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H Hirt

Publications and source records attributed to H Hirt.

At least 37 records · Page 2Linked to original sources
PubMed2000

SIMKK, a mitogen-activated protein kinase (MAPK) kinase, is a specific activator of the salt stress-induced MAPK, SIMK.

In eukaryotes, mitogen-activated protein kinases (MAPKs) play key roles in the transmission of external signals, such as mitogens, hormones, and different stresses. MAPKs are activated by MAPK kinases through phosphorylation of MAPKs at both the threonine and tyrosine residues of the conserved TXY activation motif. In plants, several MAPKs are involved in signaling of hormones, stresses, cell cycle, and developmental cues. Recently, we showed that salt stress-induced MAPK (SIMK) is activated when alfalfa cells are exposed to hyperosmotic conditions. Here, we report the isolation and characterization of the alfalfa MAPK kinase SIMKK (SIMK kinase). SIMKK encodes an active protein kinase that interacts specifically with SIMK, but not with three other MAPKs, in the yeast two-hybrid system. Recombinant SIMKK specifically activates SIMK by phosphorylating both the threonine and tyrosine residues in the activation loop of SIMK. SIMKK contains a putative MAPK docking site at the N terminus that is conserved in mammalian MAPK kinases, transcription factors, and phosphatases. Removal of the MAPK docking site of SIMKK partially compromises but does not completely abolish interaction with SIMK, suggesting that other domains of SIMKK also are involved in MAPK binding. In transient expression assays, SIMKK specifically activates SIMK but not two other MAPKs. Moreover, SIMKK enhances the salt-induced activation of SIMK. These data suggest that the salt-induced activation of SIMK is mediated by the dual-specificity protein kinase SIMKK.

Amino Acid Sequence↗
PubMed2000

Wound-induced expression and activation of WIG, a novel glycogen synthase kinase 3.

Glycogen synthase kinase 3 (GSK-3) is involved in the regulation of several physiological processes, including glycogen metabolism, protein synthesis, transcription factor activity, and developmental control. Although GSK-3-like genes have been isolated from plants, no function for any of these kinases has been defined. We report here that the alfalfa wound-induced gene (WIG, for wound-induced GSK-3), lencoding a functional plant GSK-3-like kinase, is activated when the alfalfa leaves are wounded. Although WIG transcripts are hardly detectable in mature leaves, WIG mRNA accumulates rapidly after wounding. Using a peptide antibody that specifically recognizes p53(WIG), we show that p53(WIG) kinase is activated immediately after wounding. Wound-induced activation of p53(WIG) kinase is a post-translational process, because the concentrations of p53(WIG) protein do not change in intact and wounded leaves, and inhibition of transcription or translation does not block activation by wounding. However, inactivation of p53(WIG) kinase, which usually occurs within 60 min after wounding, is dependent on transcription and translation of one or more protein factors. These data suggest that the WIG kinase is involved in wound signaling in plants.

Amino Acid Sequence↗
PubMed2000

Inducible expression of Enterococcus faecalis aggregation substance surface protein facilitates bacterial internalization by cultured enterocytes.

Aggregation substance (AS) is an Enterococcus faecalis surface protein that may contribute to virulence. Using a recently described system for controlled expression of AS in E. faecalis and the heterologous host Lactococcus lactis, experiments were designed to assess the effect of AS on bacterial internalization by HT-29 and Caco-2 enterocytes. AS expression was associated with increased internalization of E. faecalis by HT-29 enterocytes and of L. lactis by HT-29 and Caco-2 enterocytes. Compared to enterocytes cultivated under standard conditions, either cultivation in hypoxia or 1-h pretreatment of enterocytes with calcium-free medium resulted in increased internalization of both E. faecalis and L. lactis (with and without AS expression). Also, AS expression augmented these increases when E. faecalis was incubated with pretreated HT-29 enterocytes and when L. lactis was incubated with pretreated Caco-2 and HT-29 enterocytes. These data indicated that AS might facilitate E. faecalis internalization by cultured enterocytes.

