Motoko Ohnishi

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.

Protein Phosphatase 2C Inactivates F-actin Binding of Human Platelet Moesin

During activation of platelets by thrombin phosphorylation of Thr558 in the C-terminal domain of the membrane-F-actin linking protein moesin increases transiently, and this correlates with protrusion of filopodial structures. Calyculin A enhances phosphorylation of moesin by inhibition of phosphatases. To measure this moesin-specific activity, a nonradioactive enzyme-linked immunosorbent assay method was developed with the synthetic peptideCys-Lys555-Tyr-Lys-Thr(P)-Leu-Arg560coupled to bovine serum albumin as the substrate and moesin phosphorylation state-specific polyclonal antibodies for the detection and quantitation of dephosphorylation. Calyculin A-sensitive and -insensitive protein-threonine phosphatase activities were detected in platelet lysates and separated by DEAE-cellulose chromatography. The calyculin A-sensitive enzyme was identified as a type 1 protein phosphatase. The calyculin A-insensitive enzyme activity was purified to homogeneity by phenyl- Sepharose, protamine-, and phosphonic acid peptide-agarose chromatography and characterized biochemically and immunologically as a 53-kDa protein(s) and a type 2C protein phosphatase (PP2C). Phosphorylation of Thr558 is necessary for F-actin binding of moesin in vitro. The purified enzyme, as well as bacterially made PP2Cα and PP2Cβ, efficiently dephosphorylate(s) highly purified platelet phospho-moesin. This reverses the activating effect of phosphorylation, and moesin no longer co-sediments with actin filaments. In vivo, regulation of these phosphatase activities are likely to influence dynamic interactions between the actin cytoskeleton and membrane constituents linked to moesin.

Sanguinarine as a Potent and Specific Inhibitor of Protein Phosphatase 2C in Vitro and Induces Apoptosis via Phosphorylation of p38 in HL60 Cells

Sanguinarine, a plant alkaloid, was identified as a potent and specific protein phosphatase (PP) 2C inhibitor. It inhibited PP2C competitively with respect to α-casein (Ki=0.68 μM) and showed selectivity for PP2C as compared with PP1, PP2A, and PP2B in vitro. In vivo, sanguinarine showed cytotoxicity toward human promyelocytic leukemia cell line HL60, with an IC50 value of 0.37 μM, and induced apoptosis through a caspase-3/7-dependent mechanism involving the phosphorylation of p38, a PP2Cα substrate. The apoptosis activity induced by sanguinarine was partially inhibited by a p38 inhibitor, SB203580, and was involved in the phospho-p38 protein in HL60 cells.

Activation of c-Jun amino-terminal kinase is required for retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells

P19 embryonal carcinoma cells are known to differentiate into neurons and glia when treated with relatively high concentrations (>100 nM) of retinoic acid (RA). Concomitant with this RA‐induced neural differentiation, we observed an activation of the c‐Jun amino‐terminal kinase (JNK). JNK was required for the RA‐induced neural differentiation, because dominant‐negative JNK blocked the differentiation. Studies using protein phosphatase inhibitors and protein kinase inhibitors suggested that both okadaic acid‐sensitive protein phosphatase(s) and protein kinase C participate in the RA‐induced activation of JNK.

Pisiferdiol and pisiferic acid isolated from Chamaecyparis pisifera activate protein phosphatase 2C in vitro and induce caspase-3/7-dependent apoptosis via dephosphorylation of Bad in HL60 cells

Protein phosphatase 2C (PP2C) dephosphorylates a broad range of substrates and regulates apoptosis, stress response and growth-related pathways. In the course of screening for PP2C activators from natural sources, we isolated abietane-type diterpenes, pisiferdiol and pisiferic acid from Chamaecyparis pisifera. Pisiferdiol having a unique seven-membered ring showed more specific PP2C activation activity (1.3-fold at 100 microM) than pisiferic acid having a normal six-membered ring and oleic acid, which is known to activate PP2C. Pisiferdiol and pisiferic acid showed mixed-type activation with respect to alpha-casein, and this differed from the non-competitive activation of oleic acid in vitro. In vivo, the cytotoxicity of pisiferdiol toward human promyelocytic leukemia cell line HL60 with an IC(50) value of 18.3 microM was 2-fold and 7-fold stronger than those of pisiferic acid and oleic acid, and pisiferdiol induced apoptosis through a caspase 3/7-dependent mechanism involving the dephosphorylation of Bad(1), which is a PP2C substrate. We thus conclude that pisiferdiol and pisiferic acid are novel PP2C activators, and the more specific activator, pisiferdiol, may be a useful chemical probe to study PP2C-mediated signaling pathways, and a lead compound for pharmaceutical agents.

Disruption of the mouse protein Ser/Thr phosphatase 2Cβ gene leads to early pre-implantation lethality

Protein phosphatase 2Cbeta (PP2Cbeta) is a member of a family of protein Ser/Thr phosphatases (PP2C) that is composed of at least twelve different gene products. Recent studies have revealed that PP2Cbeta mRNA accumulates in mature sperm, unfertilized metaphase II-arrested oocytes and zygotes, but that the mRNA level then decreases sharply between the early two-cell and eight-cell stages, remaining at low levels during the 16-cell to blastocyst stages of mice. These observations raised the possibility that PP2Cbeta plays a crucial role during gametogenesis, fertilization, and/or early stages of embryonic development. In this study, we employed a gene knockout technique in mice to test this possibility. We found that PP2Cbeta(Delta/wt) mice generate normal mature gametes. However, PP2Cbeta(Delta/Delta) embryos die between the two-cell and eight-cell stages. To our interest, PP2Cbeta(Delta/Delta) ES cells which had been generated by transfecting PP2Cbeta(3lox/3lox) ES cells with Cre-expressing plasmid were viable. In addition, knockdown of PP2Cbeta using siRNA did not affect the proliferation of wild-type ES cells. These observations suggest that relatively high PP2Cbeta expression is specifically required during the early stages of pre-implantation development. The possible mechanisms for the early pre-implantation lethality of PP2Cbeta(Delta/Delta) mice are discussed.

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.

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.