Zsigmond Fehér

Protein phosphatase 2A is involved in hyphal growth of Neurospora crassa

Abstract Cantharidin and calyculin A, natural toxins that are inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A, respectively), inhibit Neurospora crassa hyphal growth. When N. cassa was grown in the presence of either drug, abnormalities were observed at hyphal tips. In addition, both drugs induced an increase in hyphal branching. Cantharidin inhibited N. crassa hyphal growth in a temperature-dependent manner, as the effect of the drug was more pronounced at 34° C than at 25° C. In addition to the drug-mediated inhibition of phosphatase activity, a genetic approach was used to determine the phenotypic consequences of reduced PP2A activity. Two strains with subnormal PP2A activity were constructed. The first, in which the original pph-1 gene (encoding the PP2A catalytic subunit) was replaced with an ectopically integrated copy of pph-1, exhibited lower levels of pph-1 transcript, lower PP2A activity and increased sensitivity to cantharidin. Similarly, in a second strain, in which the pph-1 gene was cloned in an antisense orientation downstream of the inducible isocitrate lyase promoter, lower levels of pph-1 transcript, as well as of PP2A activity, and a reduction in hyphal growth were observed. The results of this study indicate that PP2A, and probably other Ser/Thr phosphatases, are involved in the regulation of hyphal growth in N. crassa.

Characterization of inl+ transformants of Neurospora crassa obtained with a recombinant cosmid-pool

SummaryWe constructed a Neurospora crassa gene library in a cosmid vector and used the cosmid-pool DNA to transform an inl, rg Neurospora crassa strain to inositol prototrophy. The inl+ colonies obtained in this experiment proved to be integrative type transformants. Genetic analysis revealed that the integration event occurred at or near the inl locus. In one of the transformants the inl+ trait exhibited mitotic and meiotic instability. In hybridization experiments free plasmids were detected in the F1 progeny of the transformants. We were able to recover eleven different plasmids from the F1 progeny of the transformants. None of these plasmids proved to carry a functional copy of the inl+ gene as judged by its transforming ability. Possible explanations for the observed phenomena are discussed.

pzl-1 encodes a novel protein phosphatase-Z-like Ser/Thr protein phosphatase in Neurospora crassa.

The gene and cDNA of a novel protein phosphatase were cloned from Neurospora crassa. The pzl-1 gene encompasses three introns and is localized to the left arm of chromosome I between cyt-21 and Fsr-12. It encodes a protein of 58.3 kDa containing a Ser/Pro rich N-terminal segment, and a C-terminal domain that is similar to the catalytic subunit of type 1 protein phosphatases. The first 51 amino acid residues, including a potential N-myristoylation site, as well as the C-terminal domain (about 300 residues) have a high level of sequence identity with yeast PPZ phosphatases. However, residues 52-208 do not share high similarity with other proteins. The mRNA of pzl-1 was detected in all phases of asexual development of the filamentous fungus.

ISOLATION AND CHARACTERIZATION OF THE CATALYTIC SUBUNIT OF PROTEIN PHOSPHATASE 2A FROM NEUROSPORA CRASSA

The catalytic subunit of protein phosphatase 2A (PP2Ac) was purified from Neurospora crassa extract by (NH4)2SO4-ethanol precipitation followed by DEAE-Sephacel, heparin-Sepharose, and MonoQ chromatography steps about 900-fold to a specific activity of 1200 U/g with a 2% yield. The apparent M(r) of PP2Ac was estimated to be 35 kDa by gel filtration and 33 kDa by SDS polyacrylamide gel electrophoresis. Half maximal inhibition of PP2Ac was achieved at 0.3 nM okadaic acid, 0.1 nM microcystin-LR, 56 nM cantharidin and 280 nM endothall concentrations. The preparation was completely inhibited by 20 mM NaF, was insensitive to rabbit muscle inhibitor-2, and was specific for the alpha-subunit of rabbit muscle phosphorylase kinase. According to its biochemical properties, N. crassa PP2Ac is very similar to its mammalian counterparts. Antipeptide antibodies raised against the N-terminal and C-terminal ends of human PP2Ac did not cross-react with N. crassa PP2Ac, indicating sequence differences outside the catalytic core of the enzyme.

