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• W1986301417 abstract "The Atf1 transcription factor plays a vital role in the ability of Schizosaccharomyces pombe cells to respond to various stress conditions. It regulates the expression of many genes in a stress-dependent manner, and its function is dependent upon the stress-activated MAPK, Sty1/Spc1. Moreover, Atf1 is directly phosphorylated by Sty1. Here we have investigated the role of such phosphorylation. Atf1 protein accumulates following stress, and this accumulation is lost in a strain defective in the Sty1 signaling pathway. In addition, accumulation of a mutant Atf1 protein that can no longer be phosphorylated is lost. Measurement of the half-life of Atf1 demonstrates that changes in Atf1 stability are responsible for this accumulation. Atf1 stability is also regulated by its heterodimeric partner, Pcr1. Similarly, Pcr1 levels are regulated by Atf1. Thus multiple pathways exist that ensure that Atf1 levels are appropriately regulated. Phosphorylation of Atf1 is important for cells to mount a robust response to H2O2 stress, because the Atf1 phospho-mutant displays sensitivity to this stress, and induction of gene expression is lower than that observed in wild-type cells. Surprisingly, however, loss of Atf1 phosphorylation does not lead to the complete loss of stress-activated expression of Atf1 target genes. Accordingly, the Atf1 phospho-mutant does not display the same overall stress sensitivities as the atf1 deletion mutant. Taken together, these data suggest that Sty1 phosphorylation of Atf1 is not required for activation of Atf1 per se but rather for modulating its stability. The Atf1 transcription factor plays a vital role in the ability of Schizosaccharomyces pombe cells to respond to various stress conditions. It regulates the expression of many genes in a stress-dependent manner, and its function is dependent upon the stress-activated MAPK, Sty1/Spc1. Moreover, Atf1 is directly phosphorylated by Sty1. Here we have investigated the role of such phosphorylation. Atf1 protein accumulates following stress, and this accumulation is lost in a strain defective in the Sty1 signaling pathway. In addition, accumulation of a mutant Atf1 protein that can no longer be phosphorylated is lost. Measurement of the half-life of Atf1 demonstrates that changes in Atf1 stability are responsible for this accumulation. Atf1 stability is also regulated by its heterodimeric partner, Pcr1. Similarly, Pcr1 levels are regulated by Atf1. Thus multiple pathways exist that ensure that Atf1 levels are appropriately regulated. Phosphorylation of Atf1 is important for cells to mount a robust response to H2O2 stress, because the Atf1 phospho-mutant displays sensitivity to this stress, and induction of gene expression is lower than that observed in wild-type cells. Surprisingly, however, loss of Atf1 phosphorylation does not lead to the complete loss of stress-activated expression of Atf1 target genes. Accordingly, the Atf1 phospho-mutant does not display the same overall stress sensitivities as the atf1 deletion mutant. Taken together, these data suggest that Sty1 phosphorylation of Atf1 is not required for activation of Atf1 per se but rather for modulating its stability. Eukaryotic cells have developed response mechanisms to combat the harmful effects of a variety of stress conditions. In the majority of cases, such responses involve changes in the gene expression pattern of the cell, leading to increased levels and activities of proteins that have stress-protective functions. Central to these responses are the sensing and signaling pathways that communicate with the nucleus and facilitate necessary changes in gene expression. Of particular importance are the pathways