Koji Katsura

Selective suppression of stress-activated protein kinase pathway by protein phosphatase 2C in mammalian cells

Protein phosphatase 2Cα (PP2Cα) or PP2Cβ‐1 expressed in COS7 cells suppressed anisomycin‐ and NaCl‐enhanced phosphorylations of p38 co‐expressed in the cells. PP2Cα or PP2Cβ‐1 expression also suppressed both basal and stress‐enhanced phosphorylations of MKK3b and MKK6b, which are upstream protein kinases of p38, and of MKK4, which is one of the major upstream protein kinases of JNK. Basal activity of MKK7, another upstream protein kinase of JNK, was also suppressed by PP2Cα or PP2Cβ‐1 expression. However, basal as well as serum‐activated phosphorylation of MKK1a, an upstream protein kinase of ERKs, was not affected by PP2Cβ or PP2Cβ‐1. A catalytically inactive mutant of PP2Cβ‐1 further enhanced the NaCl‐stimulated phosphorylations of MMK3b, MKK4 and MKK6b, suggesting that this mutant PP2Cβ‐1 works as a dominant negative form. These results suggest that PP2C selectively inhibits the SAPK pathways through suppression of MKK3b, MKK4, MKK6b and MKK7 activities in mammalian cells.

A novel protein phosphatase 2C family member (PP2Cζ) is able to associate with ubiquitin conjugating enzyme 91

In this study we have cloned a novel member of mouse protein phosphatase 2C family, PP2Cζ, which is composed of 507 amino acids and has a unique N‐terminal region. The overall similarity of the amino acid sequence between PP2Cζ and PP2Cα was 22%. On Northern blot analysis PP2Cζ was found to be expressed specifically in the testicular germ cells. PP2Cζ expressed in COS7 cells was able to associate with ubiquitin conjugating enzyme 9 (UBC9) and the association was enhanced by co‐expression of small ubiquitin‐related modifier‐1 (SUMO‐1), suggesting that PP2Cζ exhibits its specific role through its SUMO‐induced recruitment to UBC9.

The complete nucleotide sequence and characterization of the linear DNA plasmid pRS64-2 from the plant pathogenic fungus Rhizoctonia solani

Abstract The complete nucleotide sequence of one of three linear DNA plasmids (pRS64-2) from the plant pathogenic fungus Rhizoctonia solani was determined. The pRS64-2 DNA consisted of 2877 nucleotides. The nucleotide sequences of the middle 2.2-kb regions of the other two plasmids (pRS64-1 and pRS64-3) were also determined. Comparison of the nucleotide sequences among the three plasmid DNAs indicated the presence of four regions with more than 86% sequence homology, suggesting the development of three plasmid DNAs from a common ancestor. A computer-based study of the pRS64-2 DNA-folding at both termini predicted hairpin loop structures. The hairpin loops consisted of the left- and right-hand terminal 113 and 105 nucleotides, respectively, and had no sequence homology. They had not undergone flip-flop inversion. The hairpins form cruciform base-paired structures.

Expression and localization of the linear DNA plasmid-encoded protein (RS224) in Rhizoctonia solani AG2-2

Expression of the linear DNA plasmid-encoded protein (RS224) from the plant-pathogenic fungus Rhizoctonia solani isolate H-16, anastomosis group 2-2, and its localization were studied. Extracts from Escherichia coli cells expressing the open reading frame (ORF) of RS224 (RS224ORF in pRS224) contain a 92-kDa T7.Tag-RS224orf fusion protein. Antisera raised against the fusion protein obtained from E. coli cells cross-reacted with a 90-kDa protein in the mycelia. To analyze the subcellular localization of the 92-kDa protein, mycelia of R. solani were disrupted and fractionated. Antibodies against RS224 proteins specifically reacted to the mitochondrial fraction, suggesting that RS224 is localized in mitochondria.

Protoplast Fusion and DNA Plasmid Characterisation in Rhizoctonia Solani

Protoplasts have been used routinely for physiological and biological investigations. More recently, protoplasts have been frequently used for genetic studies. Two methods have been developed for introducing favorable genes into microbial protoplasts (Figure 1). One method, protoplast fusion (Figure 1) serves as a convenient method for introducing genes from one fungal isolate to another. The protoplast fusion process has been shown to generate a large number of random recombinants. Furthermore, because the protoplast fusion is observed not only among different strains but also different species, this method of gene transfer may be advantageous to improve fungal strains. The other method, genetic transformation of protoplasts by the uptake of recombinant DNA (Figure 1) is widely used.