Yuchio Yanagawa

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.

Enrichment and efficient screening of ES cells containing a targeted mutation: the use of DT‐A gene with the polyadenylation signal as a negative selection maker

Gene targeting in embryonic stem (ES) cells via homologous recombination can occur at very low frequency. In order to enrich homologous recombinants before screening, a negative selection marker, such as thymidine kinase (TK) and diphtheria toxin A fragment (DT‐A), has been commonly used. In this study, we developed a negative selection marker using DT‐A gene with polyadenylation signal and it was designated DT‐ApA. To determine the difference in targeting efficiency of the negative selections, we constructed three different targeting vectors for each negative selection (first, TK at the 3′ end, second, TK at both the 5′ and 3′ ends <2 X TK>, and third, DT‐ApA at the 5′ end of the homologous sequences). Gene targeting experiments using these constructs clearly showed that negative selection using DT‐ApA was more efficient than that using TK for homologous recombination and that negative selection using DT‐ApA was as efficient as that using 2 X TK. Considering the fact that the use of DT‐ApA is more convenient for construction of targeting vectors than that of 2 X TK, DT‐ApA is an efficient negative selection marker.In addition, we examined long and accurate PCR (LA‐PCR) for screening gene targeted clones. The use of LA‐PCR with genomic DNAs from ES cell clones facilitated simple detection of homologous recombinants, suggesting that the screening with LA‐PCR is compatible with the use of longer homologous sequences of both arms in vector design. Our results indicate that the use of DT‐ApA for negative selection together with the application of LA‐PCR for screening ensures efficient and time‐saving screening for homologous recombinants.

Differentiation‐dependent enhanced expression of protein phosphatase 2Cβ in germ cells of mouse seminiferous tubules

The presence of five distinct isoforms of protein phosphatase 2Cβ (PP2Cβ1∼‐5) is known. In this study, we demonstrate that the mRNA levels of PP2Cβ‐3, ‐4 and ‐5 and PP2Cβ protein level increased during the course of the first wave of spermatogenesis in neonatal mouse testis. Northern blot and in situ hybridization analyses revealed that PP2Cβ‐3, ‐4 and ‐5 were expressed predominantly in pachytene spermatocytes and in more highly differentiated germ cells. The substrate specificity of PP2Cβ‐4 determined with artificial substrates differed from those of PP2Cβ‐3 and ‐5, suggesting that the difference in the structure of PP2Cβ‐3, ‐4 and ‐5 reflect their unique physiological functions in testicular germ cells.

Isoform specific phosphorylation of protein phosphatase 2C expressed in COS7 cells

Of the six distinct isoforms of mouse protein phosphatase 2C (PP2C) (α, β‐1, β‐2, β‐3, β‐4 and β‐5), PP2Cα was specifically phosphorylated on the serine residue(s) when expressed in COS7 cells. Analysis of phosphorylation sites using site‐directed mutagenesis demonstrated that Ser‐375 and/or Ser‐377 were phosphorylated in vivo. These serine residues were the sites of phosphorylation by casein kinase II in vitro. Phosphorylation of PP2Cα was enhanced two‐fold by the addition of okadaic acid to the culture medium, but addition of cyclosporin A had no such effect. These results suggest that the expressed PP2Cα is phosphorylated by a casein kinase II‐like protein kinase and dephosphorylated by PP1 and/or PP2A in COS7 cells.

Enhanced UV sensitivity of yeast cells induced by overexpression of Mg2+-dependent protein phosphatase α (type 2C α)

The UV sensitivity of wild-type Saccharomyces cerevisiae cells was increased 2-fold when rat Mg(2+)-dependent protein phosphatase alpha (protein phosphatase type 2C alpha) was overexpressed in the cells. The overexpression of this enzyme rendered the rad 18 mutant (defective in postreplication repair) more UV-sensitive than was observed in the wild-type cells. However, this increase in UV sensitivity disappeared when the host cells had a rad 1 mutation (defective in excision repair). These results suggest that the Mg(2+)-dependent protein phosphatase overexpressed in the yeast cells inhibited their excision repair system.