collectively known as MAPK 5The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; SAPK, stress-activated protein kinase; JNK, c-Jun NH2-terminal kinase; ORF, open reading frame; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; E3, ubiquitin-protein isopeptide ligase; MM, minimal medium. pathways (1Marshall C.J. Curr. Opin. Genet. Dev. 1994; 4: 82-89Crossref PubMed Scopus (902) Google Scholar). The best characterized of these is a mammalian pathway that activates ERK1 and ERK2 in response to a variety of growth factors and mitogens and has been shown to be involved in the control of cell proliferation and differentiation (2Waskiewicz A.J. Cooper J.A. Curr. Opin. Cell Biol. 1995; 7: 798-805Crossref PubMed Scopus (535) Google Scholar, 3Kyriakis J.M. Avruch J. BioEssays. 1996; 18: 567-577Crossref PubMed Scopus (661) Google Scholar). Another subset of the MAPK pathways within a cell, the stress-activated protein kinases (SAPKs), responds to stress. In mammalian cells, two such pathways exist that lead to the activation of the JNK and p38 kinases (2Waskiewicz A.J. Cooper J.A. Curr. Opin. Cell Biol. 1995; 7: 798-805Crossref PubMed Scopus (535) Google Scholar, 3Kyriakis J.M. Avruch J. BioEssays. 1996; 18: 567-577Crossref PubMed Scopus (661) Google Scholar). A number of transcription factors are phosphorylated in response to SAPK activation, examples being c-Jun, which is regulated by JNK, and ATF2, which can be regulated by both JNK and p38 (4Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1710) Google Scholar, 5Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2957) Google Scholar, 6Livingstone C. Patel G. Jones N. EMBO J. 1995; 14: 1785-1797Crossref PubMed Scopus (476) Google Scholar, 7Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2046) Google Scholar, 8van Dam H. Wilhelm D. Herr I. Steffen A. Herrlich P. Angel P. EMBO J. 1995; 14: 1798-1811Crossref PubMed Scopus (571) Google Scholar). In Schizosaccharomyces pombe, a single member of the SAPK family called Sty1/Spc1/Phh1 (hereafter referred to as Sty1) has been identified (9Shiozaki K. Russell P. Nature. 1995; 378: 739-743Crossref PubMed Scopus (397) Google Scholar, 10Millar J.B. Buck V. Wilkinson M.G. Genes Dev. 1995; 9: 2117-2130Crossref PubMed Scopus (311) Google Scholar, 11Kato Jr., T. Okazaki K. Murakami H. Stettler S. Fantes P.A. Okayama H. FEBS Lett. 1996; 378: 207-212Crossref PubMed Scopus (155) Google Scholar). The Sty1 MAPK stimulates gene expression via the Atf1 transcription factor, which is similar to the human ATF2 factor, and binds to closely related DNA sequences (12Shiozaki K. Russell P. Genes Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (367) Google Scholar, 13Wilkinson M.G. Samuels M. Takeda T. Toone W.M. Shieh J.C. Toda T. Millar J.B. Jones N. Genes Dev. 1996; 10: 2289-2301Crossref PubMed Scopus (314) Google Scholar). Thus, the transcription factor targets of the S. pombe pathway are closely related to a subset of those phosphorylated and regulated by SAPKs in mammalian cells. The Atf1 factor was identified independently through the S. pombe DNA-sequencing project, by a genetic screen for sterile mutants that demonstrated a defect in G1 arrest following nitrogen starvation and through a genetic screen for genes that could suppress the mating defect of sty1– cells (12Shiozaki K. Russell P. Genes Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (367) Google Scholar, 14Takeda T. Toda T. Kominami K. Kohnosu A. Yanagida M. Jones N. EMBO J. 1995; 14: 6193-6208Crossref PubMed Scopus (230) Google Scholar, 15Kanoh J. Watanabe Y. Ohsugi M. Iino Y. Yamamoto M. Genes Cells. 