Protein phosphatase Siw14 controls intracellular localization of Gln3 in cooperation with Npr1 kinase in Saccharomyces cerevisiae

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Gene 409 (2008) 34 – 43 www.elsevier.com/locate/gene

Protein phosphatase Siw14 controls intracellular localization of Gln3 in cooperation with Npr1 kinase in Saccharomyces cerevisiae Masataka Hirasaki, Yoshinobu Kaneko, Satoshi Harashima ⁎ Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan Received 6 September 2007; received in revised form 1 November 2007; accepted 5 November 2007 Available online 22 November 2007 Received by J.A. Engler

Abstract Saccharomyces cerevisiae Δsiw14 disruptant exhibits caffeine sensitivity. To understand the function of Siw14, double disruptants for SIW14 and each of 102 viable protein kinases (PKase) genes were constructed and examined for suppression of caffeine sensitivity based on the premise that the sensitivity was caused either by accumulation of an unknown phosphorylated Siw14 substrate(s) or by depletion of an unphosphorylated substrate(s) of Siw14 in the Δsiw14 disruptant. Among 102 pkase disruptions, only one, Δnpr1, suppressed the caffeine sensitivity of the Δsiw14 disruptant. Because Gln3 (a phosphorylated transcriptional activator)-dependent transcription is induced by disruption of NPR1, we further examined the effect of disruption and overexpression of GLN3 on the caffeine sensitivity of the Δsiw14 disruptant. Disruption of GLN3 was found to partially suppress the caffeine sensitivity of the Δsiw14 disruptant, while overexpression of GLN3 in wild-type cells caused caffeine sensitivity, providing the first evidence that Siw14 functions in the Gln3 regulatory network. We also found that, unlike in a wild-type background, Gln3 accumulates in the nucleus whether cells are exposed or not to caffeine in the Δsiw14 disruptant, and that this nuclear localization was abolished by disruption of NPR1. Interestingly, the level of Gln3 phosphorylation in both the Δsiw14 and Δnpr1 disruptants decreased relative to wild type, independent of exposure to caffeine. We conclude that Siw14 controls the intracellular localization of Gln3 in combination with Npr1, and one of the causes for the caffeine sensitivity of the Δsiw14 disruptant was an accumulation of dephosphorylated Gln3 in the nucleus. © 2007 Elsevier B.V. All rights reserved. Keywords: Yeast; Caffeine; Gln3-target genes; Genetic interaction; Phosphorylation state

1. Introduction Protein phosphorylation/dephosphorylation plays an important role in regulating many cellular processes such as signal transduction, cell cycle progression and gene expression (Zolnierowicz and Bollen, 2000). While the physiological importance of this regulation has been well established, an appreciation Abbreviations: PPase, protein phosphatase; PKase, protein kinase; MAP, mitogen-activated protein; TOR, target of rapamycin; DAPI, 4′,6-diamidino-2phenylindole; PCR, polymerase chain reaction; RT-PCR, reverse transcriptasepolymerase chain reaction. ⁎ Corresponding author. Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: +81 6 6879 7420; fax: +81 6 6879 7421. E-mail address: [email protected] (S. Harashima). 0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2007.11.005

for the complexity of the protein phosphorylation/dephosphorylation network continues to grow. Protein kinase (PKase) catalyzes the phosphorylation of protein while protein phosphatase (PPase) catalyzes the dephosphorylation of phosphorylated protein. Although the physiology of protein phosphorylation catalyzed by PKase has been the subject of intense study, only recently has the regulation of protein phosphatases been studied in the same detail (Aggen et al., 2000). In Saccharomyces cerevisiae, the nucleotide sequence of the entire genome predicts 117 PKase and 32 PPase genes among approximately 6000 genes (Sakumoto et al., 1999). Uncovering the role of the entire suite of protein phosphatases in a singlecelled model eukaryote is being undertaken to understand their possible physiological roles in higher eukaryotes. To this end, we previously constructed 30 Δppase disruptants (in all but the

M. Hirasaki et al. / Gene 409 (2008) 34–43

two essential PPase genes) and 435 Δppase double disruptants in all possible combinations, and examined them for a variety of phenotypes (Sakumoto et al., 1999). These analyses led to the discovery of a variety of new phenotypes. One of the phenotypes found from analysis of the single disruptants was that the Δsiw14 disruptant exhibits caffeine sensitivity (Sakumoto et al., 2002). SIW14 was originally identified by synthetic interaction with WHI2 (Care et al., 2004) (SGD: http://www.yeastgenome. org/) that acts in the stress–response pathway (Kaida et al., 2002). SIW14 is believed to encode a protein tyrosine phosphatase (PTPase) (SGD: http://www.yeastgenome.org/), but the cellular function of SIW14 including its substrate is unknown. Caffeine is a purine analog that affects many cellular processes. Caffeine inhibits mammalian cAMP phosphodiesterase although it is not clear whether caffeine has a similar inhibitory effect on PDE1 or PDE2 encoding the same enzyme in S. cerevisiae (Hampsey, 1997). In S. cerevisiae, growth sensitivity to caffeine is often associated with defects in components of the MAP kinase pathway. For example, an SLT2 disruptant, lacking a protein kinase component of the SLT2–MAP kinase system,

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exhibits caffeine sensitivity (Hampsey, 1997). Caffeine sensitivity has also been observed in a variety of other disruptants including PPZ1 which encodes PP1-related PPP members (Sakumoto et al., 2002). However, the mechanism responsible for the caffeine sensitivity in these disruptants is unknown. Recently, it was reported that caffeine targets the TOR complex 1, and rapamycin and caffeine displayed remarkably similar effects on global gene expression (Kuranda et al., 2006; Reinke et al., 2006). Therefore, TOR pathway may be involved in the caffeine sensitivity in these disruptants. In this report, we aimed to clarify the function of Siw14 and used a caffeine-sensitive phenotype to identify a PKase which interacts genetically with Siw14. We anticipate that this genetic interaction may lead to identification of the physiological substrate(s) of Siw14. Siw14, in combination with the protein kinase Npr1, was found to regulate directly or indirectly the intracellular localization of Gln3, a phosphorylated transcriptional activator. One possible cause for the caffeine sensitivity of the Δsiw14 disruptant is suggested to be an accumulation of dephosphorylated Gln3 in the nucleus.