Bacterial Proteins↗
PubMed2000

Heterologous inducible expression of Enterococcus faecalis pCF10 aggregation substance asc10 in Lactococcus lactis and Streptococcus gordonii contributes to cell hydrophobicity and adhesion to fibrin.

Aggregation substance proteins encoded by the sex pheromone plasmid family of Enterococcus faecalis have been shown previously to contribute to the formation of a stable mating complex between donor and recipient cells and have been implicated in the virulence of this increasingly important nosocomial pathogen. In an effort to characterize the protein further, prgB, the gene encoding the aggregation substance Asc10 on pCF10, was cloned in a vector containing the nisin-inducible nisA promoter and its two-component regulatory system. Expression of aggregation substance after nisin addition to cultures of E. faecalis and the heterologous bacteria Lactococcus lactis and Streptococcus gordonii was demonstrated. Electron microscopy revealed that Asc10 was presented on the cell surfaces of E. faecalis and L. lactis but not on that of S. gordonii. The protein was also found in the cell culture supernatants of all three species. Characterization of Asc10 on the cell surfaces of E. faecalis and L. lactis revealed a significant increase in cell surface hydrophobicity upon expression of the protein. Heterologous expression of Asc10 on L. lactis also allowed the recognition of its binding ligand (EBS) on the enterococcal cell surface, as indicated by increased transfer of a conjugative transposon. We also found that adhesion of Asc10-expressing bacterial cells to fibrin was elevated, consistent with a role for the protein in the pathogenesis of enterococcal endocarditis. The data demonstrate that Asc10 expressed under the control of the nisA promoter in heterologous species will be an useful tool in the detailed characterization of this important enterococcal conjugation protein and virulence factor.

Bacterial Adhesion↗
PubMed1999

MAP kinases in plant signal transduction.

Mitogen-activated protein kinase (MAPK) pathways are modules involved in the transduction of extracellular signals to intracellular targets in all eukaryotes. Distinct MAPK pathways are regulated by different extracellular stimuli and are implicated in a wide variety of biological processes. In plants there is evidence for MAPKs playing a role in the signaling of abiotic stresses, pathogens and plant hormones. The large number and divergence of plant MAPKs indicates that this ancient mechanism of bioinformatics is extensively used in plants and may provide a new molecular handle on old questions.

Amino Acid Sequence↗
PubMed1999

Short communication: unsaturated fatty acids inhibit MP2C, a protein phosphatase 2C involved in the wound-induced MAP kinase pathway regulation.

When mechanically injured, plants develop multiple defense systems including the activation of specific genes. These responses are triggered by a complex network of signalling events that include Ca2+ fluxes, the production of free fatty acids from membrane lipids, as well as the activation of mitogen-activated protein kinases (MAPK). In the present paper, we address the question of the regulation of the MAPK pathway by wound-induced Ca2+ and fatty acid signals. We report that MP2C, a serine/threonine protein phosphatase 2C from alfalfa involved in MAPK pathway inactivation, is inhibited specifically in vitro by long-carbon-chain polyunsaturated fatty acids, and alpha-linolenic acid, the primary product of the octadecanoid pathway, was found to be the most potent inhibitor. Ca2+ also inhibits MP2C, but only at high concentrations, and other divalent cations show similar inhibitory effect, making it unlikely that Ca2+ is involved in the regulation of MP2C in vivo. Overall, our data suggest that cross-talk between wound-induced MAPK and octadecanoid pathways may occur at the level of protein phosphatase 2C and linolenic acid.

