Redox regulation of the calcium/calmodulin-dependent protein kinases

Reactive oxygen intermediates (ROI) have been viewed traditionally as damaging to the cell. However, a predominance of evidence has shown that ROI can also function as important activators of key cellular processes, and ROI have been shown to play a vital role in cell signaling networks. The calcium/calmodulin-dependent protein kinases (CaM kinases) are a family of related kinases that are activated in response to increased intracellular calcium concentrations. In this report we demonstrate that hydrogen peroxide treatment results in the activation of both CaM kinase II and IV in Jurkat T lymphocytes. Surprisingly, this activation occurs in the absence of any detectable calcium flux, suggesting a novel means for the activation of these kinases. Treatment of Jurkat cells with phorbol 12-myristate 13-acetate (PMA), which does not cause a calcium flux, also activated the CaM kinases. The addition of catalase to the cultures inhibited PMA-induced activation of the CaM kinases, suggesting that similar to hydrogen peroxide, PMA also activates the CaM kinases via the production of ROI. One mechanism by which this likely occurs is through oxidation and consequential inactivation of cellular phosphatases. In support of this concept, okadaic acid and microcystin-LR, which are inhibitors of protein phosphatase 2A (PP2A), induced CaM kinase II and IV activity in these cells. Overall, these results demonstrate a novel mechanism by which ROI can induce CaM kinase activation in T lymphocytes.     

Specificity of calcium/calmodulin-dependent protein kinases in mouse egg activation

CaMKIIγ, the predominant CaMKII isoform in mouse eggs, controls egg activation by regulating cell cycle resumption. In this study we further characterize the involvement and specificity of CaMKIIγ in mouse egg activation. Using exogenous expression of different cRNAs in Camk2g^−/− eggs, we show that the other multifunctional CaM kinases, CaMKI, and CaMKIV, are not capable of substituting CaMKIIγ to initiate cell cycle resumption in response to a rise in intracellular Ca²⁺. Exogenous expression of Camk2g or Camk2d results in activation of nearly 80% of Camk2g^−/− MII eggs after stimulation with SrCl₂, which does not differ from the incidence of activation of wild-type eggs expressing exogenous Egfp. In contrast, none of the Camk2g^−/− MII eggs expressing Camk1 or Camk4 activate in response to SrCl₂ treatment. Expression of a constitutively active form of Camk4 (ca-Camk4), but not Camk1, triggers egg activation. EMI2, an APC/C repressor, is a key component in regulating egg activation downstream of CaMKII in both Xenopus laevis and mouse. We show that exogenous expression of either Camk2g, Camk2d, or ca-Camk4, but not Camk1, Camk4, or a catalytically inactive mutant form of CaMKIIγ (kinase-dead) in Camk2g^−/− mouse eggs leads to almost complete degradation (~90%) of exogenously expressed EMI2 followed by cell cycle resumption. Thus, degradation of EMI2 following its phosphorylation specifically by CaMKII is mechanistically linked to and promotes cell cycle resumption in MII eggs.     

Stagonospora nodorum: cause of stagonospora nodorum blotch of wheat

Stagonospora nodorum is an important pathogen of wheat and related cereals, causing both a leaf and glume blotch. This review summarizes recent advances in our understanding of taxonomy, control and pathogenicity of this species.
Taxonomy:   Stagonospora (syn. Septoria ) nodorum (Berk.) Castell. and Germano [teleomorph: Phaeosphaeria (syn. Leptosphaeria ) nodorum (Müll.) Hedjar.], kingdom Fungi, phylum Ascomycota, subphylum Euascomycota, class Dothideomycetes, order Pleosporales, family Phaeosphaeriaceae, genus Phaeosphaeria , species nodorum .
Host range:   Wheat, Triticum aestivum , T. durum , Triticale, are the main hosts but other cereals and wild grasses have been reported to harbour S. nodorum. Disease symptoms are lens-shaped necrotic lesions on leaves, girdling necrosis on stems (especially the nodes, hence ' nodorum ') and lesions on glumes. Mature lesions produce pycnidia scattered throughout the lesions, especially as tissue senesces.
Useful websites:   http://ocid.nacse.org/research/deephyphae/htmls/asco_taxlist_spat.html (taxonomic information), http://ohioline.osu.edu/ac-fact/0002.html (disease information), http://wwwacnfp.murdoch.edu.au/  (ACNFP homepage), http://www.broad.mit.edu/annotation/fungi/stagonospora_nodorum/index.html (genome sequence homepage), http://cogeme.ex.ac.uk/efungi/ (genome sequence annotation and analysis).     