Expression of protein phosphatase 1 during the asexual development of Neurospora crassa

We cloned and sequenced the cDNA and the gene encoding the catalytic subunit of protein phosphatase 1 from the filamentous fungus Neurospora crassa. The gene, designated ppp-1 (phosphoprotein phosphatase 1), was mapped by restriction fragment length polymorphism to linkage group III, in the vicinity of con-7 and trp-1. The expression of the gene was monitored by reverse transcriptase and polymerase chain reactions, by Western blotting, and by protein phosphatase activity assays in synchronized cultures. Transcripts of ppp-1 were detected in the dormant conidia. The abundance of ppp-1 mRNA, Ppp-1 protein, and the activity of protein phosphatase 1 increased during germination and subsequent hyphal elongation as well as during the early stages of aerial mycelium formation.

Detection of Ser/Thr protein phosphatases in Neurospora crassa

Protein phosphorylation is a frequent posttranslational modification regulating cellular processes in eukaryotes. The phosphate content of a protein is determined by the conflicting activities of protein kinases and phosphatases. Protein phosphatases were divided into Ser/Thr and Tyr specific groups, depending on the phosphorylated residue in the substrate molecules. The former group was further classified based on enzymatic criteria (reviewed in Cohen 1989 Ann. Rev. Biochem. 58:453-508). Protein phosphatase 1 (PP1) is inhibited by two heat stable proteins termed inhibitor-1 and -2. Protein phosphatase 2A is inhibited by nanomolar concentration of the tumor promoter okadaic acid. Protein phosphatase 2B (PP2B) also called calcineurin is stimulated by Ca-calmodulin, and protein phosphatase 2C (PP2C) is a Mg2+ dependent enzyme. Molecular cloning of the catalytic subunits revealed that PP1-PP2A-PP2B consist of a highly conserved superfamily of proteins. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol41/iss1/26 Detection of Ser/Thr protein phosphatases in Neurospora crassa B. Szoor,1 Z. Feher,2 G. Szabo,2 P. Gergely,1 and V. Dombradi,1 Departments of Medical Chemistry1 and Biology2, University Medical School, Debrecen, Hungary Protein phosphorylation is a frequent posttranslational modification regulating cellular processes in eukaryotes. The phosphate content of a protein is determined by the conflicting activities of protein kinases and phosphatases. Protein phosphatases were divided into Ser/Thr and Tyr specific groups, depending on the phosphorylated residue in the substrate molecules. The former group was further classified based on enzymatic criteria (reviewed in Cohen 1989 Ann. Rev. Biochem. 58:453-508). Protein phosphatase 1 (PP1) is inhibited by two heat stable proteins termed inhibitor-1 and -2. Protein phosphatase 2A is inhibited by nanomolar concentration of the tumor promoter okadaic acid. Protein phosphatase 2B (PP2B) also called calcineurin is stimulated by Ca-calmodulin, and protein phosphatase 2C (PP2C) is a Mg2+ dependent enzyme. Molecular cloning of the catalytic subunits revealed that PP1-PP2A-PP2B consist of a highly conserved superfamily of proteins. The catalytic subunit of PP2B from N. crassa was cloned and expressed (Higuchi et al. 1991 J. Biol. Chem. 266:18104-18112). We assumed that PP1 and PP2A were also present in this organisms. In order to test this possibility we assayed phosphatase activities in N. crassa. For the detection of PP1 and PP2A we adapted a method originally described for S. cerevisiae (Cohen et al. 1989 FEBS Lett. 250:601-606). PP2B activity was measured according to Higuchi et al. (1991 J. Biol. Chem. 266:18104-18112). Neurospora crassa strain RL3-8 (FGSC stock number 2218) was cultured in Vogels minimal medium for 48 h at 27oC. Two agar slants (one week old) were used to inoculate 400 ml of medium. Mycelia were harvested by filtering through cheesecloth. The mycelial mat was weighed, frozen in liquid nitrogen and stored at -70oC. Homogenization was carried out by disintegrating the mat at the temperature of the liquid nitrogen in mortar with pestle, then by mixing the powder with 4 volumes/weight extraction buffer (50 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.1% vol/vol. beta-mercaptoethanol) containing a mixture of freshly diluted protease inhibitors (5 mM benzamidine, 0.2 mM phenylmethyl sulfonyl fluoride, 1 mM ophenanthroline). Extraction was completed by mixing the ice cold homogenate at full speed for 1 min in an MSE blender. The extract was filtered through glass wool and was centrifuged at 4oC and 15,000 g for 15 min. The supernatant was used as crude extract in the phosphatase assays. We found that small aliquots (0.1-0.5 ml) of extract can be stored, after being frozen in liquid nitrogen, at -70oC for 7 days without significant loss of phosphatase activity. PP1 and