Calcium↗
PubMed1999

Distinct osmo-sensing protein kinase pathways are involved in signalling moderate and severe hyper-osmotic stress

Plant growth is severely affected by hyper-osmotic salt conditions. Although a number of salt-induced genes have been isolated, the sensing and signal transduction of salt stress is little understood. We provide evidence that alfalfa cells have two osmo-sensing protein kinase pathways that are able to distinguish between moderate and extreme hyper-osmotic conditions. A 46 kDa protein kinase was found to be activated by elevated salt concentrations (above 125 mM NaCl). In contrast, at high salt concentrations (above 750 mM NaCl), a 38 kDa protein kinase, but not the 46 kDa kinase, became activated. By biochemical and immunological analysis, the 46 kDa kinase was identified as SIMK, a member of the family of MAPKs (mitogen-activated protein kinases). SIMK is not only activated by NaCl, but also by KCl and sorbitol, indicating that the SIMK pathway is involved in mediating general hyper-osmotic conditions. Salt stress induces rapid but transient activation of SIMK, showing maximal activity between 8 and 16 min before slow inactivation. When inactive, most mammalian and yeast MAPKs are cytoplasmic but undergo nuclear transloca- tion upon activation. By contrast, SIMK was found to be a constitutively nuclear protein and the activity of the kinase was not correlated with changes in its intra-cellular compartmentation, suggesting an intra-nuclear mechanism for the regulation of SIMK activity.

Journal Article↗
PubMed1999

Rapid Avr9- and Cf-9 -dependent activation of MAP kinases in tobacco cell cultures and leaves: convergence of resistance gene, elicitor, wound, and salicylate responses.

The Cf-9 resistance (R) gene from tomato confers resistance to the fungal pathogen Cladosporium fulvum expressing the corresponding, pathogen-derived avirulence gene product Avr9. To understand how an initial R/Avr recognition event is transmitted and triggers the induction of plant defenses, we investigated early Avr9/Cf-9-dependent activation of protein kinases in transgenic tobacco expressing the Cf-9 gene. We identified two protein kinases of 46 and 48 kD, using myelin basic protein as substrate, that became rapidly activated in a strictly gene-for-gene manner within 2 to 5 min after Avr9 elicitation in both Cf9 tobacco plants and derived cell cultures. Studies with pharmacological inhibitors and effectors revealed that Ca2+ influx and a phosphorylation event(s) are required for kinase activation, but neither enzyme is involved in the Avr9-dependent synthesis of active oxygen species. The activation of both kinases is achieved via post-translational mechanisms, and the activation but not inactivation step includes tyrosine phosphorylation. Using specific antibodies, we found that the 46- and 48-kD kinases were similiar to WIPK (for wound-induced protein kinase) and SIPK (for salicylic acid-induced protein kinase), two previously characterized mitogen-activated protein (MAP) kinases from tobacco. In addition, Cf9 tobacco plants and cell cultures showed an Avr9-dependent accumulation of the WIPK transcript. Cf9 tobacco suspension cultures are thus a unique system in which to analyze the earliest events in R gene function. These data indicate that (1) the R/Avr-mediated induction of plant defense is accomplished via several parallel signaling mechanisms, and (2) R/Avr-dependent signal transduction pathways are interlinked at MAP kinases with responses of plants not only to non-race-specific elicitors but also to abiotic stimuli, such as wounding and mechanical stress.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

A MAP kinase is activated late in plant mitosis and becomes localized to the plane of cell division.

In eukaryotes, mitogen-activated protein kinases (MAPKs) are part of signaling modules that transmit diverse stimuli, such as mitogens, developmental cues, or various stresses. Here, we report a novel alfalfa MAPK, Medicago MAP kinase 3 (MMK3). Using an MMK3-specific antibody, we detected the MMK3 protein and its associated activity only in dividing cells. The MMK3 protein could be found during all stages of the cell cycle, but its protein kinase activity was transient in mitosis and correlated with the timing of phragmoplast formation. Depolymerization of microtubules by short treatments with the drug amiprophosmethyl during anaphase and telophase abolished MMK3 activity, indicating that intact microtubules are required for MMK3 activation. During anaphase, MMK3 was found to be concentrated in between the segregating chromosomes; later, it localized at the midplane of cell division in the phragmoplast. As the phragmoplast microtubules were redistributed from the center to the periphery during telophase, MMK3 still localized to the whole plane of division; thus, phragmoplast microtubules are not required to keep MMK3 at this location. Together, these data strongly support a role for MMK3 in the regulation of plant cytokinesis.

