Rcn1, the fission yeast homolog of human DSCR1, regulates arsenite tolerance independently from calcineurin.

Calcineurin (CN) is a conserved Ca2+/calmodulin-dependent phosphoprotein phosphatase that plays a key role in Ca2+ signaling. Regulator of calcineurin 1 (RCAN1), also known as Down syndrome critical region gene 1 (DSCR1), interacts with calcineurin and inhibits calcineurin-dependent signaling in various organisms. Ppb1, the fission yeast calcineurin regulates Cl--homeostasis, and Ppb1 deletion induces MgCl2 hypersensitivity. Here, we characterize the conserved and novel roles of the fission yeast RCAN1 homolog rcn1+. Consistent with its role as an endogenous calcineurin inhibitor, Rcn1 overproduction reproduced the calcineurin-null phenotypes, including MgCl2 hypersensitivity and inhibition of calcineurin signaling upon extracellular Ca2+ stimuli as evaluated by the nuclear translocation and transcriptional activation of the calcineurin substrate Prz1. Notably, overexpression of rcn1+ causes hypersensitivity to arsenite, whereas calcineurin deletion induces arsenite tolerance, showing a phenotypic discrepancy between Rcn1 overexpression and calcineurin deletion. Importantly, although Rcn1 deletion induces modest sensitivities to arsenite and MgCl2 in wild-type cells, the arsenite tolerance, but not MgCl2 sensitivity, associated with Ppb1 deletion was markedly suppressed by Rcn1 deletion. Collectively, our findings reveal a previously unrecognized functional collaboration between Rcn1 and calcineurin, wherein Rcn1 not only negatively regulates calcineurin in the Cl- homeostasis, but also Rcn1 mediates calcineurin signaling to modulate arsenite cytotoxicity.

ACAGT-007a, an anti-cancer compound that modulates ERK MAPK signaling, induces nuclear enrichment of phosphorylated ERK in T3M4 pancreatic cancer cells.

The extracellular-signal-regulated-kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as pancreatic cancers. ACAGT-007a (GT-7), an anti-cancer compound, stimulates ERK phosphorylation, thereby inducing growth inhibition and apoptosis in T3M4 pancreatic cancer cells. However, how GT-7 stimulates ERK phosphorylation and induces apoptosis in ERK-active T3M4 cells remains unclear. To look into the mechanism, we performed a spatiotemporal analysis of ERK phosphorylation mediated by GT-7 in T3M4 cells. The immunoblotting showed that GT-7 stimulates ERK phosphorylation within 1 h, which was more remarkable after 2 h. Importantly, apoptosis induction as evaluated by the cleaved Caspase-3 was observed only after 2-h incubation with GT-7. The immunofluorescence staining revealed the enrichment of phosphorylated ERK (phospho-ERK) in the nucleus upon 1-h incubation with GT-7. Fractionation experiments showed that GT-7 increases phospho-ERK levels in the cytoplasm within 1 h, whereas nuclear phospho-ERK accumulation is observed after 2-h incubation with GT-7. MEK inhibition by U0126 significantly diminishes nuclear phospho-ERK distribution and apoptosis induction stimulated by GT-7. Thus, GT-7 may initiate the induction of ERK phosphorylation in the cytoplasm, which leads to phospho-ERK enrichment in the nucleus. This nuclear phospho-ERK accumulation by GT-7 precedes and may underlie apoptosis induction in T3M4.

Sequestration of the PKC ortholog Pck2 in stress granules as a feedback mechanism of MAPK signaling in fission yeast.

Protein kinase C (PKC) signaling is a highly conserved signaling module that plays a central role in a myriad of physiological processes, ranging from cell proliferation to cell death, via various signaling pathways, including MAPK signaling. Stress granules (SGs) are non-membranous cytoplasmic foci that aggregate in cells exposed to environmental stresses. Here, we explored the role of SGs in PKC/MAPK signaling activation in fission yeast. High-heat stress (HHS) induced Pmk1 MAPK activation and Pck2 translocation from the cell tips into poly(A)-binding protein (Pabp)-positive SGs. Pck2 dispersal from the cell tips required Pck2 kinase activity, and constitutively active Pck2 exhibited increased translocation to SGs. Importantly, Pmk1 deletion impaired Pck2 recruitment to SGs, indicating that MAPK activation stimulates Pck2 SG translocation. Consistently, HHS-induced SGs delayed Pck2 relocalization at the cell tips, thereby blocking subsequent Pmk1 reactivation after recovery from HHS. HHS partitioned Pck2 into the Pabp-positive SG-containing fraction, which resulted in reduced Pck2 abundance and kinase activity in the soluble fraction. Taken together, these results indicate that MAPK-dependent Pck2 SG recruitment serves as a feedback mechanism to intercept PKC/MAPK activation induced by HHS, which might underlie PKC-related diseases.

