein contained a signal peptide, two DUF26 domains, a transmembrane domain, and a serine/threonine protein kinase domain. Transient expression of a green fluorescence protein fused with TaCRK1 in wheat and onion indicated that TaCRK1 may localize to plasma membranes. Characterization of TaCRK1 silencing induced by virus-mediated method in CI12633 showed that the downregulation of TaCRK1 transcript did not obviously impair resistance to R. cerealis. This study paves the way to further CRK research in wheat.

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

    PubMed

    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

  2. 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.

  3. 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

  4. Identification of PAN2 by quantitative proteomics as a leucine-rich repeat-receptor-like kinase acting upstream of PAN1 to polarize cell division in maize.

    PubMed

    Zhang, Xiaoguo; Facette, Michelle; Humphries, John A; Shen, Zhouxin; Park, Yeri; Sutimantanapi, Dena; Sylvester, Anne W; Briggs, Steven P; Smith, Laurie G

    2012-11-01

    Mechanisms governing the polarization of plant cell division are poorly understood. Previously, we identified pangloss1 (PAN1) as a leucine-rich repeat-receptor-like kinase (LRR-RLK) that promotes the polarization of subsidiary mother cell (SMC) divisions toward the adjacent guard mother cell (GMC) during stomatal development in maize (Zea mays). Here, we identify pangloss2 (PAN2) as a second LRR-RLK promoting SMC polarization. Quantitative proteomic analysis identified a PAN2 candidate by its depletion from membranes of pan2 single and pan1;pan2 double mutants. Genetic mapping and sequencing of mutant alleles confirmed the identity of this protein as PAN2. Like PAN1, PAN2 has a catalytically inactive kinase domain and accumulates in SMCs at sites of GMC contact before nuclear polarization. The timing of polarized PAN1 and PAN2 localization is very similar, but PAN2 acts upstream because it is required for polarized accumulation of PAN1 but is independent of PAN1 for its own localization. We find no evidence that PAN2 recruits PAN1 to the GMC contact site via a direct or indirect physical interaction, but PAN2 interacts with itself. Together, these results place PAN2 at the top of a cascade of events promoting the polarization of SMC divisions, potentially functioning to perceive or amplify GMC-derived polarizing cues.

  5. Thermoreversible gel for delivery of activin receptor-like kinase 5 inhibitor SB-505124 for glaucoma filtration surgery.

    PubMed

    Sutariya, Vijaykumar; Miladore, Nicholas; Geldenhuys, Werner; Bhatia, Deepak; Wehrung, Daniel; Nakamura, Hiroshi

    2013-01-01

    The purpose of this study is to investigate a thermoreversible gel using Pluronic F-127 to deliver an activin receptor-like kinase 5 (ALK-5) inhibitor SB-505124 in glaucoma filtration surgery (GFS). The gel was characterized for in vitro drug release and viscosity studies. Cytotoxicity of Pluronic F-127 was examined by MTT assay using cultured rabbit subconjunctival fibroblasts. In addition, Pluronic F-127 gel (18% w/v) containing 5 mg of SB-505124 was applied at the surgical site in an in vivo rabbit GFS model. In the in vitro viscosity study, the gel showed a change in viscosity (from 1000 cps to 45,000 cps) from low temperature (10°C) to body temperature (37°C). The in vitro drug release study demonstrated 100% drug release within 12 h. The gel did not show cytotoxicity to the cultured rabbit subconjunctival cells by MTT assay. In the in vivo rabbit GFS model, the drug was successfully delivered by injection and no severe post-surgical complications were observed. A thermoreversible gel system with SB-505124 was successfully prepared and delivered for the rabbit GFS model, and it may provide a novel delivery system in GFS.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26446555

  7. 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

    2010-03-01

    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.

  8. Heterotrimeric G Proteins Serve as a Converging Point in Plant Defense Signaling Activated by Multiple Receptor-Like Kinases1[C][W][OA

    PubMed Central

    Liu, Jinman; Ding, Pingtao; Sun, Tongjun; Nitta, Yukino; Dong, Oliver; Huang, Xingchuan; Yang, Wei; Li, Xin; Botella, José Ramón; Zhang, Yuelin

    2013-01-01

    In fungi and metazoans, extracellular signals are often perceived by G-protein-coupled receptors (GPCRs) and transduced through heterotrimeric G-protein complexes to downstream targets. Plant heterotrimeric G proteins are also involved in diverse biological processes, but little is known about their upstream receptors. Moreover, the presence of bona fide GPCRs in plants is yet to be established. In Arabidopsis (Arabidopsis thaliana), heterotrimeric G protein consists of one Gα subunit (G PROTEIN α-SUBUNIT1), one Gβ subunit (ARABIDOPSIS G PROTEIN β-SUBUNIT1 [AGB1]), and three Gγs subunits (ARABIDOPSIS G PROTEIN γ-SUBUNIT1 [AGG1], AGG2, and AGG3). We identified AGB1 from a suppressor screen of BAK1-interacting receptor-like kinase1-1 (bir1-1), a mutant that activates cell death and defense responses mediated by the receptor-like kinase (RLK) SUPPRESSOR OF BIR1-1. Mutations in AGB1 suppress the cell death and defense responses in bir1-1 and transgenic plants overexpressing SUPPRESSOR OF BIR1-1. In addition, agb1 mutant plants were severely compromised in immunity mediated by three other RLKs, FLAGELLIN-SENSITIVE2 (FLS2), Elongation Factor-TU RECEPTOR (EFR), and CHITIN ELICITOR RECEPTOR KINASE1 (CERK1), respectively. By contrast, G PROTEIN α-SUBUNIT1 is not required for either cell death in bir1-1 or pathogen-associated molecular pattern-triggered immunity mediated by FLS2, EFR, and CERK1. Further analysis of agg1 and agg2 mutant plants indicates that AGG1 and AGG2 are also required for pathogen-associated molecular pattern-triggered immune responses mediated by FLS2, EFR, and CERK1, as well as cell death and defense responses in bir1-1. We hypothesize that the Arabidopsis heterotrimeric G proteins function as a converging point of plant defense signaling by mediating responses initiated by multiple RLKs, which may fulfill equivalent roles to GPCRs in fungi and animals. PMID:23424249

  9. 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.

  10. Heterozygous disruption of activin receptor-like kinase 1 is associated with increased arterial pressure in mice

    PubMed Central

    González-Núñez, María; Riolobos, Adela S.; Castellano, Orlando; Fuentes-Calvo, Isabel; de los Ángeles Sevilla, María; Oujo, Bárbara; Pericacho, Miguel; Cruz-Gonzalez, Ignacio; Pérez-Barriocanal, Fernando; ten Dijke, Peter; López-Novoa, Jose M.

    2015-01-01

    ABSTRACT The activin receptor-like kinase 1 (ALK-1) is a type I cell-surface receptor for the transforming growth factor-β (TGF-β) family of proteins. Hypertension is related to TGF-β1, because increased TGF-β1 expression is correlated with an elevation in arterial pressure (AP) and TGF-β expression is upregulated by the renin-angiotensin-aldosterone system. The purpose of this study was to assess the role of ALK-1 in regulation of AP using Alk1 haploinsufficient mice (Alk1+/−). We observed that systolic and diastolic AP were significantly higher in Alk1+/− than in Alk1+/+ mice, and all functional and structural cardiac parameters (echocardiography and electrocardiography) were similar in both groups. Alk1+/− mice showed alterations in the circadian rhythm of AP, with higher AP than Alk1+/+ mice during most of the light period. Higher AP in Alk1+/− mice is not a result of a reduction in the NO-dependent vasodilator response or of overactivation of the peripheral renin-angiotensin system. However, intracerebroventricular administration of losartan had a hypotensive effect in Alk1+/− and not in Alk1+/+ mice. Alk1+/− mice showed a greater hypotensive response to the β-adrenergic antagonist atenolol and higher concentrations of epinephrine and norepinephrine in plasma than Alk1+/+ mice. The number of brain cholinergic neurons in the anterior basal forebrain was reduced in Alk1+/− mice. Thus, we concluded that the ALK-1 receptor is involved in the control of AP, and the high AP of Alk1+/− mice is explained mainly by the sympathetic overactivation shown by these animals, which is probably related to the decreased number of cholinergic neurons. PMID:26398936

  11. 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. PMID:24585212

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

    PubMed Central

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

    2015-01-01

    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. PMID:25969551

  13. 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.

  14. A genetic screen identifies a requirement for cysteine-rich–receptor-like kinases in rice NH1 (OsNPR1)-mediated immunity

    DOE PAGESBeta

    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.; et al

    2016-05-13

    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.more » 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. Furthermore, these experiments demonstrate a requirement for CRKs in NH1-mediated immunity and establish a molecular link between NH1 and induction of CRK10 expression.« less

  15. 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

  16. Lotus japonicus E3 Ligase SEVEN IN ABSENTIA4 Destabilizes the Symbiosis Receptor-Like Kinase SYMRK and Negatively Regulates Rhizobial Infection[C][W

    PubMed Central

    Den Herder, Griet; Yoshida, Satoko; Antolín-Llovera, Meritxell; Ried, Martina K.; Parniske, Martin

    2012-01-01

    The Lotus japonicus SYMBIOSIS RECEPTOR-LIKE KINASE (SYMRK) is required for symbiotic signal transduction upon stimulation of root cells by microbial signaling molecules. Here, we identified members of the SEVEN IN ABSENTIA (SINA) E3 ubiquitin-ligase family as SYMRK interactors and confirmed their predicted ubiquitin-ligase activity. In Nicotiana benthamiana leaves, SYMRK–yellow fluorescent protein was localized at the plasma membrane, and interaction with SINAs, as determined by bimolecular fluorescence complementation, was observed in small punctae at the cytosolic interface of the plasma membrane. Moreover, fluorescence-tagged SINA4 partially colocalized with SYMRK and caused SYMRK relocalization as well as disappearance of SYMRK from the plasma membrane. Neither the localization nor the abundance of Nod-factor receptor1 was altered by the presence of SINA4. SINA4 was transcriptionally upregulated during root symbiosis, and rhizobia inoculated roots ectopically expressing SINA4 showed reduced SYMRK protein levels. In accordance with a negative regulatory role in symbiosis, infection thread development was impaired upon ectopic expression of SINA4. Our results implicate SINA4 E3 ubiquitin ligase in the turnover of SYMRK and provide a conceptual mechanism for its symbiosis-appropriate spatio-temporal containment. PMID:22534128

  17. Two Redundant Receptor-Like Cytoplasmic Kinases Function Downstream of Pattern Recognition Receptors to Regulate Activation of SA Biosynthesis1[OPEN

    PubMed Central

    Kong, Qing; Qu, Na; Ma, Junling; Li, Meng; Cheng, Yu-ti; Zhang, Qian; Wu, Di; Zhang, Zhibin; Zhang, Yuelin

    2016-01-01

    Salicylic acid (SA) serves as a critical signaling molecule in plant defense. Two transcription factors, SARD1 and CBP60g, control SA biosynthesis through regulating pathogen-induced expression of Isochorismate Synthase1, which encodes a key enzyme for SA biosynthesis. Here, we report that Pattern-Triggered Immunity Compromised Receptor-like Cytoplasmic Kinase1 (PCRK1) and PCRK2 function as key regulators of SA biosynthesis. In the pcrk1 pcrk2 double mutant, pathogen-induced expression of SARD1, CBP60g, and ICS1 is greatly reduced. The pcrk1 pcrk2 double mutant, but neither of the single mutants, exhibits reduced accumulation of SA and enhanced disease susceptibility to bacterial pathogens. Both PCRK1 and PCRK2 interact with the pattern recognition receptor FLS2, and treatment with pathogen-associated molecular patterns leads to rapid phosphorylation of PCRK2. Our data suggest that PCRK1 and PCRK2 function downstream of pattern recognition receptor in a signal relay leading to the activation of SA biosynthesis. PMID:27208222

  18. 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.

  19. Receptor-like protein kinase HvLysMR1 of barley (Hordeum vulgare L.) is induced during leaf senescence and heavy metal stress.

    PubMed

    Ouelhadj, Akli; Kaminski, Marc; Mittag, Maria; Humbeck, Klaus

    2007-01-01

    The Hordeum vulgare cDNA clone HvLysMR1 that encodes a putative receptor-like protein kinase was identified by restriction fragment differential display-polymerase chain reaction (PCR) comparing cDNA populations derived from mRNAs of primary leaves stressed with chromium for 48 h with controls. The full-length sequence codes for a protein with 622 amino acids which includes characteristic domains of lysine motif receptor-like kinases: an N-terminal signal peptide, two lysine motifs, a transmembrane region, and a serine/threonine kinase domain at the C-terminal end. The expression of HvLysMR1 is induced during exposure to different heavy metals and its transcript accumulates during leaf senescence. Addition of the calcium ionophore A23187 induces HvLysMR1 expression, indicating the involvement of Ca2+ in the regulation of HvLysMR1. In vitro phosphorylation of HvLysMR1 was analysed with [32P]ATP. Using the overexpressed and purified HvLysMR1-kinase domain, the phosphorylation of HvLysMR1 could be confirmed by nano-liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) with neutral loss-triggered MS-MS-MS spectra at amino acids localized at the juxtamembrane region. The involvement of HvLysMR1 during heavy metal stress and leaf senescence is discussed. PMID:17322551

  20. The Arabidopsis LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE3 Regulates the Cross Talk between Immunity and Abscisic Acid Responses1[W][OPEN

    PubMed Central

    Paparella, Chiara; Savatin, Daniel Valentin; Marti, Lucia; De Lorenzo, Giulia; Ferrari, Simone

    2014-01-01

    Transmembrane receptor-like kinases characterized by the presence of one or more lysin motif (LysM) domains in the extracytoplasmic portion (LysM-containing receptor-like kinases [LYKs]) mediate recognition of symbiotic and pathogenic microorganisms in plants. The Arabidopsis (Arabidopsis thaliana) genome encodes five putative LYKs; among them, AtLYK1/CHITIN ELICITOR RECEPTOR KINASE1 is required for response to chitin and peptidoglycan, and AtLYK4 contributes to chitin perception. More recently, AtLYK3 has been shown to be required for full repression, mediated by Nod factors, of Arabidopsis innate immune responses. In this work, we show that AtLYK3 also negatively regulates basal expression of defense genes and resistance to Botrytis cinerea and Pectobacterium carotovorum infection. Enhanced resistance of atlyk3 mutants requires PHYTOALEXIN-DEFICIENT3, which is crucial for camalexin biosynthesis. The expression of AtLYK3 is strongly repressed by elicitors and fungal infection and is induced by the hormone abscisic acid (ABA), which has a negative impact on resistance against B. cinerea and P. carotovorum. Plants lacking a functional AtLYK3 also show reduced physiological responses to ABA and are partially resistant to ABA-induced inhibition of PHYTOALEXIN-DEFICIENT3 expression. These results indicate that AtLYK3 is important for the cross talk between signaling pathways activated by ABA and pathogens. PMID:24639336

  1. 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

  2. The Arabidopsis LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE3 regulates the cross talk between immunity and abscisic acid responses.

    PubMed

    Paparella, Chiara; Savatin, Daniel Valentin; Marti, Lucia; De Lorenzo, Giulia; Ferrari, Simone

    2014-05-01

    Transmembrane receptor-like kinases characterized by the presence of one or more lysin motif (LysM) domains in the extracytoplasmic portion (LysM-containing receptor-like kinases [LYKs]) mediate recognition of symbiotic and pathogenic microorganisms in plants. The Arabidopsis (Arabidopsis thaliana) genome encodes five putative LYKs; among them, AtLYK1/CHITIN ELICITOR RECEPTOR KINASE1 is required for response to chitin and peptidoglycan, and AtLYK4 contributes to chitin perception. More recently, AtLYK3 has been shown to be required for full repression, mediated by Nod factors, of Arabidopsis innate immune responses. In this work, we show that AtLYK3 also negatively regulates basal expression of defense genes and resistance to Botrytis cinerea and Pectobacterium carotovorum infection. Enhanced resistance of atlyk3 mutants requires PHYTOALEXIN-DEFICIENT3, which is crucial for camalexin biosynthesis. The expression of AtLYK3 is strongly repressed by elicitors and fungal infection and is induced by the hormone abscisic acid (ABA), which has a negative impact on resistance against B. cinerea and P. carotovorum. Plants lacking a functional AtLYK3 also show reduced physiological responses to ABA and are partially resistant to ABA-induced inhibition of PHYTOALEXIN-DEFICIENT3 expression. These results indicate that AtLYK3 is important for the cross talk between signaling pathways activated by ABA and pathogens.

  3. 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.

  4. Activation of Plant Immune Responses by a Gain-of-Function Mutation in an Atypical Receptor-Like Kinase1[C][W][OA

    PubMed Central

    Bi, Dongling; Cheng, Yu Ti; Li, Xin; Zhang, Yuelin

    2010-01-01

    Arabidopsis (Arabidopsis thaliana) suppressor of npr1-1, constitutive1 (snc1) contains a gain-of-function mutation in a Toll/interleukin receptor-nucleotide binding site-leucine-rich repeat Resistance (R) protein and it has been a useful tool for dissecting R-protein-mediated immunity. Here we report the identification and characterization of snc4-1D, a semidominant mutant with snc1-like phenotypes. snc4-1D constitutively expresses defense marker genes PR1, PR2, and PDF1.2, and displays enhanced pathogen resistance. Map-based cloning of SNC4 revealed that it encodes an atypical receptor-like kinase with two predicted extracellular glycerophosphoryl diester phosphodiesterase domains. The snc4-1D mutation changes an alanine to threonine in the predicted cytoplasmic kinase domain. Wild-type plants transformed with the mutant snc4-1D gene displayed similar phenotypes as snc4-1D, suggesting that the mutation is a gain-of-function mutation. Epistasis analysis showed that NON-RACE-SPECIFIC DISEASE RESISTANCE1 is required for the snc4-1D mutant phenotypes. In addition, the snc4-1D mutant phenotypes are partially suppressed by knocking out MAP KINASE SUBSTRATE1, a positive defense regulator associated with MAP KINASE4. Furthermore, both the morphology and constitutive pathogen resistance of snc4-1D are partially suppressed by blocking jasmonic acid synthesis, suggesting that jasmonic acid plays an important role in snc4-1D-mediated resistance. Identification of snc4-1D provides us a unique genetic system for analyzing the signal transduction pathways downstream of receptor-like kinases. PMID:20508139

  5. BAM 1 and RECEPTOR-LIKE PROTEIN KINASE 2 constitute a signaling pathway and modulate CLE peptide-triggered growth inhibition in Arabidopsis root.

    PubMed

    Shimizu, Noriko; Ishida, Takashi; Yamada, Masashi; Shigenobu, Shuji; Tabata, Ryo; Kinoshita, Atsuko; Yamaguchi, Katsushi; Hasebe, Mitsuyasu; Mitsumasu, Kanako; Sawa, Shinichiro

    2015-12-01

    Ligand receptor-based signaling is a means of cell-to-cell communication for coordinating developmental and physiological processes in multicellular organisms. In plants, cell-producing meristems utilize this signaling to regulate their activities and ensure for proper development. Shoot and root systems share common requirements for carrying out this process; however, its molecular basis is largely unclear. It has been suggested that synthetic CLV3/EMBRYO SURROUNDING REGION (CLE) peptide shrinks the root meristem through the actions of CLAVATA2 (CLV2) and the RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) pathway in Arabidopsis thaliana. Our genetic screening for mutations that resist CLE peptide signaling in roots determined that BAM1, which is a member of the leucine-rich repeat receptor-like kinase (LRR-RLK) family, is also involved in this pathway. BAM1 is preferentially expressed in the root tip, including the quiescent center and its surrounding stem cells. Our genetic analysis revealed that BAM1 functions together with RPK2. Using coimmunoprecipitation assay, we showed that BAM1 is capable of forming heteromeric complexes with RPK2. These findings suggest that the BAM1 and RPK2 receptors constitute a signaling pathway that modulates cell proliferation in the root meristem and that related molecules are employed in root and shoot meristems. PMID:26083273

  6. Over-expression of an S-domain receptor-like kinase extracellular domain improves panicle architecture and grain yield in rice

    PubMed Central

    Zou, Xiaohua; Qin, Zhengrui; Zhang, Chunyu; Liu, Bin; Liu, Jun; Zhang, Chengsheng; Lin, Chentao; Li, Hongyu; Zhao, Tao

    2015-01-01

    The S-domain receptor kinase (SRK) comprises a highly polymorphic subfamily of receptor-like kinases (RLKs) originally found to be involved in the self-incompatibility response in Brassica. Although several members have been identified to play roles in developmental control and disease responses, the correlation between SRKs and yield components in rice is still unclear. The utility of transgenic expression of a dominant negative form of SRK, OsLSK1 (Large spike S-domain receptor like Kinase 1), is reported here for the improvement of grain yield components in rice. OsLSK1 was highly expressed in nodes of rice and is a plasma membrane protein. The expression of OsLSK1 responded to the exogenous application of growth hormones, to abiotic stresses, and its extracellular domain could form homodimers or heterodimers with other related SRKs. Over-expression of a truncated version of OsLSK1 (including the extracellular and transmembrane domain of OsLSK1 without the intracellular kinase domain) increased plant height and improve yield components, including primary branches per panicle and grains per primary branch, resulting in about a 55.8% increase of the total grain yield per plot (10 plants). Transcriptional analysis indicated that several key genes involved in the GA biosynthetic and signalling pathway were up-regulated in transgenic plants. However, full-length cDNA over-expression and RNAi of OsLSK1 transgenic plants did not exhibit a detectable visual phenotype and possible reasons for this were discussed. These results indicate that OsLSK1 may act redundantly with its homologues to affect yield traits in rice and manipulation of OsLSK1 by the dominant negative method is a practicable strategy to improve grain yield in rice and other crops. PMID:26428067

  7. An S-domain receptor-like kinase, OsSIK2, confers abiotic stress tolerance and delays dark-induced leaf senescence in rice.

    PubMed

    Chen, Li-Juan; Wuriyanghan, Hada; Zhang, Yu-Qin; Duan, Kai-Xuan; Chen, Hao-Wei; Li, Qing-Tian; Lu, Xiang; He, Si-Jie; Ma, Biao; Zhang, Wan-Ke; Lin, Qing; Chen, Shou-Yi; Zhang, Jin-Song

    2013-12-01

    Receptor-like kinases play important roles in plant development and defense responses; however, their functions in other processes remain unclear. Here, we report that OsSIK2, an S-domain receptor-like kinase from rice (Oryza sativa), is involved in abiotic stress and the senescence process. OsSIK2 is a plasma membrane-localized protein with kinase activity in the presence of Mn(2+). OsSIK2 is expressed mainly in rice leaf and sheath and can be induced by NaCl, drought, cold, dark, and abscisic acid treatment. Transgenic plants overexpressing OsSIK2 and mutant sik2 exhibit enhanced and reduced tolerance to salt and drought stress, respectively, compared with the controls. Interestingly, a truncated version of OsSIK2 without most of the extracellular region confers higher salt tolerance than the full-length OsSIK2, likely through the activation of different sets of downstream genes. Moreover, seedlings of OsSIK2-overexpressing transgenic plants exhibit early leaf development and a delayed dark-induced senescence phenotype, while mutant sik2 shows the opposite phenotype. The downstream PR-related genes specifically up-regulated by full-length OsSIK2 or the DREB-like genes solely enhanced by truncated OsSIK2 are all induced by salt, drought, and dark treatments. These results indicate that OsSIK2 may integrate stress signals into a developmental program for better adaptive growth under unfavorable conditions. Manipulation of OsSIK2 should facilitate the improvement of production in rice and other crops.

  8. Identification of a receptor-like protein kinase gene rapidly induced by abscisic acid, dehydration, high salt, and cold treatments in Arabidopsis thaliana.

    PubMed Central

    Hong, S W; Jon, J H; Kwak, J M; Nam, H G

    1997-01-01

    A cDNA clone for a receptor-like protein kinase gene (RPK1) was isolated from Arabidopsis thaliana. The clone is 1952 bp long with 1623 bp of an open reading frame encoding a peptide of 540 amino acids. The deduced peptide (RPK1) contains four distinctive domains characteristic of receptor kinases: (a) a putative amino-terminal signal sequence domain; (b) a domain with five extracellular leucine-rich repeat sequences; (c) a membrane-spanning domain; and (d) a cytoplasmic protein kinase domain that contains all of the 11 subdomains conserved among protein kinases. The RPK1 gene is expressed in flowers, stems, leaves, and roots. Expression of the RPK1 gene is induced within 1 h after treatment with abscisic acid (ABA). The gene is also rapidly induced by several environmental stresses such as dehydration, high salt, and low temperature, suggesting that the gene is involved in a general stress response. The dehydration-induced expression is not impaired in aba-1, abi1-1, abi2-1, and abi3-1 mutants, suggesting that the dehydration-induced expression of the RPK1 gene is ABA-independent. A possible role of this gene in the signal transduction pathway of ABA and the environmental stresses is discussed. PMID:9112773

  9. Antisense-oligonucleotide mediated exon skipping in activin-receptor-like kinase 2: inhibiting the receptor that is overactive in fibrodysplasia ossificans progressiva.

    PubMed

    Shi, Songting; Cai, Jie; de Gorter, David J J; Sanchez-Duffhues, Gonzalo; Kemaladewi, Dwi U; Hoogaars, Willem M H; Aartsma-Rus, Annemieke; 't Hoen, Peter A C; ten Dijke, Peter

    2013-01-01

    Fibrodysplasia ossificans progressiva (FOP) is a rare heritable disease characterized by progressive heterotopic ossification of connective tissues, for which there is presently no definite treatment. A recurrent activating mutation (c.617G→A; R206H) of activin receptor-like kinase 2 (ACVR1/ALK2), a BMP type I receptor, has been shown as the main cause of FOP. This mutation constitutively activates the BMP signaling pathway and initiates the formation of heterotopic bone. In this study, we have designed antisense oligonucleotides (AONs) to knockdown mouse ALK2 expression by means of exon skipping. The ALK2 AON could induce exon skipping in cells, which was accompanied by decreased ALK2 mRNA levels and impaired BMP signaling. In addition, the ALK2 AON potentiated muscle differentiation and repressed BMP6-induced osteoblast differentiation. Our results therefore provide a potential therapeutic approach for the treatment of FOP disease by reducing the excessive ALK2 activity in FOP patients.

  10. 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

  11. 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

  12. GsSRK, a G-type lectin S-receptor-like serine/threonine protein kinase, is a positive regulator of plant tolerance to salt stress.

    PubMed

    Sun, Xiao-Li; Yu, Qing-Yue; Tang, Li-Li; Ji, Wei; Bai, Xi; Cai, Hua; Liu, Xiao-Fei; Ding, Xiao-Dong; Zhu, Yan-Ming

    2013-03-15

    Receptor-like protein kinases (RLKs) play vital roles in sensing outside signals, yet little is known about RLKs functions and roles in stress signal perception and transduction in plants, especially in wild soybean. Through the microarray analysis, GsSRK was identified as an alkaline (NaHCO3)-responsive gene, and was subsequently isolated from Glycine soja by homologous cloning. GsSRK encodes a 93.22kDa protein with a highly conserved serine/threonine protein kinase catalytic domain, a G-type lectin region, and an S-locus region. Real-time PCR results showed that the expression levels of GsSRK were largely induced by ABA, salt, and drought stresses. Over expression of GsSRK in Arabidopsis promoted seed germination, as well as primary root and rosette leaf growth during the early stages of salt stress. Compared to the wild type Arabidopsis, GsSRK overexpressors exhibited enhanced salt tolerance and higher yields under salt stress, with higher chlorophyll content, lower ion leakage, higher plant height, and more siliques at the adult developmental stage. Our studies suggest that GsSRK plays a crucial role in plant response to salt stress.

  13. 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.

  14. The Receptor-Like Kinase SIT1 Mediates Salt Sensitivity by Activating MAPK3/6 and Regulating Ethylene Homeostasis in Rice.

    PubMed

    Li, Chen-Hui; Wang, Geng; Zhao, Ji-Long; Zhang, Li-Qing; Ai, Lian-Feng; Han, Yong-Feng; Sun, Da-Ye; Zhang, Sheng-Wei; Sun, Ying

    2014-06-01

    High salinity causes growth inhibition and shoot bleaching in plants that do not tolerate high salt (glycophytes), including most crops. The molecules affected directly by salt and linking the extracellular stimulus to intracellular responses remain largely unknown. Here, we demonstrate that rice (Oryza sativa) Salt Intolerance 1 (SIT1), a lectin receptor-like kinase expressed mainly in root epidermal cells, mediates salt sensitivity. NaCl rapidly activates SIT1, and in the presence of salt, as SIT1 kinase activity increased, plant survival decreased. Rice MPK3 and MPK6 function as the downstream effectors of SIT1. SIT1 phosphorylates MPK3 and 6, and their activation by salt requires SIT1. SIT1 mediates ethylene production and salt-induced ethylene signaling. SIT1 promotes accumulation of reactive oxygen species (ROS), leading to growth inhibition and plant death under salt stress, which occurred in an MPK3/6- and ethylene signaling-dependent manner in Arabidopsis thaliana. Our findings demonstrate the existence of a SIT1-MPK3/6 cascade that mediates salt sensitivity by affecting ROS and ethylene homeostasis and signaling. These results provide important information for engineering salt-tolerant crops. PMID:24907341

  15. Splicing of Receptor-Like Kinase-Encoding SNC4 and CERK1 is Regulated by Two Conserved Splicing Factors that Are Required for Plant Immunity

    PubMed Central

    Zhang, Zhibin; Liu, Yanan; Ding, Pingtao; Li, Yan; Kong, Qing; Zhang, Yuelin

    2014-01-01

    Plant immune receptors belonging to the receptor-like kinase (RLK) family play important roles in the recognition of microbial pathogens and activation of downstream defense responses. The Arabidopsis mutant snc4-1D contains a gain-of-function mutation in the RLK SNC4 (SUPPRESSOR OF NPR1-1, CONSTITUTIVE4), which leads to constitutive activation of defense responses. Analysis of suppressor mutants of snc4-1D identified two conserved splicing factors, SUA (SUPPRESSOR OF ABI3-5) and RSN2 (REQUIRED FOR SNC4-1D 2), that are required for the constitutive defense responses in snc4-1D. In sua and rsn2 mutants, SNC4 splicing is altered and the amount of SNC4 transcripts is reduced. Further analysis showed that SUA and RSN2 are also required for the proper splicing of CERK1 (CHITIN ELICITOR RECEPTOR KINASE1), which encodes another RLK that functions as a receptor for chitin. In sua and rsn2 mutants, induction of reactive oxygen species by chitin is reduced and the non-pathogenic bacteria Pseudomonas syringae pv. tomato DC3000hrcC grows to higher titers than in wild-type plants. Our study suggests that pre-mRNA splicing plays important roles in the regulation of plant immunity mediated by the RLKs SNC4 and CERK1. PMID:25267732

  16. Splicing of receptor-like kinase-encoding SNC4 and CERK1 is regulated by two conserved splicing factors that are required for plant immunity.

    PubMed

    Zhang, Zhibin; Liu, Yanan; Ding, Pingtao; Li, Yan; Kong, Qing; Zhang, Yuelin

    2014-12-01

    Plant immune receptors belonging to the receptor-like kinase (RLK) family play important roles in the recognition of microbial pathogens and activation of downstream defense responses. The Arabidopsis mutant snc4-1D contains a gain-of-function mutation in the RLK SNC4 (SUPPRESSOR OF NPR1-1, CONSTITUTIVE4), which leads to constitutive activation of defense responses. Analysis of suppressor mutants of snc4-1D identified two conserved splicing factors, SUA (SUPPRESSOR OF ABI3-5) and RSN2 (REQUIRED FOR SNC4-1D 2), that are required for the constitutive defense responses in snc4-1D. In sua and rsn2 mutants, SNC4 splicing is altered and the amount of SNC4 transcripts is reduced. Further analysis showed that SUA and RSN2 are also required for the proper splicing of CERK1 (CHITIN ELICITOR RECEPTOR KINASE1), which encodes another RLK that functions as a receptor for chitin. In sua and rsn2 mutants, induction of reactive oxygen species by chitin is reduced and the non-pathogenic bacteria Pseudomonas syringae pv. tomato DC3000hrcC grows to higher titers than in wild-type plants. Our study suggests that pre-mRNA splicing plays important roles in the regulation of plant immunity mediated by the RLKs SNC4 and CERK1. PMID:25267732

  17. A plant receptor-like gene, the S-locus receptor kinase of Brassica oleracea L. , encodes a functional serine/threonine kinase

    SciTech Connect

    Stein, J.C.; Nasrallah, J.B. )

    1993-03-01

    To investigate the catalytic properties of the Brassica oleracea S-locus receptor kinase (SRK), the authors have expressed the domain that is homologous to protein kinases as a fusion protein in Escherichia coli. Following in vivo labeling of cultures with [sup 32]P-labeled inorganic phosphate, they observed phosphorylation of the fusion protein on serine and threonine, but not on tyrosine. In contrast, labeling was not observed when lysine-524, a residue conserved among all protein kinases, was mutated to arginine, thus confirmed that SRK phosphorylation was the result of intrinsic serine/threonine kinase activity. 26 refs., 3 figs.

  18. Characterization of temperature-sensitive mutants reveals a role for receptor-like kinase SCRAMBLED/STRUBBELIG in coordinating cell proliferation and differentiation during Arabidopsis leaf development.

    PubMed

    Lin, Lin; Zhong, Si-Hui; Cui, Xiao-Feng; Li, Jianming; He, Zu-Hua

    2012-12-01

    The balance between cell proliferation and cell differentiation is essential for leaf patterning. However, identification of the factors coordinating leaf patterning and cell growth behavior is challenging. Here, we characterized a temperature-sensitive Arabidopsis mutant with leaf blade and venation defects. We mapped the mutation to the sub-2 allele of the SCRAMBLED/STRUBBELIG (SCM/SUB) receptor-like kinase gene whose functions in leaf development have not been demonstrated. The sub-2 mutant displayed impaired blade development, asymmetric leaf shape and altered venation patterning under high ambient temperature (30°C), but these defects were less pronounced at normal growth temperature (22°C). Loss of SCM/SUB function results in reduced cell proliferation and abnormal cell expansion, as well as altered auxin patterning. SCM/SUB is initially expressed throughout leaf primordia and becomes restricted to the vascular cells, coinciding with its roles in early leaf patterning and venation formation. Furthermore, constitutive expression of the SCM/SUB gene also restricts organ growth by inhibiting the transition from cell proliferation to expansion. We propose the existence of a SCM/SUB-mediated developmental stage-specific signal for leaf patterning, and highlight the importance of the balance between cell proliferation and differentiation for leaf morphogenesis.

  19. The leucine-rich repeat receptor-like kinase BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 and the cytochrome P450 PHYTOALEXIN DEFICIENT3 contribute to innate immunity to aphids in Arabidopsis.

    PubMed

    Prince, David C; Drurey, Claire; Zipfel, Cyril; Hogenhout, Saskia A

    2014-04-01

    The importance of pathogen-associated molecular pattern-triggered immunity (PTI) against microbial pathogens has been recently demonstrated. However, it is currently unclear if this layer of immunity mediated by surface-localized pattern recognition receptors (PRRs) also plays a role in basal resistance to insects, such as aphids. Here, we show that PTI is an important component of plant innate immunity to insects. Extract of the green peach aphid (GPA; Myzus persicae) triggers responses characteristic of PTI in Arabidopsis (Arabidopsis thaliana). Two separate eliciting GPA-derived fractions trigger induced resistance to GPA that is dependent on the leucine-rich repeat receptor-like kinase BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1)/SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE3, which is a key regulator of several leucine-rich repeat-containing PRRs. BAK1 is required for GPA elicitor-mediated induction of reactive oxygen species and callose deposition. Arabidopsis bak1 mutant plants are also compromised in immunity to the pea aphid (Acyrthosiphon pisum), for which Arabidopsis is normally a nonhost. Aphid-derived elicitors induce expression of PHYTOALEXIN DEFICIENT3 (PAD3), a key cytochrome P450 involved in the biosynthesis of camalexin, which is a major Arabidopsis phytoalexin that is toxic to GPA. PAD3 is also required for induced resistance to GPA, independently of BAK1 and reactive oxygen species production. Our results reveal that plant innate immunity to insects may involve early perception of elicitors by cell surface-localized PRRs, leading to subsequent downstream immune signaling.

  20. The Leucine-Rich Repeat Receptor-Like Kinase BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 and the Cytochrome P450 PHYTOALEXIN DEFICIENT3 Contribute to Innate Immunity to Aphids in Arabidopsis1[C][W][OPEN

    PubMed Central

    Prince, David C.; Drurey, Claire; Zipfel, Cyril; Hogenhout, Saskia A.

    2014-01-01

    The importance of pathogen-associated molecular pattern-triggered immunity (PTI) against microbial pathogens has been recently demonstrated. However, it is currently unclear if this layer of immunity mediated by surface-localized pattern recognition receptors (PRRs) also plays a role in basal resistance to insects, such as aphids. Here, we show that PTI is an important component of plant innate immunity to insects. Extract of the green peach aphid (GPA; Myzus persicae) triggers responses characteristic of PTI in Arabidopsis (Arabidopsis thaliana). Two separate eliciting GPA-derived fractions trigger induced resistance to GPA that is dependent on the leucine-rich repeat receptor-like kinase BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1)/SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE3, which is a key regulator of several leucine-rich repeat-containing PRRs. BAK1 is required for GPA elicitor-mediated induction of reactive oxygen species and callose deposition. Arabidopsis bak1 mutant plants are also compromised in immunity to the pea aphid (Acyrthosiphon pisum), for which Arabidopsis is normally a nonhost. Aphid-derived elicitors induce expression of PHYTOALEXIN DEFICIENT3 (PAD3), a key cytochrome P450 involved in the biosynthesis of camalexin, which is a major Arabidopsis phytoalexin that is toxic to GPA. PAD3 is also required for induced resistance to GPA, independently of BAK1 and reactive oxygen species production. Our results reveal that plant innate immunity to insects may involve early perception of elicitors by cell surface-localized PRRs, leading to subsequent downstream immune signaling. PMID:24586042

  1. Isolation and identification of Triticeae chromosome 1 receptor-like kinase genes (Lrk10) from diploid, tetraploid, and hexaploid species of the genus Avena.

    PubMed

    Cheng, D W; Armstrong, K C; Drouin, G; McElroy, A; Fedak, G; Molnar, S D

    2003-02-01

    The DNA sequence of an extracellular (EXC) domain of an oat (Avena sativa L.) receptor-like kinase (ALrk10) gene was amplified from 23 accessions of 15 Avena species (6 diploid, 6 tetraploid, and 3 hexaploid). Primers were designed from one partial oat ALrk10 clone that had been used to map the gene in hexaploid oat to linkage groups syntenic to Triticeae chromosome 1 and 3. Cluster (phylogenetic) analyses showed that all of the oat DNA sequences amplified with these primers are orthologous to the wheat and barley sequences that are located on chromosome 1 of the Triticeae species. Triticeae chromosome 3 Lrk10 sequences were not amplified using these primers. Cluster analyses provided evidence for multiple copies at a locus. The analysis divided the ALrk EXC sequences into two groups, one of which included AA and AABB genome species and the other CC, AACC, and CCCC genome species. Both groups of sequences were found in hexaploid AACCDD genome species, but not in all accessions. The C genome group was divided into 3 subgroups: (i) the CC diploids and the perennial autotetraploid, Avena macrostachya (this supports other evidence for the presence of the C in this autotetraploid species); (ii) a sequence from Avena maroccana and Avena murphyi and several sequences from different accessions of A. sativa; and (iii) A. murphyi and sequences from A. sativa and Avena sterilis. This suggests a possible polyphyletic origin for A. sativa from the AACC progenitor tetraploids or an origin from a progenitor of the AACC tetraploids. The sequences of the A genome group were not as clearly divided into subgroups. Although a group of sequences from the accession 'SunII' and a sequence from line Pg3, are clearly different from the others, the A genome diploid sequences were interspersed with tetraploid and hexaploid sequences.

  2. 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

  3. 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.

  4. 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...

  5. Subcellular localization and functions of the barley stem rust resistance receptor-like serine/threonine-specific protein kinase Rpg1.

    PubMed

    Nirmala, Jayaveeramuthu; Brueggeman, Robert; Maier, Christina; Clay, Christine; Rostoks, Nils; Kannangara, C Gamini; von Wettstein, Diter; Steffenson, Brian J; Kleinhofs, Andris

    2006-05-01

    The Rpg1 gene confers resistance to many pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici and has protected barley from serious disease losses for over 60 years. Rpg1 encodes a constitutively expressed protein with two tandem kinase domains. Fractionation by differential centrifugation and aqueous two-phase separation of the microsome proteins located Rpg1 mainly in the cytosol but also in the plasma membrane and intracellular membranes. Recombinant Rpg1 autophosphorylates in vitro intramolecularly only serine and threonine amino acids with a preference for Mn(2+) cations and a K(m) of 0.15 and a V(max) of 0.47 nmol.min(-1).mg(-1) protein. The inability of wild-type Rpg1 to transphosphorylate a recombinant Rpg1 inactivated by site-directed mutation confirmed that Rpg1 autophosphorylation proceeds exclusively via an intramolecular mechanism. Site-directed mutagenesis of the two adjacent lysine residues in the ATP anchor of the two-kinase domains established that the first of the two tandem kinase domains is nonfunctional and that lysine 461 of the second domain is the catalytically active residue. Transgenic barley, expressing Rpg1 mutated in either the kinase 1 or 2 domains, were fully susceptible to P. graminis f. sp. tritici revealing requirement of both kinase domains for resistance. In planta-expressed Rpg1 mutant protein confirmed that mutation in domain 2, but not 1, rendered the protein incapable of autophosphorylation.

  6. 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. PMID:25941232

  7. 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

    PubMed Central

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

    2007-01-01

    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 P213, with unit-cell parameters a = b = c = 143.2 Å. PMID:17768341

  8. 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.

  9. IDENTIFICATION OF IN VITRO AUTOPHOSPHORYLATION SITES AND THE EFFECTS OF PHOSPHORYLATION ON THE ARABIDOPSIS CRINKLY4 (ACR4) RECEPTOR-LIKE KINASE INTRACELLULAR DOMAIN: INSIGHTS INTO CONFORMATION, OLIGOMERIZATION AND ACTIVITY

    PubMed Central

    Meyer, Matthew R.; Lichti, Cheryl F.; Townsend, R. Reid; Rao, A. Gururaj

    2011-01-01

    Arabidopsis CRINKLY4 (ACR4) is a receptor-like kinase (RLK) that consists of an extracellular domain and an intracellular domain (ICD) with serine/threonine kinase activity. While genetic and cell biology experiments have demonstrated that ACR4 is important in cell fate specification and overall development of the plant, little is known about the biochemical properties of the kinase domain and the mechanisms that underlie the overall function of the receptor. To complement in planta studies on the function of ACR4, we have expressed the ICD in Escherichia coli as a soluble C-terminal fusion to the N-utilization substance A (NusA) protein, purified the recombinant protein and characterized the enzymatic and conformational properties. The protein autophosphorylates via an intramolecular mechanism, prefers Mn2+ over Mg2+ as the divalent cation and displays typical Michaelis-Menten kinetics with respect to ATP with an apparent Km of 6.67 ± 2.07 μM and Vmax of 1.83 ± 0.18 nmol/min/mg. Autophosphorylation is accompanied by a conformational change as demonstrated by circular dichroism, fluorescence spectroscopy and limited proteolysis with trypsin. Analysis by nano-liquid chromatography mass spectrometry (nano-LC-MS) revealed 16 confirmed sites of phosphorylation at Ser and Thr residues. Sedimentation velocity and gel-filtration experiments indicate that the ICD has a propensity to oligomerize and that this property is lost upon autophosphorylation. PMID:21294549

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

    PubMed

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

    2016-03-01

    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

  11. 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

  12. 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

    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

  13. 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

  14. 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.

