María T. Téllez-Iñón

A Calcium-Dependent Protein Kinase Is Systemically Induced upon Wounding in Tomato Plants

A full-length cDNA clone (LeCDPK1) from tomato (Lycopersicon esculentum) encoding a calcium-dependent protein kinase (CDPK) was isolated by screening a cDNA library from tomato cell cultures exposed to Cladosporium fulvum elicitor preparations. The predicted amino acid sequence of the cDNA reveals a high degree of similarity with other members of the CDPK family. LeCDPK1 has a putative N-terminal myristoylation sequence and presents a possible palmitoylation site. The in vitro translated protein conserves the biochemical properties of a member of the CDPK family. In addition, CDPK activity was detected in soluble and particulate extracts of tomato leaves. Basal levels of LeCDPK1 mRNA were detected by northern-blot analysis in roots, stems, leaves, and flowers of tomato plants. The expression of LeCDPK1 was rapidly and transiently enhanced in detached tomato leaves treated with pathogen elicitors and H2O2. Moreover, when tomato greenhouse plants were subjected to mechanical wounding, a transient increase of LeCDPK1 steady-state mRNA levels was detected locally at the site of the injury and systemically in distant non-wounded leaves. The increase observed in LeCDPK1 mRNA upon wounding correlates with an increase in the amount and in the activity of a soluble CDPK detected in extracts of tomato leaves, suggesting that this kinase is part of physiological plant defense mechanisms against biotic or abiotic attacks.

Protein kinase C in Trypanosoma cruzi epimastigote forms: partial purification and characterization

A protein kinase C activity from epimastigote forms of Trypanosoma cruzi was characterized. Cytosolic extracts were chromatographed on DEAE-cellulose columns giving two peaks of kinase activity which were eluted at 0.1 and 0.15 M NaCl. The first activity peak requires Ca2+ and phosphatidylserine for activity. Further kinase purification was performed by chromatography on phenyl Sepharose columns. In these columns the enzyme activity was adsorbed in the presence of Ca2+ and eluted with a EGTA-containing buffer. T. cruzi protein kinase C activity preferentially phosphorylated histone H1. It was stimulated by diacylglycerol and phorbol myristate acetate, and inhibited by polymyxin B and staurosporine. After subcellular fractionation and epimastigote cells, the kinase was found to be associated with microsomal and cytosolic fractions.

Calmodulin and Ca2+-dependent cyclic AMP phosphodiesterase activity in Trypanosoma cruzi

Calmodulin has been purified from Trypanosoma cruzi epimastigote forms by ion-exchange chromatography, gel filtration and affinity chromatography on 2-chloro-10-(3-aminopropyl)phenotiazine-Sepharose. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the factor showed a polypeptide band with an apparent molecular weight of 16 000. In addition, cyclic AMP phosphodiesterase activity from T. cruzi epimastigote forms was purified by ion-exchange chromatography and affinity chromatography on a brain calmodulin-Sepharose column. The enzyme was activated by homologous calmodulin as well as by bovine brain and Neurospora crassa calmodulins. The activation required micromolar concentrations of Ca2+ and it was blocked by EGTA and by some neuroleptic drugs such as chlorpromazine, fluphenazine and compound 48/80. Activations were observed at micromolar concentrations of cyclic AMP as substrate. In addition, T. cruzi calmodulin was also active in bringing about the stimulation of brain phosphodiesterase.

Changes in Calcium-Dependent Protein Kinase Activity during in Vitro Tuberization in Potato

A soluble Ca2+-dependent protein kinase (CDPK) was purified to homogeneity in potato (Solanum tuberosum L.) plants. Potato CDPK was strictly dependent on Ca2+ (one-half maximal activation 0.6 [mu]M) and phosphorylated a wide diversity of substrates, in which Syntide 2 was the best phosphate acceptor (Michaelis constant = 30 [mu]M). The kinase was inhibited by Ca2+-chelating agents, phenotiazine derivatives, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (one-half maximal inhibition = 0.25 mM). Polyclonal antibodies directed against the regulatory region of the soybean CDPK recognized a 53-kD polypeptide. In an autophosphorylation assay, this same band was strongly labeled with [[gamma]-32P]ATP in the presence of Ca2+. CDPK activity was high in nontuberized plants, but increased 2.5-fold at the onset of tuber development and was reduced to one-half of its original activity when the tuber had completed formation. In the early stages of tuberization, Ca2+-dependent phosphorylation of endogenous targets (specific bands of 68, 51, and 46 kD) was observed. These polypeptides were not labeled in nontuberizing plants or in completely formed tubers, indicating that this phosphorylation is a stage-specific event. In addition, dephosphorylation of specific polypeptides was detected in tuberizing plants, suggesting the involvement of a phosphatase. Preincubation of crude extracts with phosphatase inhibitors rendered a 100% increase in CDPK activity.

Serine/Threonine Protein Phosphatases type 2A and their roles in stress signaling

Serine/Threonine protein phosphatases are ubiquitous enzymes in all eukaryotes but many of their physiological roles in plants remain unknown. The available results have demonstrated critical functions for these enzymes in the regulation of adaptive stress responses, and recent studies have directed attention to the functional roles of Ser/Thr phosphatases type 2A (PP2A) as components of stress signaling pathways. This review is focused primarily on plant PP2As and their participation in the control of biotic and abiotic stress responses.