PP2A were assayed with rabbit muscle phosphorylase a substrate. Phosphorylase a was prepared from phosphorylase b with [gamma-32P]ATP and phosphorylase kinase. The assay mixture contained 5 uM 32P-phosphorylase a dimer and 5 mM caffeine in the extraction buffer in a total volume of 30 ul. The reaction was initiated by the addition of the substrate and was terminated after 10 min incubation at 30oC by the addition of 100 ul 10% TCA. TCA soluble radioactivity was counted by Cherenkov radiation. Maximal specific activity was obtained if the Published by New Prairie Press, 2017 extract was diluted with extraction buffer, so that less than 20% of the total 32P was released from the substrate. To test if the liberated radioactivity was in Pi or small phosphopeptide(s) we converted Pi into phosphomolybdic acid and extracted it with organic solvents (Antoniw and Cohen 1976 Eur. J. Biochem. 68:45-54). More than 95% of the radioactivity was recovered in the organic phase. In addition, 20 mM NaF (a well known phosphatase inhibitor) reduced the activity to 5-8%. These facts demonstrate that the assay measures phosphatase activity and the effect of proteases can be neglected. After establishing the assay system we investigated the effect of specific phosphatase inhibitors. One micromolar okadaic acid caused a nearly complete inhibition with the half maximal effect at 10 nM (Fig.1.). Since the IC50 of okadaic acid is 0.1 nM for PP2A and 1520 nM for PP1 in mammalian tissue extracts (Cohen et al. 1989 FEBS Lett. 250:596-600) we assumed that most of the phosphorylase phosphatase activity in N. crassa extract was due to PP1. To test this hypothesis we measured the effect of okadaic acid in the presence of an excess of inhibitor-2. As shown in Fig. 1, under these conditions the activity was half maximally inhibited by 0.3 nM okadaic acid and 90% inhibition was observed at 1 nM concentration. The sensitivity of N. crassa PP1 to the rabbit muscle inhibitor-2 was demonstrated in Fig.2. When PP2A was inactivated with 1 nM okadaic acid, 10,000 U/ml inhibitor-2 caused complete inhibition. 50% inhibition was observed at 30 U/ml concentration i.e. around 1 U inhibitor-2 in the 30 æl assay mixture elicited half maximal inhibition. http://newprairiepress.org/fgr/vol41/iss1/26 DOI: 10.4148/1941-4765.1388 Figure 1. Inhibition of phosphorylase phosphatase activity by okadaic acid in the presence and absence of inhibitor-2 (I-2) Published by New Prairie Press, 2017 Figure 2. Inhibition of phosphorylase phosphatase activity by inhibitor-2 in the presence of okadaic acid (OA) Our results indicate that the phosphorylase phosphatase activity inhibited by 1 nM okadaic acid can be attributed to PP2A and the one inhibited by 10,000 U/ml inhibitor-2 belongs to PP1. Protein concentration was measured according to Read and Northcote (1981 Anal. Biochem. 116:53-64). Using these methods we found 4.95 +/0.9 mU/mg protein (n=10) of total phosphorylase phosphatase activity in N. crassa mycelial extract, 20% of which was PP2A and 65% PP1. By definition one unit of the enzyme liberated 1 umol Pi in 1 min under the above conditions. PP2B was assayed with rabbit muscle inhibitor-1. The substrate was phosphorylated with [gamma-32P]ATP and bovine cAMP-dependent protein kinase. The reaction mixture contained 3 uM 32P-inhibitor-1, 40 mM Tris/HCl pH 7.0, 0.4 mM DTT, 0.2 mM CaCl2, 1 mM MnCl2 and 12 uM calmodulin in 30 ul total volume. The reaction was started by the addition of substrate and after 10 min incubation at 30oC it was terminated by the addition of 100 ul 20% TCA and 100 ul 6% BSA. The released radioactivity was counted as above. Using the extraction method, we proved that more than 95% of the TCA soluble radioactivity was due to the presence of 32Pi. About 40% of the total activity was inhibited by 0.5 mM EDTA. The activity stimulated by http://newprairiepress.org/fgr/vol41/iss1/26 DOI: 10.4148/1941-4765.1388 Ca2+-calmodulin in the presence of Mn2+ was attributed to PP2B, thus the specific activity of PP2B was estimated to be 0.044 +/0.01 U/mg protein (n=6) in the extracts. One unit of PP2B liberated 1 nmol Pi in 1 min in the above assay. The assays described in the present communication are suitable to test protein phosphatase mutants by measuring crude N. crassa extracts, and provide a handle for the purification and characterization of the respective enzymes. The results show considerable similarity between the N. crassa and animal protein phosphatases suggesting that cloning of PP1 and PP2A catalytic subunits could be possible using heterologous cDNA probes. Acknowledgements. This work was supported by the grants OTKA 6005 and 1501. Sz.B. is a recipient of a fellowship from the Hungarian Academy of Sciences, and the Foundation for the Hungarian Science. Published by New Prairie Press, 2017