  15. 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...

  16. P7170: A Novel Molecule with Unique Profile of mTORC1/C2 and Activin Receptor-like Kinase 1 Inhibition Leading to Antitumor and Antiangiogenic Activity.

    PubMed

    Jalota-Badhwar, Archana; Bhatia, Dimple R; Boreddy, Srinivas; Joshi, Asavari; Venkatraman, Magesh; Desai, Nikesh; Chaudhari, Sarika; Bose, Julie; Kolla, Lakshmi S; Deore, Vijaykumar; Yewalkar, Nilambari; Kumar, Sanjay; Sharma, Rajiv; Damre, Anagha; More, Avinash; Sharma, Somesh; Agarwal, Veena R

    2015-05-01

    The mTOR pathway is often upregulated in cancer and thus intensively pursued as a target to design novel anticancer therapies. Approved and emerging drugs targeting the mTOR pathway have positively affected the clinical landscape. Recently, activin receptor-like kinase 1 (ALK1), belonging to the TGFβ receptor family, has been reported as an emerging target for antiangiogenic cancer therapy. Here, we describe a novel orally efficacious compound, P7170, that inhibits mTORC1/mTORC2/ALK1 activity with a potent cell growth inhibition. In cell-based assays, P7170 strongly inhibited (IC50 < 10 nmol/L) the phosphorylation of p70S6K (T389) and pAKT (S473). In many cancer cell lines, such as prostate, ovarian, colon, and renal, P7170 treatment resulted in marked cell growth inhibition. Furthermore, it induced G1-S cell-cycle arrest and autophagy. In vitro HUVEC tube formation, in vivo Matrigel plug, and rat aorta ring assays demonstrated that P7170 exhibited significant antiangiogenic activity. In addition, ALK1 knockdown studies in HUVEC confirmed that the antiangiogenic activity of P7170 was primarily due to ALK1 inhibition. Strong inhibition of ALK1 in addition to mTORC1/mTORC2 differentiates P7170 in its mechanism of action in comparison with existing inhibitors. In vivo mouse xenograft studies revealed P7170 to exhibit a significant dose-dependent tumor growth inhibition in a broad range of human tumor types when administered orally at 10 to 20 mg/kg doses. The distinctive pharmacological profile with favorable pharmacokinetic parameters and in vivo efficacy makes P7170 an attractive candidate for clinical development. It is currently being tested in phase I clinical studies.

  17. 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.

  18. The receptor like kinase at Rhg1-a/Rfs2 caused pleiotropic resistance to sudden death syndrome and soybean cyst nematode as a transgene by altering signaling responses

    PubMed Central

    2012-01-01

    Background Soybean (Glycine max (L. Merr.)) resistance to any population of Heterodera glycines (I.), or Fusarium virguliforme (Akoi, O’Donnell, Homma & Lattanzi) required a functional allele at Rhg1/Rfs2. H. glycines, the soybean cyst nematode (SCN) was an ancient, endemic, pest of soybean whereas F. virguliforme causal agent of sudden death syndrome (SDS), was a recent, regional, pest. This study examined the role of a receptor like kinase (RLK) GmRLK18-1 (gene model Glyma_18_02680 at 1,071 kbp on chromosome 18 of the genome sequence) within the Rhg1/Rfs2 locus in causing resistance to SCN and SDS. Results A BAC (B73p06) encompassing the Rhg1/Rfs2 locus was sequenced from a resistant cultivar and compared to the sequences of two susceptible cultivars from which 800 SNPs were found. Sequence alignments inferred that the resistance allele was an introgressed region of about 59 kbp at the center of which the GmRLK18-1 was the most polymorphic gene and encoded protein. Analyses were made of plants that were either heterozygous at, or transgenic (and so hemizygous at a new location) with, the resistance allele of GmRLK18-1. Those plants infested with either H. glycines or F. virguliforme showed that the allele for resistance was dominant. In the absence of Rhg4 the GmRLK18-1 was sufficient to confer nearly complete resistance to both root and leaf symptoms of SDS caused by F. virguliforme and provided partial resistance to three different populations of nematodes (mature female cysts were reduced by 30–50%). In the presence of Rhg4 the plants with the transgene were nearly classed as fully resistant to SCN (females reduced to 11% of the susceptible control) as well as SDS. A reduction in the rate of early seedling root development was also shown to be caused by the resistance allele of the GmRLK18-1. Field trials of transgenic plants showed an increase in foliar susceptibility to insect herbivory. Conclusions The inference that soybean has adapted part of an

  19. 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

  20. A receptor-like protein mediates the response to pectin modification by activating brassinosteroid signaling.

    PubMed

    Wolf, Sebastian; van der Does, Dieuwertje; Ladwig, Friederike; Sticht, Carsten; Kolbeck, Andreas; Schürholz, Ann-Kathrin; Augustin, Sebastian; Keinath, Nana; Rausch, Thomas; Greiner, Steffen; Schumacher, Karin; Harter, Klaus; Zipfel, Cyril; Höfte, Herman

    2014-10-21

    The brassinosteroid (BR) signaling module is a central regulator of plant morphogenesis, as indicated by the large number of BR-responsive cell wall-related genes and the severe growth defects of BR mutants. Despite a detailed knowledge of the signaling components, the logic of this auto-/paracrine signaling module in growth control remains poorly understood. Recently, extensive cross-talk with other signaling pathways has been shown, suggesting that the outputs of BR signaling, such as gene-expression changes, are subject to complex control mechanisms. We previously provided evidence for a role of BR signaling in a feedback loop controlling the integrity of the cell wall. Here, we identify the first dedicated component of this feedback loop: a receptor-like protein (RLP44), which is essential for the compensatory triggering of BR signaling upon inhibition of pectin de-methylesterification in the cell wall. RLP44 is required for normal growth and stress responses and connects with the BR signaling pathway, presumably through a direct interaction with the regulatory receptor-like kinase BAK1. These findings corroborate a role for BR in controlling the sensitivity of a feedback signaling module involved in maintaining the physico-chemical homeostasis of the cell wall during cell expansion.

  1. 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

  2. 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

  3. Regulation of mouse gamete interaction by a sperm tyrosine kinase.

    PubMed

    Leyton, L; LeGuen, P; Bunch, D; Saling, P M

    1992-12-15

    A 95-kDa mouse sperm protein has been previously identified as a putative receptor involved in the sperm-egg interactions that lead to fertilization. The ligand for this receptor is the zona pellucida glycoprotein ZP3. This constituent of the oocyte-specific extracellular matrix mediates not only sperm binding to the zona but also triggers acrosomal exocytosis. The latter, also termed the acrosome reaction, is a key regulatory event upon which fertilization is absolutely dependent. Previously, we showed that the 95-kDa protein that binds ZP3 is a substrate for tyrosine kinase, and its phosphotyrosine content increases after sperm-zona pellucida binding. Here, we show the presence of protein tyrosine kinase activity in sperm plasma membranes and in electroeluted 95-kDa protein. The tyrosine kinase activity of the isolated protein is stimulated by solubilized zona pellucida and inhibited by tyrphostin RG-50864, a membrane-permeable tyrosine kinase inhibitor. Furthermore, tyrphostin inhibits zona-triggered acrosomal exocytosis in a dose-dependent manner. These findings indicate that the 95-kDa protein participates in a critical regulatory event of gamete interaction; moreover, our experiments suggest that sperm protein tyrosine kinase may be an excellent target for the control of fertility.

  4. 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...

  5. Beacon interacts with cdc2/cdc28-like kinases.

    PubMed

    Kantham, Lakshmi; Kerr-Bayles, Lyndal; Godde, Nathan; Quick, Melissa; Webb, Ryan; Sunderland, Terry; Bond, Judy; Walder, Ken; Augert, Guy; Collier, Greg

    2003-04-25

    Previously we found elevated beacon gene expression in the hypothalamus of obese Psammomys obesus. Beacon administration into the lateral ventricle of P. obesus stimulated food intake and body weight gain. In the current study we used yeast two-hybrid technology to screen for proteins in the human brain that interact with beacon. CLK4, an isoform of cdc2/cdc28-like kinase family of proteins, was identified as a strong interacting partner for beacon. Using active recombinant proteins and a surface plasmon resonance based detection technique, we demonstrated that the three members of this subfamily of kinases (CLK1, 2, and 4) all interact with beacon. Based on the known sequence and functional properties of beacon and CLKs, we speculate that beacon could either modulate the function of key regulatory molecules such as PTP1B or control the expression patterns of specific genes involved in the central regulation of energy metabolism.

  6. KLIFS: a knowledge-based structural database to navigate kinase-ligand interaction space.

    PubMed

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

    2014-01-23

    Protein kinases regulate the majority of signal transduction pathways in cells and have become important targets for the development of designer drugs. We present a systematic analysis of kinase-ligand interactions in all regions of the catalytic cleft of all 1252 human kinase-ligand cocrystal structures present in the Protein Data Bank (PDB). The kinase-ligand interaction fingerprints and structure database (KLIFS) contains a consistent alignment of 85 kinase ligand binding site residues that enables the identification of family specific interaction features and classification of ligands according to their binding modes. We illustrate how systematic mining of kinase-ligand interaction space gives new insights into how conserved and selective kinase interaction hot spots can accommodate the large diversity of chemical scaffolds in kinase ligands. These analyses lead to an improved understanding of the structural requirements of kinase binding that will be useful in ligand discovery and design studies.

  7. Novel interaction partners of the TPR/MET tyrosine kinase.

    PubMed

    Schaaf, Christian P; Benzing, Jörg; Schmitt, Thomas; Erz, Dorothee H R; Tewes, Magdalena; Bartram, Claus R; Janssen, Johannes W G

    2005-02-01

    A large variety of biological processes is mediated by stimulation of the receptor tyrosine kinase MET. Screening a mouse embryo cDNA library, we were able to identify several novel, putative intracellular TPR/MET-substrates: SNAPIN, DCOHM, VAV-1, Sorting nexin 2, Death associated protein kinase 3, SMC-1, Centromeric protein C, and hTID-1. Interactions as identified by yeast two-hybrid analysis were validated in vitro and in vivo by mammalian two-hybrid studies, a far-western assay and coimmunoprecipitation. Participation in apoptosis-regulating mechanisms through interaction with DAPK-3 and cell cycle control via binding to nuclear proteins such as CENPC and SMC-1 are possible new aspects of intracellular MET signaling.

  8. 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

  9. Pneumococcal phosphoglycerate kinase interacts with plasminogen and its tissue activator.

    PubMed

    Fulde, M; Bernardo-García, N; Rohde, M; Nachtigall, N; Frank, R; Preissner, K T; Klett, J; Morreale, A; Chhatwal, G S; Hermoso, J A; Bergmann, S

    2014-03-01

    Streptococcus pneumoniae is not only a commensal of the nasopharyngeal epithelium, but may also cause life-threatening diseases. Immune-electron microscopy studies revealed that the bacterial glycolytic enzyme, phosphoglycerate kinase (PGK), is localised on the pneumococcal surface of both capsulated and non-capsulated strains and colocalises with plasminogen. Since pneumococci may concentrate host plasminogen (PLG) together with its activators on the bacterial cell surface to facilitate the formation of plasmin, the involvement of PGK in this process was studied. Specific binding of human or murine PLG to strain-independent PGK was documented, and surface plasmon resonance analyses indicated a high affinity interaction with the kringle domains 1-4 of PLG. Crystal structure determination of pneumococcal PGK together with peptide array analysis revealed localisation of PLG-binding site in the N-terminal region and provided structural motifs for the interaction with PLG. Based on structural analysis data, a potential interaction of PGK with tissue plasminogen activator (tPA) was proposed and experimentally confirmed by binding studies, plasmin activity assays and thrombus degradation analyses. PMID:24196407

  10. Twitchin kinase interacts with MAPKAP kinase 2 in Caenorhabditis elegans striated muscle

    PubMed Central

    Matsunaga, Yohei; Qadota, Hiroshi; Furukawa, Miho; Choe, Heejoo (Helen); Benian, Guy M.

    2015-01-01

    In Caenorhabditis elegans, twitchin is a giant polypeptide located in muscle A-bands. The protein kinase of twitchin is autoinhibited by 45 residues upstream (NL) and 60 residues downstream (CRD) of the kinase catalytic core. Molecular dynamics simulation on a twitchin fragment revealed that the NL is released by pulling force. However, it is unclear how the CRD is removed. To identify proteins that may remove the CRD, we performed a yeast two-hybrid screen using twitchin kinase as bait. One interactor is MAK-1, C. elegans orthologue of MAPKAP kinase 2. MAPKAP kinase 2 is phosphorylated and activated by p38 MAP kinase. We demonstrate that the CRD of twitchin is important for binding to MAK-1. mak-1 is expressed in nematode body wall muscle, and antibodies to MAK-1 localize between and around Z-disk analogues and to the edge of A-bands. Whereas unc-22 mutants are completely resistant, mak-1 mutants are partially resistant to nicotine. MAK-1 can phosphorylate twitchin NL-Kin-CRD in vitro. Genetic data suggest the involvement of two other mak-1 paralogues and two orthologues of p38 MAP kinase. These results suggest that MAK-1 is an activator of twitchin kinase and that the p38 MAP kinase pathway may be involved in the regulation of twitchin. PMID:25851606

  11. Chaperones as thermodynamic sensors of drug-target interactions reveal kinase inhibitor specificities in living cells.

    PubMed

    Taipale, Mikko; Krykbaeva, Irina; Whitesell, Luke; Santagata, Sandro; Zhang, Jianming; Liu, Qingsong; Gray, Nathanael S; Lindquist, Susan

    2013-07-01

    The interaction between the HSP90 chaperone and its client kinases is sensitive to the conformational status of the kinase, and stabilization of the kinase fold by small molecules strongly decreases chaperone interaction. Here we exploit this observation and assay small-molecule binding to kinases in living cells, using chaperones as 'thermodynamic sensors'. The method allows determination of target specificities of both ATP-competitive and allosteric inhibitors in the kinases' native cellular context in high throughput. We profile target specificities of 30 diverse kinase inhibitors against >300 kinases. Demonstrating the value of the assay, we identify ETV6-NTRK3 as a target of the FDA-approved drug crizotinib (Xalkori). Crizotinib inhibits proliferation of ETV6-NTRK3-dependent tumor cells with nanomolar potency and induces the regression of established tumor xenografts in mice. Finally, we show that our approach is applicable to other chaperone and target classes by assaying HSP70/steroid hormone receptor and CDC37/kinase interactions, suggesting that chaperone interactions will have broad application in detecting drug-target interactions in vivo.

  12. Serum amyloid A induces contrary immune responses via formyl peptide receptor-like 1 in human monocytes.

    PubMed

    Lee, Ha Young; Kim, Mi-Kyoung; Park, Kyoung Sun; Shin, Eun Ha; Jo, Seong Ho; Kim, Sang Doo; Jo, Eun Jin; Lee, Youl-Nam; Lee, Chuhee; Baek, Suk-Hwan; Bae, Yoe-Sik

    2006-07-01

    Although the level of serum amyloid A has been reported to be up-regulated during inflammatory response, the role of serum amyloid A on the regulation of inflammation and immune response has not been elucidated. We found that serum amyloid A stimulated the production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-10, which are proinflammatory and anti-inflammatory cytokines, respectively, in human monocytes. Low concentrations of serum amyloid A stimulated TNF-alpha production with maximal activity at 6 h after stimulation, whereas high concentrations of serum amyloid A stimulated IL-10 production with maximal activity at 12 h. The activations of the two cytokines by serum amyloid A occurred at both the transcription and translational levels. Signaling events induced by serum amyloid A included the activation of two mitogen-activated protein kinases (extracellular signal-regulated kinase and p38 kinase), which were found to be required for TNF-alpha and IL-10 production, respectively. The stimulation of formyl peptide receptor-like-1-expressing RBL-2H3 cells, but not of vector-expressing RBL-2H3 cells with serum amyloid A, induced mitogen-activated protein kinases activation and the accumulation of the RNAs of these two cytokines. Together, our findings suggest that serum amyloid A modulates contrary immune responses via formyl peptide receptor-like 1, by inducing TNF-alpha or IL-10, and demonstrate that extracellular signal-regulated kinase and p38 kinase play counteracting roles in this process.

  13. 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.

  14. Structural Bioinformatics and Protein Docking Analysis of the Molecular Chaperone-Kinase Interactions: Towards Allosteric Inhibition of Protein Kinases by Targeting the Hsp90-Cdc37 Chaperone Machinery

    PubMed Central

    Lawless, Nathan; Blacklock, Kristin; Berrigan, Elizabeth; Verkhivker, Gennady

    2013-01-01

    A fundamental role of the Hsp90-Cdc37 chaperone system in mediating maturation of protein kinase clients and supporting kinase functional activity is essential for the integrity and viability of signaling pathways involved in cell cycle control and organism development. Despite significant advances in understanding structure and function of molecular chaperones, the molecular mechanisms and guiding principles of kinase recruitment to the chaperone system are lacking quantitative characterization. Structural and thermodynamic characterization of Hsp90-Cdc37 binding with protein kinase clients by modern experimental techniques is highly challenging, owing to a transient nature of chaperone-mediated interactions. In this work, we used experimentally-guided protein docking to probe the allosteric nature of the Hsp90-Cdc37 binding with the cyclin-dependent kinase 4 (Cdk4) kinase clients. The results of docking simulations suggest that the kinase recognition and recruitment to the chaperone system may be primarily determined by Cdc37 targeting of the N-terminal kinase lobe. The interactions of Hsp90 with the C-terminal kinase lobe may provide additional “molecular brakes” that can lock (or unlock) kinase from the system during client loading (release) stages. The results of this study support a central role of the Cdc37 chaperone in recognition and recruitment of the kinase clients. Structural analysis may have useful implications in developing strategies for allosteric inhibition of protein kinases by targeting the Hsp90-Cdc37 chaperone machinery. PMID:24287464

  15. Citron rho-interacting kinase, a novel tissue-specific ser/thr kinase encompassing the Rho-Rac-binding protein Citron.

    PubMed

    Di Cunto, F; Calautti, E; Hsiao, J; Ong, L; Topley, G; Turco, E; Dotto, G P

    1998-11-01

    We have identified a novel serine/threonine kinase belonging to the myotonic dystrophy kinase family. The kinase can be produced in at least two different isoforms: a approximately 240-kDa protein (Citron Rho-interacting kinase, CRIK), in which the kinase domain is followed by the sequence of Citron, a previously identified Rho/Rac binding protein; a approximately 54-kDa protein (CRIK-short kinase (SK)), which consists mostly of the kinase domain. CRIK and CRIK-SK proteins are capable of phosphorylating exogenous substrates as well as of autophosphorylation, when tested by in vitro kinase assays after expression into COS7 cells. CRIK kinase activity is increased severalfold by coexpression of costitutively active Rho, while active Rac has more limited effects. Kinase activity of endogenous CRIK is indicated by in vitro kinase assays after immunoprecipitation with antibodies recognizing the Citron moiety of the protein. When expressed in keratinocytes, full-length CRIK, but not CRIK-SK, localizes into corpuscular cytoplasmic structures and elicits recruitment of actin into these structures. The previously reported Rho-associated kinases ROCK I and II are ubiquitously expressed. In contrast, CRIK exhibits a restricted pattern of expression, suggesting that this kinase may fulfill a more specialized function in specific cell types.

  16. Suppressor of cytokine signaling 1 interacts with oncogenic lymphocyte-specific protein tyrosine kinase.

    PubMed

    Venkitachalam, Srividya; Chueh, Fu-Yu; Leong, King-Fu; Pabich, Samantha; Yu, Chao-Lan

    2011-03-01

    Lymphocyte-specific protein tyrosine kinase (Lck) plays a key role in T cell signal transduction and is tightly regulated by phosphorylation and dephosphorylation. Lck can function as an oncoprotein when overexpressed or constantly activated by mutations. Our previous studies showed that Lck-induced cellular transformation could be suppressed by enforced expression of suppressor of cytokine signaling 1 (SOCS1), a SOCS family member involved in the negative feedback control of cytokine signaling. We observed attenuated Lck kinase activity in SOCS1-expressing cells, suggesting an important role of SOCS in regulating Lck functions. It remains largely unknown whether and how SOCS proteins interact with the oncogenic Lck kinase. Here, we report that among four SOCS family proteins, SOCS1, SOCS2, SOCS3 and CIS (cytokine-inducible SH2 domain containing protein), SOCS1 has the highest affinity in binding to the oncogenic Lck kinase. We identified the positive regulatory phosphotyrosine 394 residue in the kinase domain as the key interacting determinant in Lck. Additionally, the Lck kinase domain alone is sufficient to bind SOCS1. While the SH2 domain in SOCS1 is important in its association with the oncogenic Lck kinase, other functional domains may also contribute to overall binding affinity. These findings provide important mechanistic insights into the role of SOCS proteins as tumor suppressors in cells transformed by oncogenic protein tyrosine kinases.

  17. Pim Kinase Interacts with Nonstructural 5A Protein and Regulates Hepatitis C Virus Entry

    PubMed Central

    Park, Chorong; Min, Saehong; Park, Eun-Mee; Lim, Yun-Sook; Kang, Sangmin; Suzuki, Tetsuro; Shin, Eui-Cheol

    2015-01-01

    ABSTRACT The life cycle of hepatitis C virus (HCV) is highly dependent on host cellular proteins for virus propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assay using the HCV nonstructural 5A (NS5A) protein as a probe. Of ∼9,000 human cellular proteins immobilized in a microarray, approximately 90 cellular proteins were identified as NS5A interactors. Of these candidates, Pim1, a member of serine/threonine kinase family composed of three different isoforms (Pim1, Pim2, and Pim3), was selected for further study. Pim kinases share a consensus sequence which overlaps with kinase activity. Pim kinase activity has been implicated in tumorigenesis. In the present study, we verified the physical interaction between NS5A and Pim1 by both in vitro pulldown and coimmunoprecipitation assays. Pim1 interacted with NS5A through amino acid residues 141 to 180 of Pim1. We demonstrated that protein stability of Pim1 was increased by NS5A protein and this increase was mediated by protein interplay. Small interfering RNA (siRNA)-mediated knockdown or pharmacological inhibition of Pim kinase abrogated HCV propagation. By employing HCV pseudoparticle entry and single-cycle HCV infection assays, we further demonstrated that Pim kinase was involved in HCV entry at a postbinding step. These data suggest that Pim kinase may represent a new host factor for HCV entry. IMPORTANCE Pim1 is an oncogenic serine/threonine kinase. HCV NS5A protein physically interacts with Pim1 and contributes to Pim1 protein stability. Since Pim1 protein expression level is upregulated in many cancers, NS5A-mediated protein stability may be associated with HCV pathogenesis. Either gene silencing or chemical inhibition of Pim kinase abrogated HCV propagation in HCV-infected cells. We further showed that Pim kinase was specifically required at an early entry step of the HCV life cycle. Thus, we have identified Pim kinase not only as an HCV cell

  18. Divergent modulation of Src-family kinase regulatory interactions with ATP-competitive inhibitors.

    PubMed

    Leonard, Stephen E; Register, A C; Krishnamurty, Ratika; Brighty, Gabriel J; Maly, Dustin J

    2014-08-15

    Multidomain protein kinases, central controllers of signal transduction, use regulatory domains to modulate catalytic activity in a complex cellular environment. Additionally, these domains regulate noncatalytic functions, including cellular localization and protein-protein interactions. Src-family kinases (SFKs) are promising therapeutic targets for a number of diseases and are an excellent model for studying the regulation of multidomain kinases. Here, we demonstrate that the regulatory domains of the SFKs Src and Hck are divergently affected by ligands that stabilize two distinct inactive ATP-binding site conformations. Conformation-selective, ATP-competitive inhibitors differentially modulate the ability of the SH3 and SH2 domains of Src and Hck to engage in intermolecular interactions and the ability of the kinase-inhibitor complex to undergo post-translational modification by effector enzymes. This surprising divergence in regulatory domain behavior by two classes of inhibitors that each stabilize inactive ATP-binding site conformations is found to occur through perturbation or stabilization of the αC helix. These studies provide insight into how conformation-selective, ATP-competitive inhibitors can be designed to modulate domain interactions and post-translational modifications distal to the ATP-binding site of kinases.

  19. 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. PMID:27408918

  20. 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.

  1. 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.

  2. The Interaction between Cyclin B1 and Cytomegalovirus Protein Kinase pUL97 is Determined by an Active Kinase Domain.

    PubMed

    Steingruber, Mirjam; Socher, Eileen; Hutterer, Corina; Webel, Rike; Bergbrede, Tim; Lenac, Tihana; Sticht, Heinrich; Marschall, Manfred

    2015-08-11

    Replication of human cytomegalovirus (HCMV) is characterized by a tight virus-host cell interaction. Cyclin-dependent protein kinases (CDKs) are functionally integrated into viral gene expression and protein modification. The HCMV-encoded protein kinase pUL97 acts as a CDK ortholog showing structural and functional similarities. Recently, we reported an interaction between pUL97 kinase with a subset of host cyclins, in particular with cyclin T1. Here, we describe an interaction of pUL97 at an even higher affinity with cyclin B1. As a striking feature, the interaction between pUL97 and cyclin B1 proved to be strictly dependent on pUL97 activity, as interaction could be abrogated by treatment with pUL97 inhibitors or by inserting mutations into the conserved kinase domain or the nonconserved C-terminus of pUL97, both producing loss of activity. Thus, we postulate that the mechanism of pUL97-cyclin B1 interaction is determined by an active pUL97 kinase domain.

  3. How to awaken your nanomachines: Site-specific activation of focal adhesion kinases through ligand interactions.

    PubMed

    Walkiewicz, Katarzyna W; Girault, Jean-Antoine; Arold, Stefan T

    2015-10-01

    The focal adhesion kinase (FAK) and the related protein-tyrosine kinase 2-beta (Pyk2) are highly versatile multidomain scaffolds central to cell adhesion, migration, and survival. Due to their key role in cancer metastasis, understanding and inhibiting their functions are important for the development of targeted therapy. Because FAK and Pyk2 are involved in many different cellular functions, designing drugs with partial and function-specific inhibitory effects would be desirable. Here, we summarise recent progress in understanding the structural mechanism of how the tug-of-war between intramolecular and intermolecular interactions allows these protein 'nanomachines' to become activated in a site-specific manner.

  4. 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

    2016-09-01

    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.

  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. Suppression of death-associated protein kinase 2 by interaction with 14-3-3 proteins.

    PubMed

    Yuasa, Keizo; Ota, Reina; Matsuda, Shinya; Isshiki, Kinuka; Inoue, Masahiro; Tsuji, Akihiko

    2015-08-14

    Death-associated protein kinase 2 (DAPK2), a Ca(2+)/calmodulin-regulated serine/threonine kinase, induces apoptosis. However, the signaling mechanisms involved in this process are unknown. Using a proteomic approach, we identified 14-3-3 proteins as novel DAPK2-interacting proteins. The 14-3-3 family has the ability to bind to phosphorylated proteins via recognition of three conserved amino acid motifs (mode 1-3 motifs), and DAPK2 contains the mode 3 motif ((pS/pT)X1-2-COOH). The interaction of 14-3-3 proteins with DAPK2 was dependent on the phosphorylation of Thr(369), and effectively suppressed DAPK2 kinase activity and DAPK2-induced apoptosis. Furthermore, we revealed that the 14-3-3 binding site Thr(369) of DAPK2 was phosphorylated by the survival kinase Akt. Our findings suggest that DAPK2-induced apoptosis is negatively regulated by Akt and 14-3-3 proteins.

  7. 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.

  8. Intrinsic cleavage of receptor-interacting protein kinase-1 by caspase-6.

    PubMed

    van Raam, B J; Ehrnhoefer, D E; Hayden, M R; Salvesen, G S

    2013-01-01

    Necroptosis is a form of programmed cell death that occurs in the absence of caspase activation and depends on the activity of the receptor-interacting protein kinases. Inactivation of these kinases by caspase-mediated cleavage has been shown to be essential for successful embryonic development, survival and activation of certain cell types. The initiator of extrinsic apoptosis, caspase-8, which has a pro-death as well as a pro-life function, has been assigned this role. In the present study we demonstrate that caspase-6, an executioner caspase, performs this role during apoptosis induced through the intrinsic pathway. In addition, we demonstrate that in the absence of caspase activity, intrinsic triggers of apoptosis induce the receptor-interacting-kinase-1-dependent production of pro-inflammatory cytokines. We show that ubiquitously expressed caspase-6 has a supporting role in apoptosis by cleaving this kinase, thus preventing production of inflammatory cytokines as well as inhibiting the necroptotic pathway. These findings shed new light on the regulation of necroptosis as well as cell death in an inflammatory environment wherein cells receive both intrinsic and extrinsic death signals.

  9. 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.

  10. Intermolecular and Intramolecular Interactions Regulate Catalytic Activity of Myotonic Dystrophy Kinase-Related Cdc42-Binding Kinase α

    PubMed Central

    Tan, Ivan; Seow, Kah Tong; Lim, Louis; Leung, Thomas

    2001-01-01

    Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) is a Cdc42-binding serine/threonine kinase with multiple functional domains. We had previously shown MRCKα to be implicated in Cdc42-mediated peripheral actin formation and neurite outgrowth in HeLa and PC12 cells, respectively. Here we demonstrate that native MRCK exists in high-molecular-weight complexes. We further show that the three independent coiled-coil (CC) domains and the N-terminal region preceding the kinase domain are responsible for intermolecular interactions leading to MRCKα multimerization. N terminus-mediated dimerization and consequent transautophosphorylation are critical processes regulating MRCKα catalytic activities. A region containing the two distal CC domains (CC2 and CC3; residues 658 to 930) was found to interact intramolecularly with the kinase domain and negatively regulates its activity. Its deletion also resulted in an active kinase, confirming a negative autoregulatory role. We provide evidence that the N terminus-mediated dimerization and activation of MRCK and the negative autoregulatory kinase–distal CC interaction are two mutually exclusive events that tightly regulate the catalytic state of the kinase. Disruption of this interaction by a mutant kinase domain resulted in increased kinase activity. MRCK kinase activity was also elevated when cells were treated with phorbol ester, which can interact directly with a cysteine-rich domain next to the distal CC domain. We therefore suggest that binding of phorbol ester to MRCK releases its autoinhibition, allowing N-terminal dimerization and subsequent kinase activation. PMID:11283256

  11. 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.

  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. PMID:19836940

  13. 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.

  14. Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases.

    PubMed

    Zeke, András; Bastys, Tomas; Alexa, Anita; Garai, Ágnes; Mészáros, Bálint; Kirsch, Klára; Dosztányi, Zsuzsanna; Kalinina, Olga V; Reményi, Attila

    2015-11-01

    Mitogen-activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less-characterized disordered regions. We used a structurally consistent model on kinase-docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under-explored part of the human proteome and applied experimental tools specifically tailored to detect low-affinity protein-protein interactions for their validation in vitro and in cell-based assays. The combined computational and experimental approach enabled the identification of many novel MAPK-docking motifs that were elusive for other large-scale protein-protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase-mediated partnerships evolved over time. The analysis suggests that most human MAPK-binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK-binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles. PMID:26538579

  15. HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck

    PubMed Central

    2012-01-01

    Background Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association. Results To test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the Km for ATP as well as enhanced inhibitor potency. Conclusions These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting. PMID:22420777

  16. 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.

  17. 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-...

  18. Oestrogen receptors interact with the α-catalytic subunit of AMP-activated protein kinase

    PubMed Central

    Lipovka, Yulia; Chen, Hao; Vagner, Josef; Price, Theodore J.; Tsao, Tsu-Shuen; Konhilas, John P.

    2015-01-01

    Normal and pathological stressors engage the AMP-activated protein kinase (AMPK) signalling axis to protect the cell from energetic pressures. Sex steroid hormones also play a critical role in energy metabolism and significantly modify pathological progression of cardiac disease, diabetes/obesity and cancer. AMPK is targeted by 17β-oestradiol (E2), the main circulating oestrogen, but the mechanism by which E2 activates AMPK is currently unknown. Using an oestrogen receptor α/β (ERα/β) positive (T47D) breast cancer cell line, we validated E2-dependent activation of AMPK that was mediated through ERα (not ERβ) by using three experimental strategies. A series of co-immunoprecipitation experiments showed that both ERs associated with AMPK in cancer and striated (skeletal and cardiac) muscle cells. We further demonstrated direct binding of ERs to the α-catalytic subunit of AMPK within the βγ-subunit-binding domain. Finally, both ERs interacted with the upstream liver kinase B 1 (LKB1) kinase complex, which is required for E2-dependent activation of AMPK. We conclude that E2 activates AMPK through ERα by direct interaction with the βγ-binding domain of AMPKα. PMID:26374855

  19. Focal Adhesion Kinase Is Involved in Rabies Virus Infection through Its Interaction with Viral Phosphoprotein P

    PubMed Central

    Fouquet, Baptiste; Nikolic, Jovan; Larrous, Florence; Bourhy, Hervé; Wirblich, Christoph

    2014-01-01

    ABSTRACT The rabies virus (RABV) phosphoprotein P is a multifunctional protein: it plays an essential role in viral transcription and replication, and in addition, RABV P has been identified as an interferon antagonist. Here, a yeast two-hybrid screen revealed that RABV P interacts with the focal adhesion kinase (FAK). The binding involved the 106-to-131 domain, corresponding to the dimerization domain of P and the C-terminal domain of FAK containing the proline-rich domains PRR2 and PRR3. The P-FAK interaction was confirmed in infected cells by coimmunoprecipitation and colocalization of FAK with P in Negri bodies. By alanine scanning, we identified a single mutation in the P protein that abolishes this interaction. The mutant virus containing a substitution of Ala for Arg in position 109 in P (P.R109A), which did not interact with FAK, is affected at a posttranscriptional step involving protein synthesis and viral RNA replication. Furthermore, FAK depletion inhibited viral protein expression in infected cells. This provides the first evidence of an interaction of RABV with FAK that positively regulates infection. IMPORTANCE Rabies virus exhibits a small genome that encodes a limited number of viral proteins. To maintain efficient virus replication, some of them are multifunctional, such as the phosphoprotein P. We and others have shown that P establishes complex networks of interactions with host cell components. These interactions have revealed much about the role of P and about host-pathogen interactions in infected cells. Here, we identified another cellular partner of P, the focal adhesion kinase (FAK). Our data shed light on the implication of FAK in RABV infection and provide evidence that P-FAK interaction has a proviral function. PMID:25410852

  20. 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

  1. Mapping of protein-protein interactions within the DNA-dependent protein kinase complex.

    PubMed Central

    Gell, D; Jackson, S P

    1999-01-01

    In mammalian cells, the Ku and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) proteins are required for the correct and efficient repair of DNA double-strand breaks. Ku comprises two tightly-associated subunits of approximately 69 and approximately 83 kDa, which are termed Ku70 and Ku80 (or Ku86), respectively. Previously, a number of regions of both Ku subunits have been demonstrated to be involved in their interaction, but the molecular mechanism of this interaction remains unknown. We have identified a region in Ku70 (amino acid residues 449-578) and a region in Ku80 (residues 439-592) that participate in Ku subunit interaction. Sequence analysis reveals that these interaction regions share sequence homology and suggests that the Ku subunits are structurally related. On binding to a DNA double-strand break, Ku is able to interact with DNA-PKcs, but how this interaction is mediated has not been defined. We show that the extreme C-terminus of Ku80, specifically the final 12 amino acid residues, mediates a highly specific interaction with DNA-PKcs. Strikingly, these residues appear to be conserved only in Ku80 sequences from vertebrate organisms. These data suggest that Ku has evolved to become part of the DNA-PK holo-enzyme by acquisition of a protein-protein interaction motif at the C-terminus of Ku80. PMID:10446239

  2. 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.

  3. Targeting protein–protein interactions in complexes organized by A kinase anchoring proteins

    PubMed Central

    Calejo, Ana I.; Taskén, Kjetil

    2015-01-01

    Cyclic AMP is a ubiquitous intracellular second messenger involved in the regulation of a wide variety of cellular processes, a majority of which act through the cAMP – protein kinase A (PKA) signaling pathway and involve PKA phosphorylation of specific substrates. PKA phosphorylation events are typically spatially restricted and temporally well controlled. A-kinase anchoring proteins (AKAPs) directly bind PKA and recruit it to specific subcellular loci targeting the kinase activity toward particular substrates, and thereby provide discrete spatiotemporal control of downstream phosphorylation events. AKAPs also scaffold other signaling molecules into multi-protein complexes that function as crossroads between different signaling pathways. Targeting AKAP coordinated protein complexes with high-affinity peptidomimetics or small molecules to tease apart distinct protein–protein interactions (PPIs) therefore offers important means to disrupt binding of specific components of the complex to better understand the molecular mechanisms involved in the function of individual signalosomes and their pathophysiological role. Furthermore, development of novel classes of small molecules involved in displacement of AKAP-bound signal molecules is now emerging. Here, we will focus on mechanisms for targeting PPI, disruptors that modulate downstream cAMP signaling and their role, especially in the heart. PMID:26441649

  4. Drosophila Tribbles Antagonizes Insulin Signaling-Mediated Growth and Metabolism via Interactions with Akt Kinase

    PubMed Central

    Das, Rahul; Sebo, Zachary; Pence, Laramie; Dobens, Leonard L.

    2014-01-01

    Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation. PMID:25329475

  5. Allosteric Interactions between the Myristate- and ATP-Site of the Abl Kinase

    PubMed Central

    Iacob, Roxana E.; Zhang, Jianming; Gray, Nathanael S.; Engen, John R.

    2011-01-01

    Abl kinase inhibitors targeting the ATP binding pocket are currently employed as potent anti-leukemogenic agents but drug resistance has become a significant clinical limitation. Recently, a compound that binds to the myristate pocket of Abl (GNF-5) was shown to act cooperatively with nilotinib, an ATP-competitive inhibitor to target the recalcitrant “T315I” gatekeeper mutant of Bcr-Abl. To uncover an explanation for how drug binding at a distance from the kinase active site could lead to inhibition and how inhibitors could combine their effects, hydrogen exchange mass spectrometry (HX MS) was employed to monitor conformational effects in the presence of both dasatinib, a clinically approved ATP-site inhibitor, and GNF-5. While dasatinib binding to wild type Abl clearly influenced Abl conformation, no binding was detected between dasatinib and T315I. GNF-5, however, elicited the same conformational changes in both wild type and T315I, including changes to dynamics within the ATP site located approximately 25 Å from the site of GNF-5 interaction. Simultaneous binding of dasatinib and GNF-5 to T315I caused conformational and/or dynamics changes in Abl such that effects of dasatinib on T315I were the same as when it bound to wild type Abl. These results provide strong biophysical evidence that allosteric interactions play a role in Abl kinase downregulation and that targeting sites outside the ATP binding site can provide an important pharmacological tool to overcome mutations that cause resistance to ATP-competitive inhibitors. PMID:21264348

  6. Protein-Protein Interaction for the De Novo Design of Cyclin-Dependent Kinase Peptide Inhibitors.

    PubMed

    Arumugasamy, Karthiga; Tripathi, Sunil Kumar; Singh, Poonam; Singh, Sanjeev Kumar

    2016-01-01

    The homology of the inhibitor binding site regions on the surface of cyclin-dependent kinases (CDKs) makes actual CDK inhibitors unable to bind specifically to their molecular targets. Most of them are ATP competitive inhibitors with low specificity that also affect the phosphorylation mechanisms of other nontarget kinases giving rise to harmful side effects. So, the search of specific and potent inhibitors able to bind to the desired CDK target is still a pending issue. Structure based drug design minimized the erroneous binding and increased the affinity of the inhibitor interaction. In the case of CDKs their activation and regulation mechanisms mainly depend on protein-protein interactions (PPIs). The design of drugs targeting these PPIs makes feasible and promising towards the discovery of new and specific CDK inhibitors. Development of peptide inhibitors for a target protein is an emerging approach in computer aided drug designing. This chapter describes in detail methodology for use of the VitAL-Viterbi algorithm for de novo peptide design of CDK2 inhibitors.

  7. Impact of kinase activating and inactivating patient mutations on binary PKA interactions

    PubMed Central

    Röck, Ruth; Mayrhofer, Johanna E.; Bachmann, Verena; Stefan, Eduard

    2015-01-01

    The second messenger molecule cAMP links extracellular signals to intracellular responses. The main cellular cAMP effector is the compartmentalized protein kinase A (PKA). Upon receptor initiated cAMP-mobilization, PKA regulatory subunits (R) bind cAMP thereby triggering dissociation and activation of bound PKA catalytic subunits (PKAc). Mutations in PKAc or RIa subunits manipulate PKA dynamics and activities which contribute to specific disease patterns. Mutations activating cAMP/PKA signaling contribute to carcinogenesis or hormone excess, while inactivating mutations cause hormone deficiency or resistance. Here we extended the application spectrum of a Protein-fragment Complementation Assay based on the Renilla Luciferase to determine binary protein:protein interactions (PPIs) of the PKA network. We compared time- and dose-dependent influences of cAMP-elevation on mutually exclusive PPIs of PKAc with the phosphotransferase inhibiting RIIb and RIa subunits and the protein kinase inhibitor peptide (PKI). We analyzed PKA dynamics following integration of patient mutations into PKAc and RIa. We observed that oncogenic modifications of PKAc(L206R) and RIa(Δ184-236) as well as rare disease mutations in RIa(R368X) affect complex formation of PKA and its responsiveness to cAMP elevation. With the cell-based PKA PPI reporter platform we precisely quantified the mechanistic details how inhibitory PKA interactions and defined patient mutations contribute to PKA functions. PMID:26347651

  8. 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.

  9. A conserved protein interaction network involving the yeast MAP kinases Fus3 and Kss1.

    PubMed

    Kusari, Anasua B; Molina, Douglas M; Sabbagh, Walid; Lau, Chang S; Bardwell, Lee

    2004-01-19

    The Saccharomyces cerevisiae mitogen-activated protein kinases (MAPKs) Fus3 and Kss1 bind to multiple regulators and substrates. We show that mutations in a conserved docking site in these MAPKs (the CD/7m region) disrupt binding to an important subset of their binding partners, including the Ste7 MAPK kinase, the Ste5 adaptor/scaffold protein, and the Dig1 and Dig2 transcriptional repressors. Supporting the possibility that Ste5 and Ste7 bind to the same region of the MAPKs, they partially competed for Fus3 binding. In vivo, some of the MAPK mutants displayed reduced Ste7-dependent phosphorylation, and all of them exhibited multiple defects in mating and pheromone response. The Kss1 mutants were also defective in Kss1-imposed repression of Ste12. We conclude that MAPKs contain a structurally and functionally conserved docking site that mediates an overall positively acting network of interactions with cognate docking sites on their regulators and substrates. Key features of this interaction network appear to have been conserved from yeast to humans. PMID:14734536

  10. 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.