Fission Yeast PUF Proteins Puf3 and Puf4 Are Novel Regulators of PI4P5K Signaling.

Phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) is a highly conserved enzyme that generates phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) by phosphorylating phosphatidylinositol 4-phosphate (PI(4)P). Schizosaccharomyces pombe (S. pombe) its3-1 is a loss-of-function mutation in the essential its3+ gene that encodes a PI4P5K. Its3 regulates cell proliferation, cytokinesis, cell integrity, and membrane trafficking, but little is known about the regulatory mechanisms of Its3. To identify regulators of Its3, we performed a genetic screening utilizing the high-temperature sensitivity (TS) of its3-1 and identified puf3+ and puf4+, encoding Pumilio/PUF family RNA-binding proteins as multicopy suppressors of its3-1 cells. The deletions of the PUF domains in the puf3+ and puf4+ genes resulted in the reduced ability to suppress its3-1, suggesting that the suppression by Puf3 and Puf4 may involve their RNA-binding activities. The gene knockout of Puf4, but not that of Puf3, exacerbated the TS of its3-1. Interestingly, mutant Its3 expression levels both at mRNA and protein levels were lower than those of the wild-type (WT) Its3. Consistently, the overexpression of the mutant its3-1 gene suppressed the its3-1 phenotypes. Notably, Puf3 and Puf4 overexpression increased the mRNA and protein expression levels of both Its3 and Its3-1. Collectively, our genetic screening revealed a functional relationship between the Pumilio/PUF family RNA-binding proteins and PI4P5K.

Down-regulation of dual-specificity phosphatase 6, a negative regulator of oncogenic ERK signaling, by ACA-28 induces apoptosis in NIH/3T3 cells overexpressing HER2/ErbB2.

Dual-specificity phosphatase 6 (DUSP6) is a key negative feedback regulator of the member of the RAS-ERK MAPK signaling pathway that is associated with cellular proliferation and differentiation. Deterioration of DUSP6 expression could therefore result in deregulated growth activity. We have previously discovered ACA-28, a novel anticancer compound with a unique property to stimulate ERK phosphorylation and induce apoptosis in ERK-active melanoma cells. However, the mechanism of cancer cell-specific-apoptosis by ACA-28 remains obscure. Here, we investigated the involvement of DUSP6 in the mechanisms of the ACA-28-mediated apoptosis by using the NIH/3T3 cells overexpressing HER2/ErbB2 (A4-15 cells), as A4-15 exhibited higher ERK phosphorylation and are more susceptible to ACA-28 than NIH/3T3. We showed that A4-15 exhibited high DUSP6 protein levels, which require ERK activation. Notably, the silencing of the DUDSP6 gene by siRNA inhibited proliferation and induced apoptosis in A4-15, but not in NIH/3T3, indicating that A4-15 requires high DUSP6 expression for growth. Importantly, ACA-28 preferentially down-regulated the DUSP6 protein and proliferation in A4-15 via the proteasome, while it stimulated ERK phosphorylation. Collectively, the up-regulation of DUSP6 may exert a growth-promoting role in cancer cells overexpressing HER2. DUSP6 down-regulation in ERK-active cancer cells might have the potential as a novel cancer measure.

Protein Kinase N Family Negatively Regulates Constitutive Androstane Receptor-Mediated Transcriptional Induction of Cytochrome P450 2b10 in the Livers of Mice.