Signal transduction mechanisms in Trypanosoma cruzi

Trypanosoma cruzi, the etiological agent of Chagas disease, is an adequate model for studies on the evolution of signal transduction pathways. These pathways involve molecular entities such as membrane receptors, transduction G proteins, protein kinases and second messengers (Ca(2+), cyclic AMP, cyclic GMP, nitric oxide). In this article, Mirtha M. Flawiá, María T. Téllez-Iñón and Héctor N. Torres describe the studies performed on T. cruzi transduction pathways and their role in the control of metacyclogenesis and cell motility.

Characterization of the Trypanosoma cruzi Cdc2p-related protein kinase 1 and identification of three novel associating cyclins.

Several Cdc2p-related protein kinases (CRKs) have been described in trypanosomatids but their role in the control of the cell cycle nor their biological functions have been addressed. In Trypanosoma cruzi two CRKs have been identified, TzCRK1 and TzCRK3. In this work we further characterize T. cruzi CRK1 and report the identification of three novel associating cyclins. We demonstrate that CRK1 levels and localization do not vary during the cell cycle, and show that it is localized in the cytoplasm, discrete regions of the nucleus, and is highly concentrated in the mitochondrion DNA (kinetoplast), suggesting a putative control function in this organelle. Using purified anti-CRK1 IgGs, we immunoprecipitated from the soluble fraction of T. cruzi epimastigote forms a protein kinase activity which is not inhibited by CDK inhibitors. In addition, we co-precipitated with p13Suc1p beads a kinase activity that was inhibited by the CDK inhibitor flavopiridol and olomoucine. Lastly, using the yeast two-hybrid system we identified three novel cyclin-like proteins able to associate with TzCRK1, and demonstrate that two of these cyclins also bind the T. cruzi CRK3 protein, indicating that these two CRKs are cyclin-dependent kinases.

Evidence for CRK3 participation in the cell division cycle of Trypanosoma cruzi.

Trypanosoma cruzi CRK3 gene encodes a Cdc2p related protein kinase (CRK). To establish if it has a role in the regulation of the parasite cell cycle we studied CRK3 expression and activity throughout three life cycle stages. CRK3 from epimastigote soluble extracts interacted with p13(suc1)-beads. Endogenous CRK3 phosphorylated histone H1 and this activity was inhibited by specific CDK inhibitors: Olomoucine, Flavopiridol and Roscovitine. Flavopiridol partially inhibited the growth of T. cruzi epimastigotes at 50 nM, the lowest concentration used, but even with the highest (5 microM), cell growth was not completely arrested. CRK3 from Flavopiridol-inhibited epimastigote extracts exhibited a dose dependent inhibition of histone H1 phosphorylation. T. cruzi p13(suc1)-binding CRK displayed the same inhibition profile. This suggests that CRK3 is the enzyme responsible for the majority of the kinase activity associated with p13(suc1). CRK3 activity of hydroxyurea (HU) synchronized epimastigotes peaked in G2/M boundary while the kinase activity associated to p13(suc1)-beads increased at the same time point but remained high until late G2/M. In addition, CRK3 expression was constant during the cell cycle. This is a common pattern of CDK activity regulation. Taken together, these results support the idea that CRK3 is involved in control of the cell cycle in T. cruzi.

Adenylate cyclase activity in Cyanobacteria: activation by Ca2+-calmodulin and a calmodulin-like activity

An adenylate cyclase activity was partially characterized in the cyanobacterium Anabaena sp. The enzyme activity is found in soluble cell fractions and shows an apparent molecular weight of about 183,400. This adenylate cyclase is activated by Ca2+ and bovine brain or spinach calmodulin and it is inhibited by EGTA and some phenothiazine derivatives. Furthermore, Anabaena sp. extracts contain a calmodulin-like activity which stimulates bovine brain cyclic AMP phosphodiesterase and the Anabaena adenylate cyclase. EGTA and phenothiazine derivatives block the cyanobacterial modulator effect.

Inhibition of Early Endosome Fusion by Trypansom cruzi-Infected Macrophage Cytosol

ABSTRACT. Trypanosoma cruzi trypomastigotes survive inside macrophages by promoting fusion between the parasitophorous vacuole and mature host lysosomes upon internalization. Since trypomastigotes can evade the lytic pathway, the earliest steps of endocytosis, such as early endosome fusion, may be affected. To test this hypothesis, we used an in vitro early endosome fusion assay. Our results show that trypomastigote‐infected macrophage cytosols cannot promote fusion between early endosomes, compared to mock‐infected cytosols (heat‐killed trypomastigotes were used in the parasite‐macrophage interaction assay). GTPγS addition potentiates the fusogenic activity driven by trypomastigote‐infected macrophage cytosol‐mediated assays, unlike the biphasic fusogenic effect obtained with GTPγS treatment of macrophage cytosol controls. Calcium‐stimulated early endosome fusogenic processes are not affected in the assays mediated by infected macrophage cytosol. We conclude that GTP‐regulated factors, and not calcium‐regulated elements, are involved in the inhibition of the early endosome fusogenic process by the trypomastigote‐infected macrophage cytosol. This primary impediment to the progress of a normal endocytosis may be a relevant step required for the lysosomal recruitment‐fusion of the host lysosmes upon trypomastigote infection and further survival of the parasite within its host.