Takayasu Kobayashi

Functional counterparts of mammalian protein kinases PDK1 and SGK in budding yeast

BACKGROUND In animal cells, recruitment of phosphatidylinositol 3-kinase by growth factor receptors generates 3-phosphoinositides, which stimulate 3-phosphoinositide-dependent protein kinase-1 (PDK1). Activated PDK1 then phosphorylates and activates downstream protein kinases, including protein kinase B (PKB)/c-Akt, p70 S6 kinase, PKC isoforms, and serum- and glucocorticoid-inducible kinase (SGK), thereby eliciting physiological responses. RESULTS We found that two previously uncharacterised genes of Saccharomyces cerevisiae, which we term PKH1 and PKH2, encode protein kinases with catalytic domains closely resembling those of human and Drosophila PDK1. Both Pkh1 and Pkh2 were essential for cell viability. Expression of human PDK1 in otherwise inviable pkh1Delta pkh2Delta cells permitted growth. In addition, the yeast YPK1 and YKR2 genes were found to encode protein kinases each with a catalytic domain closely resembling that of SGK; both Ypk1 and Ykr2 were also essential for viability. Otherwise inviable ypk1Delta ykr2Delta cells were fully rescued by expression of rat SGK, but not mouse PKB or rat p70 S6 kinase. Purified Pkh1 activated mammalian SGK and PKBalpha in vitro by phosphorylating the same residue as PDK1. Pkh1 activated purified Ypk1 by phosphorylating the equivalent residue (Thr504) and was required for maximal Ypk1 phosphorylation in vivo. Unlike PKB, activation of Ypk1 and SGK by Pkh1 did not require phosphatidylinositol 3,4,5-trisphosphate, consistent with the absence of pleckstrin homology domains in these proteins. The phosphorylation consensus sequence for Ypk1 was similar to that for PKBalpha and SGK. CONCLUSIONS Pkh1 and Pkh2 function similarly to PDK1, and Ypk1 and Ykr2 to SGK. As in animal cells, these two groups of yeast kinases constitute two tiers of a signalling cascade required for yeast cell growth.

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.

PP2C family members play key roles in regulation of cell survival and apoptosis

Although unlimited proliferation of cancer cells is supported by multiple signaling pathways involved in the regulation of proliferation, survival, and apoptosis, the molecular mechanisms coordinating these different pathways to promote the proliferation and survival of cancer cells have remained unclear. SAPK and integrin‐ILK signaling pathways play key roles in the promotion of apoptosis and cell proliferation/survival, respectively. Studies of TNFα‐ and H2O2‐induced apoptosis revealed that ASK1, a component of the SAPK system, mediates the TNFα and H2O2 signaling of apoptosis. ASK1 is activated by autophosphorylation of a specific threonine residue (T845) following TNFα stimulation. Our recent studies indicate that PP2Cɛ, a member of the PP2C family, associates with and inactivates ASK1 by dephosphorylating T845. In contrast, PP2Cδ/ILKAP, a second PP2C family member, activates ASK1 by enhancing cellular phosphorylation of T845. PP2Cδ/ILKAP also forms a complex with ILK1 to inhibit the GSK3β‐mediated integrin‐ILK1 signaling in vivo, inhibiting cell cycle progression. These observations raise the possibility that PP2Cδ/ILKAP acts to control the cross‐talk between integrin‐induced and TNFα‐induced signaling pathways, inhibiting the former and stimulating the latter, thereby inhibiting proliferation and survival and promoting the apoptosis of cancer cells. (Cancer Sci 2006; 97: 563–567)

Protein Phosphatase 2C∈ Is an Endoplasmic Reticulum Integral Membrane Protein That Dephosphorylates the Ceramide Transport Protein CERT to Enhance Its Association with Organelle Membranes

Protein phosphatase 2Cϵ (PP2Cϵ), a mammalian PP2C family member, is expressed in various tissues and is implicated in the negative regulation of stress-activated protein kinase pathways. We show that PP2Cϵ is an endoplasmic reticulum (ER) transmembrane protein with a transmembrane domain at the amino terminus and the catalytic domain facing the cytoplasm. Yeast two-hybrid screening of a human brain library using PP2Cϵ as bait resulted in the isolation of a cDNA that encoded vesicle-associated membrane protein-associated protein A (VAPA). VAPA is an ER resident integral membrane protein involved in recruiting lipid-binding proteins such as the ceramide transport protein CERT to the ER membrane. Expression of PP2Cϵ resulted in dephosphorylation of CERT in a VAPA expression-dependent manner, which was accompanied by redistribution of CERT from the cytoplasm to the Golgi apparatus. The expression of PP2Cϵ also enhanced the association between CERT and VAPA. In addition, knockdown of PP2Cϵ expression by short interference RNA attenuated the interaction between CERT and VAPA and the sphingomyelin synthesis. These results suggest that CERT is a physiological substrate of PP2Cϵ and that dephosphorylation of CERT by PP2Cϵ may play an important role in the regulation of ceramide trafficking from the ER to the Golgi apparatus.