  11. 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

  12. Ab initio MO studies of interaction mechanisms of Protein Kinase C with cell membranes

    NASA Astrophysics Data System (ADS)

    Tsuda, Ken-ichiro; Kaneko, Hiroki; Shimada, Jiro; Takada, Toshikazu

    2001-12-01

    Protein Kinase C (PKC) is a family of regulatory enzymes. It is considered that binding with phorbol ester which are PKC activators, increases affinity of PKC for the membranes and consequently induces its conformation change. Electrostatic interactions between PKC and the membrane is assumed to be important, and performed ab initio MO calculations of one domain of PKC consisting of 50 amino acids and its complex with the ester is performed to investigate how the electrostatic potential of PKC changes through docking with the substrate. From the calculation, it is shown that the electrostatic potential of PKC near the binding site is dramatically affected through the binding, suggesting attractive interactions with the cell membrane.

  13. Protein kinase–inhibitor database: Structural variability of and inhibitor interactions with the protein kinase P-loop

    PubMed Central

    Patel, Ronak Y.; Doerksen, Robert J.

    2011-01-01

    Structure based drug design of protein-kinase inhibitors has been facilitated by availability of an enormous number of structures in the Protein Databank (PDB), systematic analyses of which can provide insight into the factors which govern ligand–protein kinase interactions and into the conformational variability of the protein kinases. In this study, a non-redundant database containing 755 unique, curated and annotated PDB protein kinase–inhibitor complexes (each consisting of a single protein kinase chain, a ligand and water molecules around the ligand) was created. With this dataset, analyses were performed of protein conformational variability and interactions of ligands with 11 P-loop residues. Analysis of ligand–protein interactions included ligand atom preference, ligand–protein hydrogen bonds and the number and position of crystallographic water molecules around important P-loop residues. Analysis of variability in the conformation of the P-loop considered backbone and side-chain dihedral angles, and solvent accessible surface area (SASA). A distorted conformation of the P-loop was observed for some of the protein kinase structures. Lower SASA was observed for the hydrophobic residue in β1 of several members of the AGC family of protein kinases. Our systematic studies were performed amino-acid by amino-acid, which is unusual for analyses of protein kinase–inhibitor complexes. PMID:20681595

  14. Tousled-like kinase in a microbial eukaryote regulates spindle assembly and S-phase progression by interacting with Aurora kinase and chromatin assembly factors.

    PubMed

    Li, Ziyin; Gourguechon, Stéphane; Wang, Ching C

    2007-11-01

    The Tousled-like kinases are an evolutionarily conserved family of proteins implicated in DNA repair, DNA replication and mitosis in metazoans and plants. Their absence from the yeasts and other eukaryotic 'microbes' suggests a specific role for them in the development of multicellular organisms. In this study, two closely related Tousled-like kinase homologs, TLK1 and TLK2, were identified in Trypanosoma brucei, a unicellular protozoan parasite. Only TLK1 plays an essential role in cell growth, and a deficiency in TLK1 led to an enrichment of S-phase cells, defective spindle formation and aberrant chromosome segregation. Although both TLK proteins localize to the nucleus, only TLK1 also concentrates in the spindle poles during mitosis. Both TLK proteins are phosphorylated by the Aurora kinase (AUK1), and both can autophosphorylate and phosphorylate histone H3 and the chromatin assembly factors Asf1A and Asf1B in vitro, but only TLK1 is autophosphorylated and capable of oligomerizing and interacting with AUK1, Asf1A and Asf1B in vivo. These discrepancies between the two TLK proteins can be attributed to minor differences between their N- and C-terminal sequences. In summary, TLK1 cooperates with Aurora kinase to regulate spindle assembly and chromosome segregation, and it performs a role in DNA replication probably by regulating histone modification in trypanosomes. PMID:17940067

  15. 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

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

    PubMed

    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.

  17. 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.

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

    PubMed

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

    2015-02-01

    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. PMID:25564610

  19. 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.

  20. Receptor-like function of heparin in the binding and uptake of neutral lipids

    SciTech Connect

    Bosner, M.S.; Gulick, T.; Riley, D.J.S.; Spilburg, C.A.; Lange, L.G. III )

    1988-10-01

    Molecular mechanisms regulating the binding, amphipathic stabilization, and metabolism of the major neutral lipids 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, the authors 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 {sup 125}I-labeled cholesterol esterase and {sup 125}I-labeled triglyceride lipase. 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 K{sub d} of 100 nM and a B{sub max} of approximately 50-60 pmol per mg of vesicle protein. They 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 generalized pathway for transport of neutral lipids into endothelial and other cells.

  1. 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. PMID:14551255

  2. 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.

  3. Tomato 14-3-3 protein TFT7 interacts with a MAP kinase kinase to regulate immunity-associated programmed cell death mediated by diverse disease resistance proteins.

    PubMed

    Oh, Chang-Sik; Martin, Gregory B

    2011-04-22

    Programmed cell death (PCD) associated with immunity is triggered when a plant disease resistance (R) protein recognizes a corresponding pathogen virulence protein. In tomato, detection by the host Pto kinase of the Pseudomonas syringae proteins AvrPto or AvrPtoB causes localized PCD. Previously, we reported that both MAPKKKα (mitogen-activated protein kinase kinase kinase) and the tomato 14-3-3 protein 7 (TFT7) positively regulate Pto-mediated PCD in tomato and Nicotiana benthamiana. In addition, in contrast to MAPKKKα, TFT7 is required for PCD mediated by four other R proteins. Here we investigate why TFT7 is required for PCD induced by diverse R proteins in plants. We discovered that a MAPKK, SlMKK2, which acts downstream of SlMAPKKKα, also interacts with TFT7 in plant cells. Gene silencing experiments revealed that the orthologous genes of both SlMKK2 and TFT7 in N. benthamiana are required for PCD mediated by the same set of R proteins. SlMKK2 and its orthologs contain a 14-3-3 binding site in their N terminus, and Thr(33) in this site is required for interaction with TFT7 in vivo. Like the structurally similar human 14-3-3ε protein, TFT7 forms a homodimer in vivo. Because TFT7 interacts with both SlMAPKKKα and SlMKK2 and also forms a homodimer, we propose that TFT7 may coordinately recruit these client proteins for efficient signal transfer, leading to PCD induction. PMID:21378171

  4. 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.

  5. Facilitated interaction between the pyruvate dehydrogenase kinase isoform 2 and the dihydrolipoyl acetyltransferase.

    PubMed

    Hiromasa, Yasuaki; Roche, Thomas E

    2003-09-01

    The dihydrolipoyl acetyltransferase (E2) has an enormous impact on pyruvate dehydrogenase kinase (PDK) phosphorylation of the pyruvate dehydrogenase (E1) component by acting as a mobile binding framework and in facilitating and mediating regulation of PDK activity. Analytical ultracentrifugation (AUC) studies established that the soluble PDK2 isoform is a stable dimer. The interaction of PDK2 with the lipoyl domains of E2 (L1, L2) and the E3-binding protein (L3) were characterized by AUC. PDK2 interacted very weakly with L2 (Kd approximately 175 microM for 2 L2/PDK2) but much tighter with dimeric glutathione S-transferase (GST)-L2 (Kd approximately 3 microM), supporting the importance of bifunctional binding. Reduction of lipoyl groups resulted in approximately 8-fold tighter binding of PDK2 to GST-L2red, which was approximately 300-fold tighter than binding of 2 L2red and also much tighter than binding by GST-L1red and GST-L3red. The E2 60-mer bound approximately 18 PDK2 dimers with a Kd similar to GST-L2. E2.E1 bound more PDK2 (approximately 27.6) than E2 with approximately 2-fold tighter affinity. Lipoate reduction fostered somewhat tighter binding at more sites by E2 and severalfold tighter binding at the majority of sites on E2.E1. ATP and ADP decreased the affinity of PDK2 for E2 by 3-5-fold and adenosine 5'-(beta,gamma-imino)triphosphate or phosphorylation of E1 similarly reduced PDK2 binding to E2.E1. Reversible bifunctional binding to L2 with the mandatory singly held transition fits the proposed "hand-over-hand" movement of a kinase dimer to access E1 without dissociating from the complex. The gain in binding interactions upon lipoate reduction likely aids reduction-engendered stimulation of PDK2 activity; loosening of binding as a result of adenine nucleotides and phosphorylation may instigate movement of lipoyl domain-held kinase to a new E1 substrate. PMID:12816949

  6. 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.

  7. TOUSLED Kinase Activity Oscillates during the Cell Cycle and Interacts with Chromatin Regulators1

    PubMed Central

    Ehsan, Hashimul; Reichheld, Jean-Philippe; Durfee, Tim; Roe, Judith L.

    2004-01-01

    The TOUSLED (TSL)-like nuclear protein kinase family is highly conserved in plants and animals. tsl loss of function mutations cause pleiotropic defects in both leaf and flower development, and growth and initiation of floral organ primordia is abnormal, suggesting that basic cellular processes are affected. TSL is more highly expressed in exponentially growing Arabidopsis culture cells than in stationary, nondividing cells. While its expression remains constant throughout the cell cycle in dividing cells, TSL kinase activity is higher in enriched late G2/M-phase and G1-phase populations of Arabidopsis suspension culture cells compared to those in S-phase. tsl mutants also display an aberrant pattern and increased expression levels of the mitotic cyclin gene CycB1;1, suggesting that TSL represses CycB1;1 expression at certain times during development or that cells are delayed in mitosis. TSL interacts with and phosphorylates one of two Arabidopsis homologs of the nucleosome assembly/silencing protein Asf1 and histone H3, as in humans, and a novel plant SANT/myb-domain protein, TKI1, suggesting that TSL plays a role in chromatin metabolism. PMID:15047893

  8. Phosphorylation of LRRK2 by casein kinase 1α regulates trans-Golgi clustering via differential interaction with ARHGEF7.

    PubMed

    Chia, Ruth; Haddock, Sara; Beilina, Alexandra; Rudenko, Iakov N; Mamais, Adamantios; Kaganovich, Alice; Li, Yan; Kumaran, Ravindran; Nalls, Michael A; Cookson, Mark R

    2014-01-01

    LRRK2, a gene relevant to Parkinson's disease, encodes a scaffolding protein with both GTPase and kinase activities. LRRK2 protein is itself phosphorylated and therefore is subject to regulation by cell signalling; however, the kinase(s) responsible for this event have not been definitively identified. Here using an unbiased siRNA kinome screen, we identify and validate casein kinase 1α (CK1α) as being responsible for LRRK2 phosphorylation, including in the adult mouse striatum. We further show that LRRK2 recruitment to TGN46-positive Golgi-derived vesicles is modulated by constitutive LRRK2 phosphorylation by CK1α. These effects are mediated by differential protein interactions of LRRK2 with a guanine nucleotide exchange factor, ARHGEF7. These pathways are therefore likely involved in the physiological maintenance of the Golgi in cells, which may play a role in the pathogenesis of Parkinson's disease. PMID:25500533

  9. Energetics of Src homology domain interactions in receptor tyrosine kinase-mediated signaling.

    PubMed

    Ladbury, John E; Arold, Stefan T

    2011-01-01

    Intracellular signaling from receptor tyrosine kinases (RTK) on extracellular stimulation is fundamental to all cellular processes. The protein-protein interactions which form the basis of this signaling are mediated through a limited number of polypeptide domains. For signal transduction without corruption, based on a model where signaling pathways are considered as linear bimolecular relays, these interactions have to be highly specific. This is particularly the case when one considers that any cell may have copies of similar binding domains found in numerous proteins. In this work, an overview of the thermodynamics of binding of two of the most common of these domains (SH2 and SH3 domains) is given. This, coupled with insight from high-resolution structural detail, provides a comprehensive survey of how recognition of cognate binding sites for these domains occurs. Based on the data presented, we conclude that specificity offered by these interactions of SH2 and SH3 domains is limited and not sufficient to enforce mutual exclusivity in RTK-mediated signaling. This may explain the current lack of success in pharmaceutical intervention to inhibit the interactions of these domains when they are responsible for aberrant signaling and the resulting disease states such as cancer.

  10. Epigenetic regulation of traf2- and Nck-interacting kinase (TNIK) in polycystic ovary syndrome

    PubMed Central

    Li, Da; Jiao, Jiao; Zhou, Yi-Ming; Wang, Xiu-Xia

    2015-01-01

    Emerging evidence has led to considerable interest in the role of Traf2- and Nck-interacting kinase (TNIK) in polycystic ovary syndrome (PCOS) development. However, the epigenetic mechanism regulating TNIK transcription remains largely unknown. Here, we show that (i) TNIK mRNA expression is significantly increased in PCOS ovarian tissues, compared to normal ovarian tissues; (ii) PCOS ovarian tissues exhibit a hypermethylation pattern at the cg10180092 site, (iii) and cg10180092 is the critical site for the transcriptional regulation of TNIK. Mechanistically, hypermethylated cg10180092 site-mediated loss of holocarboxylase synthetase (HLCS)-related H3K9me enrichment activated TNIK transcription in PCOS ovarian tissues. Notably, a substantial body of evidence indicates that DNA hypermethylation is an alternative mechanism for gene inactivation, and a new role for DNA hypermethylationmediated TNIK activating was observed in this study. This may improve our understanding of divergent transcriptional regulation in the initiation and progression of TNIK-related PCOS. PMID:26279758

  11. 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.

  12. 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.

  13. 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

  14. The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans.

    PubMed

    D'Souza, Serena A; Rajendran, Luckshika; Bagg, Rachel; Barbier, Louis; van Pel, Derek M; Moshiri, Houtan; Roy, Peter J

    2016-04-01

    The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle's plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue. PMID:27123983

  15. The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans

    PubMed Central

    D’Souza, Serena A.; Rajendran, Luckshika; Bagg, Rachel; van Pel, Derek M.; Moshiri, Houtan; Roy, Peter J.

    2016-01-01

    The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle’s plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue. PMID:27123983

  16. The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans.

    PubMed

    D'Souza, Serena A; Rajendran, Luckshika; Bagg, Rachel; Barbier, Louis; van Pel, Derek M; Moshiri, Houtan; Roy, Peter J

    2016-04-01

    The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle's plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue.

  17. ABL kinases promote breast cancer osteolytic metastasis by modulating tumor-bone interactions through TAZ and STAT5 signaling

    PubMed Central

    Wang, Jun; Rouse, Clay; Jasper, Jeff S.; Pendergast, Ann Marie

    2016-01-01

    Bone metastases occur in up to 70% of advanced breast cancer. For most patients with breast cancer, bone metastases are predominantly osteolytic. Interactions between tumor cells and stromal cells in the bone microenvironment drive osteolytic bone metastasis, a process that requires the activation of osteoclasts, cells that break down bone. Here, we report that ABL kinases promoted metastasis of breast cancer cells to bone by regulating the crosstalk between tumor and the bone microenvironment. ABL kinases protected tumor cells from apoptosis induced by TRAIL (TNF-related apoptosis-inducing ligand), activated the transcription factor STAT5, and promoted osteolysis through the STAT5-dependent expression of genes encoding the osteoclast activating factors interleukin 6 (IL6) and matrix metalloproteinase-1 (MMP1). Furthermore, ABL kinases increased the abundance of the Hippo pathway mediator TAZ and the expression of TAZ-dependent target genes that promote bone metastasis. Knockdown of ABL kinases or treatment with ABL-specific allosteric inhibitor impaired osteolytic metastasis of breast cancer cells in mice. These findings revealed a role for ABL kinases in regulating tumor-bone interactions and provide a rationale for targeting both tumor and the bone microenvironment with ABL-specific inhibitors. PMID:26838548

  18. ABL kinases promote breast cancer osteolytic metastasis by modulating tumor-bone interactions through TAZ and STAT5 signaling.

    PubMed

    Wang, Jun; Rouse, Clay; Jasper, Jeff S; Pendergast, Ann Marie

    2016-02-01

    Bone metastases occur in up to 70% of advanced breast cancer. For most patients with breast cancer, bone metastases are predominantly osteolytic. Interactions between tumor cells and stromal cells in the bone microenvironment drive osteolytic bone metastasis, a process that requires the activation of osteoclasts, cells that break down bone. We report that ABL kinases promoted metastasis of breast cancer cells to bone by regulating the crosstalk between tumor cells and the bone microenvironment. ABL kinases protected tumor cells from apoptosis induced by TRAIL (TNF-related apoptosis-inducing ligand), activated the transcription factor STAT5, and promoted osteolysis through the STAT5-dependent expression of genes encoding the osteoclast-activating factors interleukin-6 (IL-6) and matrix metalloproteinase 1 (MMP1). Furthermore, in breast cancer cells, ABL kinases increased the abundance of the Hippo pathway mediator TAZ and the expression of TAZ-dependent target genes that promote bone metastasis. Knockdown of ABL kinases or treatment with ABL-specific allosteric inhibitor impaired osteolytic metastasis of breast cancer cells in mice. These findings revealed a role for ABL kinases in regulating tumor-bone interactions and provide a rationale for using ABL-specific inhibitors to limit breast cancer metastasis to bone. PMID:26838548

  19. Receptor-Interacting Protein Kinase-2 Inhibition by CYLD Impairs Antibacterial Immune Responses in Macrophages

    PubMed Central

    Wex, Katharina; Schmid, Ursula; Just, Sissy; Wang, Xu; Wurm, Rebecca; Naumann, Michael; Schlüter, Dirk; Nishanth, Gopala

    2016-01-01

    Upon infection with intracellular bacteria, nucleotide oligomerization domain protein 2 recognizes bacterial muramyl dipeptide and binds, subsequently, to receptor-interacting serine/threonine kinase 2 (RIPK2), which activates immune responses via the nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) and extracellular signal-regulated kinase (ERK) pathways. Activation of RIPK2 depends on its K63 ubiquitination by E3 ligases, whereas the deubiquitinating enzyme A20 counter regulates RIPK2 activity by cleaving K63-polyubiquitin chains from RIPK2. Here, we newly identify the deubiquitinating enzyme CYLD as a new inhibitor of RIPK2. We show that CYLD binds to and removes K63-polyubiquitin chains from RIPK2 in Listeria monocytogenes (Lm) infected murine bone marrow-derived macrophages. CYLD-mediated K63 deubiquitination of RIPK2 resulted in an impaired activation of both NF-κB and ERK1/2 pathways, reduced production of proinflammatory cytokines interleukin-6 (IL-6), IL-12, anti-listerial reactive oxygen species (ROS) and nitric oxide (NO), and, finally, impaired pathogen control. In turn, RIPK2 inhibition by siRNA prevented activation of NF-κB and ERK1/2 and completely abolished the protective effect of CYLD deficiency with respect to the production of IL-6, NO, ROS, and pathogen control. Noteworthy, CYLD also inhibited autophagy of Listeria in a RIPK2-ERK1/2-dependent manner. The protective function of CYLD deficiency was dependent on interferon gamma (IFN-γ) prestimulation of infected macrophages. Interestingly, the reduced NF-κB activation in CYLD-expressing macrophages limited the protective effect of IFN-γ by reducing NF-κB-dependent signal transducers and activators of transcription-1 (STAT1) activation. Taken together, our study identifies CYLD as an important inhibitor of RIPK2-dependent antibacterial immune responses in macrophages. PMID:26834734

  20. Death-associated protein kinase 2 mediates nocodazole-induced apoptosis through interaction with tubulin.

    PubMed

    Isshiki, Kinuka; Hirase, Taishi; Matsuda, Shinya; Miyamoto, Kenji; Tsuji, Akihiko; Yuasa, Keizo

    Death-associated protein kinase 2 (DAPK2) is a positive regulator of apoptosis. Although we recently reported that 14-3-3 proteins inhibit DAPK2 activity and its subsequent apoptotic effects via binding to DAPK2, the molecular mechanisms underlying the DAPK2-mediated apoptotic pathway remain unclear. Therefore, we attempted to further identify DAPK2-interacting proteins using pull-down assays and mass spectrometry. The microtubule β-tubulin was identified as a novel DAPK2-binding protein in HeLa cells. Pull-down assays revealed that DAPK2 interacted with the α/β-tubulin heterodimer, and that the C-terminal region of DAPK2, which differs from that of other DAPK family members, was sufficient for the association with β-tubulin. Although the microtubule-depolymerizing agent nocodazole induced apoptosis in HeLa cells, the level of apoptosis was significantly decreased in the DAPK2 knockdown cells. Furthermore, we found that treatment with nocodazole resulted in an increased binding of DAPK2 to β-tubulin. These findings indicate that DAPK2 mediates nocodazole-induced apoptosis via binding to tubulin.

  1. 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

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

    PubMed

    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.

  3. Inositol hexakisphosphate kinase-1 interacts with perilipin1 to modulate lipolysis.

    PubMed

    Ghoshal, Sarbani; Tyagi, Richa; Zhu, Qingzhang; Chakraborty, Anutosh

    2016-09-01

    Lipolysis leads to the breakdown of stored triglycerides (TAG) to release free fatty acids (FFA) and glycerol which is utilized by energy expenditure pathways to generate energy. Therefore, a decrease in lipolysis augments fat accumulation in adipocytes which promotes weight gain. Conversely, if lipolysis is not complemented by energy expenditure, it leads to FFA induced insulin resistance and type-2 diabetes. Thus, lipolysis is under stringent physiological regulation, although the precise mechanism of the regulation is not known. Deletion of inositol hexakisphosphate kinase-1 (IP6K1), the major inositol pyrophosphate biosynthetic enzyme, protects mice from high fat diet (HFD) induced obesity and insulin resistance. IP6K1-KO mice are lean due to enhanced energy expenditure. Therefore, IP6K1 is a target in obesity and type-2 diabetes. However, the mechanism/s by which IP6K1 regulates adipose tissue lipid metabolism is yet to be understood. Here, we demonstrate that IP6K1-KO mice display enhanced basal lipolysis. IP6K1 modulates lipolysis via its interaction with the lipolytic regulator protein perilipin1 (PLIN1). Furthermore, phosphorylation of IP6K1 at a PKC/PKA motif modulates its interaction with PLIN1 and lipolysis. Thus, IP6K1 is a novel regulator of PLIN1 mediated lipolysis. PMID:27373682

  4. 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

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

    PubMed

    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

  6. 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.

  7. Palmitylation of Src family tyrosine kinases regulates functional interaction with a B cell substrate.

    PubMed

    Saouaf, S J; Wolven, A; Resh, M D; Bolen, J B

    1997-05-19

    Palmitylation of Src family tyrosine kinases has been shown to play a role in directing their membrane localization. Here we demonstrate that palmitylation can also regulate recognition and tyrosine phosphorylation of the B cell Src kinase substrate Ig alpha. Blk and Src, which are not palmitylated, phosphorylate co-expressed Ig alpha in Cos cells, whereas palmitylated Src kinases do not. Addition of a palmitylation site to Blk abrogates its phosphorylation of the substrate, while mutation of Fyn's palmitylation sites results in recognition and phosphorylation of Ig alpha. These results indicate that palmitylation, a reversible protein modification, aids in regulating recognition of physiologic substrates by Src family tyrosine kinases. PMID:9177269

  8. 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

    2005-06-01

    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.

  9. Interaction of mitogen-activated protein kinases with the kinase interaction motif of the tyrosine phosphatase PTP-SL provides substrate specificity and retains ERK2 in the cytoplasm.

    PubMed

    Zúñiga, A; Torres, J; Ubeda, J; Pulido, R

    1999-07-30

    ERK1 and ERK2 associate with the tyrosine phosphatase PTP-SL through a kinase interaction motif (KIM) located in the juxtamembrane region of PTP-SL. A glutathione S-transferase (GST)-PTP-SL fusion protein containing the KIM associated with ERK1 and ERK2 as well as with p38/HOG, but not with the related JNK1 kinase or with protein kinase A or C. Accordingly, ERK2 showed in vitro substrate specificity to phosphorylate GST-PTP-SL in comparison with GST-c-Jun. Furthermore, tyrosine dephosphorylation of ERK2 by the PTP-SLDeltaKIM mutant was impaired. The in vitro association of ERK1/2 with GST-PTP-SL was highly stable; however, low concentrations of nucleotides partially dissociated the ERK1/2.PTP-SL complex. Partial deletions of the KIM abrogated the association of PTP-SL with ERK1/2, indicating that KIM integrity is required for interaction. Amino acid substitution analysis revealed that Arg and Leu residues within the KIM are essential for the interaction and suggested a regulatory role for Ser(231). Finally, coexpression of PTP-SL and ERK2 in COS-7 cells resulted in the retention of ERK2 in the cytoplasm in a KIM-dependent manner. Our results demonstrate that the noncatalytic region of PTP-SL associates with mitogen-activated protein kinases with high affinity and specificity, providing a mechanism for substrate specificity, and suggest a role for PTP-SL in the regulation of mitogen-activated protein kinase translocation to the nucleus upon activation.

  10. CLAVATA1 dominant-negative alleles reveal functional overlap between multiple receptor kinases that regulate meristem and organ development.

    PubMed

    Diévart, Anne; Dalal, Monica; Tax, Frans E; Lacey, Alexzandria D; Huttly, Alison; Li, Jianming; Clark, Steven E

    2003-05-01

    The CLAVATA1 (CLV1) receptor kinase controls stem cell number and differentiation at the Arabidopsis shoot and flower meristems. Other components of the CLV1 signaling pathway include the secreted putative ligand CLV3 and the receptor-like protein CLV2. We report evidence indicating that all intermediate and strong clv1 alleles are dominant negative and likely interfere with the activity of unknown receptor kinase(s) that have functional overlap with CLV1. clv1 dominant-negative alleles show major differences from dominant-negative alleles characterized to date in animal receptor kinase signaling systems, including the lack of a dominant-negative effect of kinase domain truncation and the ability of missense mutations in the extracellular domain to act in a dominant-negative manner. We analyzed chimeric receptor kinases by fusing CLV1 and BRASSINOSTEROID INSENSITIVE1 (BRI1) coding sequences and expressing these in clv1 null backgrounds. Constructs containing the CLV1 extracellular domain and the BRI1 kinase domain were strongly dominant negative in the regulation of meristem development. Furthermore, we show that CLV1 expressed within the pedicel can partially replace the function of the ERECTA receptor kinase. We propose the presence of multiple receptors that regulate meristem development in a functionally related manner whose interactions are driven by the extracellular domains and whose activation requires the kinase domain.

  11. CLAVATA1 Dominant-Negative Alleles Reveal Functional Overlap between Multiple Receptor Kinases That Regulate Meristem and Organ Development

    PubMed Central

    Diévart, Anne; Dalal, Monica; Tax, Frans E.; Lacey, Alexzandria D.; Huttly, Alison; Li, Jianming; Clark, Steven E.

    2003-01-01

    The CLAVATA1 (CLV1) receptor kinase controls stem cell number and differentiation at the Arabidopsis shoot and flower meristems. Other components of the CLV1 signaling pathway include the secreted putative ligand CLV3 and the receptor-like protein CLV2. We report evidence indicating that all intermediate and strong clv1 alleles are dominant negative and likely interfere with the activity of unknown receptor kinase(s) that have functional overlap with CLV1. clv1 dominant-negative alleles show major differences from dominant-negative alleles characterized to date in animal receptor kinase signaling systems, including the lack of a dominant-negative effect of kinase domain truncation and the ability of missense mutations in the extracellular domain to act in a dominant-negative manner. We analyzed chimeric receptor kinases by fusing CLV1 and BRASSINOSTEROID INSENSITIVE1 (BRI1) coding sequences and expressing these in clv1 null backgrounds. Constructs containing the CLV1 extracellular domain and the BRI1 kinase domain were strongly dominant negative in the regulation of meristem development. Furthermore, we show that CLV1 expressed within the pedicel can partially replace the function of the ERECTA receptor kinase. We propose the presence of multiple receptors that regulate meristem development in a functionally related manner whose interactions are driven by the extracellular domains and whose activation requires the kinase domain. PMID:12724544

  12. Homeodomain-Interacting Protein Kinase-2: A Critical Regulator of the DNA Damage Response and the Epigenome

    PubMed Central

    Kuwano, Yuki; Nishida, Kensei; Akaike, Yoko; Kurokawa, Ken; Nishikawa, Tatsuya; Masuda, Kiyoshi; Rokutan, Kazuhito

    2016-01-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is a serine/threonine kinase that phosphorylates and activates the apoptotic program through interaction with diverse downstream targets including tumor suppressor p53. HIPK2 is activated by genotoxic stimuli and modulates cell fate following DNA damage. The DNA damage response (DDR) is triggered by DNA lesions or chromatin alterations. The DDR regulates DNA repair, cell cycle checkpoint activation, and apoptosis to restore genome integrity and cellular homeostasis. Maintenance of the DDR is essential to prevent development of diseases caused by genomic instability, including cancer, defects of development, and neurodegenerative disorders. Recent studies reveal a novel HIPK2-mediated pathway for DDR through interaction with chromatin remodeling factor homeodomain protein 1γ. In this review, we will highlight the molecular mechanisms of HIPK2 and show its functions as a crucial DDR regulator. PMID:27689990

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

    PubMed

    Li, Shan; Ma, Yushu; Wei, Dongzhi

    2016-02-01

    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.

  14. Identification of two eukaryote-like serine/threonine kinases encoded by Chlamydia trachomatis serovar L2 and characterization of interacting partners of Pkn1.

    PubMed

    Verma, Anita; Maurelli, Anthony T

    2003-10-01

    Genome sequencing of C. trachomatis serovar D revealed the presence of three putative open reading frames (ORFs), CT145 (Pkn1), CT673 (Pkn5), and CT301 (PknD), encoding eukaryote-like serine/threonine kinases (Ser/Thr kinases). Two of these putative kinase genes, CT145 and CT301, were PCR amplified from serovar L2, cloned, and sequenced. Predicted translation products of the ORFs showed the presence of conserved kinase motifs at the N terminus of the proteins. CT145 and CT301 (encoding Pkn1 and PknD, respectively) were expressed in Escherichia coli as GST fusion proteins. In vitro kinase assays with Escherichia coli-derived glutathione S-transferase fusion proteins showed autophosphorylation of Pkn1 and PknD, indicating that they are functional kinases. Gene expression analysis of these kinase genes in Chlamydia by reverse transcriptase PCR indicated expression of these kinases at the early mid phase of the developmental cycle. Immunoprecipitated native chlamydial Pkn1 and PknD proteins also showed autophosphorylation in an in vitro kinase assay. Phosphoamino acid analysis by thin-layer chromatography confirmed that Pkn1 and PknD are phosphorylated on both serine and threonine residues. Interaction of Pkn1 and PknD with each other as well as interaction of Pkn1 with inclusion membrane protein G (IncG) was demonstrated by using a bacterial two-hybrid system. These interactions were further suggested by phosphorylation of the proteins in in vitro kinase assays. This report is the first description of the existence of functional Ser/Thr kinases in Chlamydia. The results of these findings should lead to a better understanding of how Chlamydia interact and interfere with host signaling pathways, since kinases represent potential mediators of the intimate host-pathogen interactions that are essential to the intracellular life cycle of Chlamydia.

  15. Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging.

    PubMed

    Slaughter, Brian D; Schwartz, Joel W; Li, Rong

    2007-12-18

    Fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), and photon counting histograms (PCH) are fluctuation methods that emerged recently as potentially useful tools for obtaining parameters of molecular dynamics, interactions, and oligomerization in vivo. Here, we report the successful implementation of FCS, FCCS, and PCH in live yeast cells using fluorescent protein-tagged proteins expressed from their native chromosomal loci, examining cytosolic dynamics and interactions among components of the mitogen activated protein kinase (MAPK) cascade, a widely occurring signaling motif, in response to mating pheromone. FCS analysis detailed the diffusion characteristics and mobile concentrations of MAPK proteins. FCCS analysis using EGFP and mCherry-tagged protein pairs observed the interactions of Ste7 (MAPK kinase) with the MAPKs, Fus3 or Kss1, and of the scaffold protein, Ste5, with Ste7 and Ste11 (MAPK kinase kinase) in the cytosol, providing in vivo constants of their binding equilibrium. The interaction of Ste5 with Fus3 in the cytosol was below the limit of detection, suggesting a weak interaction, if it exists, with K(d) >400-500 nM. Using PCH, we show that cytosolic Ste5 were mostly monomers. Artificial dimerization of Ste5, as confirmed by PCH, using a dimerizing tag, stimulated the interaction between Ste5 and Fus3. Native Ste5 was found to bind Fus3 preferentially at the cortex in pheromone-treated cells, as detected by fluorescence resonance energy transfer (FRET). These results provide a quantitative spatial map of MAPK complexes in vivo and directly support the model that membrane association and regulation of the Ste5 scaffold are critical steps in MAPK activation. PMID:18077328

  16. Focal adhesion kinase regulates expression of thioredoxin-interacting protein (TXNIP) in cancer cells.

    PubMed

    Ho, Baotran; Huang, Grace; Golubovskaya, Vita M

    2014-01-01

    Focal Adhesion Kinase (FAK) plays an important role in cancer cell survival. Previous microarray gene profiling study detected inverse regulation between expression of thioredoxin-interacting protein (TXNIP) and FAK, where down-regulation of FAK by siRNA in MCF-7 cells caused up-regulation of TXNIP mRNA level, and in contrast up-regulation of doxycyclin- induced FAK caused repression of TXNIP. In the present report, we show that overexpression of FAK in MCF-7 cells repressed TXNIP promoter activity. Treatment of MCF-7 cells with 1alpha, 25-dihydroxyvitamin D3 (1,25D) down-regulated endogenous FAK and up-regulated TXNIP protein level, and treatment with 5-FU decreased FAK protein expression and up-regulated TXNIP protein expression in 293 cells. Moreover, silencing of FAK with siRNA increased TXNIP protein expression, while overexpression of FAK inhibited TXNIP protein expression in 293 cells. In addition, treatment of DBTRG glioblastoma cells with FAK inhibitor Y15 increased TXNIP mRNA, decreased cancer cell viability and increased apoptosis. These results for the first time demonstrate FAK-regulated TXNIP expression which is important for apoptotic, survival and oxidative stress signaling pathways in cancer cells. PMID:23387972

  17. 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

  18. Interactions of ABCG2 (BCRP) with epidermal growth factor receptor kinase inhibitors developed for molecular imaging.

    PubMed

    Qawasmi, Israa; Shmuel, Miriam; Eyal, Sara

    2014-01-01

    The objective of this study was to investigate in vitro the interactions between novel epidermal growth factor receptor kinase inhibitors (EGFRIs) developed for positron emission tomography (PET) imaging and the major efflux transporter breast cancer resistance protein (BCRP/ABCG2). Seven compounds were evaluated, using the ATPase activity assays and Madin-Darbey canine kidney (MDCK) cells overexpressing BCRP. Five of the tested compounds activated BCRP ATPase to various extent. Overexpression of BCRP conferred resistance to ML04, ML06, methoxy-Br-ML03, and PEG6-ML05 (IC50 values for inhibition of control cell proliferation 2.1 ± 0.6, 2.2 ± 0.7, 1.8 ± 1.2, and 2.8 ± 3.1 μM, respectively, compared to >50 μM in MDCK-BCRP cells). At submicromolar concentrations, none of the EGFRIs significantly inhibited BCRP. Immunoblotting studies indicated that BCRP expression is evident in cell lines utilized for in vivo tumor grafting in small animal PET imaging studies. Thus, the intensity of EGFRIs radioactivity signals previously observed in tumor xenografts reflects an interplay between transporter-mediated distribution of the probe into tumor cells and target binding. Concomitant use of efflux transporter inhibitors may help distinguish between the contribution of efflux transport and EGFR binding to the tissue signal.

  19. 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. PMID:27057269

  20. 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

  1. Cdc42p-Interacting Protein Bem4p Regulates the Filamentous-Growth Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Pitoniak, Andrew; Chavel, Colin A.; Chow, Jacky; Smith, Jeremy; Camara, Diawoye; Karunanithi, Sheelarani; Li, Boyang; Wolfe, Kennith H.

    2014-01-01

    The ubiquitous Rho (Ras homology) GTPase Cdc42p can function in different settings to regulate cell polarity and cellular signaling. How Cdc42p and other proteins are directed to function in a particular context remains unclear. We show that the Cdc42p-interacting protein Bem4p regulates the mitogen-activated protein kinase (MAPK) pathway that controls filamentous growth in Saccharomyces cerevisiae. Bem4p controlled the filamentous-growth pathway but not other MAPK pathways (mating or high-osmolarity glycerol response [HOG]) that also require Cdc42p and other shared components. Bem4p associated with the plasma membrane (PM) protein, Sho1p, to regulate MAPK activity and cell polarization under nutrient-limiting conditions that favor filamentous growth. Bem4p also interacted with the major activator of Cdc42p, the guanine nucleotide exchange factor (GEF) Cdc24p, which we show also regulates the filamentous-growth pathway. Bem4p interacted with the pleckstrin homology (PH) domain of Cdc24p, which functions in an autoinhibitory capacity, and was required, along with other pathway regulators, to maintain Cdc24p at polarized sites during filamentous growth. Bem4p also interacted with the MAPK kinase kinase (MAPKKK) Ste11p. Thus, Bem4p is a new regulator of the filamentous-growth MAPK pathway and binds to general proteins, like Cdc42p and Ste11p, to promote a pathway-specific response. PMID:25384973

  2. Phosphatidylinositol-3 kinase activation induced upon Fc gamma RIIIA- ligand interaction

    PubMed Central

    1994-01-01

    Induced activation of protein tyrosine kinase(s) is a central event in signal transduction mediated via the low affinity receptor for IgG (Fc gamma RIIIA, CD16) in natural killer (NK) cells. Tyrosine phosphorylation may affect the function of several protein directly, or indirectly by inducing their association with other tyrosine phosphorylated proteins. Here, we report that Fc gamma RIII stimulation induces activation of phosphatidylinositol (PI)-3 kinase in NK cells. Phosphotyrosine immunoprecipitates from Fc gamma RIII-stimulated NK cells contain PI-kinase activity and PI-3 kinase can be directly precipitated from them. Conversely, a series of tyrosine-phosphorylated proteins is coprecipitated with PI-3 kinase from the stimulated, but not from control cells. Analogous results obtained using Jurkat T cells expressing transfected Fc gamma RIIIA alpha ligand binding chain in association with gamma 2 or zeta 2 homodimers indicate that both complexes transduce this effect, although the Fc gamma RIIIA-zeta 2 complexes do so with greater efficiency. Accumulation of phosphoinositide D3 phosphorylated products in stimulated cells confirms PI-3 kinase activation, indicating the participation of this enzyme in Fc gamma RIIIA-mediated signal transduction. PMID:8294866

  3. Interactions between the kinetochore complex and the protein kinase A pathway in Saccharomyces cerevisiae.

    PubMed

    Ma, Lina; Ho, Krystina; Piggott, Nina; Luo, Zongli; Measday, Vivien

    2012-07-01

    The kinetochore is a large structure composed of multiple protein subcomplexes that connect chromosomes to spindle microtubules to enable accurate chromosome segregation. Significant advances have been made in the identification of kinetochore proteins and elucidation of kinetochore structure; however, comparatively little is known about how cellular signals integrate with kinetochore function. In the budding yeast Saccharomyces cerevisiae, the cyclic AMP protein kinase A signaling pathway promotes cellular growth in response to glucose. In this study, we find that decreasing protein kinase A activity, either by overexpressing negative regulators of the pathway or deleting the upstream effector Ras2, improves the viability of ipl1 and spc24 kinetochore mutants. Ipl1/Aurora B is a highly conserved kinase that corrects attachment of sister kinetochores that have attached to the same spindle pole, whereas Spc24 is a component of the conserved Ndc80 kinetochore complex that attaches directly to microtubules. Unexpectedly, we find that kinetochore mutants have increased phosphorylation levels of protein kinase A substrates, suggesting that the cyclic AMP protein kinase A signaling pathway is stimulated. The increase in protein kinase A activity in kinetochore mutants is not induced by activation of the spindle checkpoint or a metaphase delay because protein kinase A activity remains constant during an unperturbed cell cycle. Finally, we show that lowering protein kinase A activity can rescue the chromosome loss defect of the inner kinetochore ndc10 mutant. Overall, our data suggest that the increased protein kinase A activity in kinetochore mutants is detrimental to cellular growth and chromosome transmission fidelity.

  4. IRBIT Interacts with the Catalytic Core of Phosphatidylinositol Phosphate Kinase Type Iα and IIα through Conserved Catalytic Aspartate Residues

    PubMed Central

    Ando, Hideaki; Hirose, Matsumi; Gainche, Laura; Kawaai, Katsuhiro; Bonneau, Benjamin; Ijuin, Takeshi; Itoh, Toshiki; Takenawa, Tadaomi; Mikoshiba, Katsuhiko

    2015-01-01

    Phosphatidylinositol phosphate kinases (PIPKs) are lipid kinases that generate phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a critical lipid signaling molecule that regulates diverse cellular functions, including the activities of membrane channels and transporters. IRBIT (IP3R-binding protein released with inositol 1,4,5-trisphosphate) is a multifunctional protein that regulates diverse target proteins. Here, we report that IRBIT forms signaling complexes with members of the PIPK family. IRBIT bound to all PIPK isoforms in heterologous expression systems and specifically interacted with PIPK type Iα (PIPKIα) and type IIα (PIPKIIα) in mouse cerebellum. Site-directed mutagenesis revealed that two conserved catalytic aspartate residues of PIPKIα and PIPKIIα are involved in the interaction with IRBIT. Furthermore, phosphatidylinositol 4-phosphate, Mg2+, and/or ATP interfered with the interaction, suggesting that IRBIT interacts with catalytic cores of PIPKs. Mutations of phosphorylation sites in the serine-rich region of IRBIT affected the selectivity of its interaction with PIPKIα and PIPKIIα. The structural flexibility of the serine-rich region, located in the intrinsically disordered protein region, is assumed to underlie the mechanism of this interaction. Furthermore, in vitro binding experiments and immunocytochemistry suggest that IRBIT and PIPKIα interact with the Na+/HCO3− cotransporter NBCe1-B. These results suggest that IRBIT forms signaling complexes with PIPKIα and NBCe1-B, whose activity is regulated by PI(4,5)P2. PMID:26509711

  5. Glutathione S-transferases interact with AMP-activated protein kinase: evidence for S-glutathionylation and activation in vitro.