The roles of calcium/calmodulin-dependent and Ras/mitogen-activated protein kinases in the development of psychostimulant-induced behavioral sensitization

Although the development of behavioral sensitization to psychostimulants such as cocaine and amphetamine is confined mainly to one nucleus in the brain, the ventral tegmental area (VTA), this process is nonetheless complex, involving a complicated interplay between neurotransmitters, neuropeptides and trophic factors. In the present review we present the hypothesis that calcium-stimulated second messengers, including the calcium/calmodulin-dependent protein kinases and the Ras/mitogen-activated protein kinases, represent the major biochemical pathways whereby converging extracellular signals are integrated and amplified, resulting in the biochemical and molecular changes in dopaminergic neurons in the VTA that represent the critical neuronal correlates of the development of behavioral sensitization to psychostimulants. Moreover, given the important role of calcium-stimulated second messengers in the expression of behavioral sensitization, these signal transduction systems may represent the biochemical substrate through which the transient neurochemical changes associated with the development of behavioral sensitization are translated into the persistent neurochemical, biochemical and molecular alterations in neuronal function that underlie the long-term expression of psychostimulant-induced behavioral sensitization.     

Trehalose biosynthesis is involved in sporulation of Stagonospora nodorum

Stagonospora nodorum is a necrotrophic fungal pathogen that is the causal agent of leaf and glume blotch on wheat. S. nodorum is a polycyclic pathogen, whereby rain-splashed pycnidiospores attach to and colonise wheat tissue and subsequently sporulate again within 2–3 weeks. As several cycles of infection are needed for a damaging infection, asexual sporulation is a critical phase of its infection cycle. A non-targeted metabolomics screen for sporulation-associated metabolites identified that trehalose accumulated significantly in concert with asexual sporulation both in vitro and in planta. A reverse-genetics approach was used to investigate the role of trehalose in asexual sporulation. Trehalose biosynthesis was disrupted by deletion of the gene Tps1, encoding a trehalose 6-phosphate synthase, resulting in almost total loss of trehalose during in vitro growth and in planta. In addition, lesion development and pycnidia formation were also significantly reduced in tps1 mutants. Reintroduction of the Tps1 gene restored trehalose biosynthesis, pathogenicity and sporulation to wild-type levels. Microscopic examination of tps1 infected wheat leaves showed that pycnidial formation often halted at an early stage of development. Further examination of the tps1 phenotype revealed that tps1 pycnidiospores exhibited a reduced germination rate while under heat stress, and tps1 mutants had a reduced growth rate while under oxidative stress. This study confirms a link between trehalose biosynthesis and pathogen fitness in S. nodorum.     

Mating Types of Phaeosphaeria nodorum (anamorph Stagonospora nodorum) from Central Asia

The distribution was examined of the mating type idiomorphs (MAT‐1 and ‐2) of Phaeosphaeria (anamorph Stagonospora) nodorum using DNA from 49 isolates collected from commercial and experimental fields in 2003 and 2004 in Central Asia. MAT‐1 and ‐2 isolates were present in the Kazakh and Russian origins of P. nodorum, but no MAT‐2 isolates were found in Tajikistan. The possibility of a skewed Tajik population cannot be excluded, considering that the sampled region in Tajikistan was geographically isolated from Kazakhstan and Russia.     

Metabolite profiling identifies the mycotoxin alternariol in the pathogen Stagonospora nodorum

A recent comparative proteomics study identified the short-chain dehydrogenase (Sch1) as being required for asexual sporulation (Tan et al. Eukaryotic Cell 7:1916–1929, 2008). Metabolite profiling was undertaken on the mutant strains of Stagonospora nodorum lacking the Sch1 gene to help elucidate its role. Gas chromatography-mass spectrometry of the polar metabolites in the Sch1 mutants identified a secondary metabolite at a 200-fold greater concentration than observed in the wild-type strains. Comparative analysis of the secondary metabolite and the mycotoxin alternariol using ESI-MS/MS confirmed the identity of the compound as alternariol. This is the first report to confirm the presence of a mycotoxin in S. nodorum and compelling the field to consider the health implication of this disease.     