In receptor-type transcription factors-mediated cytochrome P450 (P450) induction, few studies have attempted to clarify the roles of protein kinase N (PKN) in the transcriptional regulation of P450s. This study aimed to examine the involvement of PKN in the transcriptional regulation of P450s by receptor-type transcription factors, including the aryl hydrocarbon receptor, constitutive androstane receptor (CAR), and pregnane X receptor. The mRNA and protein levels and metabolic activity of P450s in the livers of wild-type (WT) and double-mutant (D) mice harboring both PKN1 kinase-negative knock-in and PKN3 knockout mutations [PKN1 T778A/T778A; PKN3 -/-] were determined after treatment with activators for receptor-type transcription factors. mRNA and protein levels and metabolic activity of CYP2B10 were significantly higher in D mice treated with the CAR activator phenobarbital (PB) but not with 1,4-bis((3,5-dichloropyridin-2-yl)oxy)benzene compared with WT mice. We examined the CAR-dependent pathway regulated by PKN after PB treatment because the extent of CYP2B10 induction in WT and D mice was notably different in response to treatment with different CAR activators. The mRNA levels of Cyp2b10 in primary hepatocytes from WT and D mice treated with PB alone or in combination with Src kinase inhibitor 1 (SKI-1) or U0126 (a mitogen-activated protein kinase inhibitor) were evaluated. Treatment of hepatocytes from D mice with the combination of PB with U0126 but not SKI-1 significantly increased the mRNA levels of Cyp2b10 compared with those from the corresponding WT mice. These findings suggest that PKN may have inhibitory effects on the Src-receptor for activated C kinase 1 (RACK1) pathway in the CAR-mediated induction of Cyp2b10 in mice livers. SIGNIFICANCE STATEMENT: This is the first report of involvement of PKN in the transcriptional regulation of P450s. The elucidation of mechanisms responsible for induction of P450s could help optimize the pharmacotherapy and improve drug development. We examined whether the mRNA and protein levels and activities of P450s were altered in double-mutant mice harboring both PKN1 kinase-negative knock-in and PKN3 knockout mutations. PKN1/3 negatively regulates CAR-mediated induction of Cyp2b10 through phosphorylation of a signaling molecule in the Src-RACK1 pathway.

Chemical genetic analysis of FTY720- and Ca2+ -sensitive mutants reveals a functional connection between FTY720 and membrane trafficking.

FTY720, a sphingosine-1-phosphate (S1P) analog, is used as an immune modulator to treat multiple sclerosis. Accumulating evidence has suggested the mode of action of FTY720 independent of an S1P modulator. In fission yeast, FTY720 induces an increase in intracellular Ca2+ and ROS levels. We have previously identified 49 genes of which deletion causes FTY720 sensitivity. Here, we characterized the FTY720-sensitive mutants in terms of their relevance to the Ca2+ homeostasis and identified the 16 FTY720- and Ca2+ -sensitive mutants (fcs mutants). Most of the FTY720-sensitive mutants showed elevated Ca2+ levels and exhibited Ca2+ dysregulation by FTY720 treatment. One of the functional categories among the genes whose deletion renders cells susceptible to FTY720 and Ca2+ include the Golgi/endosomal membrane trafficking. Notably, FTY720, but not phosphorylated FTY720 incapable of inducing Ca2+ increase, inhibited the secretion of acid phosphatase in the wild-type cells. Importantly, secretory defects of the Golgi/endosomal trafficking mutants, Vps45, or Ryh1 deletion, were further exacerbated by FTY720. Our fcs mutant screen also identified the adenylyl cyclase-associated protein Cap1 and a Rictor homolog Ste20, whose deletion markedly exacerbated FTY720-sensitive secretory impairment. Collectively, our data may suggest a synergistic impact of FTY720 combined with secretion perturbation on proliferation and Ca2+ homeostasis.

Discovery of new benzhydrol biscarbonate esters as potent and selective apoptosis inducers of human melanomas bearing the activated ERK pathway: SAR studies on an ERK MAPK signaling modulator, ACA-28.

The recent discovery that an ERK signaling modulator [ACA-28 (2a)] preferentially kills human melanoma cell lines by inducing ERK-dependent apoptosis has generated significant interest in the field of anti-cancer therapy. In the first SAR study on 2a, here, we successfully developed candidates (2b, 2c) both of which induce more potent and selective apoptosis towards ERK-active melanoma cells than 2a, thus revealing the structural basis for inducing the ERK-dependent apoptosis and proposing the therapeutic prospect of these candidates against ERK-dependent cancers represented by melanoma.

Distinct modes of stress granule assembly mediated by the KH-type RNA-binding protein Rnc1.