1996; 1: 391-408Crossref PubMed Scopus (116) Google Scholar). Atf1 binds to its cognate site together with a second basic-leucine zipper (b-ZIP) containing protein Pcr1 (16Watanabe Y. Yamamoto M. Mol. Cell. Biol. 1996; 16: 704-711Crossref PubMed Google Scholar); the heterodimer has a significantly higher binding affinity than either homodimer complex (17Kon N. Krawchuk M.D. Warren B.G. Smith G.R. Wahls W.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13765-13770Crossref PubMed Scopus (133) Google Scholar). Disruption of the atf1 gene results in a range of phenotypes, including defects in sexual differentiation and maintenance of viability in stationary phase and sensitivity to osmotic stress and oxidative stress (14Takeda T. Toda T. Kominami K. Kohnosu A. Yanagida M. Jones N. EMBO J. 1995; 14: 6193-6208Crossref PubMed Scopus (230) Google Scholar, 15Kanoh J. Watanabe Y. Ohsugi M. Iino Y. Yamamoto M. Genes Cells. 1996; 1: 391-408Crossref PubMed Scopus (116) Google Scholar, 18Degols G. Russell P. Mol. Cell. Biol. 1997; 17: 3356-3363Crossref PubMed Google Scholar, 19Nguyen A.N. Lee A. Place W. Shiozaki K. Mol. Biol. Cell. 2000; 11: 1169-1181Crossref PubMed Scopus (138) Google Scholar, 20Quinn J. Findlay V.J. Dawson K. Millar J.B. Jones N. Morgan B.A. Toone W.M. Mol. Biol. Cell. 2002; 13: 805-816Crossref PubMed Scopus (167) Google Scholar). Thus Atf1 is crucial for fission yeast cells to respond normally to a range of different stress conditions. Previous studies have shown that stress-induced activation of Sty1 results in its translocation to the nucleus whereby it binds to, and phosphorylates, Atf1 (21Gaits F. Degols G. Shiozaki K. Russell P. Genes Dev. 1998; 12: 1464-1473Crossref PubMed Scopus (134) Google Scholar). This finding, together with the observation that the expression of Atf1 target genes requires Sty1 activation, strongly suggested that phosphorylation of Atf1 stimulates its activity. However, the mechanism by which this regulation might occur is not known. In mammalian cells, regulation of ATF2 by JNK or p38-induced phosphorylation has been investigated in detail. Regulation appears to be exercised at a number of different levels. In the absence of stimulating conditions, ATF2 is transcriptionally inactive due to an intramolecular interaction between the DNA binding domain and the amino-terminal region. It has been suggested that this intramolecular inhibition is disrupted, and transcriptional activities restored, when ATF2 interacts with other proteins, such as E1A or c-Jun, or when it is phosphorylated by SAPKs (22Li X.Y. Green M.R. Genes Dev. 1996; 10: 517-527Crossref PubMed Scopus (107) Google Scholar). Phosphorylation has also been shown to increase the activity of the isolated transactivation domain of ATF2, although the mechanism involved remains obscure (6Livingstone C. Patel G. Jones N. EMBO J. 1995; 14: 1785-1797Crossref PubMed Scopus (476) Google Scholar, 8van Dam H. Wilhelm D. Herr I. Steffen A. Herrlich P. Angel P. EMBO J. 1995; 14: 1798-1811Crossref PubMed Scopus (571) Google Scholar, 23Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1339) Google Scholar). Yet another mode of regulation involves ubiquitin-mediated degradation by the 26 S proteasome; phosphorylation of ATF2 by SAPKs appears to protect it from ubiquitination and subsequent degradation (24Firestein R. Feuerstein N. J. Biol. Chem. 1998; 273: 5892-5902Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 25Fuchs S.Y. Ronai Z. Mol. Cell. Biol. 1999; 19: 3289-3298Crossref PubMed Scopus (52) Google Scholar, 26Fuchs S.Y. Tappin I. Ronai Z. J. Biol. Chem. 2000; 275: 12560-12564Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In this study, we sought to investigate how Atf1 may be regulated through Sty1-mediated phosphorylation. Our results indicate that phosphorylation regulates the stability of Atf1 as does heterodimerization with its binding partner Pcr1. This regulation, however, appears to be only partly responsible for the role of Sty1 in stimulating Atf1-dependent gene expression suggesting that at least one other mode of control must exist that does not involve direct Atf1 phosphorylation. Yeast Strains and General Methods—S. pombe strains used are listed in Table 1. Yeast media and general experimental methods were as described (27Moreno S. Klar A. Nurse P. Methods Enzymol. 