Variation in Aggressiveness of Stagonospora nodorum Isolates in North Dakota

Stagonospora nodorum blotch (SNB), caused by Stagonospora nodorum, is an important disease in the northern Great Plains of the United States and in other wheat‐producing regions in the world. SNB can be managed by different strategies including the use of resistant cultivars. Genetic variation in the pathogen populations is one of the important factors in the development of durable resistant cultivars. Our main objective was to determine variation in aggressiveness/virulence in the 40 isolates of S. nodorum collected from various locations in North Dakota. To achieve this goal, we tested the isolates on two susceptible wheat cultivars (cvs ‘ND495’ and ‘Alsen’) and two resistant wheat cultivars (cvs ‘Erik’ and ‘Salamouni’) – two‐leaf‐stage seedlings under controlled conditions. Aggressiveness of each isolate was characterized by the two epidemiological parameters: percent necrotic leaf area (% NLA) and lesion type (LT) 8 days post‐inoculation. The isolates differed significantly (P ≤ 0.05) for % NLA and LT, and were grouped into three aggressiveness groups (AG): low, medium and highly aggressive. Four isolates (S50, S57, S66 and S89) induced 18–26% NLA and were included into the low aggressive group (AG 1). Three isolates (S15, S39 and S89) induced 57–59% NLA and were considered highly aggressive (AG 3). Thirty‐three isolates were medium aggressive (AG 2). No relationship between AG and mating types was observed. There were significant (P ≤ 0.05) differences in % NLA and LT among wheat cultivars. Significant wheat cultivars by isolates interaction was also demonstrated, suggesting evidence for the existence of host specificity in this system. Overall, our results indicate that S. nodorum isolates prevalent in North Dakota varied greatly in their aggressiveness and that AG 3 isolates can be utilized in breeding wheat for resistance to SNB.     

The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases

The AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism in response to metabolic stress and to other regulatory signals. AMPK activity is absolutely dependent upon phosphorylation of AMPKalphaThr-172 in its activation loop by one or more AMPK kinases (AMPKKs). The tumor suppressor kinase, LKB1, is a major AMPKK present in a variety of tissues and cells, but several lines of evidence point to the existence of other AMPKKs. We have employed three cell lines deficient in LKB1 to study AMPK regulation and phosphorylation, HeLa, A549, and murine embryo fibroblasts derived from LKB(-/-) mice. In HeLa and A549 cells, mannitol, 2-deoxyglucose, and ionomycin, but not 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), treatment activates AMPK by alphaThr-172 phosphorylation. These responses, as well as the downstream effects of AMPK on the phosphorylation of acetyl-CoA carboxylase, are largely inhibited by the Ca(2+)/ calmodulin-dependent protein kinase kinase (CaMKK) inhibitor, STO-609. AMPKK activity in HeLa cell lysates measured in vitro is totally inhibited by STO-609 with an IC50 comparable with that of the known CaMKK isoforms, CaMKKalpha and CaMKKbeta. Furthermore, 2-deoxyglucose- and ionomycin-stimulated AMPK activity, alphaThr-172 phosphorylation, and acetyl-CoA carboxylase phosphorylation are substantially reduced in HeLa cells transfected with small interfering RNAs specific for CaMKKalpha and CaMKKbeta. Lastly, the activation of AMPK in response to ionomycin and 2-deoxyglucose is not impaired in LKB1(-/-) murine embryo fibroblasts. These data indicate that the CaMKKs function in intact cells as AMPKKs, predicting wider roles for these kinases in regulating AMPK activity in vivo.     

Investigating the role of Aurora kinases in RAS signaling

Activating ras mutations are frequently found in malignant tumors of the pancreas, colon, lung and other tissues. RAS activates a number of downstream pathways that ultimately cause cellular transformation. Several recent studies suggested that one of those pathways involves Aurora kinases. Overexpression of Aurora‐B kinase can augment transformation by oncogenic RAS, however the mechanism was not determined. The cooperative effect of high levels of Aurora kinase is important since this kinase is frequently overexpressed in human tumors. We have used two Aurora kinase inhibitors to test their effect on RAS signaling. We find that these inhibitors have no effect on the phosphorylation of MEK1/2 or MAPK in response to RAS. Furthermore, inhibiting Aurora kinases in human cancer cells with or without activated RAS did not change the length of the cell cycle nor induce apoptosis suggesting that these kinases do not play a direct role in these key cellular responses to activated RAS. Overexpression of Aurora B can cause cells to become polyploid. Also, inducing polyploidy with cytochalasin D was reported to induce neoplastic transformation, suggesting that Aurora overexpression may cooperate with RAS indirectly by inducing polyploidy. We find that inducing polyploidy with cytochalasin D or blebbistatin does not enhance transformation by oncogenic RAS. Our observations argue against a direct role for Aurora kinases in the RAS‐MAPK pathway, and suggest that the polyploid state does not enhance transformation by RAS. J. Cell. Biochem. 106: 33–41, 2009. © 2008 Wiley‐Liss, Inc.     