Table 1 Strains used in this study Strains

Genotype

BY4739 BY4742 SH5209 SH5210 AY102 MH1 MH2 MH3 MH4 MH5 MH6 MH8

MATα leu2Δ0 lys2Δ0 ura3Δ0 MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 MATa ura3-52 his3-Δ200 leu2Δ1 lys2Δ202 trp1Δ63 MATα ura3-52 his3-Δ200 leu2Δ1 lys2Δ202 trp1Δ63 MATa ura3-1 his3-11,15 leu2-3,112 ade2-1 can1-100 trp1-1 RAD5 RAD53-Myc13::KanMX6 MATα Δbap2::KanMX4 MATα Δgap1::KanMX4 MATα Δtat2::KanMX4 MATα Δnpr1::KanMX4 MATα Δgat1::KanMX4 MATα Δsiw14::KanMX4 MATa ura3-52 his3-Δ200 leu2Δ1 lys2Δ202 trp1Δ63 canr

MH21 MH31

MATα Δgln3::CgHIS3 ura3-52 leu2Δ1 lys2Δ202 his3-Δ200 trpΔ63 MATa Δsiw14::CgLEU2 ura3-52 his3Δ200 leu2Δ2 lys2Δ202 trp1Δ63 canr

MH49

MATα Δsiw14::CgLEU2Δbap2::KanMX4 ura3-52 (or ura3Δ1) his3-Δ200 (or his3Δ1) leu2Δ1 (or leu2Δ0) lys2Δ202 trp1Δ63 MATα Δsiw14::CgLEU2Δgap1::KanMX4 ura3-52 (or ura3Δ1) his3-Δ200 (or his3Δ1) leu2Δ1 (or leu2Δ0) lys2Δ202 trp1Δ63 MATα Δsiw14::CgLEU2Δtat2::KanMX4 ura3-52 (or ura3Δ1) his3-Δ200 (or his3Δ1) leu2Δ1 (or leu2Δ0) lys2Δ202 trp1Δ63 MATα Δsiw14::CgLEU2Δgln3::CgHIS3 ura3-52 (or ura3Δ1) his3-Δ200 (or his3Δ1) leu2Δ1 (or leu2Δ0) lys2Δ202 trp1Δ63 MATα Δsiw14::CgLEU2Δnpr1::KanMX4 ura3-52 (or ura3Δ1) his3-Δ200 (or his3Δ1) leu2Δ1 (or leu2Δ0) lys2Δ202 trp1Δ63 MATa ura3-1 his3-11,15 leu2-3,112 ade2-1 can1-100 trp1-1 RAD5 RAD53-Myc13::CgHIS3 MATα Δsiw14::CgLEU2Δgat1::KanMX4 ura3-52 (or ura3Δ1) his3-Δ200 (or his3Δ1) leu2Δ1 (or leu2Δ0) lys2Δ202 trp1Δ63 MATα GLN3-Myc13::CgHIS3 MATα Δsiw14::KanMX4 GLN3-Myc13::CgHIS3 MATα Δsiw14::CgLEU2Δnpr1::KanMX4 GLN3-Myc13::CgHIS3 ura3-52 (or ura3Δ1) his3-Δ200 (or his3Δ1) leu2Δ1 (or leu2Δ0) lys2Δ202 trp1Δ63 MAT α Δnpr1::KanMX4 GLN3-Myc13::CgHIS3

MH51 MH53 MH59 MH63 MH104 MH115 MH117 MH118 MH119 MH120

Note

Alias is FY833 Alias is FY834

Derived from BY4742 Derived from BY4742 Derived from BY4742 Derived from BY4742 Derived from BY4742 Derived from BY4742 canr mutant derived from SH5209 Derived from SH5210 canr mutant derived from MH8 Segregant derived from MH1 × MH31 Segregant derived from MH2 × MH31 Segregant derived from MH3 × MH31 Segregant derived from MH21 × MH31 Segregant derived from MH4 × MH31 Derived from AY102

Reference or source Invitrogen Invitrogen Winston et al., 1995 Winston et al., 1995 A gift from T. Hishida Invitrogen Invitrogen Invitrogen Invitrogen Invitrogen Invitrogen This study This study This study This study This study This study This study This study This study

Segregant derived from MH5 × MH31 Derived from BY4742 Derived from MH6 Derived from MH63

This study This study This study This study

Derived from MH4

This study

AY102 was constructed from W303-1A strain in which the rad5-G535R allele of W303-1A has been replaced by the wild-type RAD5 gene. Only newly added mutation was described as genotype for a series of MH strains except for MH8 and MH21 for which full genotype was described.

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2. Materials and methods 2.1. Strains and media S. cerevisiae strains used in this study are listed in Table 1. The 102 nonessential Δpkase disruptants generated by the Saccharomyces Genome Deletion Project (Winzeler et al., 1999) were obtained from Research Genetics/Invitrogen. These strains were constructed in a BY4739 (MATα) or BY4742 (MATα) background (Brachmann et al., 1998). Two isogenic strains FY833 (MATa) and FY834 (MATα) (Winston et al., 1995), were used for genetic analysis. Canavanine-resistant derivative FY833 canr was obtained from FY833 by spreading cells on an SC-Arg plate containing Lcanavanine sulfate (60 μg/ml) and picking colonies. Disruptions of open reading frames (ORF) in strains FY833 canr and FY834 were constructed as previously described (Sakumoto et al., 1999) using Candida glabrata HIS3 or LEU2 genes (CgHIS3 or CgLEU2, respectively), as a template, resulting in deletion of the entire ORF. SIW14 was disrupted using primers SIW14 KF and SIW14 KR. All primers are described in Table 2. Primers GLN3 KF and GLN3 KR were used to disrupt GLN3. Yeast transformation was performed by the lithium acetate procedure (Burke et al., 2000). SIW14 and GLN3 gene disruptions were verified by colony-PCR using SIW14 CF and SIW14 CR, and GLN3 CF −1000 and GLN3 CR +500 as primers, respectively. Double disruptants were generated by crossing the individual disruptants, sporulating the diploid, followed by tetrad dissection (Burke et al., 2000).