1991; 194: 795-823Crossref PubMed Scopus (3148) Google Scholar). For the dilution assays, exponentially growing cells at a concentration of 1 × 106 cells/ml were diluted 5-fold a total of four times, and 7.5 μl of each dilution, including the starting dilution of 1 × 106 cells/ml, was plated on yeast extract plates containing various stress-inducing agents.TABLE 1S. pombe strains used in this studyStrainGenotypeSourceNJ2h-ura4-D18 leu1-32 ade6-M210 his7-366C. HoffmanNJ71h90atf1-2HA6His:LEU2 ura4-D18 leu1-32 ade6-M216This studyNJ72h90atf1-11M-2HA6His:LEU2 gad7::ura4-ura4-D18 leu1-32 ade6-M216This studyH222h90gad7::ura4+ ura4-D18 leu1-32 ade6-M216Kanoh et al. (15Kanoh J. Watanabe Y. Ohsugi M. Iino Y. Yamamoto M. Genes Cells. 1996; 1: 391-408Crossref PubMed Scopus (116) Google Scholar)JX25h90pcr1::ura4+ura4-D18 leu1-32 ade6-M216Watanabe and Yamamoto (16Watanabe Y. Yamamoto M. Mol. Cell. Biol. 1996; 16: 704-711Crossref PubMed Google Scholar)NJ437h+ atf1::kanr pcr1::ura4+ leu1-32 ura4-D18 his7-366 ade6-M210This studyNJ55h-mts2-1 leu1-32 ura4-D18Gordon et al. (37Gordon C. McGurk G. Dillon P. Rosen C. Hastie N.D. Nature. 1993; 366: 355-357Crossref PubMed Scopus (210) Google Scholar)NJ28h90atf1::ura4+ leu1-32 ura4-D18Takeda et al. (14Takeda T. Toda T. Kominami K. Kohnosu A. Yanagida M. Jones N. EMBO J. 1995; 14: 6193-6208Crossref PubMed Scopus (230) Google Scholar)H178h90sty1-1atf1::ura4+ ura4-D18 leu1-32 ade6 his7-366Lab stockNJ24h-sty1::ura4+ ura4-D18Lab stockKK853h+wis1::his1+ ura4-D18 leu1-32 his1-102K. KitamuraKS2081h-wis1DD:12myc ura4-D18 leu1-32Shiozaki et al. (35Shiozaki K. Shiozaki M. Russell P. Mol. Biol. Cell. 1998; 9: 1339-1349Crossref PubMed Scopus (99) Google Scholar)NJ260h+atf1::kanr leu1-32 ura4-D18 ade6-M210 his7-366Lab stock Open table in a new tab For all procedures requiring harvesting of cells, mild centrifugation (2 min, 580 relative centrifugal force) was used. This procedure did not lead to activation of Sty1 (data not shown). A previous study has also shown that mild centrifugation for 2 min at 800 relative centrifugal force did not lead to a difference in the profile of gene expression compared with that obtained from cells isolated by filtration (28Lyne R. Burns G. Mata J. Penkett C.J. Rustici G. Chen D. Langford C. Vetrie D. Baöhler J. BMC Genomics. 2003; 4: 27Crossref PubMed Scopus (181) Google Scholar), consistent with our finding that mild centrifugation does not activate the stress-activated MAPK pathway in fission yeast. Cycloheximide (Sigma C4859) was added to cultures at a final concentration of 100 μg/ml to inhibit protein synthesis. Creation of an atf1-11M Allele and Integration—The atf1 cDNA was cloned into the pREP81 vector (29Basi G. Schmid E. Maundrell K. Gene (Amst.). 1993; 123: 131-136Crossref PubMed Scopus (570) Google Scholar) with two hemagglutinin epitopes and six histidine residues placed at the carboxyl terminus of the open reading frame (ORF). Point mutations were introduced into pREP81(atf1+) by a PCR-based method. Serines at positions 2, 4, 140, 152, 172, 226, and 438 and threonine at position 77 were replaced by alanine, and threonines at positions 204, 216, and 249 were replaced by isoleucine (pREP81(atf1-11M)). For atf1+, a 1.4-kb EcoRI fragment, which encompassed the carboxyl-terminal region of the atf1 cDNA and the nmt1 terminator region, was subcloned