Sucrose increases calcium-dependent protein kinase and phosphatase activities in potato plants.

The effect of sucrose on tuber formation, calcium-dependent protein kinase (CDPK) and phosphatase activities was analysed using in vitro cultured potato plants. In short treatments, sucrose induced CDPK and phosphatase activities. In long treatments, sucrose induced tuber formation in the absence of other tuber inducing stimuli. Sorbitol caused a minor increase in CDPK activity and affected plant morphology but did not induce tuber development. The addition of the protein kinase inhibitor Staurosporine precluded sucrose-induced tuberization. Altogether, our results suggest that phosphorylation/dephosphorylation events are involved in sucrose-induced tuber development.

Molecular characterization of StCDPK1, a calcium-dependent protein kinase from Solanum tuberosum that is induced at the onset of tuber development.

We isolated a full-length cDNA clone (StCDPK1) encoding a calcium-dependent protein kinase (CDPK) by screening a stolon tip cDNA library from potato plants (Solanum tuberosum L.). The predicted amino acid sequence of the cDNA reveals a high degree of similarity with other members of the CDPK family except in the N-terminal region. As described for other CDPKs, StCDPK1 has a putative N-terminal myristoylation sequence. A coupled transcription/translation system was used to demonstrate that this post-translational modification occurs in vitro. The behaviour of the myristoylated form of StCDPK1 during its purification on a phenyl-Sepharose column mimics that of the endogenous potato enzyme suggesting that this modification occurs in vivo. In addition, a possible palmitoylation site is present in StCDPK1. Southern blot analysis suggests that more than one CDPK isoform is present in potato plants. Northern blot analysis of steady-state mRNA levels for StCDPK1 in different tissues of potato plants shows that the transcript is differentially expressed in tuberizing stolons. The transcript appears in the early steps of tuber formation before the induction of other genes, such as Pin2 and patatin. This result parallels previous data on CDPK activity in potato plants which was highest at the beginning of tuberization. Our results suggest that StCDPK1 is developmentally regulated. The early and transient expression of this CDPK isoform in the tuberization process suggests that this kinase could trigger a cascade of phosphorylation events involved in tuber induction.

Protein kinase activity in different stages of potato (Solanum tuberosum L.) microtuberization.

In vitro culture was used to study morphogenetic aspects of the tuberization process under controlled conditions in potato (Solanum tuberosum L.) plants. This paper accurately defines four stages of tuber development and their correlation to external morphological characteristics and histological structures. Protein kinase activity, assayed in each stage using Historic HAS as substrate, was differentially expressed during the tuberization process. Phosphorylation was maximum in the first stages of tuber formation. The incorporation of [32PO4 -1] to endogenous peptides containing serine/threonine amino acidic residues followed the same pattern that the protein kinase activity did.

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.

Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi.

A soluble nucleoside diphosphate kinase (NDP kinase) was purified and characterized in epimastigote forms of Trypanosoma cruzi. The enzyme was purified by affinity chromatography on Blue-agarose and Q-Sepharose columns and by FPLC on a Superose 12 column. A membrane-associated NDP kinase was identified which accounts for 30% of total enzymatic activity. Western blot analysis of the soluble NDP kinase revealed a 16.5-kDa monomer recognized by polyclonal antibodies to NDP kinase from Dictyostelium discoideum, Candida albicans or human. Most of the T. cruzi NDP kinase is found in the cell as a hexamer composed of 16.5-kDa monomers. The Km values of the enzyme for ATP, GDP and dTDP were 0.2 +/- 0.008 mM, 0.125 +/- 0.012 mM and 0.4 +/- 0.009 mM, respectively. The parasite enzyme was stable, remained active at 65 degrees C and was found to tolerate up to 2.5 M urea. The 16.5-kDa subunit was phosphorylated with [gamma-32P]ATP or thiophosphorylated with [35S]GTP gamma S. The incubation of the 32P-labelled phosphoenzyme with unlabelled nucleoside 5'-diphosphates resulted in the formation of 32P-labelled nucleoside 5'-triphosphates without strict base specificity, indicating that the reaction mechanism of the T. cruzi enzyme is the same as reported for other NDP kinases. When the phosphoenzyme was incubated with a mixture of nucleoside 5'-diphosphates, GTP was preferentially formed.

Characterization of the catalytic subunit of Trypanosoma cruzi cyclic AMP-dependent protein kinase.