S. cerevisiae strains expressing Myc 13 -tagged Gln3 was constructed using homologous recombination of a PCR product by amplifying a Myc13 DNA and CgHIS3 using pMH107 (pMyc 13 CgHIS3) plasmid as template and GLN3 13Myc F2 and GLN3 13Myc R1 as primers, and then integrating the resultant PCR product in-frame at the GLN3 locus, just upstream of the stop codon. YPAD and synthetic minimal dextrose medium (SD) supplemented with appropriate nutrients were formulated as described (Burke et al., 2000). To select strains into which the KanMX4 gene was introduced, YPAD containing geneticin disulfate (Wako) (final concentration 300 μg/ml) was used. All cultures were grown at 30 °C. 2.2. Plasmids YCp-STE7 and YCp-STE11 plasmids were constructed by inserting a STE7 and STE11 fragment flanked by XhoI and BamHI restriction sites into XhoI- and BamHI-linearized p562 (pRS316: YCp-URA3), respectively (Sikorski and Hieter, 1989). The STE7 and STE11 fragments were obtained by PCR amplification using chromosomal DNA from strain BY4742 as template and STE7 CF and STE7 CR, and STE11 CF and STE11 CR as primers, respectively. Multi-copy NPR1 (pMH42) was constructed by inserting a NPR1 fragment flanked by SmaI and XhoI restriction sites into SmaI- and XhoI-linearized p566 (pYO326:YEp-URA3) (Ohya et al., 1991). The NPR1 fragment was obtained by PCR amplification using chromosomal DNA from strain BY4742 as

Table 2 Oligonucleotides used in this study Primer

Primer sequence

SIW14 KF SIW14 KR SIW14 CF SIW14 CR GLN3 KF GLN3 KR GLN3 CF − 1000 GLN3 CR +500 NPR1 CF NPR1 CR STE7 CF STE7 CR STE11 CF STE11 CR KanMX6 KF KanMX6 KR GLN3 13Myc F2 GLN3 13Myc R1 DAL1 F RT-primer DAL1 R RT-primer DAL5 F RT-primer DAL5 R RT-primer GAP1 F RT-primer GAP1 R RT-primer MEP2 F RT-primer MEP2 R RT-primer PUT1 F RT-primer PUT1 R RT-primer ACT1-1 RT-primer ACT1-2 RT-primer

5′-CAGCAAGATTGACGATTTGATAGAAAATGAGGCAGAAATCCACAGGAAACAGCTATGACC-3′ 5′-ATTTCGTCATCGTCATACATCTCGATAACTGCTGATCAAGTTGTAAAACGACGGCCAGT-3′ 5′-CTACTCTCTTCTGGATCAAT-3′ 5′-TTTCGAAGAGACTAGTTACG-3′ 5′-TCTGGACGTGCATGGTCGAAGTAATGAAGAGCCGAGACAACACAGGAAACAGCTATGACC-3′ 5′-TAGAAGATCCTTTTGTGGAATTATCCTCACTGATCTTTCCGTTGTAAAACGACGGCCAGT-3′ 5′-GAGGGATCCGACTAGCAGCAAAGGCAARGC-3′ 5′-CCCGTCGACATTCCCATATTTGTGAGACAC-3′ 5′-GGGCCCGGGTGCACGAAAAGCTGTACGAGG-3′ 5′-GGGCTCGAGTTACTATCACGTTTTTTGCTG-3′ 5′-CTCCTCGAGACCAGTGGTCACCATACATG-3′ 5′-CTCGGATCCACTATGGATTGCGTTCCTTG-3′ 5′-CTCCTCGAGCGTGTGATATCGGGAGTGCA-3′ 5′-CTCGGATCCTACAGGCGAATTGTGGTCCC-3′ 5′-ACCGGCAGATCCGCTAGGGATAACAGGGTAATATAGATCTCACAGGAAACAGCTATGACC-3′ 5′-AGTTCTTGTAAAGAAGAATCTTTTTATTGTCAGTACTGATGTTGTAAAACGACGGCCAGT-3′ 5′-AGCAATTGCTGACGAATTGGATTGGTTAAAATTTGGTATACGGATCCCCGGGTTAATTAA-3′ 5′-TTATTAACATAATAAGAATAATGATAATGATAATACGCGGGAATTCGAGCTCGTTTAAAC-3′ 5′-CACCCATCCGCTCTGAGTCT-3′ 5′-AGCCTATAACACCTTCACGCAAA-3′ 5′-GGCGGTCATGGGATTAAGAA-3′ 5′-AAGGCGGAAGTAACCCATGAA-3′ 5′-CCCAAGGAAGAGGCTTGGAT-3′ 5′-CCCCAGTAGGAACCCCAAAC-3′ 5′-TCGCTGGCCTAGTGGGTATC-3′ 5′-AATGACAACGGCTGACCAGAT-3′ 5′-ACACACTCTAACACGGCTGCTAAT-3′ 5′-CACACTATTGCCGTTGGCATT-3′ 5′-TGGTATGTGTAAAGCCGGTTTTG-3′ 5′-CATGATACCTTGGTGTCTTGGTCTA-3′

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Fig. 1. Caffeine sensitivity of the Δsiw14 disruptant is suppressed by disruption of NPR1. Wild type (BY4742), Δsiw14 (BY4724, Δsiw14::KanMX4), Δnpr1 (BY4742, Δnpr1::KanMX4) and Δsiw14Δnpr1 (Δsiw14::CgLEU2Δnpr1:: KanMX4) disruptants were grown to saturation in YPAD. The strains were serially diluted ten times in sterile water and spotted onto YPAD plates with or without 11 mM caffeine. Images were taken after 2 days of incubation at 30 °C.