    PubMed

    Klaus, Anna; Zorman, Sarah; Berthier, Alexandre; Polge, Cécile; Ramirez, Sacnicte; Michelland, Sylvie; Sève, Michel; Vertommen, Didier; Rider, Mark; Lentze, Nicolas; Auerbach, Daniel; Schlattner, Uwe

    2013-01-01

    AMP-activated protein kinase (AMPK) is a cellular and whole body energy sensor with manifold functions in regulating energy homeostasis, cell morphology and proliferation in health and disease. Here we apply multiple, complementary in vitro and in vivo interaction assays to identify several isoforms of glutathione S-transferase (GST) as direct AMPK binding partners: Pi-family member rat GSTP1 and Mu-family members rat GSTM1, as well as Schistosoma japonicum GST. GST/AMPK interaction is direct and involves the N-terminal domain of the AMPK β-subunit. Complex formation of the mammalian GSTP1 and -M1 with AMPK leads to their enzymatic activation and in turn facilitates glutathionylation and activation of AMPK in vitro. GST-facilitated S-glutathionylation of AMPK may be involved in rapid, full activation of the kinase under mildly oxidative physiological conditions.

  6. Fenretinide Perturbs Focal Adhesion Kinase in Premalignant and Malignant Human Oral Keratinocytes. Fenretinide’s chemopreventive mechanisms include ECM interactions

    PubMed Central

    Han, Byungdo B.; Li, Suyang; Tong, Meng; Holpuch, Andrew S.; Spinney, Richard; Wang, Daren; Border, Michael B.; Liu, Zhongfa; Sarode, Sachin; Pei, Ping; Schwendeman, Steven; Mallery, Susan R.

    2015-01-01

    The membrane-associated protein, focal adhesion kinase (FAK), modulates cell-extracellular matrix interactions and also conveys pro-survival and proliferative signals. Notably, increased intraepithelial FAK levels accompany transformation of premalignant oral intraepithelial neoplasia (OIN) to oral squamous cell carcinoma (OSCC). OIN chemoprevention is a patient-centric, optimal strategy to prevent OSCC’s co-morbidities and mortality. The cancer chemopreventive and synthetic vitamin A derivative, fenretinide, has demonstrated protein-binding capacities e.g. mTOR and retinol binding protein interactions. These studies employed a continuum of human oral keratinocytes (normal-HPV E6/E7-transduced-OSCC) to assess potential fenretinide-FAK drug protein interactions and functional consequences on cellular growth regulation and motility. Molecular modeling studies demonstrated fenretinide has ~200-fold greater binding affinity relative to the natural ligand (ATP) at FAK’s kinase domain. Fenretinide also shows intermediate binding at FAK’s FERM domain and interacts at the ATP-binding site of the closest FAK analogue, Pyk2. Fenretinide significantly suppressed proliferation via induction of apoptosis and G2/M cell cycle blockade. Fenretinide-treated cells also demonstrated F-actin disruption, significant inhibition of both directed migration and invasion of a synthetic basement membrane, and decreased phosphorylation of growth-promoting kinases. A commercially available FAK inhibitor did not suppress cell invasion. Notably, while FAK’s FERM domain directs cell invasion, FAK inhibitors target the kinase domain. In addition, FAK-specific siRNA treated cells showed an intermediate cell migration capacity; data which suggest co-contribution of the established migrating-enhancing Pyk2. Our data imply that fenretinide is uniquely capable of disrupting FAK’s and Pyk2’s pro-survival and mobility-enhancing effects and further extend fenretinide’s chemopreventive

  7. 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. PMID:26198257

  8. Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis1

    PubMed Central

    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

    2015-01-01

    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. PMID:26198257

  9. Regulation of N-Formyl Peptide Receptor Signaling and Trafficking by Arrestin-Src Kinase Interaction.

    PubMed

    Wagener, Brant M; Marjon, Nicole A; Prossnitz, Eric R

    2016-01-01

    Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that N-formyl peptide receptor (FPR)-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2) mutant deficient in Src kinase binding (arr2-P91G/P121E). Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking.

  10. Regulation of N-Formyl Peptide Receptor Signaling and Trafficking by Arrestin-Src Kinase Interaction

    PubMed Central

    Wagener, Brant M.; Marjon, Nicole A.; Prossnitz, Eric R.

    2016-01-01

    Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that N-formyl peptide receptor (FPR)-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2) mutant deficient in Src kinase binding (arr2-P91G/P121E). Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking. PMID:26788723

  11. 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

  12. 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.

  13. Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins.

    PubMed

    Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

    2016-01-01

    The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities.

  14. Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins

    PubMed Central

    Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

    2016-01-01

    The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities. PMID:27548200

  15. Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins.

    PubMed

    Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

    2016-01-01

    The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities. PMID:27548200

  16. Yeast two-hybrid interactions between Arabidopsis lyrata S Receptor Kinase and the ARC1 E3 ligase.

    PubMed

    Indriolo, Emily; Goring, Daphne R

    2016-06-01

    Here we describe protein-protein interactions between signaling components in the conserved self-incompatibility pathway from Brassica spp. and Arabidopsis lyrata. Previously, we had demonstrated that ARC1 is necessary in A. lyrata for the rejection of self-pollen by the self-incompatibility pathway. The results described here demonstrate that A. lyrata ARC1 interacts with A. lyrata S Receptor Kinase (SRK1) in the yeast 2-hybrid system. A. lyrata ARC1 also interacted with B. napus SRK910 illustrating that interactions in this pathway are conserved across species. Finally, we discuss how the more widely occurring interactions between SRK and ARC1-related family members may be modulated in vivo by expression and subcellular localization patterns resulting in a particular response. PMID:27175603

  17. 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

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

    PubMed

    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

  19. Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain.

    PubMed Central

    Xing, Z; Chen, H C; Nowlen, J K; Taylor, S J; Shalloway, D; Guan, J L

    1994-01-01

    The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules. Images PMID:8054685

  20. 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.

  1. Enhanced interaction between pseudokinase and kinase domains in Gcn2 stimulates eIF2α phosphorylation in starved cells.

    PubMed

    Lageix, Sebastien; Rothenburg, Stefan; Dever, Thomas E; Hinnebusch, Alan G

    2014-05-01

    The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α, from yeast to mammals. The Gcn2 kinase domain (KD) is inherently inactive and requires allosteric stimulation by adjoining regulatory domains. Gcn2 contains a pseudokinase domain (YKD) required for high-level eIF2α phosphorylation in amino acid starved yeast cells; however, the role of the YKD in KD activation was unknown. We isolated substitutions of evolutionarily conserved YKD amino acids that impair Gcn2 activation without reducing binding of the activating ligand, uncharged tRNA, to the histidyl-tRNA synthetase-related domain of Gcn2. Several such Gcn- substitutions cluster in predicted helices E and I (αE and αI) of the YKD. We also identified Gcd- substitutions, evoking constitutive activation of Gcn2, mapping in αI of the YKD. Interestingly, αI Gcd- substitutions enhance YKD-KD interactions in vitro, whereas Gcn- substitutions in αE and αI suppress both this effect and the constitutive activation of Gcn2 conferred by YKD Gcd- substitutions. These findings indicate that the YKD interacts directly with the KD for activation of kinase function and identify likely sites of direct YKD-KD contact. We propose that tRNA binding to the HisRS domain evokes a conformational change that increases access of the YKD to sites of allosteric activation in the adjoining KD.

  2. In silico analysis and experimental verification of OSR1 kinase - Peptide interaction.

    PubMed

    Austin, Thomas M; Nannemann, David P; Deluca, Samuel L; Meiler, Jens; Delpire, Eric

    2014-07-01

    The oxidative-stress-responsive kinase 1 (OSR1) and the STE20/SPS1-related proline/alanine-rich kinase (SPAK) are key enzymes in a signaling cascade regulating the activity of Na(+)-K(+)-2Cl(-) cotransporters (NKCC1-2) and Na(+)-Cl(-) cotransporter (NCC). Both kinases have a conserved carboxyl-terminal (CCT) domain, which recognizes a unique peptide motif present in OSR1- and SPAK-activating kinases (with-no-lysine kinase 1 (WNK1) and WNK4) as well as their substrates (NKCC1, NKCC2, and NCC). Utilizing various modalities of the Rosetta Molecular Modeling Software Suite including flexible peptide docking and protein design, we comprehensively explored the sequence space recognized by the CCT domain. Specifically, we studied single residue mutations as well as complete unbiased designs of a hexapeptide substrate. The computational study started from a crystal structure of the CCT domain of OSR1 in complex with a hexapeptide derived from WNK4. Point mutations predicted to be favorable include Arg to His or Trp substitutions at position 2 and a Phe to Tyr substitution at position 3 of the hexapeptide. In addition, de novo design yielded two peptides predicted to bind to the CCT domain: FRFQVT and TRFDVT. These results, which indicate a little bit more freedom in the composition of the peptide, were confirmed through the use of yeast two-hybrid screening.

  3. The effects of knockdown of rho-associated kinase 1 and zipper-interacting protein kinase on gene expression and function in cultured human arterial smooth muscle cells.

    PubMed

    Deng, Jing-Ti; Wang, Xiu-Ling; Chen, Yong-Xiang; O'Brien, Edward R; Gui, Yu; Walsh, Michael P

    2015-01-01

    Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in diverse physiological functions. ROCK1 phosphorylates and activates ZIPK suggesting that at least some of these physiological functions may require both enzymes. To test the hypothesis that sequential activation of ROCK1 and ZIPK is commonly involved in regulatory pathways, we utilized siRNA to knock down ROCK1 and ZIPK in cultured human arterial smooth muscle cells (SMC). Microarray analysis using a whole-transcript expression chip identified changes in gene expression induced by ROCK1 and ZIPK knockdown. ROCK1 knockdown affected the expression of 553 genes, while ZIPK knockdown affected the expression of 390 genes. A high incidence of regulation of transcription regulator genes was observed in both knockdowns. Other affected groups included transporters, kinases, peptidases, transmembrane and G protein-coupled receptors, growth factors, phosphatases and ion channels. Only 76 differentially expressed genes were common to ROCK1 and ZIPK knockdown. Ingenuity Pathway Analysis identified five pathways shared between the two knockdowns. We focused on cytokine signaling pathways since ROCK1 knockdown up-regulated 5 and down-regulated 4 cytokine genes, in contrast to ZIPK knockdown, which affected the expression of only two cytokine genes (both down-regulated). IL-6 gene expression and secretion of IL-6 protein were up-regulated by ROCK1 knockdown, whereas ZIPK knockdown reduced IL-6 mRNA expression and IL-6 protein secretion and increased ROCK1 protein expression, suggesting that ROCK1 may inhibit IL-6 secretion. IL-1β mRNA and protein levels were increased in response to ROCK1 knockdown. Differences in the effects of ROCK1 and ZIPK knockdown on cell cycle regulatory genes suggested that ROCK1 and ZIPK regulate the cell cycle by different mechanisms. ROCK1, but not ZIPK knockdown reduced the viability and inhibited proliferation of vascular SMC. We conclude that ROCK1 and

  4. The effects of knockdown of rho-associated kinase 1 and zipper-interacting protein kinase on gene expression and function in cultured human arterial smooth muscle cells.

    PubMed

    Deng, Jing-Ti; Wang, Xiu-Ling; Chen, Yong-Xiang; O'Brien, Edward R; Gui, Yu; Walsh, Michael P

    2015-01-01

    Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in diverse physiological functions. ROCK1 phosphorylates and activates ZIPK suggesting that at least some of these physiological functions may require both enzymes. To test the hypothesis that sequential activation of ROCK1 and ZIPK is commonly involved in regulatory pathways, we utilized siRNA to knock down ROCK1 and ZIPK in cultured human arterial smooth muscle cells (SMC). Microarray analysis using a whole-transcript expression chip identified changes in gene expression induced by ROCK1 and ZIPK knockdown. ROCK1 knockdown affected the expression of 553 genes, while ZIPK knockdown affected the expression of 390 genes. A high incidence of regulation of transcription regulator genes was observed in both knockdowns. Other affected groups included transporters, kinases, peptidases, transmembrane and G protein-coupled receptors, growth factors, phosphatases and ion channels. Only 76 differentially expressed genes were common to ROCK1 and ZIPK knockdown. Ingenuity Pathway Analysis identified five pathways shared between the two knockdowns. We focused on cytokine signaling pathways since ROCK1 knockdown up-regulated 5 and down-regulated 4 cytokine genes, in contrast to ZIPK knockdown, which affected the expression of only two cytokine genes (both down-regulated). IL-6 gene expression and secretion of IL-6 protein were up-regulated by ROCK1 knockdown, whereas ZIPK knockdown reduced IL-6 mRNA expression and IL-6 protein secretion and increased ROCK1 protein expression, suggesting that ROCK1 may inhibit IL-6 secretion. IL-1β mRNA and protein levels were increased in response to ROCK1 knockdown. Differences in the effects of ROCK1 and ZIPK knockdown on cell cycle regulatory genes suggested that ROCK1 and ZIPK regulate the cell cycle by different mechanisms. ROCK1, but not ZIPK knockdown reduced the viability and inhibited proliferation of vascular SMC. We conclude that ROCK1 and

  5. 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

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

    PubMed

    Armengot, Laia; Marquès-Bueno, Maria Mar; Jaillais, Yvon

    2016-07-01

    The directional transport of auxin, known as polar auxin transport (PAT), 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, PAT 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, placing 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 (RLKs) and two-component histidine kinase receptors in PAT, noting that there are probably RLKs involved in co-ordinating 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

  7. 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

    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

  8. 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

  9. Mitogen-activated protein kinase/extracellular signal-regulated kinase 2 regulates cytoskeletal organization and chemotaxis via catalytic and microtubule-specific interactions.

    PubMed Central

    Reszka, A A; Bulinski, J C; Krebs, E G; Fischer, E H

    1997-01-01

    The extracellular signal-regulated kinases (ERKs) 1 and 2 are mitogen-activated protein kinases that act as key components in a signaling cascade linking growth factor receptors to the cytoskeleton and the nucleus. ERK2 mutants have been used to alter cytoskeletal regulation in Chinese hamster ovary cells without affecting cell growth or feedback signaling. Mutation of the unique loop L6 (residues 91-95), which is in a portion of the molecule that is cryptic upon the binding of ERK2 to the microtubules (MTs), generated significant morphological alterations. Most notable phenotypes were observed after expression of a combined mutant incorporating changes to both L6 and the TEY phosphorylation lip, including a 70% increase in cell spreading. Actin stress fibers in these cells, which normally formed a single broad parallel array, were arranged in three or more orientations or in fan-like arrays. MTs, which ordinarily extend longitudinally from the centrosome, spread radially, covering a larger surface area. Single, but not the double, mutations of the Thr and Tyr residues of the TEY phosphorylation lip caused a ca. 25% increase in cell spreading, accompanied by a threefold increase in chemotactic cell migration. Mutation of Lys-52 triggered a 48% increase in cell spreading but no alteration to chemotaxis. These findings suggest that wild-type ERK2 inhibits the organization of the cytoskeleton, the spreading of the cell, and chemotactic migration. This involves control of the orientation of actin and MTs and the positioning of focal adhesions via regulatory interactions that may occur on the MTs. Images PMID:9243503

  10. 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

    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.

  11. ENV7 and YCK3, which encode vacuolar membrane protein kinases, genetically interact to impact cell fitness and vacuole morphology.

    PubMed

    Manandhar, Surya P; Gharakhanian, Editte

    2014-05-01

    Saccharomyces cerevisiae vacuoles serve as a model for membrane fusion and fission. Yck3, a vacuolar membrane kinase, has been implicated in regulation of vacuole fusion. Recently, we established Env7 as another vacuolar membrane protein kinase with similar but nonredundant function to Yck3. Here, we report that native Env7 localizes to the vacuole independent of Yck3, where as its phosphorylation is YCK3 dependent. We also show that env7Δyck3Δ double mutant exhibits severely compromised fitness, altered cell size and bud vacuoles, and F-class vacuolar morphology. Our results establish negative genetic interactions between ENV7 and YCK3 and suggest cooperative roles for the two conserved genes in regulation of membrane dynamics. Such genetic buffering supports a critical role for membrane flux in global cell fitness.

  12. Small-world networks of residue interactions in the Abl kinase complexes with cancer drugs: topology of allosteric communication pathways can determine drug resistance effects.

    PubMed

    Tse, A; Verkhivker, G M

    2015-07-01

    The human protein kinases play a fundamental regulatory role in orchestrating functional processes in complex cellular networks. Understanding how conformational equilibrium between functional kinase states can be modulated by ligand binding or mutations is critical for quantifying molecular basis of allosteric regulation and drug resistance. In this work, molecular dynamics simulations of the Abl kinase complexes with cancer drugs (Imatinib and Dasatinib) were combined with structure-based network modeling to characterize dynamics of the residue interaction networks in these systems. The results have demonstrated that structural architecture of kinase complexes can produce a small-world topology of the interaction networks. Our data have indicated that specific Imatinib binding to a small number of highly connected residues could lead to network-bridging effects and allow for efficient allosteric communication, which is mediated by a dominant pathway sensitive to the unphosphorylated Abl state. In contrast, Dasatinib binding to the active kinase form may activate a broader ensemble of allosteric pathways that are less dependent on the phosphorylation status of Abl and provide a better balance between the efficiency and resilience of signaling routes. Our results have unveiled how differences in the residue interaction networks and allosteric communications of the Abl kinase complexes can be directly related to drug resistance effects. This study offers a plausible perspective on how efficiency and robustness of the residue interaction networks and allosteric pathways in kinase structures may be associated with protein responses to drug binding.

  13. Intramolecular interactions stabilizing compact conformations of the intrinsically disordered kinase-inhibitor domain of Sic1: a molecular dynamics investigation

    PubMed Central

    Lambrughi, Matteo; Papaleo, Elena; Testa, Lorenzo; Brocca, Stefania; De Gioia, Luca; Grandori, Rita

    2012-01-01

    Cyclin-dependent kinase inhibitors (CKIs) are key regulatory proteins of the eukaryotic cell cycle, which modulate cyclin-dependent kinase (Cdk) activity. CKIs perform their inhibitory effect by the formation of ternary complexes with a target kinase and its cognate cyclin. These regulators generally belong to the class of intrinsically disordered proteins (IDPs), which lack a well-defined and organized three-dimensional (3D) structure in their free state, undergoing folding upon binding to specific partners. Unbound IDPs are not merely random-coil structures, but can present intrinsically folded structural units (IFSUs) and collapsed conformations. These structural features can be relevant to protein function in vivo. The yeast CKI Sic1 is a 284-amino acid IDP that binds to Cdk1 in complex with the Clb5,6 cyclins, preventing phosphorylation of G1 substrates and, therefore, entrance to the S phase. Sic1 degradation, triggered by multiple phosphorylation events, promotes cell-cycle progression. Previous experimental studies pointed out a propensity of Sic1 and its isolated domains to populate both extended and compact conformations. The present contribution provides models for compact conformations of the Sic1 kinase-inhibitory domain (KID) by all-atom molecular dynamics (MD) simulations in explicit solvent and in the absence of interactors. The results are integrated by spectroscopic and spectrometric data. Helical IFSUs are identified, along with networks of intramolecular interactions. The results identify a group of putative hub residues and networks of electrostatic interactions, which are likely to be involved in the stabilization of the globular states. PMID:23189058

  14. Receptor-Interacting Protein Kinase 3 Deficiency Delays Cutaneous Wound Healing.

    PubMed

    Godwin, Andrew; Sharma, Archna; Yang, Weng-Lang; Wang, Zhimin; Nicastro, Jeffrey; Coppa, Gene F; Wang, Ping

    2015-01-01

    Wound healing consists of a complex, dynamic and overlapping process involving inflammation, proliferation and tissue remodeling. A better understanding of wound healing process at the molecular level is needed for the development of novel therapeutic strategies. Receptor-interacting protein kinase 3 (RIPK3) controls programmed necrosis in response to TNF-α during inflammation and has been shown to be highly induced during cutaneous wound repair. However, its role in wound healing remains to be demonstrated. To study this, we created dorsal cutaneous wounds on male wild-type (WT) and RIPK3-deficient (Ripk3-/-) mice. Wound area was measured daily until day 14 post-wound and skin tissues were collected from wound sites at various days for analysis. The wound healing rate in Ripk3-/- mice was slower than the WT mice over the 14-day course; especially, at day 7, the wound size in Ripk3-/- mice was 53% larger than that of WT mice. H&E and Masson-Trichrome staining analysis showed impaired quality of wound closure in Ripk3-/- wounds with delayed re-epithelialization and angiogenesis and defected granulation tissue formation and collagen deposition compared to WT. The neutrophil infiltration pattern was altered in Ripk3-/- wounds with less neutrophils at day 1 and more neutrophils at day 3. This altered pattern was also reflected in the differential expression of IL-6, KC, IL-1β and TNF-α between WT and Ripk3-/- wounds. MMP-9 protein expression was decreased with increased Timp-1 mRNA in the Ripk3-/- wounds compared to WT. The microvascular density along with the intensity and timing of induction of proangiogenic growth factors VEGF and TGF-β1 were also decreased or delayed in the Ripk3-/- wounds. Furthermore, mouse embryonic fibroblasts (MEFs) from Ripk3-/- mice migrated less towards chemoattractants TGF-β1 and PDGF than MEFs from WT mice. These results clearly demonstrate that RIPK3 is an essential molecule to maintain the temporal manner of the normal progression

  15. Receptor-Interacting Protein Kinase 3 Deficiency Delays Cutaneous Wound Healing

    PubMed Central

    Yang, Weng-Lang; Wang, Zhimin; Nicastro, Jeffrey; Coppa, Gene F.; Wang, Ping

    2015-01-01

    Wound healing consists of a complex, dynamic and overlapping process involving inflammation, proliferation and tissue remodeling. A better understanding of wound healing process at the molecular level is needed for the development of novel therapeutic strategies. Receptor-interacting protein kinase 3 (RIPK3) controls programmed necrosis in response to TNF-α during inflammation and has been shown to be highly induced during cutaneous wound repair. However, its role in wound healing remains to be demonstrated. To study this, we created dorsal cutaneous wounds on male wild-type (WT) and RIPK3-deficient (Ripk3-/-) mice. Wound area was measured daily until day 14 post-wound and skin tissues were collected from wound sites at various days for analysis. The wound healing rate in Ripk3-/- mice was slower than the WT mice over the 14-day course; especially, at day 7, the wound size in Ripk3-/- mice was 53% larger than that of WT mice. H&E and Masson-Trichrome staining analysis showed impaired quality of wound closure in Ripk3-/- wounds with delayed re-epithelialization and angiogenesis and defected granulation tissue formation and collagen deposition compared to WT. The neutrophil infiltration pattern was altered in Ripk3-/- wounds with less neutrophils at day 1 and more neutrophils at day 3. This altered pattern was also reflected in the differential expression of IL-6, KC, IL-1β and TNF-α between WT and Ripk3-/- wounds. MMP-9 protein expression was decreased with increased Timp-1 mRNA in the Ripk3-/- wounds compared to WT. The microvascular density along with the intensity and timing of induction of proangiogenic growth factors VEGF and TGF-β1 were also decreased or delayed in the Ripk3-/- wounds. Furthermore, mouse embryonic fibroblasts (MEFs) from Ripk3-/- mice migrated less towards chemoattractants TGF-β1 and PDGF than MEFs from WT mice. These results clearly demonstrate that RIPK3 is an essential molecule to maintain the temporal manner of the normal progression

  16. Protein Kinase C ζ Interacts with a Novel Binding Region of Gαq to Act as a Functional Effector.

    PubMed

    Sánchez-Fernández, Guzmán; Cabezudo, Sofía; Caballero, Álvaro; García-Hoz, Carlota; Tall, Gregory G; Klett, Javier; Michnick, Stephen W; Mayor, Federico; Ribas, Catalina

    2016-04-29

    Heterotrimeric G proteins play an essential role in the initiation of G protein-coupled receptor (GPCR) signaling through specific interactions with a variety of cellular effectors. We have recently reported that GPCR activation promotes a direct interaction between Gαq and protein kinase C ζ (PKCζ), leading to the stimulation of the ERK5 pathway independent of the canonical effector PLCβ. We report herein that the activation-dependent Gαq/PKCζ complex involves the basic PB1-type II domain of PKCζ and a novel interaction module in Gαq different from the classical effector-binding site. Point mutations in this Gαq region completely abrogate ERK5 phosphorylation, indicating that Gαq/PKCζ association is required for the activation of the pathway. Indeed, PKCζ was demonstrated to directly bind ERK5 thus acting as a scaffold between Gαq and ERK5 upon GPCR activation. The inhibition of these protein complexes by G protein-coupled receptor kinase 2, a known Gαq modulator, led to a complete abrogation of ERK5 stimulation. Finally, we reveal that Gαq/PKCζ complexes link Gαq to apoptotic cell death pathways. Our data suggest that the interaction between this novel region in Gαq and the effector PKCζ is a key event in Gαq signaling.

  17. Mitogen-activated protein kinase p38b interaction with delta class glutathione transferases from the fruit fly, Drosophila melanogaster.

    PubMed

    Wongtrakul, Jeerang; Sukittikul, Suchada; Saisawang, Chonticha; Ketterman, Albert J

    2012-01-01

    Glutathione transferases (GSTs) are a family of multifunctional enzymes involved in xenobiotic biotransformation, drug metabolism, and protection against oxidative damage. The p38b mitogen-activated protein kinase is involved in cellular stress response. This study screened interactions between Drosophila melanogaster Meigen (Diptera: Drosophilidae) Delta class glutathione transferases (DmGSTs) and the D. melanogaster p38b MAPK. Therefore, 12 DmGSTs and p38b kinase were obtained as recombinant proteins. The study showed that DmGSTD8 and DmGSTD11b significantly increased p38b activity toward ATF2 and jun, which are transcription factor substrates. DmGSTD3 and DmGSTD5 moderately increased p38b activity for jun. In addition, GST activity in the presence of p38b was also measured. It was found that p38b affected substrate specificity toward CDNB (1-chloro-2,4-dinitrobenzene) and DCNB (1,2-dichloro-4-nitrobenzene) of several GST isoforms, i.e., DmGSTD2, DmGSTD5, DmGSTD8, and DmGSTD11b. The interaction of a GST and p38b can affect the substrate specificity of either enzyme, which suggests induced conformational changes affecting catalysis. Similar interactions do not occur for all the Delta enzymes and p38b, which suggests that these interactions could be specific. PMID:23438069

  18. Protein kinase CK2 interacts with Chk2 and phosphorylates Mre11 on serine 649

    SciTech Connect

    Kim, Seong-Tae . E-mail: stkim@med.skku.ac.kr

    2005-05-27

    The Mre11-Rad50-Nbs1 protein complex has been known to be involved in a variety of DNA metabolic events that involve DNA double-strand breaks (DSBs). The phosphorylation of Mre11 is increased in response to ionizing radiation, which suggests that phosphorylation of Mre11 may be an important regulatory mechanism of this complex. Mre11-phosphorylating kinase activities were observed in Chk2 immunoprecipitates and HeLa nuclear extracts. Through the tandem affinity tagging system and conventional chromatography, this kinase was purified and identified as protein kinase CK2. CK2 phosphorylates Mre11 in vitro. In vitro kinase assay with a series of truncated Mre11 proteins as substrates for CK2 and site-directed mutagenesis showed that serine 649 of Mre11 is mainly phosphorylated by CK2 in vitro. In vivo labeling and phosphopeptide mapping analysis revealed that this phosphorylation occurs in vivo. These data implicate CK2 as a potential upstream regulator of Mre11 function.

  19. Molecular dynamics simulations and modelling of the residue interaction networks in the BRAF kinase complexes with small molecule inhibitors: probing the allosteric effects of ligand-induced kinase dimerization and paradoxical activation.

    PubMed

    Verkhivker, G M

    2016-10-20

    Protein kinases are central to proper functioning of cellular networks and are an integral part of many signal transduction pathways. The family of protein kinases represents by far the largest and most important class of therapeutic targets in oncology. Dimerization-induced activation has emerged as a common mechanism of allosteric regulation in BRAF kinases, which play an important role in growth factor signalling and human diseases. Recent studies have revealed that most of the BRAF inhibitors can induce dimerization and paradoxically stimulate enzyme transactivation by conferring an active conformation in the second monomer of the kinase dimer. The emerging connections between inhibitor binding and BRAF kinase domain dimerization have suggested a molecular basis of the activation mechanism in which BRAF inhibitors may allosterically modulate the stability of the dimerization interface and affect the organization of residue interaction networks in BRAF kinase dimers. In this work, we integrated structural bioinformatics analysis, molecular dynamics and binding free energy simulations with the protein structure network analysis of the BRAF crystal structures to determine dynamic signatures of BRAF conformations in complexes with different types of inhibitors and probe the mechanisms of the inhibitor-induced dimerization and paradoxical activation. The results of this study highlight previously unexplored relationships between types of BRAF inhibitors, inhibitor-induced changes in the residue interaction networks and allosteric modulation of the kinase activity. This study suggests a mechanism by which BRAF inhibitors could promote or interfere with the paradoxical activation of BRAF kinases, which may be useful in informing discovery efforts to minimize the unanticipated adverse biological consequences of these therapeutic agents.

  20. A conserved streptococcal membrane protein, LsrS, exhibits a receptor-like function for lantibiotics.

    PubMed

    Biswas, Saswati; Biswas, Indranil

    2014-04-01

    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. 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.

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

    PubMed

    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

  3. 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

  4. Dehydroglyasperin D Inhibits the Proliferation of HT-29 Human Colorectal Cancer Cells Through Direct Interaction With Phosphatidylinositol 3-kinase

    PubMed Central

    Jung, Sung Keun; Jeong, Chul-Ho

    2016-01-01

    Background: Despite recent advances in therapy, colorectal cancer still has a grim prognosis. Although licorice has been used in East Asian traditional medicine, the molecular properties of its constituents including dehydroglyasperin D (DHGA-D) remain unknown. We sought to evaluate the inhibitory effect of DHGA-D on colorectal cancer cell proliferation and identify the primary signaling molecule targeted by DHGA-D. Methods: We evaluated anchorage-dependent and -independent cell growth in HT-29 human colorectal adenocarcinoma cells. The target protein of DHGA-D was identified by Western blot analysis with a specific antibody, and direct interaction between DHGA-D and the target protein was confirmed by kinase and pull-down assays. Cell cycle analysis by flow cytometry and further Western blot analysis was performed to identify the signaling pathway involved. Results: DHGA-D significantly suppressed anchorage-dependent and -independent HT-29 colorectal cancer cell proliferation. DHGA-D directly suppressed phosphatidylinositol 3-kinase (PI3K) activity and subsequent Akt phosphorylation and bound to the p110 subunit of PI3K. DHGA-D also significantly induced G1 cell cycle arrest, together with the suppression of glycogen synthase kinase 3β and retinoblastoma phosphorylation and cyclin D1 expression. Conclusions: DHGA-D has potent anticancer activity and targets PI3K in human colorectal adenocarcinoma HT-29 cells. To our knowledge, this is the first report to detail the molecular basis of DHGA-D in suppressing colorectal cancer cell growth. PMID:27051646

  5. Receptor Interacting Protein 3 Suppresses Vascular Smooth Muscle Cell Growth by Inhibition of the Phosphoinositide 3-Kinase-Akt Axis*

    PubMed Central

    Li, Qian; Li, Geng; Lan, Xiaomei; Zheng, Ming; Chen, Kuang-Hueih; Cao, Chun-Mei; Xiao, Rui-Ping

    2010-01-01

    Proliferation of vascular smooth muscle cells (VSMCs) is a primary mechanism underlying cardiovascular proliferative disorders. Phosphoinositide 3-kinase (PI3K)-Akt (or protein kinase B) axis has been assigned at the center of pathways that regulate cell proliferation. Here we demonstrate that enhanced PI3K-Akt signaling by mitogenic stimulation or arterial injury profoundly elevates expression of receptor interacting protein 3 (RIP3) in primary cultured rat VSMCs and in vivo and that the up-regulation of RIP3 leads to VSMC growth arrest and apoptosis via inhibiting the PI3K-Akt signaling pathway, thereby alleviating balloon injury-induced neointimal formation. Specifically, mitogenic stimulation with platelet-derived growth factor-BB or angiotensin II leads to a profound increase in RIP3 expression, which is abolished by inhibition of PI3K or Akt, and increased PI3K-Akt signaling by expression of a constitutively active PI3K mutant also elevates RIP3 expression. Importantly, adenoviral overexpression of RIP3 not only triggers apoptosis but also causes cell cycle arrest at G1/G0 phases that is associated with suppressed Akt activation. In sharp contrast, RIP3 gene silencing enhances serum- and platelet-derived growth factor-induced cell proliferation and Akt activation. In vivo adenoviral gene delivery of rat RIP3 (rRIP3) increased apoptosis and reduced VSMC proliferation, thus, effectively alleviating balloon injury-induced neointimal formation. The growth-suppressive and pro-apoptotic effects are independent of rRIP3 Ser/Thr kinase activity, because overexpression of a kinase-inactive mutant of rRIP3, similar to its wild type, is sufficient to induce growth arrest and apoptosis. These findings reveal a novel growth-suppressive action of RIP3, marking RIP3 as an important factor to prevent excessive mitogenic stimulation- or injury-induced vascular smooth muscle cells hyperplasia. PMID:20042608

  6. Selective Phosphorylation Inhibitor of Delta Protein Kinase C-Pyruvate Dehydrogenase Kinase Protein-Protein Interactions: Application for Myocardial Injury in Vivo.

    PubMed

    Qvit, Nir; Disatnik, Marie-Hélène; Sho, Eiketsu; Mochly-Rosen, Daria

    2016-06-22

    Protein kinases regulate numerous cellular processes, including cell growth, metabolism, and cell death. Because the primary sequence and the three-dimensional structure of many kinases are highly similar, the development of selective inhibitors for only one kinase is challenging. Furthermore, many protein kinases are pleiotropic, mediating diverse and sometimes even opposing functions by phosphorylating multiple protein substrates. Here, we set out to develop an inhibitor of a selective protein kinase phosphorylation of only one of its substrates. Focusing on the pleiotropic delta protein kinase C (δPKC), we used a rational approach to identify a distal docking site on δPKC for its substrate, pyruvate dehydrogenase kinase (PDK). We reasoned that an inhibitor of PDK's docking should selectively inhibit the phosphorylation of only PDK without affecting phosphorylation of the other δPKC substrates. Our approach identified a selective inhibitor of PDK docking to δPKC with an in vitro Kd of ∼50 nM and reducing cardiac injury IC50 of ∼5 nM. This inhibitor, which did not affect the phosphorylation of other δPKC substrates even at 1 μM, demonstrated that PDK phosphorylation alone is critical for δPKC-mediated injury by heart attack. The approach we describe is likely applicable for the identification of other substrate-specific kinase inhibitors. PMID:27218445

  7. Evidence for new homotypic and heterotypic interactions between transmembrane helices of proteins involved in receptor tyrosine kinase and neuropilin signaling.

    PubMed

    Sawma, Paul; Roth, Lise; Blanchard, Cécile; Bagnard, Dominique; Crémel, Gérard; Bouveret, Emmanuelle; Duneau, Jean-Pierre; Sturgis, James N; Hubert, Pierre

    2014-12-12

    Signaling in eukaryotic cells frequently relies on dynamic interactions of single-pass membrane receptors involving their transmembrane (TM) domains. To search for new such interactions, we have developed a bacterial two-hybrid system to screen for both homotypic and heterotypic interactions between TM helices. We have explored the dimerization of TM domains from 16 proteins involved in both receptor tyrosine kinase and neuropilin signaling. This study has revealed several new interactions. We found that the TM domain of Mucin-4, a putative intramembrane ligand for erbB2, dimerizes not only with erbB2 but also with all four members of the erbB family. In the Neuropilin/Plexin family of receptors, we showed that the TM domains of Neuropilins 1 and 2 dimerize with themselves and also with Plexin-A1, Plexin-B1, and L1CAM, but we were unable to observe interactions with several other TM domains notably those of members of the VEGF receptor family. The potentially important Neuropilin 1/Plexin-A1 interaction was confirmed using a surface plasmon resonance assay. This work shows that TM domain interactions can be highly specific. Exploring further the propensities of TM helix-helix association in cell membrane should have important practical implications related to our understanding of the structure-function of bitopic proteins' assembly and subsequent function, especially in the regulation of signal transduction. PMID:25315821

  8. 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

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

    PubMed

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

    2016-08-01

    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

  10. 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.

  11. Kinase requirements in human cells: V. Synthetic lethal interactions between p53 and the protein kinases SGK2 and PAK3.

    PubMed

    Baldwin, Amy; Grueneberg, Dorre A; Hellner, Karin; Sawyer, Jacqueline; Grace, Miranda; Li, Wenliang; Harlow, Ed; Munger, Karl

    2010-07-13

    Cervical carcinomas are initiated through a series of well-defined stages that rely on the expression of human papillomavirus (HPV) oncogenes. A panel of 100 small hairpin RNAs that target essential kinases in many tumor types was used to study the stepwise appearance of kinase requirements during cervical tumor development. Twenty-six kinases were commonly required in three cell lines derived from frank carcinomas, and each kinase requirement was traced to the specific stage in which the requirement emerged. Six kinases became required following HPV-induced immortalization, and the requirement for two kinases, SGK2 and PAK3, was mapped to the inactivation of p53 in primary human epithelial cells. Loss of the p53 tumor suppressor in other primary epithelial cells also induced dependence on SGK2 and PAK3. Hence, SGK2 and PAK3 provide important cellular functions following p53 inactivation, fulfilling the classical definition of synthetic lethality; loss of p53, SGK2, or PAK3 alone has little effect on cell viability, whereas loss of p53 together with either SGK2 or PAK3 loss leads to cell death. Whereas tumor suppressor gene mutations are not directly druggable, other proteins or pathways that become obligatory to cell viability following tumor suppressor loss provide theoretical targets for tumor suppressor-specific drug discovery efforts. The kinases SGK2 and PAK3 may thus represent such targets for p53-specific drug development.

  12. Rapeseed calcineurin B-like protein CBL4, interacting with CBL-interacting protein kinase CIPK24, modulates salt tolerance in plants.

    PubMed

    Liu, Wu-Zhen; Deng, Min; Li, Liang; Yang, Bo; Li, Hongwei; Deng, Hanqing; Jiang, Yuan-Qing

    2015-11-20

    Calcium is a ubiquitous intracellular secondary messenger in eukaryotes. Upon stress challenge, cytosolic Ca(2+) fluctuation could be sensed and bound by calcineurin B-like proteins (CBLs), which further regulate a group of Ser/Thr protein kinases called CBL-interacting protein kinases (CIPKs) to relay the signal and induce cellular responses. Although the CBL-CIPK network has been demonstrated to play crucial roles in plant development and responses to various environmental stresses in Arabidopsis, little is known about their function in rapeseed. In the present study, we characterized CBL4 gene from rapeseed. We found that CBL4 is localized at the plasma membrane and it interacted with CIPK24 in both yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. Unlike the orthologs in Arabidopsis, rapeseed CIPK24 did not interact with CBL10. Furthermore, expression of rapeseed CBL4 rescued the salt-sensitive phenotype of sos3-1 mutant and overexpression of rapeseed CBL4 in Arabidopsis showed enhanced tolerance of salt stress than wild-type. Overall, the results clarified the function of CBL4 in rapeseed.

  13. 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

  14. Rip1 (receptor-interacting protein kinase 1) mediates necroptosis and contributes to renal ischemia/reperfusion injury.

    PubMed

    Linkermann, Andreas; Bräsen, Jan H; Himmerkus, Nina; Liu, Shuya; Huber, Tobias B; Kunzendorf, Ulrich; Krautwald, Stefan

    2012-04-01

    Loss of kidney function in renal ischemia/reperfusion injury is due to programmed cell death, but the contribution of necroptosis, a newly discovered form of programmed necrosis, has not been evaluated. Here, we identified the presence of death receptor-mediated but caspase-independent cell death in murine tubular cells and characterized it as necroptosis by the addition of necrostatin-1, a highly specific receptor-interacting protein kinase 1 inhibitor. The detection of receptor-interacting protein kinase 1 and 3 in whole-kidney lysates and freshly isolated murine proximal tubules led us to investigate the contribution of necroptosis in a mouse model of renal ischemia/reperfusion injury. Treatment with necrostatin-1 reduced organ damage and renal failure, even when administered after reperfusion, resulting in a significant survival benefit in a model of lethal renal ischemia/reperfusion injury. Unexpectedly, specific blockade of apoptosis by zVAD, a pan-caspase inhibitor, did not prevent the organ damage or the increase in urea and creatinine in vivo in renal ischemia/reperfusion injury. Thus, necroptosis is present and has functional relevance in the pathophysiological course of ischemic kidney injury and shows the predominance of necroptosis over apoptosis in this setting. Necrostatin-1 may have therapeutic potential to prevent and treat renal ischemia/reperfusion injury. PMID:22237751

  15. The tomato NBARC-LRR protein Prf interacts with Pto kinase in vivo to regulate specific plant immunity.

    PubMed

    Mucyn, Tatiana S; Clemente, Alfonso; Andriotis, Vasilios M E; Balmuth, Alexi L; Oldroyd, Giles E D; Staskawicz, Brian J; Rathjen, John P

    2006-10-01

    Immunity in tomato (Solanum lycopersicum) to Pseudomonas syringae bacteria expressing the effector proteins AvrPto and AvrPtoB requires both Pto kinase and the NBARC-LRR (for nucleotide binding domain shared by Apaf-1, certain R gene products, and CED-4 fused to C-terminal leucine-rich repeats) protein Prf. Pto plays a direct role in effector recognition within the host cytoplasm, but the role of Prf is unknown. We show that Pto and Prf are coincident in the signal transduction pathway that controls ligand-independent signaling. Pto and Prf associate in a coregulatory interaction that requires Pto kinase activity and N-myristoylation for signaling. Pto interacts with a unique Prf N-terminal domain outside of the NBARC-LRR domain and resides in a high molecular weight recognition complex dependent on the presence of Prf. In this complex, both Pto and Prf contribute to specific recognition of AvrPtoB. The data suggest that the role of Pto is confined to the regulation of Prf and that the bacterial effectors have evolved to target this coregulatory molecular switch.

  16. A Cyclic di-GMP-binding Adaptor Protein Interacts with Histidine Kinase to Regulate Two-component Signaling.

    PubMed

    Xu, Linghui; Venkataramani, Prabhadevi; Ding, Yichen; Liu, Yang; Deng, Yinyue; Yong, Grace Lisi; Xin, Lingyi; Ye, Ruijuan; Zhang, Lianhui; Yang, Liang; Liang, Zhao-Xun

    2016-07-29

    The bacterial messenger cyclic di-GMP (c-di-GMP) binds to a diverse range of effectors to exert its biological effect. Despite the fact that free-standing PilZ proteins are by far the most prevalent c-di-GMP effectors known to date, their physiological function and mechanism of action remain largely unknown. Here we report that the free-standing PilZ protein PA2799 from the opportunistic pathogen Pseudomonas aeruginosa interacts directly with the hybrid histidine kinase SagS. We show that PA2799 (named as HapZ: histidine kinase associated PilZ) binds directly to the phosphoreceiver (REC) domain of SagS, and that the SagS-HapZ interaction is further enhanced at elevated c-di-GMP concentration. We demonstrate that binding of HapZ to SagS inhibits the phosphotransfer between SagS and the downstream protein HptB in a c-di-GMP-dependent manner. In accordance with the role of SagS as a motile-sessile switch and biofilm growth factor, we show that HapZ impacts surface attachment and biofilm formation most likely by regulating the expression of a large number of genes. The observations suggest a previously unknown mechanism whereby c-di-GMP mediates two-component signaling through a PilZ adaptor protein.