Amino Acid Sequence↗
PubMed1998

MP2C, a plant protein phosphatase 2C, functions as a negative regulator of mitogen-activated protein kinase pathways in yeast and plants.

By interference of the yeast pheromone mitogen-activated protein kinase (MAPK) pathway with an alfalfa cDNA expression library, we have isolated the MP2C gene encoding a functional protein phosphatase type 2C. Epistasis analysis in yeast indicated that the molecular target of the MP2C phosphatase is Ste11, a MAPK kinase kinase that is a central regulator of the pheromone and osmosensing pathways. In plants, MP2C functions as a negative regulator of the stress-activated MAPK (SAMK) pathway that is activated by cold, drought, touch, and wounding. Although activation of the SAMK pathway occurs by a posttranslational mechanism, de novo transcription and translation of protein factor(s) are necessary for its inactivation. MP2C is likely to be this or one of these factors, because wound-induced activation of SAMK is followed by MP2C gene expression and recombinant glutathione S-transferase-MP2C is able to inactivate extracts containing wound-induced SAMK. Wound-induced MP2C expression is a transient event and correlates with the refractory period, i.e., the time when restimulation of the SAMK pathway is not possible by a second stimulation. These data suggest that MP2C is part of a negative feedback mechanism that is responsible for resetting the SAMK cascade in plants.

Amino Acid Sequence↗
PubMed1998

Aggregation and binding substances enhance pathogenicity in rabbit models of Enterococcus faecalis endocarditis.

We investigated the importance of enterococcal aggregation substance (AS) and enterococcal binding substance (EBS) in rabbit models of Enterococcus faecalis cardiac infections. First, American Dutch belted rabbits were injected intraventricularly with 10(8) CFU and observed for 2 days. No clinical signs of illness developed in animals given AS- EBS- organisms, and all survived. All rabbits given AS- EBS+ organisms developed signs of illness, including significant pericardial inflammation, but only one of six died. All animals given AS+ EBS- organisms developed signs of illness, including pericardial inflammation, and survived. All rabbits given AS+ EBS+ organisms developed signs of illness and died. None of the rabbits receiving AS+ EBS+ organisms showed gross pericardial inflammation. The lethality and lack of inflammation are consistent with the presence of a superantigen. Rabbit and human lymphocytes were highly stimulated in vitro by cell extracts, but not cell-free culture fluids, of AS+ EBS+ organisms. In contrast, cell extracts from AS- EBS- organisms weakly stimulated lymphocyte proliferation. Culture fluids from human lymphocytes stimulated with AS+/EBS+ enterococci contained high levels of gamma interferon and tumor necrosis factor alpha (TNF-alpha) and TNF-beta, which is consistent with functional stimulation of T-lymphocyte proliferation and macrophage activation. Subsequent experiments examined the abilities of the same strains to cause endocarditis in a catheterization model. New Zealand White rabbits underwent transaortic catheterization for 2 h, at which time catheters were removed and animals were injected with 2 x 10(9) CFU of test organisms. None of the animals given AS- EBS- organisms developed vegetations or showed autopsy evidence of tissue damage. Rabbits given AS- EBS+ or AS+ EBS- organisms developed small vegetations and had splenomegaly at autopsy. All rabbits given AS+ EBS+ organisms developed large vegetations and had splenomegaly and lung congestion at autopsy. Similar experiments that left catheters in place for 3 days revealed that all rabbits given AS- EBS- or AS+ EBS+ organisms developed vegetations, but animals given AS+ EBS+ organisms had larger vegetations and autopsy evidence of lung congestion. These experiments provide direct evidence that these two cell wall components play an important role in the pathogenesis of endocarditis as well as in conjugative plasmid transfer.

Adhesins, Bacterial↗
PubMed1997

Receptor-mediated activation of a MAP kinase in pathogen defense of plants.