template and NPR1 CF and NPR1 CR as primers. Multi-copy GLN3 (pMH100) was constructed by inserting a GLN3 fragment flanked by SalI and BamHI restriction sites into SalI- and BamHIlinearized p566 (pYO326:YEp-URA3) (Ohya et al., 1991). The GLN3 fragment was obtained by PCR amplification using chromosomal DNA from strain BY4742 as template and GLN3 CF −1000 and GLN3 CR +500 as primers. To construct strain MH104 [Rad53-Myc13::CgHIS3], the Rad53-Myc13::KanMX6 construct in strain AY102 (a gift from T. Hishida) was replaced as previously described (Sakumoto et al., 1999) using C. glabrata HIS3 genes (CgHIS3) as template and KanMX6 KF and KanMX6 KR as primers. Plasmid pMH107 (Myc13::CgHIS3) was constructed by inserting a Myc13::CgHIS3 fragment into the pT7 Blue vector. The Myc13::CgHIS3 fragment was obtained by PCR amplification using chromosomal DNA from MH104 as template and GLN3 13Myc F2 and GLN3 13Myc R1 as primers.

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First-strand cDNA was synthesized using a High Capacity cDNA Archive kit (ABI). Quantitative PCR (7300 Real Time PCR system, ABI) was performed in real time on the resulting first-strand using the following primers: DAL1 F RT-primer, DAL1 R RT-primer, DAL5 F RT-primer, DAL5 R RT-primer, GAP1 F RT-primer, GAP1 R RT-primer, MEP2 F RT-primer, MEP2 R RT-primer, PUT1 F RT-primer and PUT1 R RT-primer as primers. RNA levels were determined relative to a control gene, ACT1, using ACT1-1 RT-primer and ACT1-2 RT-primer as primers. 3. Results 3.1. Caffeine sensitivity of the Δsiw14 disruptant is suppressed by disruption of NPR1 encoding a protein kinase Through previous systematic analysis of a variety of phenotypes of single PPase disruptants, we discovered that the Δsiw14 disruptant exhibited caffeine sensitivity (Sakumoto et al., 2002). Because the SIW14 deletant was the only PPase mutant exhibiting

2.3. Indirect immunofluorescence analysis Gln3-Myc13 was visualized by indirect immunofluorescence in whole fixed cells as described (Burke et al., 2000). The primary antibody was 9E10 for c-Myc (Santa Cruz) and the secondary antibody was 1104-1 J of Qdot Goat F anti-Mouse IgG (Qdot). DNA was stained for fluorescence using 4′,6-diamidino-2phenylindole (DAPI) (Vector). Cells were observed by fluorescence microscopy (BX61, Olympus) and photographed using a CCD camera (CCD-EX1, Universal Imaging Corporation, USA). 2.4. Western blot analysis Fifteen microliters of yeast protein isolated as described (Foiani et al., 1994) was loaded per lane for separation in 5% SDS-PAGE (XV PANTERA GEL KIT, DRC) and detected by use of the Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer) with horseradish peroxidase (HRP)-conjugated anti mouse monoclonal IgG1 (9E10:Santa Cruz) for Gln3-Myc13. 2.5. Transcriptional analysis Total RNA was prepared by hot phenol extraction as described (Ausubel et al., 1993) with minor modifications.

Fig. 2. Caffeine sensitivity of the Δsiw14 disruptant is suppressed by disruption of GLN3. A. Growth response to caffeine as described in the legend to Fig. 1. Wild type (BY4742), Δsiw14 (BY4724, Δsiw14::KanMX4), Δgap1 (BY4742, Δgap1:: KanMX4), Δsiw14Δgap1 (Δsiw14::CgLEU2Δgap1::KanMX4), Δbap2 (BY4742, Δbap2::KanMX4), Δsiw14Δbap2 (Δsiw14::CgLEU2Δbap2:: KanMX4), Δtat2 (BY4742, Δtat2::KanMX4), Δsiw14Δtat2 (Δsiw14:: CgLEU2Δtat2::KanMX4), Δgln3 (FY834, Δgln3::CgHIS3), Δsiw14Δgln3 (Δsiw14::CgLEU2Δgln3::CgHIS3), Δsiw14 (BY4724, Δsiw14::KanMX4), wild type (BY4742). B. Growth response to caffeine as described in the legend to Fig. 1. Wild type (BY4742), Δsiw14 (BY4724, Δsiw14::KanMX4), Δgat1 (BY4742, Δgat1::KanMX4), Δsiw14Δgat1 (Δsiw14::CgLEU2Δgat1::KanMX4).

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a previously unreported sensitivity to caffeine in this screen (the PPZ1 deletant had previously been shown to be sensitive to caffeine, Posas et al., 1993), and only one study had previously reported on Siw14 function (Care et al., 2004), we chose to investigate Siw14 function through analysis of this phenotype. We hypothesized that the caffeine-sensitive phenotype of the Δsiw14 disruptant was caused by accumulation of a phosphorylated Siw14 substrate(s), or alternatively, by depletion of an unphosphorylated Siw14 substrate(s) in the Δsiw14 disruptant. In either case, one would expect that the caffeine sensitivity of the Δsiw14 disruptant would be suppressed by disruption of the PKase gene whose product phosphorylates the Siw14 substrate. To examine this possibility, we screened for PKase genes whose disruption suppressed the caffeine sensitivity of the Δsiw14 disruptant. All 102 Δpkase disruptants in a MATα Δpkase::KanMX4 background (excluding 15 essential PKase genes, SGD: http:// www.yeastgenome.org/; Parsons et al., 2004) were crossed to MATa Δsiw14::CgLEU2 disruptant (MH31) in a FY833 canr background. One hundred diploids were obtained excluding crosses involving the sterile deletants of STE7 and STE11 (Hartwell, 1980). Heterozygous diploids of these two disruptants were constructed by introducing a single copy plasmid harboring STE7 (YCp-STE7) or STE11 (YCp-STE11) into the Δste7 or the Δste11 disruptants, respectively. Transformants were then crossed to the Δsiw14 strain (MH31). Thus, all 102 diploid hybrids were obtained. The 102 diploids thus constructed were sporulated and Leu+ KanMX4+ random spore meiotic segregants were selected (Burke et al., 2000) which were expected to have the genotype Δsiw14:: CgLEU2Δpkase::KanMX4. The 102 Δsiw14Δpkase double disruptants were then tested for caffeine sensitivity. The caffeine sensitivity of the Δsiw14 disruptant was found to be suppressible by disruption of only one PKase gene, NPR1 (Fig. 1). Suppression of the caffeine sensitivity of the Δsiw14 disruptant co-segregated with disruption of NPR1 by tetrad analysis (data not shown).