  17. A Cyclic di-GMP-binding Adaptor Protein Interacts with Histidine Kinase to Regulate Two-component Signaling.

    PubMed

    Xu, Linghui; Venkataramani, Prabhadevi; Ding, Yichen; Liu, Yang; Deng, Yinyue; Yong, Grace Lisi; Xin, Lingyi; Ye, Ruijuan; Zhang, Lianhui; Yang, Liang; Liang, Zhao-Xun

    2016-07-29

    The bacterial messenger cyclic di-GMP (c-di-GMP) binds to a diverse range of effectors to exert its biological effect. Despite the fact that free-standing PilZ proteins are by far the most prevalent c-di-GMP effectors known to date, their physiological function and mechanism of action remain largely unknown. Here we report that the free-standing PilZ protein PA2799 from the opportunistic pathogen Pseudomonas aeruginosa interacts directly with the hybrid histidine kinase SagS. We show that PA2799 (named as HapZ: histidine kinase associated PilZ) binds directly to the phosphoreceiver (REC) domain of SagS, and that the SagS-HapZ interaction is further enhanced at elevated c-di-GMP concentration. We demonstrate that binding of HapZ to SagS inhibits the phosphotransfer between SagS and the downstream protein HptB in a c-di-GMP-dependent manner. In accordance with the role of SagS as a motile-sessile switch and biofilm growth factor, we show that HapZ impacts surface attachment and biofilm formation most likely by regulating the expression of a large number of genes. The observations suggest a previously unknown mechanism whereby c-di-GMP mediates two-component signaling through a PilZ adaptor protein. PMID:27231351

  18. Signalling-dependent interactions between the kinase-coupling protein CheW and chemoreceptors in living cells.

    PubMed

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

    2014-09-01

    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 cross-linking methods to detect close interactions between the coupling protein CheW and the serine receptor Tsr in intact Escherichia 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).

  19. Metabolism-related pharmacokinetic drug−drug interactions with tyrosine kinase inhibitors: current understanding, challenges and recommendations

    PubMed Central

    Teo, Yi Ling; Ho, Han Kiat; Chan, Alexandre

    2015-01-01

    Drug−drug interactions (DDIs) occur when a patient's response to the drug is modified by administration or co-exposure to another drug. The main cytochrome P450 (CYP) enzyme, CYP3A4, is implicated in the metabolism of almost all of the tyrosine kinase inhibitors (TKIs). Therefore, there is a substantial potential for interaction between TKIs and other drugs that modulate the activity of this metabolic pathway. Cancer patients are susceptible to DDIs as they receive many medications, either for supportive care or for treatment of toxicity. Differences in DDI outcomes are generally negligible because of the wide therapeutic window of common drugs. However for anticancer agents, serious clinical consequences may occur from small changes in drug metabolism and pharmacokinetics. Therefore, the objective of this review is to highlight the current understanding of DDIs among TKIs, with a focus on metabolism, as well as to identify challenges in the prediction of DDIs and provide recommendations. PMID:25125025

  20. Exploring the interaction between human focal adhesion kinase and inhibitors: a molecular dynamic simulation and free energy calculations.

    PubMed

    Zhan, Jiu-Yu; Zhang, Ji-Long; Wang, Yan; Li, Ye; Zhang, Hong-Xing; Zheng, Qing-Chuan

    2016-11-01

    Focal adhesion kinase is an important target for the treatment of many kinds of cancers. Inhibitors of FAK are proposed to be the anticancer agents for multiple tumors. The interaction characteristic between FAK and its inhibitors is crucial to develop new inhibitors. In the present article, we used Molecular Dynamic (MD) simulation method to explore the characteristic of interaction between FAK and three inhibitors (PHM16, TAE226, and ligand3). The MD simulation results together with MM-GB/SA calculations show that the combinations are enthalpy-driven process. Cys502 and Asp564 are both essential residues due to the hydrogen bond interactions with inhibitors, which was in good agreement with experimental data. Glu500 can form a non-classical hydrogen bond with each inhibitor. Arg426 can form electrostatic interactions with PHM16 and ligand3, while weaker with TAE226. The electronic static potential was employed, and we found that the ortho-position methoxy of TAE226 has a weaker negative charge than the meta-position one in PHM16 or ligand3. Ile428, Val436, Ala452, Val484, Leu501, Glu505, Glu506, Leu553, Gly563 Leu567, Ser568 are all crucial residues in hydrophobic interactions. The key residues in this work will be available for further inhibitor design of FAK and also give assistance to further research of cancer.

  1. Phosphorylation of Voltage-Dependent Anion Channel by Serine/Threonine Kinases Governs Its Interaction with Tubulin

    PubMed Central

    Sheldon, Kely L.; Maldonado, Eduardo N.; Lemasters, John J.; Rostovtseva, Tatiana K.; Bezrukov, Sergey M.

    2011-01-01

    Tubulin was recently found to be a uniquely potent regulator of the voltage-dependent anion channel (VDAC), the most abundant channel of the mitochondrial outer membrane, which constitutes a major pathway for ATP/ADP and other metabolites across this membrane. Dimeric tubulin induces reversible blockage of VDAC reconstituted into a planar lipid membrane and dramatically reduces respiration of isolated mitochondria. Here we show that VDAC phosphorylation is an important determinant of its interaction with dimeric tubulin. We demonstrate that in vitro phosphorylation of VDAC by either glycogen synthase kinase-3β (GSK3β) or cAMP-dependent protein kinase A (PKA), increases the on-rate of tubulin binding to the reconstituted channel by orders of magnitude, but only for tubulin at the cis side of the membrane. This and the fact the basic properties of VDAC, such as single-channel conductance and selectivity, remained unaltered by phosphorylation allowed us to suggest the phosphorylation regions positioned on the cytosolic loops of VDAC and establish channel orientation in our reconstitution experiments. Experiments on human hepatoma cells HepG2 support our conjecture that VDAC permeability for the mitochondrial respiratory substrates is regulated by dimeric tubulin and channel phosphorylation. Treatment of HepG2 cells with colchicine prevents microtubule polymerization, thus increasing dimeric tubulin availability in the cytosol. Accordingly, this leads to a decrease of mitochondrial potential measured by assessing mitochondrial tetramethylrhodamine methyester uptake with confocal microscopy. Inhibition of PKA activity blocks and reverses mitochondrial depolarization induced by colchicine. Our findings suggest a novel functional link between serine/threonine kinase signaling pathways, mitochondrial respiration, and the highly dynamic microtubule network which is characteristic of cancerogenesis and cell proliferation. PMID:22022409

  2. Interactions between Eph kinases and ephrins provide a mechanism to support platelet aggregation once cell-to-cell contact has occurred

    PubMed Central

    Prevost, Nicolas; Woulfe, Donna; Tanaka, Takako; Brass, Lawrence F.

    2002-01-01

    Eph kinases are receptor tyrosine kinases whose ligands, the ephrins, are also expressed on the surface of cells. Interactions between Eph kinases and ephrins on adjacent cells play a central role in neuronal patterning and vasculogenesis. Here we examine the expression of ephrins and Eph kinases on human blood platelets and explore their role in the formation of the hemostatic plug. The results show that human platelets express EphA4 and EphB1, and the ligand, ephrinB1. Forced clustering of EphA4 or ephrinB1 led to cytoskeletal reorganization, adhesion to fibrinogen, and α-granule secretion. Clustering of ephrinB1 also caused activation of the Ras family member, Rap1B. In platelets that had been activated by ADP and allowed to aggregate, EphA4 formed complexes with two tyrosine kinases, Fyn and Lyn, and the cell adhesion molecule, L1. Blockade of Eph/ephrin interactions prevented the formation of these complexes and caused platelet aggregation at low ADP concentrations to become more readily reversible. We propose that when sustained contacts between platelets have occurred in response to agonists such as collagen, ADP, and thrombin, the binding of ephrins to Eph kinases on adjacent platelets provides a mechanism to perpetuate signaling and promote stable platelet aggregation. PMID:12084815

  3. EDR1 Physically Interacts with MKK4/MKK5 and Negatively Regulates a MAP Kinase Cascade to Modulate Plant Innate Immunity

    PubMed Central

    Zhao, Chunzhao; Nie, Haozhen; Shen, Qiujing; Zhang, Shuqun; Lukowitz, Wolfgang; Tang, Dingzhong

    2014-01-01

    Mitogen-activated protein (MAP) kinase signaling cascades play important roles in the regulation of plant defense. The Raf-like MAP kinase kinase kinase (MAPKKK) EDR1 negatively regulates plant defense responses and cell death. However, how EDR1 functions, and whether it affects the regulation of MAPK cascades, are not well understood. Here, we showed that EDR1 negatively regulates the MKK4/MKK5-MPK3/MPK6 kinase cascade in Arabidopsis. We found that edr1 mutants have highly activated MPK3/MPK6 kinase activity and higher levels of MPK3/MPK6 proteins than wild type. EDR1 physically interacts with MKK4 and MKK5, and this interaction requires the N-terminal domain of EDR1. EDR1 also negatively affects MKK4/MKK5 protein levels. In addition, the mpk3, mkk4 and mkk5 mutations suppress edr1-mediated resistance, and over-expression of MKK4 or MKK5 causes edr1-like resistance and mildew-induced cell death. Taken together, our data indicate that EDR1 physically associates with MKK4/MKK5 and negatively regulates the MAPK cascade to fine-tune plant innate immunity. PMID:24830651

  4. Phosphorylation of the amino-terminus of the AGC kinase Gad8 prevents its interaction with TORC2

    PubMed Central

    Du, Wei; Forte, Gabriella M.; Smith, Duncan; Petersen, Janni

    2016-01-01

    Cell proliferation, metabolism, migration and survival are coordinated through the tight control of two target of rapamycin (TOR) kinase complexes: TORC1 and TORC2. Here, we show that a novel phosphorylation of fission yeast Gad8 (AGC kinase) on the evolutionarily conserved threonine 6 (Thr6) prevents the physical association between Gad8 and TORC2. Accordingly, this block to protein interactions by Gad8 Thr6 phosphorylation decreases TORC2-controlled activation of Gad8. Likewise, phosphorylation of Gad8 Thr6, possibly by PKC, prevents the association of Gad8 with TORC2 thereby increasing TORC2 activity, because it reduces Gad8-mediated feedback inhibition of TORC2. Consistently, the introduction of a Gad8 T6D mutant, that mimics phosphorylation, increased TORC2 activity. Increased PKCPck2 expression prevented Gad8–TORC2 binding and so reduced the TORC2-mediated phosphorylation of Gad8 serine 546 that activates Gad8. Interestingly, independent of the Ser546 phosphorylation status, Gad8 Thr6 phosphorylation is important for remodelling the actin cytoskeleton and survival upon potassium ion and heat stresses. In contrast, Ser546 phosphorylation is required for the control of G1 arrest, mating, cell length at division and vascular size. Finally, these findings reveal a novel mode of TORC2 activation that is essential for cell survival following stress. PMID:26935949

  5. Cell cycle regulatory protein p27KIP1 is a substrate and interacts with the protein kinase CK2.

    PubMed

    Tapia, Julio C; Bolanos-Garcia, Victor M; Sayed, Muhammed; Allende, Catherine C; Allende, Jorge E

    2004-04-01

    The protein kinase CK2 is constituted by two catalytic (alpha and/or alpha') and two regulatory (beta) subunits. CK2 phosphorylates more than 300 proteins with important functions in the cell cycle. This study has looked at the relation between CK2 and p27(KIP1), which is a regulator of the cell cycle and a known inhibitor of cyclin-dependent kinases (Cdk). We demonstrated that in vitro recombinant Xenopus laevis CK2 can phosphorylate recombinant human p27(KIP1), but this phosphorylation occurs only in the presence of the regulatory beta subunit. The principal site of phosphorylation is serine-83. Analysis using pull down and surface plasmon resonance (SPR) techniques showed that p27(KIP1) interacts with the beta subunit through two domains present in the amino and carboxyl ends, while CD spectra showed that p27(KIP1) phosphorylation by CK2 affects its secondary structure. Altogether, these results suggest that p27(KIP1) phosphorylation by CK2 probably involves a docking event mediated by the CK2beta subunit. The phosphorylation of p27(KIP1) by CK2 may affect its biological activity.

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

    PubMed Central

    Kiburu, Irene N.; LaRonde-LeBlanc, Nicole

    2012-01-01

    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 Å 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. PMID:22629386

  7. Homeodomain-interacting protein kinase 2 (HIPK2) targets {beta}-catenin for phosphorylation and proteasomal degradation

    SciTech Connect

    Kim, Eun-A; Kim, Ji Eon; Sung, Ki Sa; Choi, Dong Wook; Lee, Byeong Jae; Choi, Cheol Yong

    2010-04-16

    The regulation of intracellular {beta}-catenin levels is central in the Wnt/{beta}-catenin signaling cascade and the activation of the Wnt target genes. Here, we show that homeodomain-interacting protein kinase 2 (HIPK2) acts as a negative regulator of the Wnt/{beta}-catenin pathway. Knock-down of endogenous HIPK2 increases the stability of {beta}-catenin and results in the accumulation of {beta}-catenin in the nucleus, consequently enhancing the expression of Wnt target genes and cell proliferation both in vivo and in cultured cells. HIPK2 inhibits TCF/LEF-mediated target gene activation via degradation of {beta}-catenin. HIPK2 phosphorylates {beta}-catenin at its Ser33 and Ser37 residues without the aid of a priming kinase. Substitutions of Ser33 and Ser37 for alanines abolished the degradation of {beta}-catenin associated with HIPK2. In ex vivo mouse model, HIPK2 knock-down resulted in accumulation of {beta}-catenin, thereby potentiated {beta}-catenin-mediated cell proliferation and tumor formation. Furthermore, the axis duplication induced by the ectopic expression of {beta}-catenin was blocked by co-injection of HIPK2 mRNAs into Xenopus embryos. Taken together, HIPK2 appears to function as a novel negative regulator of {beta}-catenin through its phosphorylation and proteasomal degradation.

  8. Reconstitution of interactions between tyrosine kinases and the high affinity IgE receptor which are controlled by receptor clustering.

    PubMed Central

    Scharenberg, A M; Lin, S; Cuenod, B; Yamamura, H; Kinet, J P

    1995-01-01

    High affinity IgE receptor (Fc epsilon RI) signaling after contact with antigen occurs in response to receptor clustering. This paper describes methodology, based on vaccinia virus driven protein expression, for probing signaling pathways and its application to Fc epsilon RI interactions with the lyn and syk tyrosine kinases. Reconstitution of the complete tetrameric Fc epsilon RI receptor, lyn and syk in a non-hematopoietic 'null' cell line is sufficient to reconstruct clustering-controlled receptor tyrosine phosphorylation and activation of syk, without apparent requirement for hematopoietic specific phosphatases. The src family kinase lyn phosphorylates Fc epsilon RI in response to receptor clustering, resulting in syk binding to the phosphorylated Fc epsilon RI. Lyn also participates in the tyrosine phosphorylation and activation of syk in a manner which is dependent on phosphorylated Fc epsilon RI. Using overexpression of active and dominant negative syk proteins in a mast cell line which naturally expresses Fc epsilon RI, we corroborate syk's role downstream of receptor phosphorylation, and demonstrate that syk SH2 domains protect receptor ITAMs from ongoing dephosphorylation. Based on these results, we propose that receptor clustering controls lyn-mediated Fc epsilon RI tyrosine phosphorylation by shifting a balance between phosphorylation and dephosphorylation towards accumulation of tyrosine phosphorylated Fc epsilon RI. Fc epsilon RI tyrosine phosphorylation functions to bring syk into a microenvironment where it becomes tyrosine phosphorylated and activated, thereby allowing clustering to indirectly control syk activity. Images PMID:7628439

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

    PubMed

    Tan, Zhi; Chen, Lu; Zhang, Shuxing

    2016-09-21

    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.

  10. 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

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

    PubMed

    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

  12. 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.

  13. p38 mitogen-activated protein kinase interacts with vinculin at focal adhesions during fatty acid-stimulated cell adhesion

    PubMed Central

    George, Margaret D.; Wine, Robert N.; Lackford, Brad; Kissling, Grace E.; Akiyama, Steven K.; Olden, Kenneth; Roberts, John D.

    2014-01-01

    Arachidonic acid stimulates cell adhesion by activating α2β1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. Here, we describe the interaction of cytoskeletal components with key signaling molecules that contribute to spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV as measured by electric cell-substrate impedance sensing. Fatty acid-treated cells displayed short cortical actin filaments associated with an increased number of β1 integrin-containing pseudopodia whereas untreated cells displayed elongated stress fibers and fewer clusters of β1 integrins. Confocal microscopy of arachidonic acid-treated cells showed that vinculin and phospho-p38 both appeared enriched in pseudopodia and at the tips of actin filaments, and fluorescence ratio imaging indicated the increase was specific for the phospho-(active) form of p38. Immunoprecipitates of phospho-p38 from extracts of arachidonic acid-treated cells contained vinculin, and GST-vinculin fusion proteins carrying the central region of vinculin bound phospho-p38, whereas fusion proteins expressing the terminal portions of vinculin did not. These data suggest that phospho-p38 associates with particular domains on critical focal adhesion proteins that are involved in tumor cell adhesion and spreading and that this association can be regulated by factors in the tumor microenvironment. PMID:24219282

  14. KIAA1199 interacts with glycogen phosphorylase kinase β-subunit (PHKB) to promote glycogen breakdown and cancer cell survival.

    PubMed

    Terashima, Masato; Fujita, Yoshihiko; Togashi, Yosuke; Sakai, Kazuko; De Velasco, Marco A; Tomida, Shuta; Nishio, Kazuto

    2014-08-30

    The KIAA1199 gene was first discovered to be associated with non-syndromic hearing loss. Recently, several reports have shown that the up-regulation of KIAA1199 is associated with cancer cell migration or invasion and a poor prognosis. These findings indicate that KIAA1199 may be a novel target for cancer therapy. Therefore, we explored in detail the function of KIAA1199 in cancer cells. In this study, we investigated the interaction of KIAA1199 protein with intracellular proteins in cancer cells. To this end, we expressed KIAA1199-MBP fusion protein and performed a pull-down assay. In addition, KIAA1199-overexpressing cancer cell lines were constructed using a retroviral vector and were used for further experiments. A pull-down analysis showed that the glycogen phosphorylase kinase β-subunit (PHKB) interacted with the C-terminal region of KIAA1199 protein. Furthermore, we observed the interaction of KIAA1199 with glycogen phosphorylase brain form (PYGB) under serum-free conditions. The interaction promoted glycogen breakdown and cancer cell survival. Our findings indicate that KIAA1199 plays an important role in glycogen breakdown and cancer cell survival and that it may represent a novel target for cancer therapy.

  15. KIAA1199 interacts with glycogen phosphorylase kinase β-subunit (PHKB) to promote glycogen breakdown and cancer cell survival

    PubMed Central

    Terashima, Masato; Fujita, Yoshihiko; Togashi, Yosuke; Sakai, Kazuko; De Velasco, Marco A.; Tomida, Shuta; Nishio, Kazuto

    2014-01-01

    The KIAA1199 gene was first discovered to be associated with non-syndromic hearing loss. Recently, several reports have shown that the up-regulation of KIAA1199 is associated with cancer cell migration or invasion and a poor prognosis. These findings indicate that KIAA1199 may be a novel target for cancer therapy. Therefore, we explored in detail the function of KIAA1199 in cancer cells. In this study, we investigated the interaction of KIAA1199 protein with intracellular proteins in cancer cells. To this end, we expressed KIAA1199-MBP fusion protein and performed a pull-down assay. In addition, KIAA1199-overexpressing cancer cell lines were constructed using a retroviral vector and were used for further experiments. A pull-down analysis showed that the glycogen phosphorylase kinase β-subunit (PHKB) interacted with the C-terminal region of KIAA1199 protein. Furthermore, we observed the interaction of KIAA1199 with glycogen phosphorylase brain form (PYGB) under serum-free conditions. The interaction promoted glycogen breakdown and cancer cell survival. Our findings indicate that KIAA1199 plays an important role in glycogen breakdown and cancer cell survival and that it may represent a novel target for cancer therapy. PMID:25051373

  16. Protein Kinase CK2 Interacts at the Neuromuscular Synapse with Rapsyn, Rac1, 14-3-3γ, and Dok-7 Proteins and Phosphorylates the Latter Two*

    PubMed Central

    Herrmann, Dustin; Straubinger, Marion; Hashemolhosseini, Said

    2015-01-01

    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. PMID:26198629

  17. 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.

  18. OsBRI1 Activates BR Signaling by Preventing Binding between the TPR and Kinase Domains of OsBSK3 via Phosphorylation.

    PubMed

    Zhang, Baowen; Wang, Xiaolong; Zhao, Zhiying; Wang, Ruiju; Huang, Xiahe; Zhu, Yali; Yuan, Li; Wang, Yingchun; Xu, Xiaodong; Burlingame, Alma L; Gao, Yingjie; Sun, Yu; Tang, Wenqiang

    2016-02-01

    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.

  19. 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. PMID:26853099

  20. Cloning and subcellular location of an Arabidopsis receptor-like protein that shares common features with protein-sorting receptors of eukaryotic cells.

    PubMed Central

    Ahmed, S U; Bar-Peled, M; Raikhel, N V

    1997-01-01

    Many receptors involved in clathrin-mediated protein transport through the endocytic and secretory pathways of yeast and animal cells share common features. They are all type I integral membrane proteins containing cysteine-rich lumenal domains and cytoplasmic tails with tyrosine-containing sorting signals. The cysteine-rich domains are thought to be involved in ligand binding, whereas the cytoplasmic tyrosine motifs interact with clathrin-associated adaptor proteins during protein sorting along these pathways. In addition, tyrosine-containing signals are required for the retention and recycling of some of these membrane proteins to the trans-Golgi network. Here we report the characterization of an approximately 80-kD epidermal growth factor receptor-like type I integral membrane protein containing all of these functional motifs from Arabidopsis thaliana (called AtELP for A. thaliana Epidermal growth factor receptor-Like Protein). Biochemical analysis indicates that AtELP is a membrane protein found at high levels in the roots of both monocots and dicots. Subcellular fractionation studies indicate that the AtELP protein is present in two membrane fractions corresponding to a novel, undefined compartment and a fraction enriched in vesicles containing clathrin and its associated adaptor proteins. AtELP may therefore serve as a marker for compartments involved in intracellular protein trafficking in the plant cell. PMID:9159954

  1. Specificity rendering 'hot-spots' for aurora kinase inhibitor design: the role of non-covalent interactions and conformational transitions.

    PubMed

    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

  2. Functional Role of the Interaction between Polysialic Acid and Myristoylated Alanine-rich C Kinase Substrate at the Plasma Membrane

    PubMed Central

    Theis, Thomas; Mishra, Bibhudatta; von der Ohe, Maren; Loers, Gabriele; Prondzynski, Maksymilian; Pless, Ole; Blackshear, Perry J.; Schachner, Melitta; Kleene, Ralf

    2013-01-01

    Polysialic acid (PSA) is a homopolymeric glycan that plays crucial roles in the developing and adult nervous system. So far only a few PSA-binding proteins have been identified. Here, we identify myristoylated alanine-rich C kinase substrate (MARCKS) as novel PSA binding partner. Binding assays showed a direct interaction between PSA and a peptide comprising the effector domain of MARCKS (MARCKS-ED). Co-immunoprecipitation of PSA-carrying neural cell adhesion molecule (PSA-NCAM) with MARCKS and co-immunostaining of MARCKS and PSA at the cell membrane of hippocampal neurons confirm the interaction between PSA and MARCKS. Co-localization and an intimate interaction of PSA and MARCKS at the cell surface was seen by confocal microscopy and fluorescence resonance energy transfer (FRET) analysis after the addition of fluorescently labeled PSA or PSA-NCAM to live CHO cells or hippocampal neurons expressing MARCKS as a fusion protein with green fluorescent protein (GFP). Cross-linking experiments showed that extracellularly applied PSA or PSA-NCAM and intracellularly expressed MARCKS-GFP are in close contact, suggesting that PSA and MARCKS interact with each other at the plasma membrane from opposite sides. Insertion of PSA and MARCKS-ED peptide into lipid bilayers from opposite sides alters the electric properties of the bilayer confirming the notion that PSA and the effector domain of MARCKS interact at and/or within the plane of the membrane. The MARCKS-ED peptide abolished PSA-induced enhancement of neurite outgrowth from cultured hippocampal neurons indicating an important functional role for the interaction between MARCKS and PSA in the developing and adult nervous system. PMID:23329829

  3. The N-terminal domain of the tomato immune protein Prf contains multiple homotypic and Pto kinase interaction sites.

    PubMed

    Saur, Isabel Marie-Luise; Conlan, Brendon Francis; Rathjen, John Paul

    2015-05-01

    Resistance to Pseudomonas syringae bacteria in tomato (Solanum lycopersicum) is conferred by the Prf recognition complex, composed of the nucleotide-binding leucine-rich repeats protein Prf and the protein kinase Pto. The complex is activated by recognition of the P. syringae effectors AvrPto and AvrPtoB. The N-terminal domain is responsible for Prf homodimerization, which brings two Pto kinases into close proximity and holds them in inactive conformation in the absence of either effector. Negative regulation is lost by effector binding to the catalytic cleft of Pto, leading to disruption of its P+1 loop within the activation segment. This change is translated through Prf to a second Pto molecule in the complex. Here we describe a schematic model of the unique Prf N-terminal domain dimer and its interaction with the effector binding determinant Pto. Using heterologous expression in Nicotiana benthamiana, we define multiple sites of N domain homotypic interaction and infer that it forms a parallel dimer folded centrally to enable contact between the N and C termini. Furthermore, we found independent binding sites for Pto at either end of the N-terminal domain. Using the constitutively active mutant ptoL205D, we identify a potential repression site for Pto in the first ∼100 amino acids of Prf. Finally, we find that the Prf leucine-rich repeats domain also binds the N-terminal region, highlighting a possible mechanism for transfer of the effector binding signal to the NB-LRR regulatory unit (consisting of a central nucleotide binding and C-terminal leucine-rich repeats). PMID:25792750

  4. The N-terminal domain of the tomato immune protein Prf contains multiple homotypic and Pto kinase interaction sites.

    PubMed

    Saur, Isabel Marie-Luise; Conlan, Brendon Francis; Rathjen, John Paul

    2015-05-01

    Resistance to Pseudomonas syringae bacteria in tomato (Solanum lycopersicum) is conferred by the Prf recognition complex, composed of the nucleotide-binding leucine-rich repeats protein Prf and the protein kinase Pto. The complex is activated by recognition of the P. syringae effectors AvrPto and AvrPtoB. The N-terminal domain is responsible for Prf homodimerization, which brings two Pto kinases into close proximity and holds them in inactive conformation in the absence of either effector. Negative regulation is lost by effector binding to the catalytic cleft of Pto, leading to disruption of its P+1 loop within the activation segment. This change is translated through Prf to a second Pto molecule in the complex. Here we describe a schematic model of the unique Prf N-terminal domain dimer and its interaction with the effector binding determinant Pto. Using heterologous expression in Nicotiana benthamiana, we define multiple sites of N domain homotypic interaction and infer that it forms a parallel dimer folded centrally to enable contact between the N and C termini. Furthermore, we found independent binding sites for Pto at either end of the N-terminal domain. Using the constitutively active mutant ptoL205D, we identify a potential repression site for Pto in the first ∼100 amino acids of Prf. Finally, we find that the Prf leucine-rich repeats domain also binds the N-terminal region, highlighting a possible mechanism for transfer of the effector binding signal to the NB-LRR regulatory unit (consisting of a central nucleotide binding and C-terminal leucine-rich repeats).

  5. Dbf4 regulates the Cdc5 Polo-like kinase through a distinct non-canonical binding interaction.

    PubMed

    Chen, Ying-Chou; Weinreich, Michael

    2010-12-31

    Cdc7-Dbf4 is a conserved, two-subunit kinase required for initiating eukaryotic DNA replication. Recent studies have shown that Cdc7-Dbf4 also regulates the mitotic exit network (MEN) and monopolar homolog orientation in meiosis I (Matos, J., Lipp, J. J., Bogdanova, A., Guillot, S., Okaz, E., Junqueira, M., Shevchenko, A., and Zachariae, W. (2008) Cell 135, 662-678 and Miller, C. T., Gabrielse, C., Chen, Y. C., and Weinreich, M. (2009) PLoS Genet. 5, e1000498). Both activities likely involve a Cdc7-Dbf4 interaction with Cdc5, the single Polo-like kinase in budding yeast. We previously showed that Dbf4 binds the Cdc5 polo-box domain (PBD) via an ∼40-residue N-terminal sequence, which lacks a PBD consensus binding site (S(pS/pT)(P/X)), and that Dbf4 inhibits Cdc5 function during mitosis. Here we identify a non-consensus PBD binding site within Dbf4 and demonstrate that the PBD-Dbf4 interaction occurs via a distinct PBD surface from that used to bind phosphoproteins. Genetic and biochemical analysis of multiple dbf4 mutants indicate that Dbf4 inhibits Cdc5 function through direct binding. Surprisingly, mutation of invariant Cdc5 residues required for binding phosphorylated substrates has little effect on yeast viability or growth rate. Instead, cdc5 mutants defective for binding phosphoproteins exhibit enhanced resistance to microtubule disruption and an increased rate of spindle elongation. This study, therefore, details the molecular nature of a new type of PBD binding and reveals that Cdc5 targeting to phosphorylated substrates likely regulates spindle dynamics.

  6. Identification, expression and interaction analyses of calcium-dependent protein kinase (CPK) genes in canola (Brassica napus L.)

    PubMed Central

    2014-01-01

    Background Canola (Brassica napus L.) is one of the most important oil-producing crops in China and worldwide. The yield and quality of canola is frequently threatened by environmental stresses including drought, cold and high salinity. Calcium is a well-known ubiquitous intracellular secondary messenger in plants. Calcium-dependent protein kinases (CPKs) are Ser/Thr protein kinases found only in plants and some protozoans. CPKs are Ca2+ sensors that have both Ca2+ sensing function and kinase activity within a single protein and play crucial roles in plant development and responses to various environmental stresses. Results In this study, we mined the available expressed sequence tags (ESTs) of B. napus and identified a total of 25 CPK genes, among which cDNA sequences of 23 genes were successfully cloned from a double haploid cultivar of canola. Phylogenetic analysis demonstrated that they could be clustered into four subgroups. The subcellular localization of five selected BnaCPKs was determined using green fluorescence protein (GFP) as the reporter. Furthermore, the expression levels of 21 BnaCPK genes in response to salt, drought, cold, heat, abscisic acid (ABA), low potassium (LK) and oxidative stress were studied by quantitative RT-PCR and were found to respond to multiple stimuli, suggesting that canola CPKs may be convergence points of different signaling pathways. We also identified and cloned five and eight Clade A basic leucine zipper (bZIP) and protein phosphatase type 2C (PP2C) genes from canola and, using yeast two-hybrid and bimolecular fluorescence complementation (BiFC), determined the interaction between individual BnaCPKs and BnabZIPs or BnaPP2Cs (Clade A). We identified novel, interesting interaction partners for some of the BnaCPK proteins. Conclusion We present the sequences and characterization of CPK gene family members in canola for the first time. This work provides a foundation for further crop improvement and improved understanding of

  7. Identification of interacting proteins for calcium-dependent protein kinase 8 by a novel screening system based on bimolecular fluorescence complementation.

    PubMed

    Kamimura, Mayu; Han, Yulong; Kito, Nobuki; Che, Fang-Sik

    2014-01-01

    Protein kinases are key regulators of cell function that constitute one of the largest and most functionally diverse gene families. We developed a novel assay system, based on the bimolecular fluorescence complementation (BiFC) technique in Escherichia coli, for detecting transient interactions such as those between kinases and their substrates. This system detected the interaction between OsMEK1 and its direct target OsMAP1. By contrast, BiFC fluorescence was not observed when OsMAP2 or OsMAP3, which are not substrates of OsMEK1, were used as prey proteins. We also screened for interacting proteins of calcium-dependent protein kinase 8 (OsCPK8), a regulator of plant immune responses, and identified three proteins as interacting molecules of OsCPK8. The interaction between OsCPK8 and two of these proteins (ARF-GEF and peptidyl prolyl isomerase) was confirmed in rice cells by means of BiFC technology. These results indicate that our new assay system has the potential to screen for protein kinase target molecules.

  8. Conserved interactions of a compact highly active enhancer/promoter upstream of the rhodopsin kinase (GRK1) gene.

    PubMed

    Young, Joyce E; Kasperek, Eileen M; Vogt, Todd M; Lis, Agnieszka; Khani, Shahrokh C

    2007-08-01

    Rhodopsin kinase (RK) is a conserved component of the light adaptation and recovery pathways shared among rod and cone photoreceptors of a variety of species. To gain insight into transcriptional mechanisms driving RK and potentially other genes of similar spatial profile, the components and the interactions of the highly compact enhancer/promoter region (E/P) upstream of the human RK gene were examined. Cross-species comparison outlined an active 49-bp widely shared E/P core as the major site of conservation in the entire 5' flanking sequence. The area consisted of a bicoid-type homeodomain recognition cassette and a unique T-rich module interacting with TATA-binding proteins. Homeodomain interactions involved primarily Crx and secondarily Otx2. Both strongly stimulated the E/P. In the absence of Crx, persistent E/P activity shifted from the outer retina to the inner to follow the Otx2 pattern. The spatial patterns were largely unaffected by the absence of rod transcription factors, Nrl and Nr2e3, and the RK transcriptional activity preceded the surge in rod-specific transcription. Conserved bicoid homeodomain factors thus appear to be the key factors governing localization of RK E/P activity in retina and photoreceptors.

  9. Mys protein regulates protein kinase A activity by interacting with regulatory type Ialpha subunit during vertebrate development.

    PubMed

    Kotani, Tomoya; Iemura, Shun-ichiro; Natsume, Tohru; Kawakami, Koichi; Yamashita, Masakane

    2010-02-12

    During embryonic development, protein kinase A (PKA) plays a key role in cell fate specification by antagonizing the Hedgehog (Hh) signaling pathway. However, the mechanism by which PKA activity is regulated remains unknown. Here we show that the Misty somites (Mys) protein regulates the level of PKA activity during embryonic development in zebrafish. We isolate PKA regulatory type Ialpha subunit (Prkar1a) as a protein interacting with Mys by pulldown assay in HEK293 cells followed by mass spectrometry analysis. We show an interaction between endogenous Mys and Prkar1a in the zebrafish embryo. Mys binds to Prkar1a in its C terminus region, termed PRB domain, and activates PKA in vitro. Conversely, knockdown of Mys in zebrafish embryos results in reduction in PKA activity. We also show that knockdown of Mys induces ectopic activation of Hh target genes in the eyes, neural tube, and somites downstream of Smoothened, a protein essential for transduction of Hh signaling activity. The altered patterning of gene expression is rescued by activation of PKA. Together, our results reveal a molecular mechanism of regulation of PKA activity that is dependent on a protein-protein interaction and demonstrate that PKA activity regulated by Mys is indispensable for negative regulation of the Hh signaling pathway in Hh-responsive cells. PMID:20018846

  10. Protein kinase A-anchoring protein AKAP95 interacts with MCM2, a regulator of DNA replication.

    PubMed

    Eide, Turid; Taskén, Kristin A; Carlson, Cathrine; Williams, Gareth; Jahnsen, Tore; Taskén, Kjetil; Collas, Philippe

    2003-07-18

    Protein kinase A (PKA)-anchoring protein AKAP95 is localized to the nucleus in interphase, where it primarily associates with the nuclear matrix. A yeast two-hybrid screen for AKAP95 interaction partners identified the minichromosome maintenance (MCM) 2 protein, a component of the pre-replication complex. AKAP95-MCM2 interaction was mapped to residues 1-195 of AKAP95 and corroborated by glutathione S-transferase precipitation and immunoprecipitation from chromatin. Disruption of AKAP95-MCM2 interaction with an AKAP95-(1-195) peptide within HeLa cell nuclei abolishes initiation of DNA replication in G1 phase and the elongation phase of replication in vitro without affecting global nuclear organization or import. Disruption of the C-terminal zinc finger of AKAP95 reduces efficiency of replication initiation. Disruption of the PKA-binding domain does not impair replication in G1- or S-phase nuclei, whereas a PKA inhibitor affects the initiation but not the elongation phase of replication. Depleting AKAP95 from nuclei partially depletes MCM2 and abolishes replication. Recombinant AKAP95 restores intranuclear MCM2 and replication in a dose-dependent manner. Our results suggest a role of AKAP95 in DNA replication by providing a scaffold for MCM2. PMID:12740381

  11. Two hydrophobic residues can determine the specificity of mitogen-activated protein kinase docking interactions.

    PubMed

    Bardwell, A Jane; Bardwell, Lee

    2015-10-30

    MAPKs bind to many of their upstream regulators and downstream substrates via a short docking motif (the D-site) on their binding partner. MAPKs that are in different families (e.g. ERK, JNK, and p38) can bind selectively to D-sites in their authentic substrates and regulators while discriminating against D-sites in other pathways. Here we demonstrate that the short hydrophobic region at the distal end of the D-site plays a critical role in determining the high selectivity of JNK MAPKs for docking sites in their cognate MAPK kinases. Changing just 1 or 2 key hydrophobic residues in this submotif is sufficient to turn a weak JNK-binding D-site into a strong one, or vice versa. These specificity-determining differences are also found in the D-sites of the ETS family transcription factors Elk-1 and Net. Moreover, swapping two hydrophobic residues between these D-sites switches the relative efficiency of Elk-1 and Net as substrates for ERK versus JNK, as predicted. These results provide new insights into docking specificity and suggest that this specificity can evolve rapidly by changes to just 1 or 2 amino acids.

  12. Type IV pilins regulate their own expression via direct intramembrane interactions with the sensor kinase PilS.

    PubMed

    Kilmury, Sara L N; Burrows, Lori L

    2016-05-24

    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. 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.

  14. Sorting nexin 6, a novel SNX, interacts with the transforming growth factor-beta family of receptor serine-threonine kinases.

    PubMed

    Parks, W T; Frank, D B; Huff, C; Renfrew Haft, C; Martin, J; Meng, X; de Caestecker, M P; McNally, J G; Reddi, A; Taylor, S I; Roberts, A B; Wang, T; Lechleider, R J

    2001-06-01

    Sorting nexins (SNX) comprise a family of proteins with homology to several yeast proteins, including Vps5p and Mvp1p, that are required for the sorting of proteins to the yeast vacuole. Human SNX1, -2, and -4 have been proposed to play a role in receptor trafficking and have been shown to bind to several receptor tyrosine kinases, including receptors for epidermal growth factor, platelet-derived growth factor, and insulin as well as the long form of the leptin receptor, a glycoprotein 130-associated receptor. We now describe a novel member of this family, SNX6, which interacts with members of the transforming growth factor-beta family of receptor serine-threonine kinases. These receptors belong to two classes: type II receptors that bind ligand, and type I receptors that are subsequently recruited to transduce the signal. Of the type II receptors, SNX6 was found to interact strongly with ActRIIB and more moderately with wild type and kinase-defective mutants of TbetaRII. Of the type I receptors, SNX6 was found to interact only with inactivated TbetaRI. SNXs 1-4 also interacted with the transforming growth factor-beta receptor family, showing different receptor preferences. Conversely, SNX6 behaved similarly to the other SNX proteins in its interactions with receptor tyrosine kinases. Strong heteromeric interactions were also seen among SNX1, -2, -4, and -6, suggesting the formation in vivo of oligomeric complexes. These findings are the first evidence for the association of the SNX family of molecules with receptor serine-threonine kinases.

  15. PIM kinases are essential for chronic lymphocytic leukemia cell survival (PIM2/3) and CXCR4-mediated microenvironmental interactions (PIM1).

    PubMed

    Decker, Sarah; Finter, Johannes; Forde, Aaron James; Kissel, Sandra; Schwaller, Juerg; Mack, Thomas Sebastian; Kuhn, Anabel; Gray, Nathanael; Follo, Marie; Jumaa, Hassan; Burger, Meike; Zirlik, Katja; Pfeifer, Dietmar; Miduturu, Chandrasekhar V; Eibel, Hermann; Veelken, Hendrik; Dierks, Christine

    2014-05-01

    Overexpression of the CXCR4 receptor is a hallmark of chronic lymphocytic leukemia (CLL) and is important for CLL cell survival, migration, and interaction with their protective microenvironment. In acute myelogenous leukemia (AML), PIM1 was shown to regulate the surface expression of the CXCR4 receptor. Here, we show that PIM (proviral integration site for Moloney murine leukemia virus) kinases 1-3 are overexpressed and that the CXCR4 receptor is hyperphosphorylated on Ser339 in CLL compared with normal lymphocytes. Furthermore, CXCR4 phosphorylation correlates with PIM1 protein expression and PIM1 transcript levels in CLL. PIM kinase inhibition with three different PIM kinase inhibitors induced apoptosis in CLL cells independent of the presence of protective stromal cells. In addition, PIM inhibition caused dephosphorylation of the CXCR4 receptor on Ser339, resulting in enhanced ligand-dependent CXCR4 internalization and reduced re-externalization after withdrawal of CXCL12. Furthermore, PIM inhibition in CLL cells blocked CXCR4 functions, such as migration toward CXCL12- or CXCL12-induced extracellular signal-regulated kinase (ERK) phosphorylation. In concordance, pretreatment of CLL cells with PIM kinase inhibitors strongly reduced homing of CLL cells toward the bone marrow and the spleen of Rag2(-/-)γc(-/-) mice in vivo. Interestingly, the knockdown of PIM kinases in CLL cells demonstrated diverging functions, with PIM1 regulating CXCR4 surface expression and PIM2 and PIM3 as important for the survival of CLL cells. Our results show that PIM kinase inhibitors are an effective therapeutic option for CLL, not only by impairing PIM2/3-mediated CLL cell survival, but also by blocking the PIM1/CXCR4-mediated interaction of CLL cells with their protective microenvironment. PMID:24659821

  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

    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.