Parsley cells recognize the fungal plant pathogen Phytophthora sojae through a plasma membrane receptor. A pathogen-derived oligopeptide elicitor binds to this receptor and thereby stimulates a multicomponent defense response through sequential activation of ion channels and an oxidative burst. An elicitor-responsive mitogen-activated protein (MAP) kinase was identified that acts downstream of the ion channels but independently or upstream of the oxidative burst. Upon receptor-mediated activation, the MAP kinase is translocated to the nucleus where it might interact with transcription factors that induce expression of defense genes.

Amino Acid Sequence↗
PubMed1996

Comparative analysis of 18 sex pheromone plasmids from Enterococcus faecalis: detection of a new insertion element on pPD1 and implications for the evolution of this plasmid family.

A new IS element, IS1062, related to the enterococcal IS elements IS6770 and IS1252, was detected in the 3'-terminus of the surface exclusion gene, sep1, of sex pheromone plasmid pPD1 in Enterococcus faecalis. pPD1-bearing cells lack the surface exclusion function, probably as a consequence of this insertion. Analysis of pAD1 and pPD1 sequences (7.5 kb and 2.7 kb, respectively) downstream of their aggregation substance genes revealed no similarity in these DNA regions. Detailed DNA/DNA hybridization studies using DNA probes specific for various pAD1-encoded genes needed for plasmid transfer indicated that the sex pheromone plasmids have evolved by repeated recombination and insertion of diverse transposable elements which presumably account for recent acquisition of antibiotic resistances.

Amino Acid Sequence↗
PubMed1996

Stress signaling in plants: a mitogen-activated protein kinase pathway is activated by cold and drought.

Yeast and animals use mitogen-activated protein (MAP) kinase cascades to mediate stress and extracellular signals. We have tested whether MAP kinases are involved in mediating environmental stress responses in plants. Using specific peptide antibodies that were raised against different alfalfa MAP kinases, we found exclusive activation of p44MMK4 kinase in drought- and cold-treated plants. p44MMK4 kinase was transiently activated by these treatments and was correlated with a shift in the electrophoretic mobility of the p44MMK4 protein. Although transcript levels of the MMK4 gene accumulated after drought and cold treatment, no changes in p44MMK4 steady state protein levels were observed, indicating a posttranslational activation mechanism. Extreme temperatures, drought, and salt stress are considered to be different forms of osmotic stress. However, high salt concentrations or heat shock did not induce activation of p44MMK4, indicating the existence of distinct mechanisms to mediate different stresses in alfalfa. Stress adaptation in plants is mediated by abscisic acid (ABA)-dependent and ABA-independent processes. Although ABA rapidly induced the transcription of an ABA-inducible marker gene, MMK4 transcript levels did not increase and p44MMK4 kinase was not activated. These data indicate that the MMK4 kinase pathway mediates drought and cold signaling independently of ABA.

Base Sequence↗
PubMed1996

Plant cyclins: a unified nomenclature for plant A-, B- and D-type cyclins based on sequence organization.

The comparative analysis of a large number of plant cyclins of the A/B family has recently revealed that plants possess two distinct B-type groups and three distinct A-type groups of cyclins. Despite earlier uncertainties, this large-scale comparative analysis has allowed an unequivocal definition of plant cyclins into either A or B classes. We present here the most important results obtained in this study, and extend them to the case of plant D-type cyclins, in which three groups are identified. For each of the plant cyclin groups, consensus sequences have been established and a new, rational, plant-wide naming system is proposed in accordance with the guidelines of the Commission on Plant Gene Nomenclature. This nomenclature is based on the animal system indicating cyclin classes by an upper-case roman letter, and distinct groups within these classes by an arabic numeral suffix. The naming of plant cyclin classes is chosen to indicate homology to their closest animal class. The revised nomenclature of all described plant cyclins is presented, with their classification into groups CycA1, CycA2, CycA3, CycB1, CycB2, CycD1, CycD2 and CycD3.

Amino Acid Sequence↗