3.2. Caffeine sensitivity of the Δsiw14 disruptant is also suppressed by disruption of GLN3 Although the substrate of Npr1 has not been identified, Npr1 is known to be a positive regulator of Gap1 and Bap2 and a negative regulator of Tat2 and Gln3 (Chen et al., 1994; Schmidt et al., 1998; De Craene et al., 2001; Crespo et al., 2004). Specifically, Npr1 is required for stabilization of Gap1 (De Craene et al., 2001), while Bap2 becomes more susceptible to rapamycin-induced degradation in an Δnpr1 disruptant (Omura and Kodama, 2004). Therefore, we initially predicted that suppression of the caffeine sensitivity of the Δsiw14 disruptant by disruption of NPR1 was caused by enhanced degradation or inactivation of Gap1 and Bap2. If this were the case, disruption of these genes in the Δsiw14 disruptant would be expected to suppress the caffeine sensitivity. However, the caffeine sensitivity of the Δsiw14 disruptant was not suppressed by disruption of GAP1 or BAP2 (Fig. 2A). Alternatively, because Gln3-dependent transcription is induced by disruption of Npr1 (Foiani et al., 1994; Crespo et al., 2004) and Tat2 is degraded via Npr1 function (Schmidt et al., 1998), we reasoned that suppression of the caffeine sensitivity of the Δsiw14 disruptant by disruption of NPR1 was caused by induced Gln3dependent transcription or accumulation of Tat2 in the Δsiw14Δnpr1 double disruptant. If either were the case, both Δgln3 and Δtat2 single disruptants would be expected to exhibit caffeine sensitivity. However, neither the Δgln3 nor the Δtat2 single disruptant exhibited caffeine sensitivity (Fig. 2A). Surprisingly, we found that the caffeine sensitivity of the Δsiw14 disruptant was partially suppressed by disruption of GLN3 (Fig. 2A), suggesting that suppression of caffeine sensitivity of the Δsiw14 disruptant is due to abolishment of Gln3-dependent transcription. Because disruption of GLN3, one of two known GATA transcriptional activators, suppressed the caffeine sensitivity of

Fig. 3. Multi-copy plasmids harboring NPR1 and GLN3 in wild-type cells cause caffeine sensitivity. A. Wild type (FY833) and the Δgln3 disruptant (FY834, Δgln3:: HIS3) bearing multi-copy plasmid YEpU-NPR1 (pMH042) were grown on SC-Ura plates with and without 11 mM caffeine. Images were taken after 3 days of incubation at 30 °C. B. The caffeine sensitivity of wild type (BY4742) and the Δnpr1 disruptant (BY4724, Δnpr1::KanMX4) bearing multi-copy plasmid YEpUGLN3 (pMH100) was assessed as described in A.

M. Hirasaki et al. / Gene 409 (2008) 34–43

the Δsiw14 disruptant, we also examined the effect of disrupting the other GATA transcriptional activator, GAT1 (Magasanik and Kaiser, 2002). Disruption of GAT1 was not found to suppress the caffeine sensitivity of the Δsiw14 disruptant (Fig. 2B). 3.3. Multi-copy plasmid harboring either NPR1 or GLN3 causes caffeine sensitivity in wild-type cells It was predicted that the caffeine sensitivity of the Δsiw14 disruptant was caused either by accumulation of substrate(s) phosphorylated by Npr1 or by induced Gln3-dependent tran-

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scription. If so, overexpression of either NPR1 or GLN3 in wildtype cells should lead to caffeine sensitivity. Consistent with this expectation, overexpression of either NPR1 or GLN3 in wild-type cells led to caffeine sensitivity (Fig. 3A and B). Although Gln3dependent transcription is induced by disruption of NPR1 in ammonium-grown cells (Crespo et al., 2004), interestingly, overexpression of GLN3 in the Δnpr1 disruptant did not lead to caffeine sensitivity (Fig. 3B). Moreover, overexpression of NPR1 in the Δgln3 disruptant also did not lead to caffeine sensitivity (Fig. 3A). These observations suggest that both Npr1 and Gln3 are indispensable for caffeine sensitivity.

Fig. 4. Gln3-target genes are significantly upregulated by disruption of SIW14 after caffeine addition. Early-log-phase cells of wild type (BY4742), Δsiw14 (BY4742, Δsiw14::KanMX4), Δnpr1 (BY4742, Δnpr1::KanMX4), Δsiw14Δnpr1 (Δsiw14::CgLEU2Δnpr1::KanMX4), Δgln3 (FY834, Δgln3::CgHIS3) and Δsiw14Δgln3 (Δsiw14::CgLEU2Δgln3::CgHIS3) disruptants (OD600 = 0.8) in YPAD were transferred to YPAD (solid bars) or YPAD containing 14 mM caffeine (open bars) for 1 h after which RNA was isolated as described (Ausubel et al., 1993). First-strand cDNA was synthesized using High Capacity cDNA Archive kit (ABI) and quantitative real time PCR was performed on the resulting first-strand DNA. RNA levels were determined relative to a control gene, ACT1.