  17. Structural Analysis of Pseudomonas syringae AvrPtoB Bound to Host BAK1 Reveals Two Similar Kinase-Interacting Domains in a Type III Effector

    PubMed Central

    Cheng, Wei; Munkvold, Kathy R.; Gao, Haishan; Mathieu, Johannes; Schwizer, Simon; Wang, Sha; Yan, Yong-bin; Wang, Jinjing; Martin, Gregory B.; Chai, Jijie

    2013-01-01

    SUMMARY To infect plants, Pseudomonas syringae pv. tomato delivers ~30 type III effector proteins into host cells, many of which interfere with PAMP-triggered immunity (PTI). One effector, AvrPtoB, suppresses PTI using a central domain to bind host BAK1, a kinase that acts with several pattern recognition receptors to activate defense signaling. A second AvrPtoB domain binds and suppresses the PTI-associated kinase Bti9 but is conversely recognized by the protein kinase Pto to activate effector-triggered immunity. We report the crystal structure of the AvrPtoB-BAK1 complex, which revealed structural similarity between these two AvrPtoB domains, suggesting that they arose by intragenic duplication. The BAK1 kinase domain is structurally similar to Pto, and a conserved region within both BAK1 and Pto interacts with AvrPtoB. BAK1 kinase activity is inhibited by AvrPtoB, and mutations at the interaction interface disrupt AvrPtoB virulence activity. These results shed light on a structural mechanism underlying host-pathogen coevolution. PMID:22169508

  18. 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

  19. GLUTAMATE RECEPTOR-LIKE genes mediate leaf-to-leaf wound signalling.

    PubMed

    Mousavi, Seyed A R; Chauvin, Adeline; Pascaud, François; Kellenberger, Stephan; Farmer, Edward E

    2013-08-22

    Wounded leaves communicate their damage status to one another through a poorly understood process of long-distance signalling. This stimulates the distal production of jasmonates, potent regulators of defence responses. Using non-invasive electrodes we mapped surface potential changes in Arabidopsis thaliana after wounding leaf eight and found that membrane depolarizations correlated with jasmonate signalling domains in undamaged leaves. Furthermore, current injection elicited jasmonoyl-isoleucine accumulation, resulting in a transcriptome enriched in RNAs encoding key jasmonate signalling regulators. From among 34 screened membrane protein mutant lines, mutations in several clade 3 GLUTAMATE RECEPTOR-LIKE genes (GLRs 3.2, 3.3 and 3.6) attenuated wound-induced surface potential changes. Jasmonate-response gene expression in leaves distal to wounds was reduced in a glr3.3 glr3.6 double mutant. This work provides a genetic basis for investigating mechanisms of long-distance wound signalling in plants and indicates that plant genes related to those important for synaptic activity in animals function in organ-to-organ wound signalling.

  20. RLKs orchestrate the signaling in plant male-female interaction.

    PubMed

    Li, Hongju; Yang, Wei-Cai

    2016-09-01

    Different from animals, sessile plants are equipped with a large receptor-like kinase (RLK) superfamily. RLKs are a family of single trans-membrane proteins with divergent N-terminal extracellular domains capped by a signal peptide and C-terminal intracellular kinase. Researches in the last two decades have uncovered an increasing number of RLKs that regulate plant development, stress response and sexual reproduction, highlighting a dominant role of RLK signaling in cell-to-cell communications. Sexual reproduction in flowering plants is featured by interactions between the male gametophyte and the female tissues to facilitate sperm delivery and fertilization. Emerging evidences suggest that RLKs regulate almost every aspect of plant reproductive process, especially during pollination. Therefore, in this review we will focus mainly on the function and signaling of RLKs in plant male-female interaction and discuss the future prospects on these topics. PMID:27525988

  1. Modulation of kinase-inhibitor interactions by auxiliary protein binding: Crystallography studies on Aurora A interactions with VX-680 and with TPX2

    SciTech Connect

    Zhao, Baoguang; Smallwood, Angela; Yang, Jingsong; Koretke, Kristin; Nurse, Kelvin; Calamari, Amy; Kirkpatrick, Robert B.; Lai, Zhihong

    2008-10-24

    VX-680, also known as MK-0457, is an ATP-competitive small molecule inhibitor of the Aurora kinases that has entered phase II clinical trials for the treatment of cancer. We have solved the cocrystal structure of AurA/TPX2/VX-680 at 2.3 {angstrom} resolution. In the crystal structure, VX-680 binds to the active conformation of AurA. The glycine-rich loop in AurA adopts a unique bent conformation, forming a {pi}-{pi} interaction with the phenyl group of VX-680. In contrast, in the published AurA/VX-680 structure, VX-680 binds to AurA in the inactive conformation, interacting with a hydrophobic pocket only present in the inactive conformation. These data suggest that TPX2, a protein cofactor, can alter the binding mode of VX-680 with AurA. More generally, the presence of physiologically relevant cofactor proteins can alter the kinetics, binding interactions, and inhibition of enzymes, and studies with these multiprotein complexes may be beneficial to the discovery and optimization of enzyme inhibitors as therapeutic agents.

  2. Interaction of hepatitis C virus core protein with janus kinase is required for efficient production of infectious viruses.

    PubMed

    Lee, Choongho

    2013-03-01

    Chronic hepatitis C virus (HCV) infection is responsible for the development of liver cirrhosis and hepatocellular carcinoma. HCV core protein plays not only a structural role in the virion morphogenesis by encapsidating a virus RNA genome but also a non-structural role in HCV-induced pathogenesis by blocking innate immunity. Especially, it has been shown to regulate JAK-STAT signaling pathway through its direct interaction with Janus kinase (JAK) via its proline-rich JAK-binding motif ((79)PGYPWP(84)). However, little is known about the physiological significance of this HCV core-JAK association in the context of the virus life cycle. In order to gain an insight, a mutant HCV genome (J6/JFH1-79A82A) was constructed to express the mutant core with a defective JAK-binding motif ((79)AGYAWP(84)) using an HCV genotype 2a infectious clone (J6/JFH1). When this mutant HCV genome was introduced into hepatocarcinoma cells, it was found to be severely impaired in its ability to produce infectious viruses in spite of its robust RNA genome replication. Taken together, all these results suggest an essential requirement of HCV core-JAK protein interaction for efficient production of infectious viruses and the potential of using core-JAK blockers as a new anti-HCV therapy.

  3. 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

  4. 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

  5. TaCIPK29, a CBL-interacting protein kinase gene from wheat, confers salt stress tolerance in transgenic tobacco.

    PubMed

    Deng, Xiaomin; Hu, Wei; Wei, Shuya; Zhou, Shiyi; Zhang, Fan; Han, Jiapeng; Chen, Lihong; Li, Yin; Feng, Jialu; Fang, Bin; Luo, Qingchen; Li, Shasha; Liu, Yunyi; Yang, Guangxiao; He, Guangyuan

    2013-01-01

    Calcineurin B-like protein-interacting protein kinases (CIPKs) have been found to be responsive to abiotic stress. However, their precise functions and the related molecular mechanisms in abiotic stress tolerance are not completely understood, especially in wheat. In the present study, TaCIPK29 was identified as a new member of CIPK gene family in wheat. TaCIPK29 transcript increased after NaCl, cold, methyl viologen (MV), abscisic acid (ABA) and ethylene treatments. Over-expression of TaCIPK29 in tobacco resulted in increased salt tolerance, which was demonstrated by higher germination rates, longer root lengths and better growth status of transgenic tobacco plants compared to controls when both were treated with salt stress. Physiological measurements indicated that transgenic tobacco seedlings retained high K(+)/Na(+) ratios and Ca(2+) content by up-regulating some transporter genes expression and also possessed lower H2O2 levels and reduced membrane injury by increasing the expression and activities of catalase (CAT) and peroxidase (POD) under salt stress. Moreover, transgenic lines conferred tolerance to oxidative stress by increasing the activity and expression of CAT. Finally, TaCIPK29 was located throughout cells and it preferentially interacted with TaCBL2, TaCBL3, NtCBL2, NtCBL3 and NtCAT1. Taken together, our results showed that TaCIPK29 functions as a positive factor under salt stress and is involved in regulating cations and reactive oxygen species (ROS) homeostasis.

  6. Structure-Activity Relationship Studies of Mitogen Activated Protein Kinase Interacting Kinase (MNK) 1 and 2 and BCR-ABL1 Inhibitors Targeting Chronic Myeloid Leukemic Cells.

    PubMed

    Cherian, Joseph; Nacro, Kassoum; Poh, Zhi Ying; Guo, Samantha; Jeyaraj, Duraiswamy A; Wong, Yun Xuan; Ho, Melvyn; Yang, Hai Yan; Joy, Joma Kanikadu; Kwek, Zekui Perlyn; Liu, Boping; Wee, John Liang Kuan; Ong, Esther H Q; Choong, Meng Ling; Poulsen, Anders; Lee, May Ann; Pendharkar, Vishal; Ding, Li Jun; Manoharan, Vithya; Chew, Yun Shan; Sangthongpitag, Kanda; Lim, Sharon; Ong, S Tiong; Hill, Jeffrey; Keller, Thomas H

    2016-04-14

    Clinically used BCR-ABL1 inhibitors for the treatment of chronic myeloid leukemia do not eliminate leukemic stem cells (LSC). It has been shown that MNK1 and 2 inhibitors prevent phosphorylation of eIF4E and eliminate the self-renewal capacity of LSCs. Herein, we describe the identification of novel dual MNK1 and 2 and BCR-ABL1 inhibitors, starting from the known kinase inhibitor 2. Initial structure-activity relationship studies resulted in compound 27 with loss of BCR-ABL1 inhibition. Further modification led to orally bioavailable dual MNK1 and 2 and BCR-ABL1 inhibitors 53 and 54, which are efficacious in a mouse xenograft model and also reduce the level of phosphorylated eukaryotic translation initiation factor 4E in the tumor tissues. Kinase selectivity of these compounds is also presented. PMID:27011159

  7. p21-activated kinase 2 regulates HSPC cytoskeleton, migration, and homing via CDC42 activation and interaction with β-Pix.

    PubMed

    Reddy, Pavankumar N G; Radu, Maria; Xu, Ke; Wood, Jenna; Harris, Chad E; Chernoff, Jonathan; Williams, David A

    2016-04-21

    Cytoskeletal remodeling of hematopoietic stem and progenitor cells (HSPCs) is essential for homing to the bone marrow (BM). The Ras-related C3 botulinum toxin substrate (Rac)/cell division control protein 42 homolog (CDC42) effector p21-activated kinase (Pak2) has been implicated in HSPC homing and engraftment. However, the molecular pathways mediating Pak2 functions in HSPCs are unknown. Here, we demonstrate that both Pak2 kinase activity and its interaction with the PAK-interacting exchange factor-β (β-Pix) are required to reconstitute defective ITALIC! Pak2 (ITALIC! Δ/Δ)HSPC homing to the BM. Pak2 serine/threonine kinase activity is required for stromal-derived factor-1 (SDF1α) chemokine-induced HSPC directional migration, whereas Pak2 interaction with β-Pix is required to regulate the velocity of HSPC migration and precise F-actin assembly. Lack of SDF1α-induced filopodia and associated abnormal cell protrusions seen in ITALIC! Pak2 (ITALIC! Δ/Δ)HSPCs were rescued by wild-type (WT) Pak2 but not by a Pak2-kinase dead mutant (KD). Expression of a β-Pix interaction-defective mutant of Pak2 rescued filopodia formation but led to abnormal F-actin bundles. Although CDC42 has previously been considered an upstream regulator of Pak2, we found a paradoxical decrease in baseline activation of CDC42 in ITALIC! Pak2 (ITALIC! Δ/Δ)HSPCs, which was rescued by expression of Pak2-WT but not by Pak2-KD; defective homing of ITALIC! Pak2-deleted HSPCs was rescued by constitutive active CDC42. These data demonstrate that both Pak2 kinase activity and its interaction with β-Pix are essential for HSPC filopodia formation, cytoskeletal integrity, and homing via activation of CDC42. Taken together, we provide mechanistic insights into the role of Pak2 in HSPC migration and homing.

  8. 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

    Ben-Addi, Abduelhakem; Mambole-Dema, Agnes; Brender, Christine; Martin, Stephen R; Janzen, Julia; Kjaer, Sven; Smerdon, Stephen J; Ley, Steven C

    2014-06-10

    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.

  9. Interaction with the Src Homology (SH3-SH2) Region of the Src-family Kinase Hck Structures the HIV-1 Nef Dimer for Kinase Activation and Effector Recruitment*

    PubMed Central

    Alvarado, John Jeff; Tarafdar, Sreya; Yeh, Joanne I.; Smithgall, Thomas E.

    2014-01-01

    HIV-1 Nef supports high titer viral replication in vivo and is essential for AIDS progression. Nef function depends on interactions with multiple host cell effectors, including Hck and other Src-family kinases. Here we describe the x-ray crystal structure of Nef in complex with the Hck SH3-SH2 regulatory region to a resolution of 1.86 Å. The complex crystallized as a dimer of complexes, with the conserved Nef PXXPXR motif engaging the Hck SH3 domain. A new intercomplex contact was found between SH3 Glu-93, and Nef Arg-105. Mutagenesis of Hck SH3 Glu-93 interfered with Nef·Hck complex formation and kinase activation in cells. The Hck SH2 domains impinge on the N-terminal region of Nef to stabilize a dimer conformation that exposes Asp-123, a residue critical for Nef function. Our results suggest that in addition to serving as a kinase effector for Nef, Hck binding may reorganize the Nef dimer for functional interaction with other signaling partners. PMID:25122770

  10. 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

  11. The Autophagy-related Protein Kinase Atg1 Interacts with the Ubiquitin-like Protein Atg8 via the Atg8 Family Interacting Motif to Facilitate Autophagosome Formation*

    PubMed Central

    Nakatogawa, Hitoshi; Ohbayashi, Shiran; Sakoh-Nakatogawa, Machiko; Kakuta, Soichiro; Suzuki, Sho W.; Kirisako, Hiromi; Kondo-Kakuta, Chika; Noda, Nobuo N.; Yamamoto, Hayashi; Ohsumi, Yoshinori

    2012-01-01

    In autophagy, a cup-shaped membrane called the isolation membrane is formed, expanded, and sealed to complete a double membrane-bound vesicle called the autophagosome that encapsulates cellular constituents to be transported to and degraded in the lysosome/vacuole. The formation of the autophagosome requires autophagy-related (Atg) proteins. Atg8 is a ubiquitin-like protein that localizes to the isolation membrane; a subpopulation of this protein remains inside the autophagosome and is transported to the lysosome/vacuole. In the budding yeast Saccharomyces cerevisiae, Atg1 is a serine/threonine kinase that functions in the initial step of autophagosome formation and is also efficiently transported to the vacuole via autophagy. Here, we explore the mechanism and significance of this autophagic transport of Atg1. In selective types of autophagy, receptor proteins recognize degradation targets and also interact with Atg8, via the Atg8 family interacting motif (AIM), to link the targets to the isolation membrane. We find that Atg1 contains an AIM and directly interacts with Atg8. Mutations in the AIM disrupt this interaction and abolish vacuolar transport of Atg1. These results suggest that Atg1 associates with the isolation membrane by binding to Atg8, resulting in its incorporation into the autophagosome. We also show that mutations in the Atg1 AIM cause a significant defect in autophagy, without affecting the functions of Atg1 implicated in triggering autophagosome formation. We propose that in addition to its essential function in the initial stage, Atg1 also associates with the isolation membrane to promote its maturation into the autophagosome. PMID:22778255

  12. 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

  13. Comparison of FDA Approved Kinase Targets to Clinical Trial Ones: Insights from Their System Profiles and Drug-Target Interaction Networks.

    PubMed

    Xu, Jingyu; Wang, Panpan; Yang, Hong; Zhou, Jin; Li, Yinghong; Li, Xiaoxu; Xue, Weiwei; Yu, Chunyan; Tian, Yubin; Zhu, Feng

    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.

  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. Zcchc8 is a glycogen synthase kinase-3 substrate that interacts with RNA-binding proteins

    SciTech Connect

    Gustafson, Michael P.; Welcker, Markus; Hwang, Harry C.; Clurman, Bruce E. . E-mail: bclurman@fhcrc.org

    2005-12-23

    Phosphorylation of c-Myc on threonine 58 (T58) stimulates its degradation by the Fbw7-SCF ubiquitin ligase. We used a phosphorylation-specific antibody raised against the c-Myc T58 region to attempt to identify other proteins regulated by the Fbw7 pathway. We identified two predominant proteins recognized by this antibody. The first is Ebna1 binding protein 2, a nucleolar protein that, in contrast with a previous report, is likely responsible for the nucleolar staining exhibited by this antibody. The second is Zcchc8, a nuclear protein that is highly phosphorylated in cells treated with nocodazole. We show that Zcchc8 is directly phosphorylated by GSK-3 in vitro and that GSK-3 inhibition prevents Zcchc8 phosphorylation in vivo. Moreover, we found that Zcchc8 interacts with proteins involved in RNA processing/degradation. We suggest that Zcchc8 is a GSK-3 substrate with a role in RNA metabolism.

  16. Vascular Endothelial Growth Factor Receptor-3 Directly Interacts with Phosphatidylinositol 3-Kinase to Regulate Lymphangiogenesis

    PubMed Central

    Coso, Sanja; Zeng, Yiping; Opeskin, Kenneth; Williams, Elizabeth D.

    2012-01-01

    Background Dysfunctional lymphatic vessel formation has been implicated in a number of pathological conditions including cancer metastasis, lymphedema, and impaired wound healing. The vascular endothelial growth factor (VEGF) family is a major regulator of lymphatic endothelial cell (LEC) function and lymphangiogenesis. Indeed, dissemination of malignant cells into the regional lymph nodes, a common occurrence in many cancers, is stimulated by VEGF family members. This effect is generally considered to be mediated via VEGFR-2 and VEGFR-3. However, the role of specific receptors and their downstream signaling pathways is not well understood. Methods and Results Here we delineate the VEGF-C/VEGF receptor (VEGFR)-3 signaling pathway in LECs and show that VEGF-C induces activation of PI3K/Akt and MEK/Erk. Furthermore, activation of PI3K/Akt by VEGF-C/VEGFR-3 resulted in phosphorylation of P70S6K, eNOS, PLCγ1, and Erk1/2. Importantly, a direct interaction between PI3K and VEGFR-3 in LECs was demonstrated both in vitro and in clinical cancer specimens. This interaction was strongly associated with the presence of lymph node metastases in primary small cell carcinoma of the lung in clinical specimens. Blocking PI3K activity abolished VEGF-C-stimulated LEC tube formation and migration. Conclusions Our findings demonstrate that specific VEGFR-3 signaling pathways are activated in LECs by VEGF-C. The importance of PI3K in VEGF-C/VEGFR-3-mediated lymphangiogenesis provides a potential therapeutic target for the inhibition of lymphatic metastasis. PMID:22745786

  17. Autoantibodies that stabilize the molecular interaction of Ku antigen with DNA-dependent protein kinase catalytic subunit.

    PubMed

    Satoh, M; Ajmani, A K; Stojanov, L; Langdon, J J; Ogasawara, T; Wang, J; Dooley, M A; Richards, H B; Winfield, J B; Carter, T H; Reeves, W H

    1996-09-01

    DNA-dependent protein kinase (DNA-PK) consists of a DNA binding subunit (Ku autoantigen), and a catalytic subunit (DNA-PKcs). In the present study, human autoantibodies that recognize novel antigenic determinants of DNA-PK were identified. One type of autoantibody stabilized the interaction of DNA-PKcs with Ku and recognized the DNA-PKcs -Ku complex, but not bio-chemically purified DNA-PKcs. Another type recognized purified DNA-PKcs. Autoantibodies to Ku (p70/p80 heterodimer), 'stabilizing' antibodies, and antibodies to DNA-PKcs comprise a linked autoantibody set, since antibodies recognizing purified DNA-PKcs were strongly associated with stabilizing antibodies, whereas stabilizing antibodies were strongly associated with anti-Ku. This hierarchical pattern of autoantibodies specific for components of DNA-PK (anti-Ku > stabilizing antibodies > anti-DNA-PKcs) may have implications for the pathogenesis of autoimmunity to DNA-PK and other chromatin particles. The data raise the possibility that altered antigen processing and/or stabilization of the DNA-PKcs-Ku complex due to autoantibody binding could play a role in spreading autoimmunity from Ku to the weakly associated antigen DNA-PKcs.

  18. 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

  19. 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.

  20. 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. PMID:26474642

  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. Mitogen-Activated Protein Kinase Signaling in Plant-Interacting Fungi: Distinct Messages from Conserved Messengers[W

    PubMed Central

    Hamel, Louis-Philippe; Nicole, Marie-Claude; Duplessis, Sébastien; Ellis, Brian E.

    2012-01-01

    Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved proteins that function as key signal transduction components in fungi, plants, and mammals. During interaction between phytopathogenic fungi and plants, fungal MAPKs help to promote mechanical and/or enzymatic penetration of host tissues, while plant MAPKs are required for activation of plant immunity. However, new insights suggest that MAPK cascades in both organisms do not operate independently but that they mutually contribute to a highly interconnected molecular dialogue between the plant and the fungus. As a result, some pathogenesis-related processes controlled by fungal MAPKs lead to the activation of plant signaling, including the recruitment of plant MAPK cascades. Conversely, plant MAPKs promote defense mechanisms that threaten the survival of fungal cells, leading to a stress response mediated in part by fungal MAPK cascades. In this review, we make use of the genomic data available following completion of whole-genome sequencing projects to analyze the structure of MAPK protein families in 24 fungal taxa, including both plant pathogens and mycorrhizal symbionts. Based on conserved patterns of sequence diversification, we also propose the adoption of a unified fungal MAPK nomenclature derived from that established for the model species Saccharomyces cerevisiae. Finally, we summarize current knowledge of the functions of MAPK cascades in phytopathogenic fungi and highlight the central role played by MAPK signaling during the molecular dialogue between plants and invading fungal pathogens. PMID:22517321

  3. Genetic Interactions among AMPK Catalytic Subunit Ssp2 and Glycogen Synthase Kinases Gsk3 and Gsk31 in Schizosaccharomyces Pombe.

    PubMed

    Qingyun; Ma, Yan; Kato, Toshiaki; Furuyashiki, Tomoyuki

    2016-01-01

    In Schizosaccharomyces pombe, Ssp2, an ortholog of AMP-activated protein kinase (AMPK), is critical for cell growth at restrictive temperatures and under glucose depletion as well as sexual differentiation under nitrogen depletion. To identify genes genetically related to Ssp2, we performed a genetic screening to search for the genes whose overexpression rescued the growth defects in Δssp2 cells at restrictive temperatures, and identified 35 cosmids as multicopy suppressor genes. In Southern blot analyses, 22 out of these cosmids were hybridized to an ssp2+ probe. Using nucleotide sequencing, we identified the gsk3+ gene in one of the cosmids, and the remaining 12 cosmids were hybridized to a gsk3+ probe. Overexpression of the gsk3+ gene or the gsk31+ gene, another GSK3 member, rescues defective growth of Δssp2 cells at restrictive temperatures and under glucose depletion as well as sexual differentiation under nitrogen depletion. Δgsk3Δgsk31 double knockout cells, but neither Δgsk3 nor Δgsk31 single knockout cells, phenocopy Δssp2 cells. The deletion of the gsk3+ or gsk31+ gene augments the phenotypes of Δssp2 cells. These findings suggest that Gsk3 and Gsk31 are critical and interact with Ssp2 in multiple cellular functions. PMID:27604537

  4. 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

  5. Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions.

    PubMed

    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

    2016-06-15

    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

  6. Interaction between fidgetin and protein kinase A-anchoring protein AKAP95 is critical for palatogenesis in the mouse.

    PubMed

    Yang, Yan; Mahaffey, Connie L; Bérubé, Nathalie; Frankel, Wayne N

    2006-08-01

    The gene defective in fidget mice encodes fidgetin, a member of the AAA (ATPases associated with diverse cellular activities) family of ATPases. Using a yeast two-hybrid screen, we identified cAMP-dependent protein kinase A anchoring protein 95 kDa (AKAP95) as a potential fidgetin-binding protein. Epitope-tagged fidgetin co-localized with endogenous AKAP95 in the nuclear matrix, and the physical interaction between fidgetin and AKAP95 was further confirmed by reciprocal immunoprecipitation. To evaluate the biological significance of the fidgetin-AKAP95 binding, we created AKAP95 mutant mice through a gene trap strategy. Akap95 mutant mice are surprisingly viable with no overt phenotype. However, a significant number of mice carrying both Akap95 and fidget mutations die soon after birth due to cleft palate, consistent with the overlapping expression of AKAP95 and fidgetin in the branchial arches during mouse embryogenesis. These results expand the spectrum of the pleiotropic phenotypes of fidget mice and provide new leads on the in vivo function of AKAP95.

  7. 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.

  8. The role of effectors and host immunity in plant–necrotrophic fungal interactions

    PubMed Central

    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. PMID:25513773

  9. Phosphatidylinositol 3-Phosphate 5-Kinase, FAB1/PIKfyve Kinase Mediates Endosome Maturation to Establish Endosome-Cortical Microtubule Interaction in Arabidopsis1[OPEN

    PubMed Central

    Hirano, Tomoko; Munnik, Teun; Sato, Masa H.

    2015-01-01

    Phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] is an important lipid in membrane trafficking in animal and yeast systems; however, its role is still largely obscure in plants. Here, we demonstrate that the phosphatidylinositol 3-phosphate 5-kinase, formation of aploid and binucleate cells1 (FAB1)/FYVE finger-containing phosphoinositide kinase (PIKfyve), and its product, PtdIns(3,5)P2, are essential for the maturation process of endosomes to mediate cortical microtubule association of endosomes, thereby controlling proper PIN-FORMED protein trafficking in young cortical and stele cells of root. We found that FAB1 predominantly localizes on the Sorting Nexin1 (SNX1)-residing late endosomes, and a loss of FAB1 function causes the release of late endosomal proteins, Ara7, and SNX1 from the endosome membrane, indicating that FAB1, or its product PtdIns(3,5)P2, mediates the maturation process of the late endosomes. We also found that loss of FAB1 function causes the release of endosomes from cortical microtubules and disturbs proper cortical microtubule organization. PMID:26353760

  10. The Sugar Kinase That Is Necessary for the Catabolism of Rhamnose in Rhizobium leguminosarum Directly Interacts with the ABC Transporter Necessary for Rhamnose Transport

    PubMed Central

    Rivers, Damien M. R.

    2015-01-01

    ABSTRACT Rhamnose catabolism in Rhizobium leguminosarum was found to be necessary for the ability of the organism to compete for nodule occupancy. Characterization of the locus necessary for the catabolism of rhamnose showed that the transport of rhamnose was dependent upon a carbohydrate uptake transporter 2 (CUT2) ABC transporter encoded by rhaSTPQ and on the presence of RhaK, a protein known to have sugar kinase activity. A linker-scanning mutagenesis analysis of rhaK showed that the kinase and transport activities of RhaK could be separated genetically. More specifically, two pentapeptide insertions defined by the alleles rhaK72 and rhaK73 were able to uncouple the transport and kinase activities of RhaK, such that the kinase activity was retained, but cells carrying these alleles did not have measurable rhamnose transport rates. These linker-scanning alleles were localized to the C terminus and N terminus of RhaK, respectively. Taken together, the data led to the hypothesis that RhaK might interact either directly or indirectly with the ABC transporter defined by rhaSTPQ. In this work, we show that both N- and C-terminal fragments of RhaK are capable of interacting with the N-terminal fragment of the ABC protein RhaT using a 2-hybrid system. Moreover, if RhaK fragments carrying either the rhaK72 or rhaK73 allele were used, this interaction was abolished. Phylogenetic and bioinformatic analysis of the RhaK fragments suggested that a conserved region in the N terminus of RhaK may represent a putative binding domain. Alanine-scanning mutagenesis of this region followed by 2-hybrid analysis revealed that a substitution of any of the conserved residues greatly affected the interaction between RhaT and RhaK fragments, suggesting that the sugar kinase RhaK and the ABC protein RhaT interact directly. IMPORTANCE ABC transporters involved in the transport of carbohydrates help define the overall physiological fitness of bacteria. The two largest groups of transporters

  11. 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.

  12. Traf2- and Nck-interacting kinase (TNIK) is involved in the anti-cancer mechanism of dovitinib in human multiple myeloma IM-9 cells.

    PubMed

    Chon, Hae Jung; Lee, Yura; Bae, Kyoung Jun; Byun, Byung Jin; Kim, Soon Ae; Kim, Jiyeon

    2016-07-01

    Traf2- and Nck-interacting kinase (TNIK) is a member of the germinal center kinase family. TNIK was first identified as a kinase that is involved in regulating cytoskeletal organization in many types of cells, and it was recently proposed as a novel therapeutic target in several types of human cancers. Although previous studies suggest that TNIK plays a pivotal role in cancer cell survival and prognosis, its function in hematological cancer cell survival has not been investigated. Here we investigated the relationship between TNIK function and cell viability in multiple myeloma IM-9 cells using TNIK small interfering RNA (siRNA) transfection and dovitinib treatment. Treatment of IM-9 cells with TNIK siRNA and dovitinib treatment reduced cell proliferation. The ATP competing kinase assay and western blot analysis showed that dovitinib strongly inhibited both the interaction of TNIK with ATP (K i, 13 nM) and the activation of Wnt signaling effectors such as β-catenin and TCF4. Dovitinib also induced caspase-dependent apoptosis in IM-9 cells without significant cytotoxicity in PBMCs. Our results provide new evidence that TNIK may be involved in the proliferation of multiple myeloma IM-9 cells and in the anti-cancer activity of dovitinib via inhibition of the endogenous Wnt signaling pathway.

  13. The COOH-terminal domain of the JIL-1 histone H3S10 kinase interacts with histone H3 and is required for correct targeting to chromatin.

    PubMed

    Bao, Xiaomin; Cai, Weili; Deng, Huai; Zhang, Weiguo; Krencik, Robert; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M

    2008-11-21

    The JIL-1 histone H3S10 kinase in Drosophila localizes specifically to euchromatic interband regions of polytene chromosomes and is enriched 2-fold on the male X chromosome. JIL-1 can be divided into four main domains including an NH(2)-terminal domain, two separate kinase domains, and a COOH-terminal domain. Our results demonstrate that the COOH-terminal domain of JIL-1 is necessary and sufficient for correct chromosome targeting to autosomes but that both COOH- and NH(2)-terminal sequences are necessary for enrichment on the male X chromosome. We furthermore show that a small 53-amino acid region within the COOH-terminal domain can interact with the tail region of histone H3, suggesting that this interaction is necessary for the correct chromatin targeting of the JIL-1 kinase. Interestingly, our data indicate that the COOH-terminal domain alone is sufficient to rescue JIL-1 null mutant polytene chromosome defects including those of the male X chromosome. Nonetheless, we also found that a truncated JIL-1 protein which was without the COOH-terminal domain but retained histone H3S10 kinase activity was able to rescue autosome as well as partially rescue male X polytene chromosome morphology. Taken together these findings indicate that JIL-1 may participate in regulating chromatin structure by multiple and partially redundant mechanisms.

  14. Interaction between the tRNA-binding and C-terminal domains of Yeast Gcn2 regulates kinase activity in vivo.

    PubMed

    Lageix, Sebastien; Zhang, Jinwei; Rothenburg, Stefan; Hinnebusch, Alan G

    2015-02-01

    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.

  15. 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.

  16. ADAM22, a Kv1 channel-interacting protein, recruits membrane-associated guanylate kinases to juxtaparanodes of myelinated axons.

    PubMed

    Ogawa, Yasuhiro; Oses-Prieto, Juan; Kim, Moon Young; Horresh, Ido; Peles, Elior; Burlingame, Alma L; Trimmer, James S; Meijer, Dies; Rasband, Matthew N

    2010-01-20

    Clustered Kv1 K(+) channels regulate neuronal excitability at juxtaparanodes of myelinated axons, axon initial segments, and cerebellar basket cell terminals (BCTs). These channels are part of a larger protein complex that includes cell adhesion molecules and scaffolding proteins. To identify proteins that regulate assembly, clustering, and/or maintenance of axonal Kv1 channel protein complexes, we immunoprecipitated Kv1.2 alpha subunits, and then used mass spectrometry to identify interacting proteins. We found that a disintegrin and metalloproteinase 22 (ADAM22) is a component of the Kv1 channel complex and that ADAM22 coimmunoprecipitates Kv1.2 and the membrane-associated guanylate kinases (MAGUKs) PSD-93 and PSD-95. When coexpressed with MAGUKs in heterologous cells, ADAM22 and Kv1 channels are recruited into membrane surface clusters. However, coexpression of Kv1.2 with ADAM22 and MAGUKs does not alter channel properties. Among all the known Kv1 channel-interacting proteins, only ADAM22 is found at every site where Kv1 channels are clustered. Analysis of Caspr-null mice showed that, like other previously described juxtaparanodal proteins, disruption of the paranodal junction resulted in redistribution of ADAM22 into paranodal zones. Analysis of Caspr2-, PSD-93-, PSD-95-, and double PSD-93/PSD-95-null mice showed ADAM22 clustering at BCTs requires PSD-95, but ADAM22 clustering at juxtaparanodes requires neither PSD-93 nor PSD-95. In direct contrast, analysis of ADAM22-null mice demonstrated juxtaparanodal clustering of PSD-93 and PSD-95 requires ADAM22, whereas Kv1.2 and Caspr2 clustering is normal in ADAM22-null mice. Thus, ADAM22 is an axonal component of the Kv1 K(+) channel complex that recruits MAGUKs to juxtaparanodes. PMID:20089912

  17. Two distinct forms of M-locus protein kinase localize to the plasma membrane and interact directly with S-locus receptor kinase to transduce self-incompatibility signaling in Brassica rapa.

    PubMed

    Kakita, Mitsuru; Murase, Kohji; Iwano, Megumi; Matsumoto, Tomohito; Watanabe, Masao; Shiba, Hiroshi; Isogai, Akira; Takayama, Seiji

    2007-12-01

    Many flowering plants possess systems of self-incompatibility (SI) to prevent inbreeding. In Brassica, SI recognition is controlled by the multiallelic gene complex (S-haplotypes) at the S-locus, which encodes both the male determinant S-locus protein 11 (SP11/SCR) and the female determinant S-receptor kinase (SRK). Upon self-pollination, the S-haplotype-specific interaction between the pollen-borne SP11 and the cognate stigmatic SRK receptor induces SI signaling in the stigmatic papilla cell and results in rejection of the self-pollen. Our genetic analysis of a self-compatible mutant revealed the involvement of a cytoplasmic protein kinase, M-locus protein kinase (MLPK), in the SI signaling, but its exact physiological function remains unknown. In this study, we identified two different MLPK transcripts, MLPKf1 and MLPKf2, which are produced using alternative transcriptional initiation sites and encode two isoforms that differ only at the N termini. While MLPKf1 and MLPKf2 exhibited distinct expression profiles, both were expressed in papilla cells. MLPKf1 localizes to the plasma membrane through its N-terminal myristoylation motif, while MLPKf2 localizes to the plasma membrane through its N-terminal hydrophobic region. Although both MLPKf1 and MLPKf2 could independently complement the mlpk/mlpk mutation, their mutant forms that lack the plasma membrane localization motifs failed to complement the mutation. Furthermore, a bimolecular fluorescence complementation assay revealed direct interactions between SRK and the MLPK isoforms in planta. These results suggest that MLPK isoforms localize to the papilla cell membrane and interact directly with SRK to transduce SI signaling.

  18. Oligomeric interactions provide alternatives to direct steric modes of control of sugar kinase/actin/hsp70 superfamily functions by heterotropic allosteric effectors: Inhibition of E. coli glycerol kinase

    PubMed Central

    Pettigrew, Donald W.

    2009-01-01

    Unlike those for monomeric superfamily members, heterotropic allosteric effectors of the tetrameric E. coli glycerol kinase (EGK) bind to only one of the two domains that define the catalytic cleft and far from the active site. An R369A amino acid substitution removes oligomeric interactions of a novel mini domain-swap loop of one subunit with the catalytic site of another subunit, and an A65T substitution perturbs oligomeric interactions in a second interface. Linked-functions enzyme kinetics, analytical ultracentrifugation, and FRET are used to assess effects of these substitutions on the allosteric control of catalysis. Inhibition by phosphotransferase system protein IIAGlc is reduced by the R369A substitution, and inhibition by fructose 1,6-bisphosphate is abolished by the A65T substitution. The oligomeric interactions enable the heterotropic allosteric effectors to act on both domains and modulate the catalytic cleft closure despite binding to only one domain. PMID:19819219

  19. Phosphorylation and interaction of Myopodin by Integrin-Link Kinase Lead to Suppression of Cell Growth and Motility in Prostate Cancer Cells

    PubMed Central

    Yu, Yan-Ping; Luo, Jian-Hua

    2011-01-01

    Myopodin is a tumor suppressor gene that suppresses growth of prostate and urothelial carcinomas. However, the mechanism of myopodin tumor suppressor activity or signaling that leads to activation of myopodin remains unclear. In this report, we showed that the N-terminus of myopodin binds integrin-linked kinase (ILK) both in vivo and in vitro. An ILK interaction motif of 78 amino acids (amino acids 82–157) was identified in the N-terminus region of myopodin. Induction of ILK dependent kinase activity by integrin α7 led to phosphorylation of myopodin both in vivo and in vitro. Knocking down ILK dramatically reduced the inhibition of cell growth and motility mediated by myopodin. A mutant of myopodin lacking the ILK interaction motif is inactive in suppressing the growth and motility of PC3 cells. As a result, this study showed a novel and critical signaling pathway that leads to activation of myopodin. PMID:21643011

  20. NUP214-ABL1-mediated cell proliferation in T-cell acute lymphoblastic leukemia is dependent on the LCK kinase and various interacting proteins

    PubMed Central

    De Keersmaecker, Kim; Porcu, Michaël; Cox, Luk; Girardi, Tiziana; Vandepoel, Roel; de Beeck, Joyce Op; Gielen, Olga; Mentens, Nicole; Bennett, Keiryn L.; Hantschel, Oliver

    2014-01-01

    The NUP214-ABL1 fusion protein is a constitutively active protein tyrosine kinase that is found in 6% of patients with T-cell acute lymphoblastic leukemia and that promotes proliferation and survival of T-lymphoblasts. Although NUP214-ABL1 is sensitive to ABL1 kinase inhibitors, development of resistance to these compounds is a major clinical problem, underlining the need for additional drug targets in the sparsely studied NUP214-ABL1 signaling network. In this work, we identify and validate the SRC family kinase LCK as a protein whose activity is absolutely required for the proliferation and survival of T-cell acute lymphoblastic leukemia cells that depend on NUP214-ABL1 activity. These findings underscore the potential of SRC kinase inhibitors and of the dual ABL1/SRC kinase inhibitors dasatinib and bosutinib for the treatment of NUP214-ABL1-positive T-cell acute lymphoblastic leukemia. In addition, we used mass spectrometry to identify protein interaction partners of NUP214-ABL1. Our results strongly support that the signaling network of NUP214-ABL1 is distinct from that previously reported for BCR-ABL1. Moreover, we found that three NUP214-ABL1-interacting proteins, MAD2L1, NUP155, and SMC4, are strictly required for the proliferation and survival of NUP214-ABL1-positive T-cell acute lymphoblastic leukemia cells. In conclusion, this work identifies LCK, MAD2L1, NUP155 and SMC4 as four new potential drug targets in NUP214-ABL1-positive T-cell acute lymphoblastic leukemia. PMID:23872305

  1. 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.

  2. 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

  3. Progesterone receptor isoforms PRA and PRB differentially contribute to breast cancer cell migration through interaction with focal adhesion kinase complexes.

    PubMed

    Bellance, Catherine; Khan, Junaid A; Meduri, Geri; Guiochon-Mantel, Anne; Lombès, Marc; Loosfelt, Hugues

    2013-05-01

    Progesterone receptor (PR) and progestins affect mammary tumorigenesis; however, the relative contributions of PR isoforms A and B (PRA and PRB, respectively) in cancer cell migration remains elusive. By using a bi-inducible MDA-MB-231 breast cancer cell line expressing PRA and/or PRB, we analyzed the effect of conditional PR isoform expression. Surprisingly, unliganded PRB but not PRA strongly enhanced cell migration as compared with PR(-) cells. 17,21-Dimethyl-19-norpregna-4,9-dien-3,20-dione (R5020) progestin limited this effect and was counteracted by the antagonist 11β-(4-dimethyl-amino)-phenyl-17β-hydroxy-17-(1-propynyl)-estra-4,9-dien-3-one (RU486). Of importance, PRA coexpression potentiated PRB-mediated migration, whereas PRA alone was ineffective. PR isoforms differentially regulated expressions of major players of cell migration, such as urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor type 1, uPA receptor (uPAR), and β1-integrin, which affect focal adhesion kinase (FAK) signaling. Moreover, unliganded PRB but not PRA enhanced FAK Tyr397 phosphorylation and colocalized with activated FAK in cell protrusions. Because PRB, as well as PRA, coimmunoprecipitated with FAK, both isoforms can interact with FAK complexes, depending on their respective nucleocytoplasmic trafficking. In addition, FAK degradation was coupled to R5020-dependent turnovers of PRA and PRB. Such an effect of PRB/PRA expression on FAK signaling might thus affect adhesion/motility, underscoring the implication of PR isoforms in breast cancer invasiveness and metastatic evolution with underlying therapeutic outcomes.

  4. Phosphorylation Alters the Interaction of the Arabidopsis Phosphotransfer Protein AHP1 with Its Sensor Kinase ETR1

    PubMed Central

    Scharein, Benjamin; Groth, Georg

    2011-01-01

    The ethylene receptor ethylene response 1 (ETR1) and the Arabidopsis histidine-containing phosphotransfer protein 1 (AHP1) form a tight complex in vitro. According to our current model ETR1 and AHP1 together with a response regulator form a phosphorelay system controlling the gene expression response to the plant hormone ethylene, similar to the two-component signaling in bacteria. The model implies that ETR1 functions as a sensor kinase and is autophosphorylated in the absence of ethylene. The phosphoryl group is then transferred onto a histidine at the canonical phosphorylation site in AHP1. For phosphoryl group transfer both binding partners need to form a tight complex. After ethylene binding the receptor is switched to the non-phosphorylated state. This switch is accompanied by a conformational change that decreases the affinity to the phosphorylated AHP1. To test this model we used fluorescence polarization and examined how the phosphorylation status of the proteins affects formation of the suggested ETR1−AHP1 signaling complex. We have employed various mutants of ETR1 and AHP1 mimicking permanent phosphorylation or preventing phosphorylation, respectively. Our results show that phosphorylation plays an important role in complex formation as affinity is dramatically reduced when the signaling partners are either both in their non-phosphorylated form or both in their phosphorylated form. On the other hand, affinity is greatly enhanced when either protein is in the phosphorylated state and the corresponding partner in its non-phosphorylated form. Our results indicate that interaction of ETR1 and AHP1 requires that ETR1 is a dimer, as in its functional state as receptor in planta. PMID:21912672

  5. Progesterone receptor isoforms PRA and PRB differentially contribute to breast cancer cell migration through interaction with focal adhesion kinase complexes

    PubMed Central

    Bellance, Catherine; Khan, Junaid A.; Meduri, Geri; Guiochon-Mantel, Anne; Lombès, Marc; Loosfelt, Hugues

    2013-01-01

    Progesterone receptor (PR) and progestins affect mammary tumorigenesis; however, the relative contributions of PR isoforms A and B (PRA and PRB, respectively) in cancer cell migration remains elusive. By using a bi-inducible MDA-MB-231 breast cancer cell line expressing PRA and/or PRB, we analyzed the effect of conditional PR isoform expression. Surprisingly, unliganded PRB but not PRA strongly enhanced cell migration as compared with PR(–) cells. 17,21-Dimethyl-19-norpregna-4,9-dien-3,20-dione (R5020) progestin limited this effect and was counteracted by the antagonist 11β-(4-dimethyl­amino)­phenyl-17β-hydroxy-17-(1-propynyl)­estra-4,9-dien-3-one (RU486). Of importance, PRA coexpression potentiated PRB-mediated migration, whereas PRA alone was ineffective. PR isoforms differentially regulated expressions of major players of cell migration, such as urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor type 1, uPA receptor (uPAR), and β1-integrin, which affect focal adhesion kinase (FAK) signaling. Moreover, unliganded PRB but not PRA enhanced FAK Tyr397 phosphorylation and colocalized with activated FAK in cell protrusions. Because PRB, as well as PRA, coimmunoprecipitated with FAK, both isoforms can interact with FAK complexes, depending on their respective nucleocytoplasmic trafficking. In addition, FAK degradation was coupled to R5020-dependent turnovers of PRA and PRB. Such an effect of PRB/PRA expression on FAK signaling might thus affect adhesion/motility, underscoring the implication of PR isoforms in breast cancer invasiveness and metastatic evolution with underlying therapeutic outcomes. PMID:23485561

  6. 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

  7. Drosophila homeodomain-interacting protein kinase inhibits the Skp1-Cul1-F-box E3 ligase complex to dually promote Wingless and Hedgehog signaling.