The UV excision-repair system of Saccharomyces cerevisiae is involved in the removal of methylcytosines formed in vivo by a cloned prokaryotic DNA methyltransferase

SummaryDNA methyltransferase activity is not normally found in yeast. To investigate the response of Saccharomyces cerevisiae to the presence of methylated bases, we introduced the Bacillus subtilis SPR phage DNA-[cytosine-5] methyltransferase gene on the shuttle vector, YEp51. The methyltransferase gene was functionally expressed in yeast under the control of the inducible yeast GAL10 promoter. Following induction we observed a time-dependent methylation of yeast DNA in RAD+ and rad2 mutant strains; the rad2 mutant is defective in excision-repair of UV-induced DNA damage. Analysis of restriction endonuclease digestion patterns revealed that the relative amount of methylated DNA was greater in the excision defective rad2 mutant than in the RAD+ strain. These data indicate that the yeast excision-repair system is capable of recognizing and removing m5C residues.

A DNA uptake‐stimulating protein increases the antiproliferative effect of c‐myb antisense oligonucleotide on leukemic cells

Proliferation of HL‐60 and MOLT4 leukemia cells was inhibited by a c‐myb antisense oligonucleotide (ASO) in the presence of a DNA uptake‐stimulating protein (DNA uptake‐stimulating factor, DUSF). The inhibitory effect was very mild in the absence of DUSF. Sense oligonucleotides or DUSF, alone or in combination, were found to be ineffective. Cellular expression of the c‐myb protein was significantly more inhibited by the c‐myb ASO in the presence than in the absence of DUSF. In the presence of DUSF, c‐myb protein practically disappeared from the nuclei of HL‐60 and MOLT4 cells treated with the ASO. Thus, DUSF appears to effectively stimulate the uptake of c‐myb ASO into tumor cells in vitro, augmenting its antiproliferative effect by decreasing c‐myb expression.

Expression of the Aspergillus bimG gene in Neurospora crassa

In A. nidulans the bimG gene codes for the catalytic subunit of protein phosphatase 1. The wild type bimG gene was transformed into N. crassa and expressed under the direction of the alcA promoter. The heterologous bimG mRNA and protein were detected in the transformants by RT-PCR and Western blotting, respectively. However, the transformation did not result in detectable changes in phenotype. This work demonstrates that the alcA promoter, a conditional gene expression system widely used in both Aspergillus and higher plants, also functions in N. crassa. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Authors B Csóka, T Kókai Zeke, J Doonan, H Fox, Z Fehér, and V Dombrádi This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol49/iss1/5