The catalytic subunit of cyclic AMP-dependent protein kinase from Trypanosoma cruzi epimastigote forms was purified by ionic-exchange chromatography, affinity chromatography and sucrose gradient centrifugation. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, the purified preparations showed a main polypeptide band with a mobility of about 40 kDa. In Western blots this band immunoreacted with a polyclonal antibody specific for the catalytic subunit of bovine heart protein kinase A. Hydrodynamic and molecular parameters of this subunit are as follows: molecular weight, 40,000 +/- 3000; sedimentation constant, 2.8 +/- 0.3 S; Stokes' radius, 2.8 +/- 0.2 nm; frictional ratio, 1.28 +/- 0.05. Purified preparations of T. cruzi catalytic and regulatory subunits reconstitute a holoenzyme with a sedimentation constant. 8.6 +/- 1.17 S. This data together with those previously reported by Ulloa et al. [8] indicate that the T. cruzi cyclic AMP-dependent protein kinase holoenzyme is a tetramer with the structure R2C2 of about 200 kDa. The apparent Km of the catalytic subunit for ATP and histone IIA or kemptide as phosphate donor and acceptor, respectively, were 40 microM, 48.6 microM and 26 microM, respectively.

Ca2+ calmodulin-dependent protein kinase activity in the ascomycetes Neurospora crassa.

DEAE-cellulose column chromatography of Neurospora crassa soluble mycelial extracts leads to the resolution of three major protein kinase activity peaks designated PKI, PKII, and PKIII. PKII activity is stimulated by Ca2+ and Neurospora or brain calmodulin. Maximal stimulation was observed at 2 microM-free Ca2+ and 1 microgram/ml of the modulator. The stimulatory effect of the Ca(2+)-calmodulin complex was blocked by EGTA and by some calmodulin antagonists such as phenothiazine drugs or compound 48/80. PKII phosphorylates different proteins, among which histone II-A at a low concentration and CDPKS, the synthetic peptide specific for Ca(2+-)-calmodulin dependent protein kinase, are the best substrates. Some phosphorylation can be detected in the absence of any exogenous acceptor. PKII activity assayed in the presence of histone II-A or in the absence of exogenous phosphate acceptor (autophosphorylation) co-elute in a DEAE-cellulose column at 0.28 NaCl. As result of the autophosphorylation reaction of the purified enzyme a main phosphorylated component of 70 kDa was resolved by SDS-polyacrylamide gel electrophoresis. It is possible that this component is an active part of this enzyme.

Cyclic nucleotide phosphodiesterase activity in Neurospora crassa. Purification by immunoaffinity chromatography and characterization.

Monoclonal antibodies to Neurospora crassa cyclic nucleotide phosphodiesterase (PDE I) were selected by their capacity to inhibit the enzyme activity. The monoclonal immunoglobulin, coupled to Sepharose 4B, was used for the affinity purification of PDE I activity. After SDS-polyacrylamide gel electrophoresis the affinity purified PDE I fractions showed a single polypeptide band of about 41 kDa. This band reacted in Western blots with the above mentioned monoclonal immunoglobulin.

Cyclic AMP-dependent protein kinase activity in Trypanosoma cruzi.

A cyclic AMP-dependent protein kinase 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 0.15 M- and 0.32 M-NaCl respectively. The second activity peak was stimulated by nanomolar concentrations of cyclic AMP. In addition, a cyclic AMP-binding protein co-eluted with the second kinase activity peak. Cyclic AMP-dependent protein kinase activity was further purified by gel filtration, affinity chromatography on histone-agarose and cyclic AMP-agarose, as well as by chromatography on CM-Sephadex. The enzyme ('holoenzyme') could be partially dissociated into two different components: 'catalytic' and 'regulatory'. The 'regulatory' component had specific binding for cyclic AMP, and it inhibited phosphotransferase activity of the homologous 'catalytic component' or of the 'catalytic subunit' from bovine heart. Cyclic AMP reversed these inhibitions. A 'holoenzyme preparation' was phosphorylated in the absence of exogenous phosphate acceptor and analysed by polyacrylamide-gel electrophoresis. A 56 kDa band was phosphorylated. The same preparation was analysed by Western blotting, by using polyclonal antibodies to the regulatory subunits of protein kinases type I or II. Both antibodies reacted with the 56 kDa band.

A novel stimulator of protein phosphorylation in Neurospora crassa.

An endogenous thermostable activator of Protein kinase III (PKIII) was purified from 100,000 X g supernatants of Neurospora crassa mycelial extracts. This 38,000 dalton polypeptide, clearly separable from calmodulin on P-60 gel filtration, specifically stimulated N. crassa PKIII activity on casein or phosvitin "in vitro" phosphorylation. The factor was only present in the initial growth phase of the fungus. The mechanism of PKIII activation and its possible regulatory role are discussed.

Characterization of Neurospora crassa cyclic AMP phosphodiesterase activated by calmodulin.

Activation of cyclic AMP phosphodiesterase I by brain or Neurospora calmodulin was studied. The stimulation required micromolar concentrations of Ca2+, and it was observed at cyclic AMP concentrations between 0.1 and 500 microM. Activation was blocked by EDTA and some neuroleptic drugs such as chlorpromazine and fluphenazine. These drugs inhibit the elongation of N. crassa wild-type aerial hyphae. These results reinforce the evidence towards the recognition of Ca2+-calmodulin as one of the systems controlling cyclic nucleotide concentrations in Neurospora.

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.