We have previously identified the KH-type RNA-binding protein Rnc1 as an important regulator of the posttranscriptional expression of the MAPK phosphatase Pmp1 in fission yeast. Rnc1 localization in response to stress has not been elucidated thus far. Here, we report the dual roles of Rnc1 in assembly of stress granules (SGs), nonmembranous cytoplasmic foci composed of messenger ribonucleoproteins. Rnc1 can localize to poly(A)-binding protein (Pabp)-positive SGs upon various stress stimuli, including heat shock (HS) and arsenite treatment. Furthermore, Rnc1 deletion results in decreased SGs, indicating that Rnc1 is a new component and a regulator of SGs. Notably, Rnc1 translocates to the dot-like structures faster than Pabp, and this stress-induced Rnc1 translocation does not require its RNA-binding ability, as the Rnc1KH1,2,3GD mutant protein with impaired RNA-binding activity forms dots rather more efficiently than the wild-type Rnc1 upon HS. Interestingly, in the absence of stress, Rnc1 overproduction induced massive aggregation of Pabp-positive SGs and eIF2α phosphorylation. In clear contrast, overproduction of the Rnc1KH1,2,3GD mutant failed to induce Pabp aggregation and eIF2α phosphorylation, indicating that Rnc1 overproduction-induced SG assembly requires Rnc1 RNA-binding activity. Collectively, Rnc1 regulates SG assembly, dependently or independently of its RNA-binding activity.

Spatial regulation of the KH domain RNA-binding protein Rnc1 mediated by a Crm1-independent nuclear export system in Schizosaccharomyces pombe.

RNA-binding proteins (RBPs) play important roles in the posttranscriptional regulation of gene expression, including mRNA stability, transport and translation. Fission yeast rnc1+ encodes a K Homology (KH)-type RBP, which binds and stabilizes the Pmp1 MAPK phosphatase mRNA thereby suppressing the Cl- hypersensitivity of calcineurin deletion and MAPK signaling mutants. Here, we analyzed the spatial regulation of Rnc1 and discovered a putative nuclear export signal (NES)Rnc1 , which dictates the cytoplasmic localization of Rnc1 in a Crm1-independent manner. Notably, mutations in the NESRnc1 altered nucleocytoplasmic distribution of Rnc1 and abolished its function to suppress calcineurin deletion, although the Rnc1 NES mutant maintains the ability to bind Pmp1 mRNA. Intriguingly, the Rnc1 NES mutant destabilized Pmp1 mRNA, suggesting the functional importance of the Rnc1 cytoplasmic localization. Mutation in Rae1, but not Mex67 deletion or overproduction, induced Rnc1 accumulation in the nucleus, suggesting that Rnc1 is exported from the nucleus to the cytoplasm via the mRNA export pathway involving Rae1. Importantly, mutations in the Rnc1 KH-domains abolished the mRNA-binding ability and induced nuclear localization, suggesting that Rnc1 may be exported from the nucleus together with its target mRNAs. Collectively, the functional Rae1-dependent mRNA export system may influence the cytoplasmic localization and function of Rnc1.

Skb5, an SH3 adaptor protein, regulates Pmk1 MAPK signaling by controlling the intracellular localization of the MAPKKK Mkh1.

The mitogen-activated protein kinase (MAPK) cascade is a highly conserved signaling module composed of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKK) and MAPKs. The MAPKKK Mkh1 is an initiating kinase in Pmk1 MAPK signaling, which regulates cell integrity in fission yeast (Schizosaccharomyces pombe). Our genetic screen for regulators of Pmk1 signaling identified Shk1 kinase binding protein 5 (Skb5), an SH3-domain-containing adaptor protein. Here, we show that Skb5 serves as an inhibitor of Pmk1 MAPK signaling activation by downregulating Mkh1 localization to cell tips through its interaction with the SH3 domain. Consistent with this, the Mkh1(3PA) mutant protein, with impaired Skb5 binding, remained in the cell tips, even when Skb5 was overproduced. Intriguingly, Skb5 needs Mkh1 to localize to the growing ends as Mkh1 deletion and disruption of Mkh1 binding impairs Skb5 localization. Deletion of Pck2, an upstream activator of Mkh1, impaired the cell tip localization of Mkh1 and Skb5 as well as the Mkh1-Skb5 interaction. Interestingly, both Pck2 and Mkh1 localized to the cell tips at the G1/S phase, which coincided with Pmk1 MAPK activation. Taken together, Mkh1 localization to cell tips is important for transmitting upstream signaling to Pmk1, and Skb5 spatially regulates this process.