into the YIplac128 vector (30Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2528) Google Scholar). The vector was linearized with MscI, and the resulting fragment was transformed into a wild-type strain (NJ2). Stable leu+ clones were selected, and integration and copy number were determined by PCR and Southern blotting, respectively. This strain expressing the tagged version of Atf1 was indistinguishable from one expressing untagged Atf1. For atf1-11M, a 1.2-kb fragment upstream of the atf1 coding region was PCR-amplified, ligated to the atf1-11M ORF, and inserted into YIplac128 with the nmt1 terminator region cloned downstream. The plasmid was digested at a unique StuI site and transformed into the S. pombe atf1/gad7 disruptant (JX305) (15Kanoh J. Watanabe Y. Ohsugi M. Iino Y. Yamamoto M. Genes Cells. 1996; 1: 391-408Crossref PubMed Scopus (116) Google Scholar). Integrated clones were assessed as above. Protein Extract Preparation and Western Blot Analysis—Native S. pombe cell extracts were prepared by glass bead lysis in the following buffer (50 mm Tris-Cl pH 7.5, 0.15 m KCl, 10 mm MgCl2, 20 mm β-glycerophosphate, 5 mm EDTA, 0.1 mm sodium vanadate, 1 mm dithiothreitol, 10% glycerol, 0.2% Nonidet P-40) containing 1 mm phenylmethylsulfonyl fluoride and protein inhibitor Complete™ (Roche Applied Science). Before lysis, cells were washed once in stop buffer (150 ml of NaCl, 50 mm NaF, 10 mm EDTA, 1 mm NaN3, pH8). Total cell extracts were resolved by SDS-PAGE and transferred electrophoretically to Immobilon-P™ (Millipore) or nitrocellulose. For the analysis of Atf1, 8% gels were prepared. Pcr1 was detected after extracts had been separated by 15% SDS-PAGE. Polyclonal anti-Atf1 and anti-Pcr1 antisera were obtained by immunizing rabbits with GST-Atf1 or GST-Pcr1. Anti-Atf1 and Anti-Pcr1 antisera were purified using antigen-spotted membranes and used at dilutions of 1 in 750 and 1 in 500, respectively. Anti-tubulin antiserum was obtained from Sigma (T5168) and used at a dilution of 1 in 1000, Mts4 antiserum (49Wilkinson C.R. Wallace M. Seeger M. Dubiel W. Gordon C. J. Biol. Chem. 1997; 272: 25768-25777Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) was used at a dilution of 1 in 5000, and a monoclonal anti-HA antibody (12CA5) obtained from Roche Applied Science was used at a dilution of 1 in 1000. Detection was performed using a peroxidase-conjugated anti-rabbit or anti-mouse IgG (Amersham Biosciences Pharmacia) and chemiluminescence visualization (ECL+, Amersham Biosciences) was used according to the manufacturer’s instructions. Immunoprecipitations were carried out using an anti-hemagglutinin matrix (Roche Applied Science) according to the manufacturer’s instructions. Phosphatase analysis was carried out using λ phosphatase (New England Biolabs) for 30 min at 30 °C. Quantification of Western blot signals was performed using the Chemi Genius Bioimaging system (Syngene) and the Chemi genius gel documentation and analysis system. Denatured S. pombe extracts were prepared using the following method. A volume corresponding to A600 nm 0.5–3.0 of cells was harvested and resuspended by vortexing in 1 ml of 0.3 m NaOH. 150 μl of 55% (w/v) trichloroacetic acid was added; the mixture was vortexed and incubated on ice for 10 min. The cells were pelleted at 4 °C for 10 min at 14,000 rpm. The supernatant was removed by aspiration, and the cells were spun briefly for a second time to remove remaining trichloroacetic acid. The pellet was resuspended in 75 μl of SDS gel-loading buffer per A600 nm of cells (SDS gel loading buffer: 50 mm Tris-Cl (pH 6.8), 2% SDS (w/w), 0.1% bromphenol blue, 10% (v/v) glycerol, 10 mm dithiothreitol). Proteins were denatured for 10 min at 100 °C. Samples were centrifuged briefly, before loading onto SDS-PAGE gels. Two-dimensional Gel Electrophoresis—Total cellular protein was prepared from NJ2, NJ71, and NJ72. 