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Fig. 5. Subcellular localization of Gln3 is regulated by Siw14. Early-log-phase cells of wild type (BY4742), Δsiw14 (BY4742, Δsiw14::KanMX4), Δnpr1 (BY4742, Δnpr1::KanMX4) and Δsiw14Δnpr1 (Δsiw14::CgLEU2Δnpr1::KanMX4) disruptants (OD600 = 0.2) expressing Gln3-Myc13 grown in YPAD were switched to YPAD (A, B and C, respectively), YPAD containing 14 mM caffeine (D, F and G, respectively) or YPAD containing 200 nM rapamycin (E) for 1 h. Gln3-Myc13 localization was then analyzed by indirect immunofluorescence staining with MAb 9E10. Nuclear DNA was stained with 4′,6-diamidino-2-phenylindole. Cells were observed by fluorescence microscopy (BX61, Olympus) and photographed using a CCD camera (CCD-EX1, Universal Imaging Corporation, USA).

3.4. In presence of caffeine, transcription of the Gln3-target genes is strongly upregulated by disruption of SIW14 We predicted that the caffeine sensitivity of the Δsiw14 disruptant was caused by increased transcription of one or more Gln3-target genes and that the mechanism of suppression of the caffeine sensitivity of the Δsiw14 disruptant by disruption of NPR1 was due to repression of Gln3-dependent transcription. To test this prediction, we examined expression of PUT1, encoding proline oxidase, DAL1, encoding allantoinase, DAL5, encoding allantoate and ureidosuccinate permease, GAP1, encoding general amino acid permease and MEP2, encoding high affinity ammonium permease, by means of real time RT-PCR analysis in the Δsiw14, Δnpr1 and Δsiw14Δnpr1 disruptants in the presence and absence of caffeine. The transcription of all of the above genes was induced upon caffeine treatment in wild type (Fig. 4), indicating that Gln3-target genes are induced by caffeine. This was consistent with the results of a recently reported expression profile by microarray analysis (Reinke et al., 2006). Moreover, the

transcription level induced by caffeine treatment in wild type was further increased by disruption of SIW14. This increased transcription was found to decrease upon disruption of NPR1 except for GAP1, suggesting that Npr1 is a positive regulator of Gln3 (Fig. 4), a finding that contrasts with an earlier report that it is a negative regulator (Crespo et al., 2004) (see Discussion). Based on these observations, we suggest that transcription of unknown Gln3-target gene(s) responsible for the caffeine sensitivity also increases in the Δsiw14 disruptant, and that this leads to caffeine sensitivity. 3.5. Siw14 regulates localization of Gln3 We next considered the possibility that the caffeine sensitivity of the Δsiw14 disruptant was caused by accumulation of Gln3 in the nucleus upon caffeine treatment and the accumulation of Gln3 in the nucleus of the Δsiw14 disruptant is suppressed by disruption of NPR1. To examine this possibility, Gln3 was tagged at the Cterminus with a thirteen-Myc epitope (Gln3-Myc13), expressed by

Fig. 6. Caffeine induces dephosphorylation of Gln3. Wild type (BY4742), Δsiw14 (BY4742, Δsiw14::KanMX4), Δnpr1 (BY4742, Δnpr1::KanMX4) and Δsiw14Δnpr1 (Δsiw14::CgLEU2Δnpr1::KanMX4) disruptants were grown in YPAD to an A600 of 0.8 and then shifted to YPAD (lanes 1, 4 and 6), YPAD containing 200 nM rapamycin (lane 3) or YPAD containing 14 mM caffeine (lanes 2, 5 and 7) for 1 h. Gln3 was detected by immunoblotting. C means that Gln3 is in cytoplasm while N indicates that Gln3 is in nuclear, based upon localization data in Fig. 5.

M. Hirasaki et al. / Gene 409 (2008) 34–43

the native promoter, and subjected to indirect immunofluorescence staining. Results revealed that Gln3-Myc13 primarily localizes in the cytoplasm in wild-type cells cultured in nitrogen-rich medium without caffeine (Fig. 5A), but accumulates in the nucleus upon caffeine addition (Fig. 5B), or following rapamycin treatment (Fig. 5C). By contrast, in the Δsiw14 disruptant, Gln3-Myc13 accumulates in the nucleus regardless of the presence or absence of caffeine (Fig. 5D, E). This nuclear localization in the Δsiw14 disruptant was abolished by disruption of NPR1 (Fig. 5H, I). These observations indicate that Siw14 and Npr1 regulate localization of Gln3 and the mechanism of decrease of transcription level of Gln3target gene(s) by NPR1 disruption in the Δsiw14 disruptant is due to abolishment of accumulation of Gln3 in the nucleus. 3.6. Siw14 affects phosphorylation state of Gln3 Gln3 is a phosphoprotein and its intracellular localization is regulated by phosphorylation state (Beck and Hall, 1999; Bertram et al., 2002). Therefore, we predicted that the phosphorylation level of Gln3 would be regulated by caffeine treatment, by Siw14, and by Npr1. Gel mobility analysis revealed that Gln3 was shifted down by the addition of caffeine as if Gln3 was dephosphorylated (Fig. 6, lanes 1 and 2). It is known that rapamycin induces dephosphorylation of Gln3 in wild type (Beck and Hall, 1999). The mobility of Gln3-Myc13 in a wild-type background in the presence of caffeine (Fig. 6, lane 2) was intermediate between that of Gln3-Myc13 without caffeine (Fig. 6, lane 1), and with rapamycin (Fig. 6, lane 3). The electrophoretic position of Gln3Myc13 with rapamycin treatment is known to be the same as that following calf intestinal alkaline phosphatase treatment (Beck and Hall, 1999; Bertram et al., 2002). It is likely, therefore, that Gln3 in a wild-type strain treated with caffeine (Fig. 6, lane 2) is still phosphorylated, although caffeine treatment induces dephosphorylation of Gln3 in wild type. These observations are consistent with the possibility that Gln3 has multiple phosphorylation sites (Bertram et al., 2002) and that caffeine induces dephosphorylation of some of them. We also noted that the level of Gln3 phosphorylation in the Δsiw14 (Fig. 6, lanes 4 and 5), Δnpr1 (Fig. 6, lanes 6 and 7) and Δsiw14Δnpr1 (Fig. 6, lanes 8 and 9) disruptants decreased relative to wild type with or without caffeine treatment. These observations strongly suggest that Siw14 and Npr1 are both involved in regulation of the phosphorylation state of Gln3. However, the mechanism of suppression of the caffeine sensitivity of the Δsiw14 disruptant by disruption of NPR1 is not due to suppression of dephosphorylation of Gln3 by disruption of SIW14 based on the electrophoretic evidence presented above. It should be noted that Gln3 dephosphorylation caused by caffeine treatment has not been previously reported (Fig. 6, lane 2). 4. Discussion We previously reported that a Δsiw14 disruptant exhibits sensitivity to caffeine (Sakumoto et al., 2002). Although SIW14 is believed to encode a protein tyrosine phosphatase (SGD: http://www.yeastgenome.org/), the cellular function of Siw14 including its substrate is unknown. In the present study, we found that the caffeine sensitivity of the Δsiw14 disruptant is