Rae1-mediated nuclear export of Rnc1 is an important determinant in controlling MAPK signaling.

In eukaryotic cells, RNA binding proteins (RBPs) play critical roles in regulating almost every aspect of gene expression, often shuttling between the nucleus and the cytoplasm. They are also key determinants in cell fate via controlling the target mRNAs under the regulation of various signaling pathways in response to environmental stresses. Therefore, understanding the mechanisms that couple the location of mRNA and RBPs is a major challenge in the field of gene expression and signal responses. In fission yeast, a KH-type RBP Rnc1 negatively regulates MAPK signaling activation via mRNA stabilization of the dual-specificity MAPK phosphatase Pmp1, which dephosphorylates MAPK Pmk1. Rnc1 also serves as a target of MAPK phosphorylation, which makes a feedback loop mediated by an RBP. We recently discovered that the nuclear export of Rnc1 requires mRNA-binding ability and the mRNA export factor Rae1. This strongly suggested the presence of an mRNA-export system, which recognizes the mRNA/RBP complex and dictates the location and post-transcriptional regulation of mRNA cargo. Here, we briefly review the known mechanisms of general nuclear transporting systems, with an emphasis on our recent findings on the spatial regulation of Rnc1 and its impact on the regulation of the MAPK signal transduction cascade.

Identification of ACA-28, a 1'-acetoxychavicol acetate analogue compound, as a novel modulator of ERK MAPK signaling, which preferentially kills human melanoma cells.

The extracellular signal-regulated kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as melanoma. Melanoma remains incurable despite the use of conventional chemotherapy; consequently, development of new therapeutic agents for melanoma is highly desirable. Here, we carried out a chemical genetic screen using a fission yeast phenotypic assay and showed that ACA-28, a synthetic derivative of 1'-acetoxychavicol acetate (ACA), which is a natural ginger compound, effectively inhibited the growth of melanoma cancer cells wherein ERK MAPK signaling is hyperactivated due to mutations in the upstream activating regulators. ACA-28 more potently inhibited the growth of melanoma cells than did the parental compound ACA. Importantly, the growth of normal human epidermal melanocytes (NHEM) was less affected by ACA-28 at the same 50% inhibitory concentration. In addition, ACA-28 specifically induced apoptosis in NIH/3T3 cells which were oncogenically transformed with human epidermal growth factor receptor-2 (HER2/ErbB2), but not in the parental cells. Notably, the ACA-28-induced apoptosis in melanoma and HER2-transformed cells was abrogated when ERK activation was blocked with a specific MEK inhibitor U0126. Consistently, ACA-28 more strongly stimulated ERK phosphorylation in melanoma cells, as compared in NHEM. ACA-28 might serve as a promising seed compound for melanoma treatment.

Analysis of the Rice ADP-Glucose Transporter (OsBT1) Indicates the Presence of Regulatory Processes in the Amyloplast Stroma That Control ADP-Glucose Flux into Starch.

Previous studies showed that efforts to further elevate starch synthesis in rice (Oryza sativa) seeds overproducing ADP-glucose (ADPglc) were prevented by processes downstream of ADPglc synthesis. Here, we identified the major ADPglc transporter by studying the shrunken3 locus of the EM1093 rice line, which harbors a mutation in the BRITTLE1 (BT1) adenylate transporter (OsBt1) gene. Despite containing elevated ADPglc levels (approximately 10-fold) compared with the wild-type, EM1093 grains are small and shriveled due to the reduction in the amounts and size of starch granules. Increases in ADPglc levels in EM1093 were due to their poor uptake of ADP-[(14)C]glc by amyloplasts. To assess the potential role of BT1 as a rate-determining step in starch biosynthesis, the maize ZmBt1 gene was overexpressed in the wild-type and the GlgC (CS8) transgenic line expressing a bacterial glgC-TM gene. ADPglc transport assays indicated that transgenic lines expressing ZmBT1 alone or combined with GlgC exhibited higher rates of transport (approximately 2-fold), with the GlgC (CS8) and GlgC/ZmBT1 (CS8/AT5) lines showing elevated ADPglc levels in amyloplasts. These increases, however, did not lead to further enhancement in seed weights even when these plant lines were grown under elevated CO2. Overall, our results indicate that rice lines with enhanced ADPglc synthesis and import into amyloplasts reveal additional barriers within the stroma that restrict maximum carbon flow into starch.