Protein Phosphatase-2Cα as a Positive Regulator of Insulin Sensitivity through Direct Activation of Phosphatidylinositol 3-Kinase in 3T3-L1 Adipocytes

During differentiation, expression of protein phosphatase-2Cα (PP2Cα) is increased in 3T3-L1 adipocytes. To elucidate the role of PP2Cα in insulin signaling, we overexpressed wild-type (WT) PP2Cα by adenovirus-mediated gene transfer in 3T3-L1 adipocytes. Overexpression of PP2Cα-WT enhanced the insulin sensitivity of glucose uptake without any changes in the early steps of insulin signaling. Infection with adenovirus 5 expressing PP2Cα-WT increased phosphatidylinositol 3-kinase (PI3K) activities in the immunoprecipitate using antibody against the p85 or p110 subunit under both basal and insulin-stimulated conditions, followed by activation of downstream steps in the PI3K pathway, such as phosphorylation of Akt, glycogen synthase kinase-3, and atypical protein kinase C. In contrast, overexpression of the phosphatase-defective mutant PP2Cα(R174G) did not produce such effects. Furthermore, overexpression of PP2Cα-WT (but not PP2Cα(R174G)) decreased the 32P-labeled phosphorylation state as well as the gel mobility shift of the p85 subunit, suggesting that dephosphorylation of the p85 subunit by PP2Cα activation might stimulate PI3K catalytic activity. Moreover, knockdown of PP2Cα by transfection of small interfering RNA led to a significant decrease in Akt phosphorylation. In addition, microinjection of anti-PP2Cα antibody or PP2Cα small interfering RNA led to decreased insulin-stimulated GLUT4 translocation. In conclusion, PP2Cα is a new positive regulator of insulin sensitivity that acts through a direct activation of PI3K in 3T3-L1 adipocytes.

Negative regulation of protein phosphatase 2Cβ by ISG15 conjugation

ISG15, an interferon‐upregulated ubiquitin‐like protein, is covalently conjugated to various cellular proteins (ISGylation). In this study, we found that protein phosphatase 2Cβ (PP2Cβ), which functions in the nuclear factor κB (NF‐κB) pathway via dephosphorylation of TGF‐β‐activated kinase, was ISGylated, and analysis by NF‐κB luciferase reporter assay revealed that PP2Cβ activity was suppressed by co‐expression of ISG15, UBE1L, and UbcH8. We determined the ISGylation sites of PP2Cβ and constructed its ISGylation‐resistant mutant. In contrast to the wild type, this mutant suppressed the NF‐κB pathway even in the presence of ISG15, UBE1L, and UbcH8. Thus, we propose that ISGylation negatively regulates PP2Cβ activity.

Regulation of apoptosis signal-regulating kinase 1 by protein phosphatase 2Cϵ

ASK1 (apoptosis signal-regulating kinase 1), a MKKK (mitogen-activated protein kinase kinase kinase), is activated in response to cytotoxic stresses, such as H2O2 and TNFalpha (tumour necrosis factor alpha). ASK1 induction initiates a signalling cascade leading to apoptosis. After exposure of cells to H2O2, ASK1 is transiently activated by autophosphorylation at Thr845. The protein then associates with PP5 (protein serine/threonine phosphatase 5), which inactivates ASK1 by dephosphorylation of Thr845. Although this feedback regulation mechanism has been elucidated, it remains unclear how ASK1 is maintained in the dephosphorylated state under non-stressed conditions. In the present study, we have examined the possible role of PP2Cepsilon (protein phosphatase 2Cepsilon), a member of PP2C family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2Cepsilon inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conversely, a dominant-negative PP2Cepsilon mutant enhanced AP-1 activity. Exogenous PP2Cepsilon associated with exogenous ASK1 in HEK-293 cells under non-stressed conditions, inactivating ASK1 by decreasing Thr845 phosphorylation. The association of endogenous PP2Cepsilon and ASK1 was also observed in mouse brain extracts. PP2Cepsilon directly dephosphorylated ASK1 at Thr845 in vitro. In contrast with PP5, PP2Cepsilon transiently dissociated from ASK1 within cells upon H2O2 treatment. These results suggest that PP2Cepsilon maintains ASK1 in an inactive state by dephosphorylation in quiescent cells, supporting the possibility that PP2Cepsilon and PP5 play different roles in H2O2-induced regulation of ASK1 activity.

Mitochondrial DNA repair by photolyase

Photolyase genes of Saccharomyces cerevisiae and Escherichia coli were expressed in S. cerevisiae and photoreactivation in nuclei and mitochondria of the host cells was analyzed by determination of survival and petit rates. Yeast photolyase was able to repair mitochondrial DNA effectively, whereas E. coli photolyase could reduce only a small fraction of the petit rate produced by UV irradiation. Analysis using fusion between yeast photolyase and E. coli lacZ genes as well as a chimeric gene between yeast and E. coli photolyase genes suggests the importance of the protruding amino terminal region of the yeast photolyase for its transport into mitochondria. A significant similarity between the protruding amino termini of yeast photolyase and yeast uracil-DNA-glycosylase suggests a common functional importance of the terminal sequences for both DNA repair enzymes.

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.