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suppressed by disruption of NPR1, encoding a protein kinase. We also found that the caffeine sensitivity of the Δsiw14 disruptant was partially suppressed by disruption of GLN3, providing the first evidence that Siw14 functions in the Gln3 regulatory network relative to caffeine signal transduction. Gln3 bound to Ure2 is sequestered in the cytoplasm, thereby preventing Gln3-dependent activation of nitrogen-regulated genes (Beck and Hall, 1999). Upon treatment with rapamycin, a condition which inactivates the TOR kinase complex, Gln3 is released from Ure2 and transferred to the nucleus in an active form (Beck and Hall, 1999). Because Ure2 is a phosphoprotein (Cardenas et al., 1999) and a Δure2 disruptant exhibits caffeine sensitivity (Parsons et al., 2004), we reasoned that Siw14 and Npr1 directly dephosphorylate and phosphorylate Ure2, respectively, to control localization of Gln3, and that caffeine is involved in this regulation. However, this possibility seems unlikely because it is known that Gln3 dissociates from the Gln3/Ure2 complex and moves to the nucleus when Ure2 is dephosphorylated (Bertram et al., 2000). If this is the case, Gln3 localization would be expected to be cytoplasmic in the Δsiw14 disruptant and nuclear in the Δnpr1 disruptant. Both of these possibilities are inconsistent with the results shown in Fig. 5 (panel E for the Δswi14 disruptant and panel G for the Δnpr1 disruptant). Localization of Gln3 is known to be regulated by phosphorylation state (Beck and Hall, 1999; Bertram et al., 2002). Although it has been reported that the phosphorylation level of Gln3 did not change upon disruption of NPR1 (Crespo et al., 2004), we found in this study that the phosphorylation level of Gln3 decreased significantly in a Δnpr1 disruptant (Fig. 6, lanes 6 and 7). These inconsistent results relative to phosphorylation state between the two studies might be caused by differences in growth conditions used (YPAD versus SD containing NH4+ as nitrogen source) (Crespo et al., 2004). This possibility is consistent with the recent finding that in a Δnpr1 disruptant, Gln3 localized in the cytoplasm in YPD grown cells, but in the nucleus in ammoniumgrown cells (Tate et al., 2006). Moreover, Gln3-target genes were induced by disruption of NPR1 in the ammonium medium (Crespo et al., 2004) but not in the YPAD medium (Fig. 4). We propose that Npr1 acts as a positive regulator of Gln3 in the caffeine response. This idea is supported by the following observations. i) Overexpression of GLN3 in the Δnpr1 disruptant did not lead to caffeine sensitivity, but did in wild type (Fig. 3B), and ii) increased expression of Gln3-dependent genes, e.g., PUT1, by disruption of SIW14 did not occur in the caffeine-treated Δnpr1 disruptant (Fig. 4). It is thought that Gln3 plays a general role in rapamycin toxicity because the Δgln3 disruptant exhibits rapamycin resistance (Cardenas et al., 1999). However, the mechanism has not been clarified. One approach to understanding the mechanism is to identify target gene(s) whose increased or decreased expression leads to rapamycin sensitivity. Recently, it was reported that caffeine acts as a novel small molecule inhibitor of TOR complex 1 (Reinke et al., 2006). Rapamycin and caffeine have also been reported to display remarkably similar effects on global gene expression, specifically, on expression of the DAL cluster genes, RTG target genes and NDP genes (Reinke et al., 2006). In the present study, we found that the caffeine sensitivity of the Δsiw14