Microchip electrophoresis utilizing an in situ photopolymerized Phos-tag binding polyacrylamide gel for specific entrapment and analysis of phosphorylated compounds.

A method was developed for the specific entrapment and separation of phosphorylated compounds using a Phos-tag polyacrylamide gel fabricated at the channel crossing point of a microfluidic electrophoresis chip. The channel intersection of the poly(methyl methacrylate)-made microchip was filled with a solution comprising acrylamide, N,N-methylene-bis-acrylamide, Phos-tag acrylamide, and 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], which functioned as a photocatalytic initiator. In situ polymerization at the channel crossing point was performed by irradiation with a UV LED laser beam. The fabricated Phos-tag gel (100 × 100 × 30 µm) contains ca. 20 fmol of the Phos-tag group and therefore could entrap phosphorylated compounds at the femtomolar level. The electrophoretically trapped phosphorylated compounds were released from the gel by switching the voltage to deliver high concentrations of phosphate and EDTA in a background electrolyte. The broad sample band eluted from the gel was effectively reconcentrated at the boundary of a pH junction generated by sodium ions delivered from the outlet reservoir. The reconcentrated sample components were then separated and fluorometrically detected at the end of the separation channel. Under the optimized conditions, the phosphorylated compounds were concentrated by a factor of 100-fold, and the peak resolution was comparable to that obtained by pinched injection. This method was successfully utilized to preconcentrate and analyze phosphorylated peptides in a complex peptide mixture.

Geranylgeranyltransferase Cwg2-Rho4/Rho5 module is implicated in the Pmk1 MAP kinase-mediated cell wall integrity pathway in fission yeast.

Pmk1, a fission yeast homologue of mammalian ERK MAPK, regulates cell wall integrity, cytokinesis, RNA granule formation and ion homeostasis. Our screen for vic (viable in the presence of immunosuppressant and chloride ion) mutants identified regulators of the Pmk1 MAPK signaling, including Cpp1 and Rho2, based on the genetic interaction between calcineurin and Pmk1 MAPK. Here, we identified the vic2-1 mutants carrying a mis-sense mutation in the cwg2(+) gene encoding a beta subunit of geranylgeranyltransferase I (GGTase I), which participates in the post-translational C-terminal modification of several small GTPases, allowing their targeting to the membrane. Analysis of the vic2-1/cwg2-v2 mutant strain showed that the localization of Rho1, Rho4, Rho5 and Cdc42, both at the plasma and vacuolar membranes, was impaired in the vic2-1/cwg2-v2 mutant cells. In addition, Rho4 and Rho5 deletion cells exhibited the vic phenotype and cell wall integrity defects, shared phenotypes among the components of the Pmk1 MAPK pathway. Consistently, the phosphorylation of Pmk1 MAPK on heat shock was decreased in the cwg2-v2 mutants, and rho4- and rho5-null cells. Moreover, Rho4 and Rho5 associate with Pck1/Pck2. Possible roles of Cwg2, Rho4 and Rho5 in the Pmk1 signaling will be discussed.

Spatial control of calcineurin in response to heat shock in fission yeast.

In fission yeast, Ppb1, the Ca2+/calmodulin-dependent protein phosphatase calcineurin regulates multiple biological processes, such as cytokinesis, Ca2+-homeostasis, membrane trafficking and cell wall integrity. Calcineurin dephosphorylates the Prz1 transcription factor, leading to its nuclear translocation and gene expression under the control of CDRE (calcineurin-dependent response element). Although the calcineurin-mediated spatial control of downstream transcription factors has been intensively studied in many organisms, less is known about the spatial regulation of calcineurin on stresses. Here, we show that heat shock stimulates calcineurin-dependent nuclear translocation of Prz1 and CDRE-dependent gene expression. Notably, calcineurin exhibited a dramatic change in subcellular localization, translocating from diffuse cytoplasmic to dot-like structures on heat shock. The calcineurin dots colocalized with Dcp2 or Pabp, the constituent of P-bodies or stress granules, respectively, thus suggesting that calcineurin is a component of RNA granules under heat shock. Importantly, the calcineurin inhibitor FK506 markedly inhibited the accumulation of calcineurin granules, whereas the constitutively active calcineurin strongly accumulated in the granules on heat shock, suggesting that phosphatase activity is important for calcineurin localization. Notably, the depletion of calcineurin induced a rapid appearance of Nrd1- and Pabp-positive RNA granules. The possible roles of calcineurin in response to heat shock will be discussed.