    PubMed

    Swarup, Sharan; Verheyen, Esther M

    2011-06-14

    Drosophila Homeodomain-interacting protein kinase (Hipk) has been shown to regulate in vivo, the stability of Armadillo, the transcriptional effector of Wingless signaling. The Wingless pathway culminates in the stabilization of Armadillo that, in the absence of signaling, is sequentially phosphorylated, polyubiquitinated and degraded. Loss-of-function clones for hipk result in reduced stabilized Armadillo, whereas overexpression of hipk elevates Armadillo levels to promote Wingless-responsive target gene expression. Here, we show that overexpression of hipk can suppress the effects of negative regulators of Armadillo to prevent its degradation in the wing imaginal disc. Hipk acts to stabilize Armadillo by impeding the function of the E3 ubiquitin ligase Skp1-Cul1-F-box (SCF)(Slimb), thereby inhibiting Armadillo ubiquitination and subsequent degradation. Vertebrate Hipk2 displays a similar ability to prevent β-catenin ubiquitination in a functionally conserved mechanism. We find that Hipk's ability to inhibit SCF(Slimb)-mediated ubiquitination is not restricted to Armadillo and extends to other substrates of SCF(Slimb), including the Hedgehog signaling effector Ci. Thus, similar to casein kinase 1 and glycogen synthase kinase 3, Hipk dually regulates both Wingless and Hedgehog signaling by controlling the stability of their respective signaling effectors, but it is the first kinase to our knowledge identified that promotes the stability of both Armadillo and Ci.

  8. Sex differences in social interaction behaviors in rats are mediated by extracellular signal-regulated kinase 2 expression in the medial prefrontal cortex

    PubMed Central

    Carrier, Nicole; Kabbaj, Mohamed

    2012-01-01

    Considerable sex differences occur in the incidence and prevalence of anxiety disorders where women are more anxious than men, particularly in situations where social interaction is required. In preclinical studies, the social interaction test represents a valid animal model to study sex differences in social anxiety. Indeed, female rats engage less in conspecific interactions than their male counterparts, which are behaviors indicative of higher social anxiety in female rats. In this work, we implicated extracellular signal regulated kinase 2 (ERK2) in the medial prefrontal cortex (mPFC) in mediating social interaction. Indeed, female rats’ had lower ERK2 expression compared to male rats, and overexpression of ERK2 in the mPFC increases their social interaction to the level seen in their male counterparts. These data indicate that the sexually dimorphic expression of ERK2 mediates social anxiety-like behaviors. PMID:22521590

  9. 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

  10. Dimer of arfaptin 2 regulates NF-κB signaling by interacting with IKKβ/NEMO and inhibiting IKKβ kinase activity.

    PubMed

    You, Dong-Joo; Park, Cho Rong; Furlong, Michael; Koo, Okjae; Lee, Cheolju; Ahn, Curie; Seong, Jae Young; Hwang, Jong-Ik

    2015-11-01

    IκB kinases (IKKs) are a therapeutic target due to their crucial roles in various biological processes, including the immune response, the stress response, and tumor development. IKKs integrate various upstream signals that activate NF-κB by phosphorylating IκB and also regulate many proteins related to cell growth and metabolism. Although they function as a heteromeric complex comprised of kinase subunits and an adaptor, these kinases produce distinct cellular responses by phosphorylating different target molecules, suggesting that they may also be regulated in a subtype-specific manner. In this study, arfaptin 2 was identified as an IKKβ-specific binding partner. Interestingly, arfaptin 2 also interacted with NEMO. Domain mapping studies revealed that the C-terminal region, including the IKKβ HLH domain and the first coiled-coil NEMO region were respectively required for interactions with the arfaptin 2 N-terminal flexible region. Overexpression of arfaptin 2 inhibited tumor necrosis factor (TNF)-α-stimulated nuclear factor-κB (NF-κB) signaling, whereas downregulation of arfaptin 2 by small interfering RNA enhanced NF-κB activity. Dimerization of arfaptin 2 through the Bin-Amphiphysin-Rvs domain may be essential to inhibit activation of NF-κB through multimodal interactions with IKKβs or IKKβ/NEMO, as ectopic expression of the arfaptin 2 fragment responsible for IKK interactions did not change TNFα-stimulated NF-κB activation. These data indicate that arfaptin 2 is the first molecule to regulate NF-κB signaling by interacting with the functional IKK complex but not by direct inhibiting IKKβ phosphorylation. PMID:26296658

  11. Homeodomain-interacting protein kinases (Hipks) promote Wnt/Wg signaling through stabilization of beta-catenin/Arm and stimulation of target gene expression.

    PubMed

    Lee, Wendy; Swarup, Sharan; Chen, Joanna; Ishitani, Tohru; Verheyen, Esther M

    2009-01-01

    The Wnt/Wingless (Wg) pathway represents a conserved signaling cascade involved in diverse biological processes. Misregulation of Wnt/Wg signal transduction has profound effects on development. Homeodomain-interacting protein kinases (Hipks) represent a novel family of serine/threonine kinases. Members of this group (in particular Hipk2) are implicated as important factors in transcriptional regulation to control cell growth, apoptosis and development. Here, we provide genetic and phenotypic evidence that the sole Drosophila member of this family, Hipk, functions as a positive regulator in the Wg pathway. Expression of hipk in the wing rescues loss of the Wg signal, whereas loss of hipk can enhance decreased wg signaling phenotypes. Furthermore, loss of hipk leads to diminished Arm protein levels, whereas overexpression of hipk promotes the Wg signal by stabilizing Arm, resulting in activation of Wg responsive targets. In Wg transcriptional assays, Hipk enhanced Tcf/Arm-mediated gene expression in a kinase-dependent manner. In addition, Hipk can bind to Arm and Drosophila Tcf, and phosphorylate Arm. Using both in vitro and in vivo assays, Hipk was found to promote the stabilization of Arm. We observe similar molecular interactions between Lef1/beta-catenin and vertebrate Hipk2, suggesting a direct and conserved role for Hipk proteins in promoting Wnt signaling. PMID:19088090

  12. 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.

  13. The N-terminal region of the Neurospora NDR kinase COT1 regulates morphology via its interactions with MOB2A/B

    PubMed Central

    Ziv, Carmit; Feldman, Daria; Aharoni-Kats, Liran; Chen, She; Liu, Yi; Yarden, Oded

    2015-01-01

    Summary Nuclear Dbf2p-related (NDR) protein kinases are important for cell differentiation and polar morphogenesis in various organisms, yet some of their functions are still elusive. Dysfunction of the Neurospora crassa NDR kinase COT1 leads to cessation of tip extension and hyperbranching. NDR kinases require the physical interaction between the kinase’s N-terminal region (NTR) and the MPS1-binding (MOB) proteins for their activity and functions. To study the interactions between COT1 and MOB2 proteins, we mutated several conserved residues and a novel phosphorylation site within the COT1 NTR. The phenotypes of these mutants suggest that the NTR is required for COT1 functions in regulating hyphal elongation and branching, asexual conidiation and germination. Interestingly, while both MOB2A and MOB2B promote proper hyphal growth, they have distinct COT1-dependent roles in regulation of macroconidiation. Immunoprecipitation experiments indicate physical association of COT1 with both MOB2A and MOB2B, simultaneously. Furthermore, the binding of the two MOB2 proteins to COT1 is mediated by different residues at the COT1 NTR, suggesting a hetero-trimer is formed. Thus, although MOB2A/B may have some overlapping functions in regulating hyphal tip extension, their function is not redundant and they are both required for proper fungal development. PMID:23962317

  14. Fragment-Based Screening Maps Inhibitor Interactions in the ATP-Binding Site of Checkpoint Kinase 2

    PubMed Central

    Silva-Santisteban, M. Cris; Westwood, Isaac M.; Boxall, Kathy; Brown, Nathan; Peacock, Sam; McAndrew, Craig; Barrie, Elaine; Richards, Meirion; Mirza, Amin; Oliver, Antony W.; Burke, Rosemary; Hoelder, Swen; Jones, Keith; Aherne, G. Wynne; Blagg, Julian; Collins, Ian; Garrett, Michelle D.; van Montfort, Rob L. M.

    2013-01-01

    Checkpoint kinase 2 (CHK2) is an important serine/threonine kinase in the cellular response to DNA damage. A fragment-based screening campaign using a combination of a high-concentration AlphaScreen™ kinase assay and a biophysical thermal shift assay, followed by X-ray crystallography, identified a number of chemically different ligand-efficient CHK2 hinge-binding scaffolds that have not been exploited in known CHK2 inhibitors. In addition, it showed that the use of these orthogonal techniques allowed efficient discrimination between genuine hit matter and false positives from each individual assay technology. Furthermore, the CHK2 crystal structures with a quinoxaline-based fragment and its follow-up compound highlight a hydrophobic area above the hinge region not previously explored in rational CHK2 inhibitor design, but which might be exploited to enhance both potency and selectivity of CHK2 inhibitors. PMID:23776527

  15. The mitogen-activated protein kinase cascade is activated by B-Raf in response to nerve growth factor through interaction with p21ras.

    PubMed Central

    Jaiswal, R K; Moodie, S A; Wolfman, A; Landreth, G E

    1994-01-01

    Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21ras-dependent signal transduction pathway in PC12 cells. The linkage between p21ras and MEK1 was investigated to identify those elements which participate in the regulation of MEK1 activity. We have screened for MEK activators using a coupled assay in which the MAP kinase cascade has been reconstituted in vitro. We report that we have detected a single NGF-stimulated MEK-activating activity which has been identified as B-Raf. PC12 cells express both B-Raf and c-Raf1; however, the MEK-activating activity was found only in fractions containing B-Raf. c-Raf1-containing fractions did not exhibit a MEK-activating activity. Gel filtration analysis revealed that the B-Raf eluted with an apparent M(r) of 250,000 to 300,000, indicating that it is present within a stable complex with other unidentified proteins. Immunoprecipitation with B-Raf-specific antisera quantitatively precipitated all MEK activator activity from these fractions. We also demonstrate that B-Raf, as well as c-Raf1, directly interacted with activated p21ras immobilized on silica beads. NGF treatment of the cells had no effect on the ability of B-Raf or c-Raf1 to bind to activated p21ras. These data indicate that this interaction was not dependent upon the activation state of these enzymes; however, MEK kinase activity was found to be associated with p21ras following incubation with NGF-treated samples at levels higher than those obtained from unstimulated cells. These data provide direct evidence that NGF-stimulated B-Raf is responsible for the activation of the MAP kinase cascade in PC12 cells, whereas c-Raf1 activity was not found to function within this pathway. Images PMID:7935411

  16. Interaction of Omega, Sigma, and Theta glutathione transferases with p38b mitogen-activated protein kinase from the fruit fly, Drosophila melanogaster.

    PubMed

    Wongtrakul, J; Janphen, K; Saisawang, C; Ketterman, A J

    2014-05-01

    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.

  17. 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

  18. The Tomato Calcium Sensor Cbl10 and Its Interacting Protein Kinase Cipk6 Define a Signaling Pathway in Plant Immunity[C][W

    PubMed Central

    de la Torre, Fernando; Gutiérrez-Beltrán, Emilio; Pareja-Jaime, Yolanda; Chakravarthy, Suma; Martin, Gregory B.; del Pozo, Olga

    2013-01-01

    Ca2+ signaling is an early and necessary event in plant immunity. The tomato (Solanum lycopersicum) kinase Pto triggers localized programmed cell death (PCD) upon recognition of Pseudomonas syringae effectors AvrPto or AvrPtoB. In a virus-induced gene silencing screen in Nicotiana benthamiana, we independently identified two components of a Ca2+-signaling system, Cbl10 (for calcineurin B-like protein) and Cipk6 (for calcineurin B-like interacting protein kinase), as their silencing inhibited Pto/AvrPto-elicited PCD. N. benthamiana Cbl10 and Cipk6 are also required for PCD triggered by other plant resistance genes and virus, oomycete, and nematode effectors and for host susceptibility to two P. syringae pathogens. Tomato Cipk6 interacts with Cbl10 and its in vitro kinase activity is enhanced in the presence of Cbl10 and Ca2+, suggesting that tomato Cbl10 and Cipk6 constitute a Ca2+-regulated signaling module. Overexpression of tomato Cipk6 in N. benthamiana leaves causes accumulation of reactive oxygen species (ROS), which requires the respiratory burst homolog RbohB. Tomato Cbl10 and Cipk6 interact with RbohB at the plasma membrane. Finally, Cbl10 and Cipk6 contribute to ROS generated during effector-triggered immunity in the interaction of P. syringae pv tomato DC3000 and N. benthamiana. We identify a role for the Cbl/Cipk signaling module in PCD, establishing a mechanistic link between Ca2+ and ROS signaling in plant immunity. PMID:23903322

  19. 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. PMID:27129432

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. N-acetyl-D-glucosamine kinase interacts with dynein light-chain roadblock type 1 at Golgi outposts in neuronal dendritic branch points

    PubMed Central

    Islam, Md Ariful; Sharif, Syeda Ridita; Lee, HyunSook; Seog, Dae-Hyun; Moon, Il Soo

    2015-01-01

    N-acetylglucosamine kinase (GlcNAc kinase or NAGK) is a ubiquitously expressed enzyme in mammalian cells. Recent studies have shown that NAGK has an essential structural, non-enzymatic role in the upregulation of dendritogenesis. In this study, we conducted yeast two-hybrid screening to search for NAGK-binding proteins and found a specific interaction between NAGK and dynein light-chain roadblock type 1 (DYNLRB1). Immunocytochemistry (ICC) on hippocampal neurons using antibodies against NAGK and DYNLRB1 or dynein heavy chain showed some colocalization, which was increased by treating the live cells with a crosslinker. A proximity ligation assay (PLA) of NAGK-dynein followed by tubulin ICC showed the localization of PLA signals on microtubule fibers at dendritic branch points. NAGK-dynein PLA combined with Golgi ICC showed the colocalization of PLA signals with somal Golgi facing the apical dendrite and with Golgi outposts in dendritic branch points and distensions. NAGK-Golgi PLA followed by tubulin or DYNLRB1 ICC showed that PLA signals colocalize with DYNLRB1 at dendritic branch points and at somal Golgi, indicating a tripartite interaction between NAGK, dynein and Golgi. Finally, the ectopic introduction of a small peptide derived from the C-terminal amino acids 74–96 of DYNLRB1 resulted in the stunting of hippocampal neuron dendrites in culture. Our data indicate that the NAGK-dynein-Golgi tripartite interaction at dendritic branch points functions to regulate dendritic growth and/or branching. PMID:26272270

  5. Functional analysis of the interaction between the small GTP binding protein Cdc42 and the Ste20 protein kinase in yeast.

    PubMed Central

    Peter, M; Neiman, A M; Park, H O; van Lohuizen, M; Herskowitz, I

    1996-01-01

    STE20 encodes a protein kinase related to mammalian p65Pak which functions in several signal transduction pathways in yeast, including those involved in pseudohyphal and invasive growth, as well as mating. In addition, Ste20 plays an essential role in cells lacking Cla4, a kinase with significant homology to Ste20. It is not clear how the activity of Ste20 is regulated in response to these different signals in vivo, but it has been demonstrated recently that binding of the small GTP binding protein Cdc42 is able to activate Ste20 in vitro. Here we show that Ste20 functionally interacts with Cdc42 in a GTP-dependent manner in vivo: Ste20 mutants that can no longer bind Cdc42 were unable to restore growth of ste20 cla4 mutant cells. They were also defective for pseudohyphal growth and agar invasion, and displayed reduced mating efficiency when mated with themselves. Surprisingly, however, the kinase activity of such Ste20 mutants was normal when assayed in vitro. Furthermore, these alleles were able to fully activate the MAP kinase pathway triggered by mating pheromones in vivo, suggesting that binding of Cdc42 and Ste20 was not required to activate Ste20. Wild-type Ste20 protein was visualized as a crescent at emerging buds during vegetative growth and at shmoo tips in cells arrested with alpha-factor. In contrast, a Ste20 mutant protein unable to bind Cdc42 was found diffusely throughout the cytoplasm, suggesting that Cdc42 is required to localize Ste20 properly in vivo. Images PMID:9003780

  6. Evidence for aggregation of protein kinase CK2 in the cell: a novel strategy for studying CK2 holoenzyme interaction by BRET(2).

    PubMed

    Hübner, Gerda M; Larsen, Jane Nøhr; Guerra, Barbara; Niefind, Karsten; Vrecl, Milka; Issinger, Olaf-Georg

    2014-12-01

    Protein kinase CK2 is a ubiquitous pro-survival kinase whose substrate targets are involved in various cellular processes. Crystal structure analysis confirmed constitutive activity of the kinase, yet CK2 activity regulation in the cell is still obscure. In-vitro studies suggest autoinhibitory aggregation of the hetero-tetrameric CK2 holoenzyme as a basis for CK2 regulation. In this study, we applied bioluminescent resonance energy transfer (BRET) technology to investigate CK2 holoenzyme aggregation in living cells. We designed a BRET(2) pair consisting of the fusion proteins CK2α-Rluc8 and CK2α-GFP(2). This BRET(2) sensor reported specific interaction of CK2 holoenzyme complexes. Furthermore, the BRET(2) sensor was applied to study modulators of CK2 aggregation. We found that CK2 aggregation is not static and can be influenced by the CK2-binding protein alpha subunit of the heterotrimeric G-protein that stimulates adenylyl cyclase (Gαs) and the polycationic compound polylysine. Gαs, but not the CK2 substrate β-arrestin2, decreased the BRET(2) signal by up to 50%. Likewise polylysine, but not the CK2 inhibitor DRB, decreased the signal in a dose-dependent manner up to 50%. For the first time, we present direct experimental evidence for CK2 holoenzyme aggregates in the cell. Our data suggest that CK2 activity may be controlled by holoenzyme aggregation, to our knowledge a novel mechanism for protein kinase regulation. Moreover, the BRET(2) sensor used in our study is a novel tool for studying CK2 regulation by aggregation and pharmacological screening for novel allosteric CK2 effectors.

  7. The Abi-domain Protein Abx1 Interacts with the CovS Histidine Kinase to Control Virulence Gene Expression in Group B Streptococcus

    PubMed Central

    Firon, Arnaud; Tazi, Asmaa; Da Cunha, Violette; Brinster, Sophie; Sauvage, Elisabeth; Dramsi, Shaynoor; Golenbock, Douglas T.; Glaser, Philippe; Poyart, Claire; Trieu-Cuot, Patrick

    2013-01-01

    Group B Streptococcus (GBS), a common commensal of the female genital tract, is the leading cause of invasive infections in neonates. Expression of major GBS virulence factors, such as the hemolysin operon cyl, is regulated directly at the transcriptional level by the CovSR two-component system. Using a random genetic approach, we identified a multi-spanning transmembrane protein, Abx1, essential for the production of the GBS hemolysin. Despite its similarity to eukaryotic CaaX proteases, the Abx1 function is not involved in a post-translational modification of the GBS hemolysin. Instead, we demonstrate that Abx1 regulates transcription of several virulence genes, including those comprising the hemolysin operon, by a CovSR-dependent mechanism. By combining genetic analyses, transcriptome profiling, and site-directed mutagenesis, we showed that Abx1 is a regulator of the histidine kinase CovS. Overexpression of Abx1 is sufficient to activate virulence gene expression through CovS, overcoming the need for an additional signal. Conversely, the absence of Abx1 has the opposite effect on virulence gene expression consistent with CovS locked in a kinase-competent state. Using a bacterial two-hybrid system, direct interaction between Abx1 and CovS was mapped specifically to CovS domains involved in signal processing. We demonstrate that the CovSR two-component system is the core of a signaling pathway integrating the regulation of CovS by Abx1 in addition to the regulation of CovR by the serine/threonine kinase Stk1. In conclusion, our study reports a regulatory function for Abx1, a member of a large protein family with a characteristic Abi-domain, which forms a signaling complex with the histidine kinase CovS in GBS. PMID:23436996

  8. Biochemical and molecular analysis of a transmembrane protein kinase from Arabidopsis thaliana. Progress report, January 1993

    SciTech Connect

    Bleecker, A.B.

    1993-06-01

    We have isolated genomic and cDNA clones encoding a novel receptor-like protein kinase from the higher plant Arabidopsis thaliana. This kinase is being studied by combining biochemical, molecular, and genetic approaches. Domain-specific antibodies immunodecorate a polypeptide with a molecular mass of 120,000 daltons in extracts of Arabidopsis, where it has been found in all portions of the plant examined including root, stem, leaf, flower, and silique. Cytochemical analysis and initial studies using the kinase promoter with the GUS reporter gene system also indicate that the kinase is present throughout the plant. The kinase is glycosylated, like animal receptor kinases, and has been partially purified from Arabidopsis by using lectin columns. The kinase has been expressed in E coli, purified, and found to autophosphorylate on serine and threonine residues, but not on tyrosine residues. As such, it belongs to the small family of receptor-like kinases with serine/threonine specificity. Transgenic plants are now being produced that either overexpress or carry altered forms of the protein kinase gene. These experiments will help determine the natural role the kinase plays in a pathway of signal transduction.

  9. Biochemical and molecular analysis of a transmembrane protein kinase from Arabidopsis thaliana

    SciTech Connect

    Bleecker, A.B.

    1993-01-01

    We have isolated genomic and cDNA clones encoding a novel receptor-like protein kinase from the higher plant Arabidopsis thaliana. This kinase is being studied by combining biochemical, molecular, and genetic approaches. Domain-specific antibodies immunodecorate a polypeptide with a molecular mass of 120,000 daltons in extracts of Arabidopsis, where it has been found in all portions of the plant examined including root, stem, leaf, flower, and silique. Cytochemical analysis and initial studies using the kinase promoter with the GUS reporter gene system also indicate that the kinase is present throughout the plant. The kinase is glycosylated, like animal receptor kinases, and has been partially purified from Arabidopsis by using lectin columns. The kinase has been expressed in E coli, purified, and found to autophosphorylate on serine and threonine residues, but not on tyrosine residues. As such, it belongs to the small family of receptor-like kinases with serine/threonine specificity. Transgenic plants are now being produced that either overexpress or carry altered forms of the protein kinase gene. These experiments will help determine the natural role the kinase plays in a pathway of signal transduction.

  10. 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 Responses.

    PubMed

    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

    2015-09-01

    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.

  11. 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 Responses.

    PubMed

    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

    2015-09-01

    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

  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. Biochemical and Spatial Coincidence in the Provisional Ser/Thr Protein Kinase Interaction Network of Mycobacterium tuberculosis*♦

    PubMed Central

    Baer, Christina E.; Iavarone, Anthony T.; Alber, Tom; Sassetti, Christopher M.

    2014-01-01

    Many Gram-positive bacteria coordinate cellular processes by signaling through Ser/Thr protein kinases (STPKs), but the architecture of these phosphosignaling cascades is unknown. To investigate the network structure of a prokaryotic STPK system, we comprehensively explored the pattern of signal transduction in the Mycobacterium tuberculosis Ser/Thr kinome. Autophosphorylation is the dominant mode of STPK activation, but the 11 M. tuberculosis STPKs also show a specific pattern of efficient cross-phosphorylation in vitro. The biochemical specificity intrinsic to each kinase domain was used to map the provisional signaling network, revealing a three-layer architecture that includes master regulators, signal transducers, and terminal substrates. Fluorescence microscopy revealed that the STPKs are specifically localized in the cell. Master STPKs are concentrated at the same subcellular sites as their substrates, providing additional support for the biochemically defined network. Together, these studies imply a branched functional architecture of the M. tuberculosis Ser/Thr kinome that could enable horizontal signal spreading. This systems-level approach provides a biochemical and spatial framework for understanding Ser/Thr phospho-signaling in M. tuberculosis, which differs fundamentally from previously defined linear histidine kinase cascades. PMID:24928517

  14. A novel immuno-competitive capture mass spectrometry strategy for protein-protein interaction profiling reveals that LATS kinases regulate HCV replication through NS5A phosphorylation.

    PubMed

    Meistermann, Hélène; Gao, Junjun; Golling, Sabrina; Lamerz, Jens; Le Pogam, Sophie; Tzouros, Manuel; Sankabathula, Sailaja; Gruenbaum, Lore; Nájera, Isabel; Langen, Hanno; Klumpp, Klaus; Augustin, Angélique

    2014-11-01

    Mapping protein-protein interactions is essential to fully characterize the biological function of a protein and improve our understanding of diseases. Affinity purification coupled to mass spectrometry (AP-MS) using selective antibodies against a target protein has been commonly applied to study protein complexes. However, one major limitation is a lack of specificity as a substantial part of the proposed binders is due to nonspecific interactions. Here, we describe an innovative immuno-competitive capture mass spectrometry (ICC-MS) method to allow systematic investigation of protein-protein interactions. ICC-MS markedly increases the specificity of classical immunoprecipitation (IP) by introducing a competition step between free and capturing antibody prior to IP. Instead of comparing only one experimental sample with a control, the methodology generates a 12-concentration antibody competition profile. Label-free quantitation followed by a robust statistical analysis of the data is then used to extract the cellular interactome of a protein of interest and to filter out background proteins. We applied this new approach to specifically map the interactome of hepatitis C virus (HCV) nonstructural protein 5A (NS5A) in a cellular HCV replication system and uncovered eight new NS5A-interacting protein candidates along with two previously validated binding partners. Follow-up biological validation experiments revealed that large tumor suppressor homolog 1 and 2 (LATS1 and LATS2, respectively), two closely related human protein kinases, are novel host kinases responsible for NS5A phosphorylation at a highly conserved position required for optimal HCV genome replication. These results are the first illustration of the value of ICC-MS for the analysis of endogenous protein complexes to identify biologically relevant protein-protein interactions with high specificity.

  15. Role of the SH3-ligand domain of simian immunodeficiency virus Nef in interaction with Nef-associated kinase and simian AIDS in rhesus macaques.

    PubMed

    Khan, I H; Sawai, E T; Antonio, E; Weber, C J; Mandell, C P; Montbriand, P; Luciw, P A

    1998-07-01

    The nef gene of the human and simian immunodeficiency viruses (HIV and SIV) is dispensable for viral replication in T-cell lines; however, it is essential for high virus loads and progression to simian AIDS (SAIDS) in SIV-infected adult rhesus macaques. Nef proteins from HIV type 1 (HIV-1), HIV-2, and SIV contain a proline-Xaa-Xaa-proline (PxxP) motif. The region of Nef with this motif is similar to the Src homology region 3 (SH3) ligand domain found in many cell signaling proteins. In virus-infected lymphoid cells, Nef interacts with a cellular serine/threonine kinase, designated Nef-associated kinase (NAK). In this study, analysis of viral clones containing point mutations in the nef gene of the pathogenic clone SIVmac239 revealed that several strictly conserved residues in the PxxP region were essential for Nef-NAK interaction. The results of this analysis of Nef mutations in in vitro kinase assays indicated that the PxxP region in SIV Nef was strikingly similar to the consensus sequence for SH3 ligand domains possessing the minus orientation. To test the significance of the PxxP motif of Nef for viral pathogenesis, each proline was mutated to an alanine to produce the viral clone SIVmac239-P104A/P107A. This clone, expressing Nef that does not associate with NAK, was inoculated into seven juvenile rhesus macaques. In vitro kinase assays were performed on virus recovered from each animal; the ability of Nef to associate with NAK was restored in five of these animals as early as 8 weeks after infection. Analysis of nef genes from these viruses revealed patterns of genotypic reversion in the mutated PxxP motif. These revertant genotypes, which included a second-site suppressor mutation, restored the ability of Nef to interact with NAK. Additionally, the proportion of revertant viruses increased progressively during the course of infection in these animals, and two of these animals developed fatal SAIDS. Taken together, these results demonstrated that in vivo

  16. 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

  17. A contactin-receptor-like protein tyrosine phosphatase beta complex mediates adhesive communication between astroglial cells and gonadotrophin-releasing hormone neurones.

    PubMed

    Parent, A-S; Mungenast, A E; Lomniczi, A; Sandau, U S; Peles, E; Bosch, M A; Rønnekleiv, O K; Ojeda, S R

    2007-11-01

    Although it is well established that gonadotrophin-releasing hormone (GnRH) neurones and astrocytes maintain an intimate contact throughout development and adult life, the cell-surface molecules that may contribute to this adhesiveness remain largely unknown. In the peripheral nervous system, the glycosylphosphatidyl inositol (GPI)-anchored protein contactin is a cell-surface neuronal protein required for axonal-glial adhesiveness. A glial transmembrane protein recognised by neuronal contactin is receptor-like protein tyrosine phosphatase beta (RPTP beta), a phosphatase with structural similarities to cell adhesion molecules. In the present study, we show that contactin, and its preferred in cis partner Caspr1, are expressed in GnRH neurones. We also show that the RPTP beta mRNA predominantly expressed in hypothalamic astrocytes encodes an RPTP beta isoform (short RPTP beta) that uses its carbonic anhydrase (CAH) extracellular subdomain to interact with neuronal contactin. Immunoreactive contactin is most abundant in GnRH nerve terminals projecting to both the organum vasculosum of the lamina terminalis and median eminence, implying GnRH axons as an important site of contactin-dependent cell adhesiveness. GT1-7 immortalised GnRH neurones adhere to the CAH domain of RPTPbeta, and this adhesiveness is blocked when contactin GPI anchoring is disrupted or contactin binding capacity is immunoneutralised, suggesting that astrocytic RPTP beta interacts with neuronal contactin to mediate glial-GnRH neurone adhesiveness. Because the abundance of short RPTP beta mRNA increases in the female mouse hypothalamus (but not in the cerebral cortex) before puberty, it appears that an increased interaction between GnRH axons and astrocytes mediated by RPTP beta-contactin is a dynamic mechanism of neurone-glia communication during female sexual development. PMID:17927663

  18. Molecular Basis of the Interaction of the Human Protein Tyrosine Phosphatase Non-receptor Type 4 (PTPN4) with the Mitogen-activated Protein Kinase p38γ.

    PubMed

    Maisonneuve, Pierre; Caillet-Saguy, Célia; Vaney, Marie-Christine; Bibi-Zainab, Edoo; Sawyer, Kristi; Raynal, Bertrand; Haouz, Ahmed; Delepierre, Muriel; Lafon, Monique; Cordier, Florence; Wolff, Nicolas

    2016-08-01

    The human protein tyrosine phosphatase non-receptor type 4 (PTPN4) prevents cell death induction in neuroblastoma and glioblastoma cell lines in a PDZ·PDZ binding motifs-dependent manner, but the cellular partners of PTPN4 involved in cell protection are unknown. Here, we described the mitogen-activated protein kinase p38γ as a cellular partner of PTPN4. The main contribution to the p38γ·PTPN4 complex formation is the tight interaction between the C terminus of p38γ and the PDZ domain of PTPN4. We solved the crystal structure of the PDZ domain of PTPN4 bound to the p38γ C terminus. We identified the molecular basis of recognition of the C-terminal sequence of p38γ that displays the highest affinity among all endogenous partners of PTPN4. We showed that the p38γ C terminus is also an efficient inducer of cell death after its intracellular delivery. In addition to recruiting the kinase, the binding of the C-terminal sequence of p38γ to PTPN4 abolishes the catalytic autoinhibition of PTPN4 and thus activates the phosphatase, which can efficiently dephosphorylate the activation loop of p38γ. We presume that the p38γ·PTPN4 interaction promotes cellular signaling, preventing cell death induction.

  19. Focal Adhesion Kinase Directly Interacts with TSC2 Through Its FAT Domain and Regulates Cell Proliferation in Cashmere Goat Fetal Fibroblasts.

    PubMed

    Zheng, Xu; Bao, Wenlei; Yang, Jiaofu; Zhang, Tao; Sun, Dongsheng; Liang, Yan; Li, Shuyu; Wang, Yanfeng; Feng, Xue; Hao, Huifang; Wang, Zhigang

    2016-09-01

    Focal adhesion kinase (FAK) is a cytoplasmic nonreceptor tyrosine kinase that senses a variety of extracellular signals, such as growth factors and integrins, to control the process of cell proliferation and metabolism. We cloned three goat FAK transcript variants (KM655805, KM658268, and KM658269) that encode 1052, 1006, and 962 amino-acid residue proteins. Bioinformatics analysis indicated that the putative FAK protein contains an FERM domain, a PTK domain, two Proline-rich regions, and a focal adhesion-targeting (FAT) domain. All the three transcript variants of FAK were detected in seven different goat tissues, and variant 1 had the most accumulation whereas variant 2 and variant 3 had lower accumulation. Treatment of goat fetal fibroblasts (GFbs) with a specific FAK inhibitor, TAE226, inhibited cell proliferation (p < 0.05) and induced damage to the cell morphology in a dose- and time-dependent manner. Further research demonstrated that FAK directly interacted with TSC2 (Tuberous sclerosis 2) tuberin domain through its C-terminus, which contains the complete FAT domain. In conclusion, our results indicated that FAK may be widely expressed in Cashmere goat tissues and its products participate in the mammalian target of rapamycin signaling pathway and cell proliferation through a direct interaction with TSC2 in GFBs. PMID:27380318

  20. Allosteric modulation of AURKA kinase activity by a small-molecule inhibitor of its protein-protein interaction with TPX2

    PubMed Central

    Janeček, Matej; Rossmann, Maxim; Sharma, Pooja; Emery, Amy; Huggins, David J.; Stockwell, Simon R.; Stokes, Jamie E.; Tan, Yaw S.; Almeida, Estrella Guarino; Hardwick, Bryn; Narvaez, Ana J.; Hyvönen, Marko; Spring, David R.; McKenzie, Grahame J.; Venkitaraman, Ashok R.

    2016-01-01

    The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the ‘Y pocket’) that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases. PMID:27339427

  1. Translocation of glycogen synthase kinase-3β (GSK-3β), a trigger of permeability transition, is kinase activity-dependent and mediated by interaction with voltage-dependent anion channel 2 (VDAC2).

    PubMed

    Tanno, Masaya; Kuno, Atsushi; Ishikawa, Satoko; Miki, Takayuki; Kouzu, Hidemichi; Yano, Toshiyuki; Murase, Hiromichi; Tobisawa, Toshiyuki; Ogasawara, Makoto; Horio, Yoshiyuki; Miura, Tetsuji

    2014-10-17

    Glycogen synthase kinase-3β (GSK-3β) is a major positive regulator of the mitochondrial permeability transition pore (mPTP), a principle trigger of cell death, under the condition of oxidative stress. However, the mechanism by which cytosolic GSK-3β translocates to mitochondria, promoting mPTP opening, remains unclear. Here we addressed this issue by analyses of the effect of site-directed mutations in GSK-3β on mitochondrial translocation and protein/protein interactions upon oxidative stress. H9c2 cardiomyoblasts were transfected with GFP-tagged GSK-3β (WT), a mutant GSK-3β insensitive to inhibitory phosphorylation (S9A), or kinase-deficient GSK-3β (K85R). Time lapse observation revealed that WT and S9A translocated from the cytosol to the mitochondria more promptly than did K85R after exposure to oxidative stress. H2O2 increased the density of nine spots on two-dimensional gel electrophoresis of anti-GSK-3β-immunoprecipitates by more than 3-fold. MALDI-TOF/MS analysis revealed that one of the spots contained voltage-dependent anion channel 2 (VDAC2). Knockdown of VDAC2, but not VDAC1 or VDAC3, by siRNA attenuated both the mitochondrial translocation of GSK-3β and mPTP opening under stress conditions. The mitochondrial translocation of GSK-3β was attenuated also when Lys-15, but not Arg-4 or Arg-6, in the N-terminal domain of GSK-3β was replaced with alanine. The oxidative stress-induced mitochondrial translocation of GSK-3β was associated with an increase in cell death, which was suppressed by lithium chloride (LiCl), a GSK-3β inhibitor. These results demonstrate that GSK-3β translocates from the cytosol to mitochondria in a kinase activity- and VDAC2-dependent manner in which an N-terminal domain of GSK-3β may function as a mitochondrial targeting sequence.

  2. Translocation of Glycogen Synthase Kinase-3β (GSK-3β), a Trigger of Permeability Transition, Is Kinase Activity-dependent and Mediated by Interaction with Voltage-dependent Anion Channel 2 (VDAC2)*

    PubMed Central

    Tanno, Masaya; Kuno, Atsushi; Ishikawa, Satoko; Miki, Takayuki; Kouzu, Hidemichi; Yano, Toshiyuki; Murase, Hiromichi; Tobisawa, Toshiyuki; Ogasawara, Makoto; Horio, Yoshiyuki; Miura, Tetsuji

    2014-01-01

    Glycogen synthase kinase-3β (GSK-3β) is a major positive regulator of the mitochondrial permeability transition pore (mPTP), a principle trigger of cell death, under the condition of oxidative stress. However, the mechanism by which cytosolic GSK-3β translocates to mitochondria, promoting mPTP opening, remains unclear. Here we addressed this issue by analyses of the effect of site-directed mutations in GSK-3β on mitochondrial translocation and protein/protein interactions upon oxidative stress. H9c2 cardiomyoblasts were transfected with GFP-tagged GSK-3β (WT), a mutant GSK-3β insensitive to inhibitory phosphorylation (S9A), or kinase-deficient GSK-3β (K85R). Time lapse observation revealed that WT and S9A translocated from the cytosol to the mitochondria more promptly than did K85R after exposure to oxidative stress. H2O2 increased the density of nine spots on two-dimensional gel electrophoresis of anti-GSK-3β-immunoprecipitates by more than 3-fold. MALDI-TOF/MS analysis revealed that one of the spots contained voltage-dependent anion channel 2 (VDAC2). Knockdown of VDAC2, but not VDAC1 or VDAC3, by siRNA attenuated both the mitochondrial translocation of GSK-3β and mPTP opening under stress conditions. The mitochondrial translocation of GSK-3β was attenuated also when Lys-15, but not Arg-4 or Arg-6, in the N-terminal domain of GSK-3β was replaced with alanine. The oxidative stress-induced mitochondrial translocation of GSK-3β was associated with an increase in cell death, which was suppressed by lithium chloride (LiCl), a GSK-3β inhibitor. These results demonstrate that GSK-3β translocates from the cytosol to mitochondria in a kinase activity- and VDAC2-dependent manner in which an N-terminal domain of GSK-3β may function as a mitochondrial targeting sequence. PMID:25187518

  3. Putative Bioactive Motif of Tritrpticin Revealed by an Antibody with Biological Receptor-Like Properties

    PubMed Central

    Sharma, Raghava; Lomash, Suvendu; Salunke, Dinakar M.

    2013-01-01

    Antimicrobial peptides represent one of the most promising future strategies for combating infections and microbial drug resistance. Tritrpticin is a 13mer tryptophan-rich cationic antimicrobial peptide with a broad spectrum of activity whose application in antimicrobial therapy has been hampered by ambiguity about its biological target and consequently the molecular interactions necessary for its antimicrobial activity. The present study provides clues about the mechanism of action of tritripticin by using a unique monoclonal antibody (mAb) as a ‘physiological’ structural scaffold. A pool of mAbs were generated against tritrpticin and based on its high affinity and ability to bind tritrpticin analogs, mAb 6C6D7 was selected and characterized further. In a screening of phage displayed random peptides, this antibody was able to identify a novel antimicrobial peptide with low sequence homology to tritrpticin, suggesting that the mAb possessed the physico-chemical characteristics mimicking the natural receptor. Subsequently, thermodynamics and molecular modeling identified a core group of hydrophobic residues in tritrpticin arranged in a distorted’s’ shaped conformation as critical for antibody binding. Comparison of the mAb induced conformation with the micelle bound structure of tritrpticin reveals how a common motif may be able to interact with multiple classes of biomolecules thus extending the target range of this innate immune peptide. Based on the concurrence between thermodynamic and structural data our results reveal a template that can be used to design novel antimicrobial pharmacophores while simultaneously demonstrating at a more fundamental level the potential of mAbs to act as receptor surrogates. PMID:24086578

  4. CSR, a scavenger receptor-like protein with a protective role against cellular damage causedby UV irradiation and oxidative stress.

    PubMed

    Han, H J; Tokino, T; Nakamura, Y

    1998-06-01

    Oxidative stress is a pathogenic condition that causes cellular damage and, in a normally functioning cell, several transcription factors respond to this threat by modulating expression of genes whose products ameliorate the altered redox status in some way. We have isolated a novel macrophage scavenger receptor-like gene, CSR (cellular stress response), whose transcription in normal fibroblasts was significantly elevated by exposure to UV radiation or hydrogen peroxide, and pre-treatment with antioxidants prevented induction of CSR . Under conditions of oxidative stress, reactive oxygen species were significantly depleted in CSR -overexpressing cells, indicating that the CSR product protects cells by scavenging oxidative molecules or harmful products of oxidation. Further investigations into the regulation and function of CSR should open a way to understanding the cellular response and the pathogenic processes caused by oxidative stress.

  5. White Spot Syndrome Virus Protein Kinase 1 Defeats the Host Cell's Iron-Withholding Defense Mechanism by Interacting with Host Ferritin

    PubMed Central

    Lin, Shin-Jen; Lee, Der-Yen; Wang, Hao-Ching; Kang, Shih-Ting; Hwang, Pung-Pung; Kou, Guang-Hsiung; Huang, Ming-Fen

    2014-01-01

    ABSTRACT Iron is an essential nutrient for nearly all living organisms, including both hosts and invaders. Proteins such as ferritin regulate the iron levels in a cell, and in the event of a pathogenic invasion, the host can use an iron-withholding mechanism to restrict the availability of this essential nutrient to the invading pathogens. However, pathogens use various strategies to overcome this host defense. In this study, we demonstrated that white spot syndrome virus (WSSV) protein kinase 1 (PK1) interacted with shrimp ferritin in the yeast two-hybrid system. A pulldown assay and 27-MHz quartz crystal microbalance (QCM) analysis confirmed the interaction between PK1 and both ferritin and apoferritin. PK1 did not promote the release of iron ions from ferritin, but it prevented apoferritin from binding ferrous ions. When PK1 was overexpressed in Sf9 cells, the cellular labile iron pool (LIP) levels were elevated significantly. Immunoprecipitation and atomic absorption spectrophotometry (AAS) further showed that the number of iron ions bound by ferritin decreased significantly at 24 h post-WSSV infection. Taken together, these results suggest that PK1 prevents apoferritin from iron loading, and thus stabilizes the cellular LIP levels, and that WSSV uses this novel mechanism to counteract the host cell's iron-withholding defense mechanism. IMPORTANCE We show here that white spot syndrome virus (WSSV) ensures the availability of iron by using a previously unreported mechanism to defeat the host cell's iron-withholding defense mechanism. This defense is often implemented by ferritin, which can bind up to 4,500 iron atoms and acts to sequester free iron within the cell. WSSV's novel counterstrategy is mediated by a direct protein-protein interaction between viral protein kinase 1 (PK1) and host ferritin. PK1 interacts with both ferritin and apoferritin, suppresses apoferritin's ability to sequester free iron ions, and maintains the intracellular labile iron pool (LIP

  6. Sak1 kinase interacts with Pso2 nuclease in response to DNA damage induced by interstrand crosslink-inducing agents in Saccharomyces cerevisiae.