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disruptant is suppressed by disruption of GLN3, and that in the presence of caffeine, transcription of Gln3-target genes is strongly upregulated by disruption of SIW14. From these observations, we suggest that one of the causes for the caffeine sensitivity of the Δsiw14 disruptant is increased transcription of Gln3-target gene(s). In this report, we suggest that the mechanism of suppression of caffeine sensitivity of the Δsiw14 disruptant by NPR1 disruption is caused by a decreased expression level of Gln3target gene(s) because increased expression of Gln3-target gene (s) in the Δsiw14 disruptant after caffeine addition was abolished by disruption of NPR1 (Fig. 4). On the other hand, it is known that Npr1 plays a pleiotropic role in transport (Schmidt et al., 1998; De Craene et al., 2001; Omura and Kodama, 2004) and the Δsiw14Δnpr1Δgln3 triple disruptant displayed greater caffeine resistance than the Δsiw14Δnpr1 or the Δsiw14Δgln3 double disruptants (data not shown). These observations suggest the additional possibility that suppression of the caffeine sensitivity of the Δsiw14 disruptant by NPR1 disruption is also caused by defective caffeine uptake through inactivation or destabilization of the unknown Npr1-dependent caffeine transporter. Gln3 is a well known phosphorylated protein (Beck and Hall, 1999). However, the specific amino acid residue(s) subject to phosphorylation has yet to be determined. In the present study, we found that disruption of SIW14 and NPR1, and caffeine addition, significantly affected transcriptional activity (Fig. 4), intracellular localization (Fig. 5) and phosphorylation state of Gln3 (Fig. 6). These observations were informative with respect to the phosphorylation/dephosphorylation state, nuclear localization and transcriptional activity of Gln3. i) Dephosphorylation of Gln3 by caffeine addition is necessary for both nuclear localization of Gln3 and induction of Gln3-dependent genes (Fig. 6, lane 2 compared to lane 1, Gln3 is in the nucleus). Upon rapamycin treatment, the TOR-controlled protein phosphatase Sit4 is known to dephosphorylate Gln3 (Beck and Hall, 1999) which then translocates into the nucleus to activate its target genes (Bertram et al., 2000). Therefore, we suggest that dephosphorylation of Gln3 by caffeine treatment is caused by TORcontrolled Sit4. ii) Dephosphorylation of Gln3 by disruption of SIW14 is necessary only for nuclear localization of Gln3, but not induction of Gln3-target genes (Fig. 6, lane 4 compared to lane 1, Gln3 is within nucleus). However, it is unlikely that Siw14 is directly involved in dephosphorylation of Gln3 because disruption of SIW14 caused the phosphorylation level of Gln3 to decrease rather than increase. Therefore, we suggest that Siw14 inactivates another PPase that is responsible for dephosphorylation of Gln3, or alternatively, activates a PKase that is responsible for phosphorylation of the Gln3. iii) Because Npr1 may be epistatic to either caffeine (Fig. 6, lane 7 compared to lane 2, Gln3 is in cytoplasm) or Siw14 (Fig. 6, lane 8 compared to lane 4, Gln3 is in cytoplasm) with respect to their effects on Gln3 localization, dephosphorylation of Gln3 by disruption of NPR1 is responsible for allowing Gln3 to accumulate in the cytoplasm. It is known that dephosphorylated Gln3 interacts with the importin Srp1 in the presence of rapamycin (Carvalho et al., 2001). However, it is possible that Gln3 phosphorylated by Npr1 may interact with Srp1 in the case of caffeine signal transduction, and thus enter the nucleus depending on dephosphorylation state as a function of

caffeine addition or Δsiw14 disruption. While Gln3 could be assumed to be directly phosphorylated by Npr1, it was recently reported that the Δnpr1 disruptant is impaired in ammonium uptake, suggesting that the derepression of Gln3-dependent genes and nuclear localization of Gln3 in the Δnpr1 disruptant could be a consequence of the reduced uptake rate of the repressing nitrogen compound (Tate et al., 2006). Therefore, it is possible that the change in phosphorylation state of Gln3 induced by disruption of Npr1 is caused by destabilization of ammonium transporters that prevents ammonium uptake. Verification of these possibilities at the molecular level will be indispensable for clarifying the detailed mechanism of caffeine signal transduction, as well as that of a variety of other signal transduction pathways regulated by this very interesting transcriptional activator, Gln3. Acknowledgements We thank Takashi Hishida for providing the strain and Yukio Mukai and Minetaka Sugiyama for helpful discussions. We also acknowledge the skillful technical support of Masaya Horiguchi. References Aggen, J.B., Nairn, A.C., Chamberlin, R., 2000. Regulation of protein phosphatase-1. Chem. Biol. 7, R13–R23. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., 1993. Current Protocols in Molecular Biology, vol. 2. John Wiley & Sons, Inc., Boston, Mass. Beck, T., Hall, M.N., 1999. The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature 402, 689–692. Bertram, P.G., Choi, J.H., Carvalho, J., Ai, W., Zeng, C., Chan, T.F., Zheng, X.F., 2000. Tripartite regulation of Gln3p by TOR, Ure2p, and phosphatases. J. Biol. Chem. 275, 35727–35733. Bertram, P.G., Choi, J.H., Carvalho, J., Chan, T.F., Ai, W., Zheng, X.F., 2002. Convergence of TOR–nitrogen and Snf1–glucose signaling pathways onto Gln3. Mol. Cell. Biol. 22, 1246–1252. Brachmann, C.B., Davies, A., Cost, G.J., Caputo, E., Li, J., Hieter, P., Boeke, J.D., 1998. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132. Burke, D., Dawson, D., Stearns, T., 2000. Methods in Yeast Genetics: a Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Cardenas, M.E., Cutler, N.S., Lorenz, M.C., Di Como, C.J., Heitman, J., 1999. The TOR signaling cascade regulates gene expression in response to nutrients. Genes Dev. 13, 3271–3279. Care, A., Vousden, K.A., Binley, K., Radcliffe, M.P., Trevethick, J., Mannazzu, I., Sudbery, P.E., 2004. A synthetic lethal screen identifies a role for the cortical actin patch/endocytosis complex in the response to nutrient deprivation in Saccharomyces cerevisiae. Genetics 166, 707–719. Chen, M.X., McPartlin, A.E., Brown, L., Chen, Y.H., Barker, H.M., Cohen, P.T., 1994. A novel human protein serine/threonine phosphatase, which possesses four tetratricopeptide repeat motifs and localizes to the nucleus. EMBO J. 13, 4278–4290. Crespo, J.L., Helliwell, S.B., Wiederkehr, C., Demougin, P., Fowler, B., Primig, M., Hall, M.N., 2004. NPR1 kinase and RSP5-BUL1/2 ubiquitin ligase control GLN3-dependent transcription in Saccharomyces cerevisiae. J. Biol. Chem. 279, 32517–37512. De Craene, J.O., Soetens, O., Andre, B., 2001. The Npr1 kinase controls biosynthetic and endocytic sorting of the yeast Gap1 permease. J. Biol. Chem. 276, 43939–43948. Foiani, M., Marini, F., Gamba, D., Lucchini, G., Plevani, P., 1994. The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae

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