Chemical genomics approach to identify genes associated with sensitivity to rapamycin in the fission yeast Schizosaccharomyces pombe.

Rapamycin and its derivatives have now emerged as an attractive therapeutic strategy with both immunosuppressant and antitumor properties. In addition, rapamycin has been proposed as a calorie restriction mimetic to extend the life span of various organisms. The fission yeast Schizosaccharomyces pombe (S. pombe) serves as a valuable genetic model system to study the mechanism(s) of drug action as well as to determine genetic contexts associated with drug sensitivity or resistance. Here, we identified genes that when deleted modulate the rapamycin-sensitive strains in S. pombe. We carried out a chemical genomics screen for rapamycin-sensitive mutants using the genome-deletion library which covers 95.3% of all nonessential fission yeast genes and confirmed 59 genes to be rapamycin sensitive. Gene Ontology (GO) enrichment analysis showed that strains sensitive to rapamycin are highly enriched in processes regulating tRNA modification and mitochondria as well as other ontologies, including cellular metabolic process, chromatin organization, cell cycle, signaling, translation, transport and other cellular processes. Analysis also showed that components of the Elongator complex are overrepresented in the sensitive strains. Here, the data obtained will provide valuable information for speculation on the actions of rapamycin as well as on TORC signaling, thereby presenting a strategy to enhance sensitivity to rapamycin.

Imp2, the PSTPIP homolog in fission yeast, affects sensitivity to the immunosuppressant FK506 and membrane trafficking in fission yeast.

Cytokinesis is a highly ordered process that divides one cell into two cells, which is functionally linked to the dynamic remodeling of the plasma membrane coordinately with various events such as membrane trafficking. Calcineurin is a highly conserved serine/threonine protein phosphatase, which regulates multiple biological functions, such as membrane trafficking and cytokinesis. Here, we isolated imp2-c3, a mutant allele of the imp2(+) gene, encoding a homolog of the mouse PSTPIP1 (proline-serine-threonine phosphatase interacting protein 1), using a genetic screen for mutations that are synthetically lethal with calcineurin deletion in fission yeast. The imp2-c3 mutants showed a defect in cytokinesis with multi-septated phenotypes, which was further enhanced upon treatment with the calcineurin inhibitor FK506. Notably, electron micrographs revealed that the imp2-c3 mutant cells accumulated aberrant multi-lamella Golgi structures and putative post-Golgi secretory vesicles, and exhibited fragmented vacuoles in addition to thickened septa. Consistently, imp2-c3 mutants showed a reduced secretion of acid phosphatase and defects in vacuole fusion. The imp2-c3 mutant cells exhibited a weakened cell wall, similar to the membrane trafficking mutants identified in the same genetic screen such as ypt3-i5. These findings implicate the PSTPIP1 homolog Imp2 in Golgi/vacuole function, thereby affecting various cellular processes, including cytokinesis and cell integrity.

Structure of the second RRM domain of Nrd1, a fission yeast MAPK target RNA binding protein, and implication for its RNA recognition and regulation.

Negative regulator of differentiation 1 (Nrd1) is known as a negative regulator of sexual differentiation in fission yeast. Recently, it has been revealed that Nrd1 also regulates cytokinesis, in which physical separation of the cell is achieved by a contractile ring comprising many proteins including actin and myosin. Cdc4, a myosin II light chain, is known to be required for cytokinesis. Nrd1 binds and stabilizes Cdc4 mRNA, and thereby suppressing the cytokinesis defects of the cdc4 mutants. Interestingly, Pmk1 MAPK phosphorylates Nrd1, resulting in markedly reduced RNA binding activity. Furthermore, Nrd1 localizes to stress granules in response to various stresses, and Pmk1 phosphorylation enhances the localization. Nrd1 consists of four RRM domains, although the mechanism by which Pmk1 regulates the RNA binding activity of Nrd1 is unknown. In an effort to delineate the relationship between Nrd1 structure and function, we prepared each RNA binding domain of Nrd1 and examined RNA binding to chemically synthesized oligo RNA using NMR. The structure of the second RRM domain of Nrd1 was determined and the RNA binding site on the second RRM domain was mapped by NMR. A plausible mechanism pertaining to the regulation of RNA binding activity by phosphorylation is also discussed.