    PubMed

    Munari, Fernanda M; Revers, Luis F; Cardone, Jacqueline M; Immich, Bruna F; Moura, Dinara J; Guecheva, Temenouga N; Bonatto, Diego; Laurino, Jomar P; Saffi, Jenifer; Brendel, Martin; Henriques, João A P

    2014-01-01

    By isolating putative binding partners through the two-hybrid system (THS) we further extended the characterization of the specific interstrand cross-link (ICL) repair gene PSO2 of Saccharomyces cerevisiae. Nine fusion protein products were isolated for Pso2p using THS, among them the Sak1 kinase, which interacted with the C-terminal β-CASP domain of Pso2p. Comparison of mutagen-sensitivity phenotypes of pso2Δ, sak1Δ and pso2Δsak1Δ disruptants revealed that SAK1 is necessary for complete WT-like repair. The epistatic interaction of both mutant alleles suggests that Sak1p and Pso2p act in the same pathway of controlling sensitivity to DNA-damaging agents. We also observed that Pso2p is phosphorylated by Sak1 kinase in vitro and co-immunoprecipitates with Sak1p after 8-MOP+UVA treatment. Survival data after treatment of pso2Δ, yku70Δ and yku70Δpso2Δ with nitrogen mustard, PSO2 and SAK1 with YKU70 or DNL4 single-, double- and triple mutants with 8-MOP+UVA indicated that ICL repair is independent of YKu70p and DNL4p in S. cerevisiae. Furthermore, a non-epistatic interaction was observed between MRE11, PSO2 and SAK1 genes after ICL induction, indicating that their encoded proteins act on the same substrate, but in distinct repair pathways. In contrast, an epistatic interaction was observed for PSO2 and RAD52, PSO2 and RAD50, PSO2 and XRS2 genes in 8-MOP+UVA treated exponentially growing cells. PMID:24362320

  7. Glucose regulates protein kinase CK2 in pancreatic β-cells and its interaction with PDX-1.

    PubMed

    Welker, Sabrina; Götz, Claudia; Servas, Christina; Laschke, Matthias W; Menger, Michael D; Montenarh, Mathias

    2013-12-01

    The pancreatic duodenal homeodomain transcription factor PDX-1 plays a pivotal role in the development of the pancreas and the maintenance of glucose homeostasis by pancreatic β-cells. Recently, we found that the highly conserved, ubiquitously expressed tetrameric Ser/Thr protein kinase CK2, which is formed by two catalytic subunits (α and/or α') and two non-catalytic subunits (β), phosphorylates PDX-1. So far, only little is known about CK2 in pancreatic β-cells and how this enzyme is regulated in these cells. In the present study, we found that (i) CK2 binds to PDX-1, (ii) the binding between CK2 and PDX-1 is regulated by glucose, (iii) glucose modulates the subcellular localization of PDX-1 and CK2 and (iv) the kinase activity is also regulated by glucose. Our novel data indicate that CK2 is a co-factor of PDX-1 in response to glucose in pancreatic β-cells.

  8. Mutations in CIT, encoding citron rho-interacting serine/threonine kinase, cause severe primary microcephaly in humans.

    PubMed

    Shaheen, Ranad; Hashem, Amal; Abdel-Salam, Ghada M H; Al-Fadhli, Fatima; Ewida, Nour; Alkuraya, Fowzan S

    2016-10-01

    Primary microcephaly is a clinical phenotype in which the head circumference is significantly reduced at birth due to abnormal brain development, primarily at the cortical level. Despite the marked genetic heterogeneity, most primary microcephaly-linked genes converge on mitosis regulation. Two consanguineous families segregating the phenotype of severe primary microcephaly, spasticity and failure to thrive had overlapping autozygomes in which exome sequencing identified homozygous splicing variants in CIT that segregate with the phenotype within each family. CIT encodes citron, an effector of the Rho signaling that is required for cytokinesis specifically in proliferating neuroprogenitors, as well as for postnatal brain development. In agreement with the critical role assigned to the kinase domain in effecting these biological roles, we show that both splicing variants predict variable disruption of this domain. The striking phenotypic overlap between CIT-mutated individuals and the knockout mice and rats that are specifically deficient in the kinase domain supports the proposed causal link between CIT mutation and primary microcephaly in humans. PMID:27503289

  9. Diacylglycerol kinase α promotes 3D cancer cell growth and limits drug sensitivity through functional interaction with Src.

    PubMed

    Torres-Ayuso, Pedro; Daza-Martín, Manuel; Martín-Pérez, Jorge; Ávila-Flores, Antonia; Mérida, Isabel

    2014-10-30

    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.

  10. High-throughput fluorescence polarization assay to identify inhibitors of Cbl(TKB)-protein tyrosine kinase interactions.

    PubMed

    Kumar, Eric A; Charvet, Casey D; Lokesh, G L; Natarajan, Amarnath

    2011-04-15

    The casitas B-lineage lymphoma (Cbl) proteins play an important role in regulating signal transduction pathways by functioning as E3 ubiquitin ligases. The Cbl proteins contain a conserved tyrosine kinase binding (TKB) domain that binds more than a dozen proteins, including protein tyrosine kinases (PTKs), in a phosphorylation-dependent manner. The cell surface expression levels of the PTKs are regulated by Cbl-mediated ubiquitination, internalization, and degradation. Dysfunction in this signaling cascade has resulted in prolonged activation of the PTKs and, therefore, has been implicated in inflammatory diseases and various cancers. Due to this negative regulatory function, Cbl has been largely ignored as a therapeutic target. However, recent studies, such as the identification of (i) gain of function c-Cbl mutations in subsets of myeloid cancer and (ii) c-Cbl as a prostate basal cell marker that correlates with poor clinical outcome, suggest otherwise. Here we report the development of a competitive high-throughput fluorescence polarization assay in a 384-well format to identify inhibitors of Cbl(TKB). The high-throughput screen readiness of the assay was demonstrated by screening the Prestwick Chemical Library. PMID:21129358

  11. 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

  12. 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.

  13. 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.

  14. 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. PMID:26851488

  15. 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.

  16. Elucidation of the interaction loci of the human pyruvate dehydrogenase complex E2·E3BP core with pyruvate dehydrogenase kinase 1 and kinase 2 by H/D exchange mass spectrometry and nuclear magnetic resonance.

    PubMed

    Wang, Junjie; Kumaran, Sowmini; Zhou, Jieyu; Nemeria, Natalia S; Tao, Hu; Kakalis, Lazaros; Park, Yun-Hee; Birkaya, Barbara; Patel, Mulchand S; Jordan, Frank

    2015-01-13

    The human pyruvate dehydrogenase complex (PDC) comprises three principal catalytic components for its mission: E1, E2, and E3. The core of the complex is a strong subcomplex between E2 and an E3-binding protein (E3BP). The PDC is subject to regulation at E1 by serine phosphorylation by four kinases (PDK1-4), an inactivation reversed by the action of two phosphatases (PDP1 and -2). We report H/D exchange mass spectrometric (HDX-MS) and nuclear magnetic resonance (NMR) studies in the first attempt to define the interaction loci between PDK1 and PDK2 with the intact E2·E3BP core and their C-terminally truncated proteins. While the three lipoyl domains (L1 and L2 on E2 and L3 on E3BP) lend themselves to NMR studies and determination of interaction maps with PDK1 and PDK2 at the individual residue level, HDX-MS allowed studies of interaction loci on both partners in the complexes, PDKs, and other regions of the E2·E3BP core, as well, at the peptide level. HDX-MS suggested that the intact E2·E3BP core enhances the binding specificity of L2 for PDK2 over PDK1, while NMR studies detected lipoyl domain residues unique to interaction with PDK1 and PDK2. The E2·E3BP core induced more changes on PDKs than any C-terminally truncated protein, with clear evidence of greater plasticity of PDK1 than of PDK2. The effect of L1L2S paralleled HDX-MS results obtained with the intact E2·E3BP core; hence, L1L2S is an excellent candidate with which to define interaction loci with these two PDKs. Surprisingly, L3S' induced moderate interaction with both PDKs according to both methods. PMID:25436986

  17. 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

  18. The tyrosine 343 residue of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) is important for its interaction with SHP1, a cytoplasmic tyrosine phosphatase with tumor suppressor functions.

    PubMed

    Hegazy, Samar A; Wang, Peng; Anand, Mona; Ingham, Robert J; Gelebart, Pascal; Lai, Raymond

    2010-06-25

    The cytoplasmic tyrosine phosphatase SHP1 has been shown to inhibit the oncogenic fusion protein nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), and loss of SHP1 contributes to NPM-ALK-mediated tumorigenesis. In this study, we aimed to further understand how SHP1 interacts and regulates NPM-ALK. We employed an in vitro model in which GP293 cells were transfected with various combinations of NPM-ALK (or mutants) and SHP1 (or mutants) expression vectors. We found that SHP1 co-immunoprecipitated with NPM-ALK, but not the enzymatically inactive NPM-ALK(K210R) mutant, or the mutant in which all three functionally important tyrosine residues (namely, Tyr(338), Tyr(342), and Tyr(343)) in the kinase activation loop (KAL) of ALK were mutated. Interestingly, whereas mutation of Tyr(338) or Tyr(342) did not result in any substantial change in the NPM-ALK/SHP1 binding (assessed by co-immunoprecipitation), mutation of Tyr(343) abrogated this interaction. Furthermore, the NPM-ALK/SHP1 binding was readily detectable when each of the remaining 8 tyrosine residues known to be phosphorylated were mutated. Although the expression of SHP1 effectively reduced the level of tyrosine phosphorylation of NPM-ALK, it did not affect that of the NPM-ALK(Y343F) mutant. In soft agar clonogenic assay, SHP1 expression significantly reduced the tumorigenicity of NPM-ALK but not that of NPM-ALK(Y343F). In conclusion, we identified Tyr(343) of NPM-ALK as the crucial site for mediating the NPM-ALK/SHP1 interaction. Our results also support the notion that the tumor suppressor effects of SHP1 on NPM-ALK are dependent on its ability to bind to this oncogenic protein.

  19. Sorafenib/regorafenib and phosphatidyl inositol 3 kinase/thymoma viral proto-oncogene inhibition interact to kill tumor cells.

    PubMed

    Sajithlal, Gangadharan B; Hamed, Hossein A; Cruickshanks, Nichola; Booth, Laurence; Tavallai, Seyedmehrad; Syed, Jahangir; Grant, Steven; Poklepovic, Andrew; Dent, Paul

    2013-10-01

    The present studies were undertaken to determine whether the multikinase inhibitors sorafenib/regorafenib cooperated with clinically relevant , phosphatidyl inositol 3 kinase (PI3K)-thymoma viral proto-oncogene (AKT) inhibitors to kill tumor cells. In liver, colorectal, lung, breast, kidney, and brain cancer cells, at clinically achievable doses, sorafenib/regorafenib and the PI3K inhibitor acetic acid (1S,4E,10R,11R,13S,14R)-[4-diallylaminomethylene-6-hydroxy-1-methoxymethyl-10,13-dimethyl-3,7,17-trioxo-1,3,4,7,10,11,12,13,14,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenanthren-11-yl ester (PX-866) cooperated in a greater than additive fashion to kill tumor cells. Cells lacking phosphatase and tensin homolog were as sensitive to the drug combination as cells expressing the protein. Similar data were obtained using the AKT inhibitors perifosine and 8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4-f] [1,6]naphthyridin-3(2H)-one hydrochloride (MK2206). PX-866 treatment abolished AKT/glycogen synthase kinase 3 (GSK3) phosphorylation, and cell killing correlated with reduced activity of AKT and mammalian target of rapamycin (mTOR). Expression of activated AKT and to a lesser extent activated mTOR reduced drug combination lethality. Expression of B-cell lymphoma-extra large or dominant negative caspase 9, but not cellular FLICE (FADD-like IL-1b-converting enzyme)-inhibitory protein short, protected cells from the drug combination. Treatment of cells with PX-866 increased protein levels of p62, lysosome-associated membrane protein 2 (LAMP2), and microtubule-associated protein light chain (LC) 3 and LC3II that correlated with a large increase in LC3-green fluorescent protein (GFP) vesicle numbers. Exposure of PX-866 treated cells to sorafenib reduced p62 and LAMP2 levels, decreased the ratio of LC3 to LC3II, and reduced LC3-GFP vesicle levels. Knockdown of Beclin1 or autophagy-related 5 suppressed drug toxicity by ∼40%. In vivo, sorafenib and PX-866 or

  20. IGF-IR tyrosine kinase interacts with NPM-ALK oncogene to induce survival of T-cell ALK+ anaplastic large-cell lymphoma cells.

    PubMed

    Shi, Ping; Lai, Raymond; Lin, Quan; Iqbal, Abid S; Young, Leah C; Kwak, Larry W; Ford, Richard J; Amin, Hesham M

    2009-07-01

    Type I insulin-like growth factor receptor (IGF-IR) tyrosine kinase plays important roles in the pathogenesis of several malignancies. Although it promotes the growth of stimulated hematopoietic cells, a direct role of IGF-IR in malignant lymphoma has not been identified. Anaplastic lymphoma kinase-positive anaplastic large-cell lymphoma (ALK(+) ALCL) is a unique type of T-cell lymphoma. Approximately 85% of ALK(+) ALCL cases harbor the translocation t(2;5)(p23;q35), which generates the chimeric oncogene NPM-ALK. In the present study, we explored a possible role of IGF-IR in ALK(+) ALCL. Our results demonstrate that IGF-IR and IGF-I are widely expressed in ALK(+) ALCL cell lines and primary tumors. Importantly, we identified novel reciprocal functional interactions between IGF-IR and NPM-ALK. Antagonism of IGF-IR decreased the viability, induced apoptosis and cell-cycle arrest, and decreased proliferation and colony formation of ALK(+) ALCL cell lines. These effects could be explained by alterations of cell survival regulatory proteins downstream of IGF-IR signaling. Our findings improve current understanding of the biology of IGF-IR and NPM-ALK and have significant therapeutic implications as they identify IGF-IR signaling as a potential therapeutic target in ALK(+) ALCL and possibly other types of malignant lymphoma.

  1. The Receiver of the Agrobacterium tumefaciens VirA Histidine Kinase Forms a Stable Interaction with VirG to Activate Virulence Gene Expression.

    PubMed

    Wise, Arlene A; Binns, Andrew N

    2015-01-01

    The plant pathogen Agrobacterium tumefaciens carries a virulence gene system that is required for the initiation of crown gall tumors on susceptible plants. Expression of the vir genes is activated by the VirA/VirG two component regulatory system. VirA is a histidine kinase which signals the presence of certain chemicals found at the site of a plant wound. The receiver domain located at its carboxyl terminus defines VirA as a hybrid histidine kinase. Here, we show that the VirA receiver interacts with the DNA-binding domain of VirG. This finding supports the hypothesis that the receiver acts as a recruiting factor for VirG. In addition, we show that removal of the VirA receiver allowed vir gene expression in response to glucose in a dose dependent manner, indicating that the receiver controls VirG activation and suggesting that the supplementary ChvE-sugar signal increases this activity. PMID:26779177

  2. The Receiver of the Agrobacterium tumefaciens VirA Histidine Kinase Forms a Stable Interaction with VirG to Activate Virulence Gene Expression

    PubMed Central

    Wise, Arlene A.; Binns, Andrew N.

    2016-01-01

    The plant pathogen Agrobacterium tumefaciens carries a virulence gene system that is required for the initiation of crown gall tumors on susceptible plants. Expression of the vir genes is activated by the VirA/VirG two component regulatory system. VirA is a histidine kinase which signals the presence of certain chemicals found at the site of a plant wound. The receiver domain located at its carboxyl terminus defines VirA as a hybrid histidine kinase. Here, we show that the VirA receiver interacts with the DNA-binding domain of VirG. This finding supports the hypothesis that the receiver acts as a recruiting factor for VirG. In addition, we show that removal of the VirA receiver allowed vir gene expression in response to glucose in a dose dependent manner, indicating that the receiver controls VirG activation and suggesting that the supplementary ChvE-sugar signal increases this activity. PMID:26779177

  3. CDC2L5, a Cdk-like kinase with RS domain, interacts with the ASF/SF2-associated protein p32 and affects splicing in vivo.

    PubMed

    Even, Yasmine; Durieux, Sandrine; Escande, Marie-Line; Lozano, Jean Claude; Peaucellier, Gérard; Weil, Dominique; Genevière, Anne-Marie

    2006-10-15

    The human CDC2L5 gene encodes a protein of unknown physiological function. This protein is closely related to the cyclin-dependent kinase (Cdks) family and contains an arginine/serine-rich (RS) domain. The Cdks were first identified as crucial regulators of cell-cycle progression, more recently they were found to be involved in transcription and mRNA processing. RS domains are mainly present in proteins regulating pre-mRNA splicing, suggesting CDC2L5 having a possible role in this process. In this study, we demonstrate that CDC2L5 is located in the nucleoplasm, at a higher concentration in speckles, the storage sites for splicing factors. Furthermore, this localization is dependent on the presence of the N-terminal sequence including the RS domain. Then, we report that CDC2L5 directly interacts with the ASF/SF2-associated protein p32, a protein involved in splicing regulation. Overexpression of CDC2L5 constructs disturbs constitutive splicing and switches alternative splice site selection in vivo. These results argue in favor of a functional role of the CDC2L5 kinase in splicing regulation.

  4. Extracellular signal-regulated kinase 2 (ERK2) phosphorylation sites and docking domain on the nuclear pore complex protein Tpr cooperatively regulate ERK2-Tpr interaction.

    PubMed

    Vomastek, Tomás; Iwanicki, Marcin P; Burack, W Richard; Tiwari, Divya; Kumar, Devanand; Parsons, J Thomas; Weber, Michael J; Nandicoori, Vinay Kumar

    2008-11-01

    Identifying direct substrates of mitogen-activated protein kinases (MAPKs) and understanding how those substrates are selected is central to understanding how these ubiquitously activated enzymes generate diverse biological responses. In previous work, we identified several new candidate substrates for the MAPK ERK2 (extracellular signal-regulated kinase 2), including the nuclear pore complex protein Tpr (translocated promoter region). In this report, we identify sites on Tpr for ERK2 phosphorylation and binding and demonstrate their functional interaction. ERK2 phosphorylation and dimerization are necessary for ERK2-Tpr binding, and this occurs through a DEF (docking site for ERK2, FXF) domain on Tpr. Surprisingly, the DEF domain and the phosphorylation sites displayed positive cooperativity to promote ERK2 binding to Tpr, in contrast to substrates where phosphorylation reduces binding. Ectopic expression or depletion of Tpr resulted in decreased movement of activated ERK2 from the cytoplasm to the nucleus, implying a role for Tpr in ERK2 translocation. Collectively, the data provide direct evidence that a component of the nuclear pore complex is a bona fide substrate of ERK2 in vivo and that activated ERK2 stably associates with this substrate after phosphorylation, where it could play a continuing role in nuclear pore function. We propose that Tpr is both a substrate and a scaffold for activated ERKs.

  5. Attenuation of DNA damage checkpoint by PBK, a novel mitotic kinase, involves protein-protein interaction with tumor suppressor p53.

    PubMed

    Nandi, Asit K; Ford, Tamara; Fleksher, Daniel; Neuman, Brian; Rapoport, Aaron P

    2007-06-22

    Pathways adopted by developing cancer cells for evasion of cellular surveillance mechanism deserve attention for therapeutic exploitation as well as for better prognosis. A novel mitotic kinase, PDZ-binding kinase or PBK, which is upregulated in a variety of neoplasms including hematological malignancies, has been the focus of our attention with a goal to understand its role in malignant conversion and to examine as a possible new therapeutic target in disparate types of cancer. Earlier, we reported that PBK expression was downregulated during macrophage differentiation of HL60 promyelocytic leukemia cells, during doxorubicin-induced growth arrest in G2/M phase and that PBK was regulated by cell cycle-specific transcription factors E2F and CREB/ATF. Here, we demonstrate that HT1080 fibrosarcoma cells become adapted to doxorubicin-induced DNA damage checkpoint upon ectopic expression of a phosphomimetic mutant of PBK as indicated by the accumulation of polyploid cells. Aberrant entry into the mitotic phase by these cells is suggested by the appearance of a mitotic phase-specific marker, MPM-2. We propose that the effect is due to downregulation of p53 caused by direct physical interaction with PBK as detected by both a biochemical means as well as by yeast two-hybrid analysis. Together, our studies provide a plausible explanation for the role of PBK augmenting tumor cell growth following transient appearance in different types of progenitor cells in vivo as reported. PMID:17482142

  6. Presence of enolase in the M-band of skeletal muscle and possible indirect interaction with the cytosolic muscle isoform of creatine kinase.

    PubMed Central

    Foucault, G; Vacher, M; Merkulova, T; Keller, A; Arrio-Dupont, M

    1999-01-01

    Glycerol-skinned skeletal muscle fibres retain the defined sarcomeric structure of the myofibrils. We show here that a small fraction of two enzymes important for energy metabolism, the cytosolic muscle isoform of creatine kinase (EC 2.7.3.2), MM-creatine kinase (MM-CK), and enolase (EC 4.2.1.11), remains bound to skinned fibres. CK is slowly exchangeable, whereas enolase is firmly bound. Two-dimensional gel electrophoresis followed by Western blot analyses demonstrates that both alpha (ubiquitous) and beta (muscle-specific) subunits of enolase are present in these preparations. Enolase and CK were co-localized at the M-band of the sarcomeres, as observed by indirect immunofluorescence and confocal microscopy. Cross-linking experiments were performed on skinned fibres with three bifunctional succinimidyl esters of different lengths and yielded a protein complex of 150 kDa that reacted with antibodies directed against either M-CK or beta-enolase. The cross-linking efficiency was greatest for the longest reagent and zero for the shortest one. The length of the cross-linker giving a covalent complex between the two enzymes does not support the notion of a direct interaction between M-CK and enolase. This is the first demonstration of the presence of an enzyme of energy metabolism other than CK at the M-band of myofibres. PMID:9931306

  7. Silencing kinase-interacting stathmin gene enhances erlotinib sensitivity by inhibiting Ser¹⁰ p27 phosphorylation in epidermal growth factor receptor-expressing breast cancer.

    PubMed

    Zhang, Dongwei; Tari, Ana M; Akar, Ugur; Arun, Banu K; LaFortune, Tiffany A; Nieves-Alicea, Rene; Hortobagyi, Gabriel N; Ueno, Naoto T

    2010-11-01

    The epidermal growth factor receptor (EGFR) signaling pathway has emerged as a promising target for cancer therapy. EGFR tyrosine kinase inhibitors (TKI) such as erlotinib have been approved for cancer treatment but have shown very limited activity in breast cancer patients. Clarifying the molecular mechanism underlying resistance to EGFR TKIs could lead to more effective treatment against breast cancer. We previously reported that the sensitivity of breast cancer cells to erlotinib is partially dependent on p27 and that cytoplasmic localization of p27 is associated with erlotinib resistance. In the present study, we found that erlotinib induces p27 phosphorylation at Ser¹⁰ (S10), and S10 p27 phosphorylation leads to erlotinib resistance in EGFR-expressing breast cancer. Inhibiting S10 phosphorylation of p27 by knocking down human kinase-interacting stathmin (KIS), a nuclear protein that can phosphorylate p27 at S10, led to p27 accumulation in the nucleus and enhanced erlotinib-mediated cytotoxicity. Further, in vivo KIS gene silencing enhanced the antitumor activity of erlotinib in an orthotopic breast cancer xenograft model. KIS depletion also enhanced erlotinib sensitivity in erlotinib-resistant EGFR-expressing triple-negative breast cancer cells. Our study provides a rationale for the development of combinations of erlotinib with KIS inhibition to overcome EGFR TKI resistance in EGFR-expressing breast cancer.

  8. IGF-IR tyrosine kinase interacts with NPM-ALK oncogene to induce survival of T-cell ALK+ anaplastic large-cell lymphoma cells

    PubMed Central

    Shi, Ping; Lai, Raymond; Lin, Quan; Iqbal, Abid S.; Young, Leah C.; Kwak, Larry W.; Ford, Richard J.

    2009-01-01

    Type I insulin-like growth factor receptor (IGF-IR) tyrosine kinase plays important roles in the pathogenesis of several malignancies. Although it promotes the growth of stimulated hematopoietic cells, a direct role of IGF-IR in malignant lymphoma has not been identified. Anaplastic lymphoma kinase-positive anaplastic large-cell lymphoma (ALK+ ALCL) is a unique type of T-cell lymphoma. Approximately 85% of ALK+ ALCL cases harbor the translocation t(2;5)(p23;q35), which generates the chimeric oncogene NPM-ALK. In the present study, we explored a possible role of IGF-IR in ALK+ ALCL. Our results demonstrate that IGF-IR and IGF-I are widely expressed in ALK+ ALCL cell lines and primary tumors. Importantly, we identified novel reciprocal functional interactions between IGF-IR and NPM-ALK. Antagonism of IGF-IR decreased the viability, induced apoptosis and cell-cycle arrest, and decreased proliferation and colony formation of ALK+ ALCL cell lines. These effects could be explained by alterations of cell survival regulatory proteins downstream of IGF-IR signaling. Our findings improve current understanding of the biology of IGF-IR and NPM-ALK and have significant therapeutic implications as they identify IGF-IR signaling as a potential therapeutic target in ALK+ ALCL and possibly other types of malignant lymphoma. PMID:19423729

  9. Pyruvate kinase M2 interacts with DNA damage-binding protein 2 and reduces cell survival upon UV irradiation.

    PubMed

    Xie, Xiao; Wang, Mingsong; Mei, Ju; Hu, Fengqing; Ding, Fangbao; Lv, Lei

    2015-11-13

    Pyruvate Kinase M2 (PKM2) is highly expressed in many solid tumors and associated with metabolism reprogramming and proliferation of tumors. Here, we report that PKM2 can bind to DNA Damage-Binding Protein 2 (DDB2), which is necessary for global nucleotide excision repair of UV induced DNA damage. The binding is promoted by UV irradiation and K433 acetylation of PKM2. Over expression of PKM2 facilitates phosphorylation of DDB2 and impairs DDB2-DDB1 binding. Furthermore, knocking down of PKM2 increases cell survival upon UV irradiation, while over expression of PKM2 reduces cell survival and over expression of DDB2-DDB1 reverts this effect. These results reveal a previously unknown regulation of PKM2 on DDB2 and provide a possible mechanism for UV induced tumorigenesis.

  10. HSP40 Interacts with Pyruvate Kinase M2 and Regulates Glycolysis and Cell Proliferation in Tumor Cells

    PubMed Central

    Huang, Liangqian; Yu, Zhenhai; Zhang, Teng; Zhao, Xiaoping; Huang, Gang

    2014-01-01

    Pyruvate kinase M2 (PKM2) is predominantly expressed in cancers, which is considered as a key regulator of the Warburg effect. In this study, HSP40 was identified as a novel binding partner of PKM2. HSP40-PKM2 association destabilized PKM2 protein through HSC70. In the presence of HSP40, PKM2 protein level and PKM2-mediated PDK1 expression were down-regulated. Moreover, HSP40 was involved in regulating glucose metabolism on PKM2 dependent way and at the mean time had an effect on mitochondrial oxygen respiration. In line with inhibition effect of HSP40 on glycolysis, the growth of cancer cells was inhibited by HSP40.Our data provided a new regulation mechanism of PKM2, which suggested a new therapeutic target for cancer therapy. PMID:24658033

  11. 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

  12. G-protein-coupled receptor kinase-interacting proteins inhibit apoptosis by inositol 1,4,5-triphosphate receptor-mediated Ca2+ signal regulation.

    PubMed

    Zhang, Songbai; Hisatsune, Chihiro; Matsu-Ura, Toru; Mikoshiba, Katsuhiko

    2009-10-16

    The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) is an intracellular IP(3)-gated calcium (Ca(2+)) release channel and plays important roles in regulation of numerous Ca(2+)-dependent cellular responses. Many intracellular modulators and IP(3)R-binding proteins regulate the IP(3)R channel function. Here we identified G-protein-coupled receptor kinase-interacting proteins (GIT), GIT1 and GIT2, as novel IP(3)R-binding proteins. We found that both GIT1 and GIT2 directly bind to all three subtypes of IP(3)R. The interaction was favored by the cytosolic Ca(2+) concentration and it functionally inhibited IP(3)R activity. Knockdown of GIT induced and accelerated caspase-dependent apoptosis in both unstimulated and staurosporine-treated cells, which was attenuated by wild-type GIT1 overexpression or pharmacological inhibitors of IP(3)R, but not by a mutant form of GIT1 that abrogates the interaction. Thus, we conclude that GIT inhibits apoptosis by modulating the IP(3)R-mediated Ca(2+) signal through a direct interaction with IP(3)R in a cytosolic Ca(2+)-dependent manner.

  13. 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, ...

  14. Diversity, classification and function of the plant protein kinase superfamily

    PubMed Central

    Lehti-Shiu, Melissa D.; Shiu, Shin-Han

    2012-01-01

    Eukaryotic protein kinases belong to a large superfamily with hundreds to thousands of copies and are components of essentially all cellular functions. The goals of this study are to classify protein kinases from 25 plant species and to assess their evolutionary history in conjunction with consideration of their molecular functions. The protein kinase superfamily has expanded in the flowering plant lineage, in part through recent duplications. As a result, the flowering plant protein kinase repertoire, or kinome, is in general significantly larger than other eukaryotes, ranging in size from 600 to 2500 members. This large variation in kinome size is mainly due to the expansion and contraction of a few families, particularly the receptor-like kinase/Pelle family. A number of protein kinases reside in highly conserved, low copy number families and often play broadly conserved regulatory roles in metabolism and cell division, although functions of plant homologues have often diverged from their metazoan counterparts. Members of expanded plant kinase families often have roles in plant-specific processes and some may have contributed to adaptive evolution. Nonetheless, non-adaptive explanations, such as kinase duplicate subfunctionalization and insufficient time for pseudogenization, may also contribute to the large number of seemingly functional protein kinases in plants. PMID:22889912

  15. Functional relevance of specific interactions between herpes simplex virus type 1 ICP4 and sequences from the promoter-regulatory domain of the viral thymidine kinase gene.

    PubMed Central

    Imbalzano, A N; Shepard, A A; DeLuca, N A

    1990-01-01

    The herpes simplex virus (HSV) type 1 immediate-early regulatory protein ICP4 is required for induced expression of HSV early and late genes, yet the mechanism by which this occurs is not known. We examined the promoter and flanking sequences of the HSV early gene that encodes thymidine kinase for the ability to interact specifically with ICP4 in gel retardation assays. Protein-DNA complexes containing ICP4 were observed with several distinct regions flanking the tk promoter. cis-Acting elements that interact with cellular transcription factors were apparently not required for these interactions to form. Purified ICP4 formed protein-DNA complexes with fragments from these regions, and Southwestern (DNA-protein blot) analysis indicated that the interaction between ICP4 and these sequences can be direct. None of the tk sequences that interact with ICP4 contains a consensus binding site for ICP4 (S. W. Faber and K. W. Wilcox, Nucleic Acids Res. 14:6067-6083, 1986), reflecting the ability of ICP4 to interact with more than one DNA sequence. A mutated ICP4 protein expressed from the viral genome that retains the ability to bind to a consensus binding site but does not bind specifically to the identified sites flanking the tk promoter results in induced transcription of the tk gene. These data support hypotheses for ICP4-mediated transactivation of the tk promoter in Vero cells that do not require the intrinsic ability of ICP4 to bind specifically in or near the promoter of the tk gene. Images PMID:2159535

  16. 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

  17. 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. PMID:26647356

  18. N-Acetyl-D-Glucosamine Kinase Interacts with Dynein-Lis1-NudE1 Complex and Regulates Cell Division.

    PubMed

    Sharif, Syeda Ridita; Islam, Ariful; Moon, Il Soo

    2016-09-01

    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

  19. Insight into the Interactions between Novel Isoquinolin-1,3-Dione Derivatives and Cyclin-Dependent Kinase 4 Combining QSAR and Molecular Docking

    PubMed Central

    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. PMID:24722522

  20. 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

  1. Carboxyl terminus of HSC70-interacting protein (CHIP) down-regulates NF-κB-inducing kinase (NIK) and suppresses NIK-induced liver injury.

    PubMed

    Jiang, Bijie; Shen, Hong; Chen, Zheng; Yin, Lei; Zan, Linsen; Rui, Liangyou

    2015-05-01

    Ser/Thr kinase NIK (NF-κB-inducing kinase) mediates the activation of the noncanonical NF-κB2 pathway, and it plays an important role in regulating immune cell development and liver homeostasis. NIK levels are extremely low in quiescent cells due to ubiquitin/proteasome-mediated degradation, and cytokines stimulate NIK activation through increasing NIK stability; however, regulation of NIK stability is not fully understood. Here we identified CHIP (carboxyl terminus of HSC70-interacting protein) as a new negative regulator of NIK. CHIP contains three N-terminal tetratricopeptide repeats (TPRs), a middle dimerization domain, and a C-terminal U-box. The U-box domain contains ubiquitin E3 ligase activity that promotes ubiquitination of CHIP-bound partners. We observed that CHIP bound to NIK via its TPR domain. In both HEK293 and primary hepatocytes, overexpression of CHIP markedly decreased NIK levels at least in part through increasing ubiquitination and degradation of NIK. Accordingly, CHIP suppressed NIK-induced activation of the noncanonical NF-κB2 pathway. CHIP also bound to TRAF3, and CHIP and TRAF3 acted coordinately to efficiently promote NIK degradation. The TPR but not the U-box domain was required for CHIP to promote NIK degradation. In mice, hepatocyte-specific overexpression of NIK resulted in liver inflammation and injury, leading to death, and liver-specific expression of CHIP reversed the detrimental effects of hepatic NIK. Our data suggest that CHIP/TRAF3/NIK interactions recruit NIK to E3 ligase complexes for ubiquitination and degradation, thus maintaining NIK at low levels. Defects in CHIP regulation of NIK may result in aberrant NIK activation in the liver, contributing to live injury, inflammation, and disease.

  2. N-Acetyl-D-Glucosamine Kinase Interacts with Dynein-Lis1-NudE1 Complex and Regulates Cell Division.

    PubMed

    Sharif, Syeda Ridita; Islam, Ariful; Moon, Il Soo

    2016-09-01

    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.

  3. 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.

  4. Interactions of the Protein-tyrosine Phosphatase-α with the Focal Adhesion Targeting Domain of Focal Adhesion Kinase Are Involved in Interleukin-1 Signaling in Fibroblasts*

    PubMed Central

    Wang, Qin; Wang, Yongqiang; Fritz, Dominik; Rajshankar, Dhaarmini; Downey, Gregory P.; McCulloch, Christopher A.

    2014-01-01

    Interleukin-1 (IL-1) signaling in fibroblasts is mediated through focal adhesions, organelles that are enriched with adaptor and cytoskeletal proteins that regulate signal transduction. We examined interactions of the focal adhesion kinase (FAK) with protein-tyrosine phosphatase-α (PTP-α) in IL-1 signaling. In wild type and FAK knock-out mouse embryonic fibroblasts, we found by immunoblotting, immunoprecipitation, immunostaining, and gene silencing that FAK is required for IL-1-mediated sequestration of PTPα to focal adhesions. Immunoprecipitation and pulldown assays of purified proteins demonstrated a direct interaction between FAK and PTPα, which was dependent on the FAT domain of FAK and by an intact membrane-proximal phosphatase domain of PTPα. Recruitment of PTPα to focal adhesions, IL-1-induced Ca2+ release from the endoplasmic reticulum, ERK activation, and IL-6, MMP-3, and MMP-9 expression were all blocked in FAK knock-out fibroblasts. These processes were restored in FAK knock-out cells transfected with wild type FAK, FAT domain, and FRNK. Our data indicate that IL-1-induced signaling through focal adhesions involves interactions between the FAT domain of FAK and PTPα. PMID:24821720

  5. Interactions of the protein-tyrosine phosphatase-α with the focal adhesion targeting domain of focal adhesion kinase are involved in interleukin-1 signaling in fibroblasts.

    PubMed

    Wang, Qin; Wang, Yongqiang; Fritz, Dominik; Rajshankar, Dhaarmini; Downey, Gregory P; McCulloch, Christopher A

    2014-06-27

    Interleukin-1 (IL-1) signaling in fibroblasts is mediated through focal adhesions, organelles that are enriched with adaptor and cytoskeletal proteins that regulate signal transduction. We examined interactions of the focal adhesion kinase (FAK) with protein-tyrosine phosphatase-α (PTP-α) in IL-1 signaling. In wild type and FAK knock-out mouse embryonic fibroblasts, we found by immunoblotting, immunoprecipitation, immunostaining, and gene silencing that FAK is required for IL-1-mediated sequestration of PTPα to focal adhesions. Immunoprecipitation and pulldown assays of purified proteins demonstrated a direct interaction between FAK and PTPα, which was dependent on the FAT domain of FAK and by an intact membrane-proximal phosphatase domain of PTPα. Recruitment of PTPα to focal adhesions, IL-1-induced Ca(2+) release from the endoplasmic reticulum, ERK activation, and IL-6, MMP-3, and MMP-9 expression were all blocked in FAK knock-out fibroblasts. These processes were restored in FAK knock-out cells transfected with wild type FAK, FAT domain, and FRNK. Our data indicate that IL-1-induced signaling through focal adhesions involves interactions between the FAT domain of FAK and PTPα.

  6. Discovery of Multitarget Antivirals Acting on Both the Dengue Virus NS5-NS3 Interaction and the Host Src/Fyn Kinases.

    PubMed

    Vincetti, Paolo; Caporuscio, Fabiana; Kaptein, Suzanne; Gioiello, Antimo; Mancino, Valentina; Suzuki, Youichi; Yamamoto, Naoki; Crespan, Emmanuele; Lossani, Andrea; Maga, Giovanni; Rastelli, Giulio; Castagnolo, Daniele; Neyts, Johan; Leyssen, Pieter; Costantino, Gabriele; Radi, Marco

    2015-06-25

    This study describes the discovery of novel dengue virus inhibitors targeting both a crucial viral protein-protein interaction and an essential host cell factor as a strategy to reduce the emergence of drug resistance. Starting from known c-Src inhibitors, a virtual screening was performed to identify molecules able to interact with a recently discovered allosteric pocket on the dengue virus NS5 polymerase. The selection of cheap-to-produce scaffolds and the exploration of the biologically relevant chemical space around them suggested promising candidates for chemical synthesis. A series of purines emerged as the most interesting candidates able to inhibit virus replication at low micromolar concentrations with no significant toxicity to the host cell. Among the identified antivirals, compound 16i proved to be 10 times more potent than ribavirin, showed a better selectivity index and represents the first-in-class DENV-NS5 allosteric inhibitor able to target both the virus NS5-NS3 interaction and the host kinases c-Src/Fyn.

  7. 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.

  8. 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.

  9. Receptor-like glycocompounds in human milk that inhibit classical and El Tor Vibrio cholerae cell adherence (hemagglutination).

    PubMed Central

    Holmgren, J; Svennerholm, A M; Lindblad, M

    1983-01-01

    The two biotypes of Vibrio cholerae were found to have cell-associated hemagglutinins which differ with regard to binding to different species of erythrocytes and inhibition by monosaccharides. A total of 12 classical V. cholerae strains (Inaba or Ogawa) strongly agglutinated human erythrocytes in a reaction specifically inhibited by L-fucose, whereas 12 El Tor strains preferably agglutinated chicken erythrocytes, a reaction reversed by D-mannose or by higher concentrations of D-fructose, D-glucose, alpha-methyl-D-mannoside, or sucrose. Milk from Swedish women inhibited both of these adherence reactions, and the predominating inhibitory activity for each reaction resisted boiling, was destroyed by periodate treatment, and bound a concanavalin A-Sepharose column, suggesting a carbohydrate structure. Further characterization indicated that the inhibitory activity for classical V. cholerae hemagglutination was distributed about equally on glycoprotein and free oligosaccharide, but was not present on glycolipid. The El Tor inhibiting activity, on the other hand, was almost exclusively of a high-molecular-weight glycoprotein nature. These results support our previous suggestion (Holmgren et al., Infect. Immun. 33:136-141, 1981) that human milk may contain receptor-like glycocompounds which can prevent bacterial adherence by competition with receptors on target cells. PMID:6295953

  10. Targeting Epstein-Barr virus-transformed B lymphoblastoid cells using antibodies with T-cell receptor-like specificities.

    PubMed

    Lai, Junyun; Tan, Wei Jian; Too, Chien Tei; Choo, Joanna Ai Ling; Wong, Lan Hiong; Mustafa, Fatimah Bte; Srinivasan, Nalini; Lim, Angeline Pei Chiew; Zhong, Youjia; Gascoigne, Nicholas R J; Hanson, Brendon J; Chan, Soh Ha; Chen, Jianzhu; MacAry, Paul A

    2016-09-01

    Epstein-Barr virus (EBV) is an oncovirus associated with several human malignancies including posttransplant lymphoproliferative disease in immunosuppressed patients. We show here that anti-EBV T-cell receptor-like monoclonal antibodies (TCR-like mAbs) E1, L1, and L2 bound to their respective HLA-A*0201-restricted EBV peptides EBNA1562-570, LMP1125-133, and LMP2A426-434 with high affinities and specificities. These mAbs recognized endogenously presented targets on EBV B lymphoblastoid cell lines (BLCLs), but not peripheral blood mononuclear cells, from which they were derived. Furthermore, these mAbs displayed similar binding activities on several BLCLs, despite inherent heterogeneity between different donor samples. A single weekly administration of the naked mAbs reduced splenomegaly, liver tumor spots, and tumor burden in BLCL-engrafted immunodeficient NOD-SCID/Il2rg(-/-) mice. In particular, mice that were treated with the E1 mAb displayed a delayed weight loss and significantly prolonged survival. In vitro, these TCR-like mAbs induced early apoptosis of BLCLs, thereby enhancing their Fc-dependent phagocytic uptake by macrophages. These data provide evidence for TCR-like mAbs as potential therapeutic modalities to target EBV-associated diseases. PMID:27338099

  11. 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.

  12. Manipulation of pro-inflammatory cytokine production by the bacterial cell-penetrating effector protein Yo