Nitric oxide-mediated modulation of calcium/calmodulin-dependent protein kinase II

The mechanisms of NO inhibition of CaMK [Ca(2+)/CaM (calmodulin)-dependent protein kinase] II activity were studied. In rat pituitary tumour GH3 cells, TRH [thyrotrophin (TSH)-releasing hormone]-stimulated phosphorylation of nNOS [neuronal NOS (NO synthase)] at Ser(847) was sensitive to an inhibitor of CaMKs, KN-93, and was enhanced by inhibition of nNOS with 7NI (7-nitroindazole). Enzyme activity of CaMKII following in situ treatment with 7NI was also increased. The in vitro activity of CaMKII was inhibited by co-incubation either with nNOS and L-arginine or with NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine) and DEA-NONOate [diethylamine-NONOate (diazeniumdiolate)]. Once inhibited by these treatments, CaMKII was observed to undergo full reactivation on the addition of a reducing reagent, DTT (dithiothreitol). In transfected cells expressing CaMKII and nNOS, treatment with the calcium ionophore A23187 further revealed nNOS phosphorylation at Ser(847), which was enhanced by 7NI and CaMKII S-nitrosylation. Mutated CaMKII (C6A), in which Cys(6) was substituted with an alanine residue, was refractory to 7NI-induced enhancement of nNOS phosphorylation or to CaMKII S-nitrosylation. Furthermore, we could identify Cys(6) as a direct target for S-nitrosylation of CaMKII using MS. In addition, treatment with glutamate caused an increase in CaMKII S-nitrosylation in rat hippocampal slices. This glutamate-induced S-nitrosylation was blocked by 7NI. These results suggest that inactivation of CaMKII mediated by S-nitrosylation at Cys(6) may contribute to NO-induced neurotoxicity in the brain.     

A functional genomics approach to dissect the mode of action of the Stagonospora nodorum effector protein SnToxA in wheat

In this study, proteomics and metabolomics were used to study the wheat response to exposure to the SnToxA effector protein secreted by the fungal pathogen Stagonospora nodorum during infection. Ninety-one different acidic and basic proteins and 101 metabolites were differentially abundant when comparing SnToxA- and control-treated wheat leaves during a 72-h time course. Proteins involved in photosynthesis were observed to increase marginally initially after exposure, before decreasing rapidly and significantly. Proteins and metabolites associated with the detoxification of reactive oxygen species in the chloroplast were also differentially abundant during SnToxA exposure, implying that the disruption of photosynthesis causes the rapid accumulation of chloroplastic reactive oxygen species. Metabolite profiling revealed major metabolic perturbations in central carbon metabolism, evidenced by significant increases in tricarboxylic acid (TCA) cycle intermediates, suggestive of an attempt by the plant to generate ATP and reducing equivalents in response to the collapse of photosynthesis caused by SnToxA. This was supported by the observation that the TCA cycle enzyme malate dehydrogenase was up-regulated in response to SnToxA. The infiltration of SnToxA also resulted in a significant increase in abundance of many pathogenicity-related proteins, even in the absence of the pathogen or other pathogen-associated molecular patterns. This approach highlights the complementary nature of proteomics and metabolomics in studying effector-host interactions, and provides further support for the hypothesis that necrotrophic pathogens, such as S. nodorum, appear to exploit existing host cell death mechanisms to promote pathogen growth and cause disease.     

Characterization, physical location and expression of the genes encoding calcium/calmodulin-dependent protein kinases in maize (Zea mays L.)

The maize genomic sequence and cDNA encoding a calcium/calmodulin-dependent protein kinase homolog were isolated and identified. The deduced peptide (MCK2) from this cDNA shared high amino acid identity (91.2%) with maize MCK1. These two genes were physically mapped onto chromosomes by fluorescence in situ hybridization using the first introns of the genes as gene-specific probes. While the MCK1 gene was assigned to a locus on the long arm of chromosome 9, the MCK2 gene was localized to a locus on the long arm of chromosome 1. Both of these genes were expressed in roots, leaves, stems and flowers, and the expression patterns of MCK were verified by RNA in situ hybridization. These results indicated that MCK expression is temporally and spatially regulated during maize growth and development.