14 mg of whole cell protein was used in immunoprecipitation reactions. After the final wash the matrix was resuspended in two-dimensional gel sample buffer (8 m urea, 2% CHAPS, 0.002% bromphenol blue) and subjected to isoelectric focusing using 7-cm strips pH 4–7 (Amersham Biosciences) and the IPGphor (Amersham Biosciences) according to the manufacturer’s instructions. Isoelectric focusing steps were as follows: rehydration for 16 h; 5000 V for 1 h, 4000 V for 1 h 30 min, and then 5000 V for 2 h. The second dimension was separated by SDS-PAGE on an 8% gel. RNA Analysis—10-μg samples of total RNA isolated at the time points indicated in the figures were denatured with formaldehyde, separated on a 1% agarose gel, and transferred to a Hybond-N+ membrane (Amersham Biosciences). Probes for RNA-DNA hybridization were PCR-generated fragments internal to the gene concerned and labeled with 32P by use of a DNA Megaprime labeling kit (Amersham Biosciences). atf1 and atf1-11M mRNA levels were quantified relative to the hmg1 transcript using the Chemi genius system. Chromatin Immunoprecipitation Assays—The chromatin immunoprecipitation procedure is based on the methods described elsewhere (31Hecht A. Strahl-Bolsinger S. Grunstein M. Methods Mol. Biol. 1999; 119: 469-479PubMed Google Scholar, 32Tanaka T. Kanpp D. Nasmyth K. Cell. 1997; 90: 649-660Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar) with some modifications. All steps were performed on ice except when indicated. 100 ml of yeast cells A595 = 0.6 were cross-linked for 10 min with 1% formaldehyde at 24 °C. After addition of 125 mm glycine and incubation for 5 min at 24 °C, cells were chilled on ice, washed with ice-cold dH2O, and suspended in 400 μl of lysis buffer (50 mm Hepes-KOH, pH 7.5, 140 mm NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 20 mm β-glycerophosphate, 50 mm NaF, 0.1 mm Na3VO4, 0.5 mm sodium pyrophosphate, Complete™ protease inhibitor EDTA free (Roche Applied Science), 1 mm EDTA). Crude extracts were prepared by 3–4 pulses (15 s at level 5) of bead beating in a FastPrep FP120 Ribolyzer (Thermo Savant, Bio 101, Inc., Vista, CA) until 70–80% of cells were broken. Extracts were sonicated four times for 10 s (level 5) using a Soniprep 150 (Sanyo) until chromatin was sheared to an average size of 500 bp and subsequently cleared of insoluble cell debris by short centrifugation at full speed. 5 μl of the whole cell extract was saved as an INPUT control. Immunoprecipitation was performed for 3–4 h with Dynal protein A-coated magnetic beads, which were previously incubated overnight with anti Atf1 antiserum. Precipitates were washed three times with 1 ml of lysis buffer, 1 ml of lysis buffer plus salt (like lysis buffer, except 500 mm NaCl), and 1 ml of wash buffer (10 mm Tris-HCl, pH 8.0, 250 mm LiCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 1 mm EDTA), respectively. Precipitates were eluted by 10 min of incubation at 65 °C in 40 μl of elution buffer (50 mm Tris-HCl, pH 8.0, 10 mm EDTA, 1% SDS). Eluates were incubated at 65 °C in a total volume of 150 μl (100 μl for INPUT control) elution buffer containing RNase (0.1 mg/ml) for overnight, then doubled in volume with TE buffer (50 mm Tris-HCl, pH 8.0, 10 mm EDTA), 0.1 mg/ml glycogen, and 0.5 mg/ml proteinase K and incubated for 2 h at 37 °C. Immunoprecipitated DNA was purified using the Qiagen PCR purification kit. We used standard PCR (data not shown) as well as real-time PCR, to quantify for each immunoprecipitate the relative amount of DNA corresponding to stress-induced (gpd1 and hsp9) or stress-independent (act1) over the stress-independent promoter (cdc2). The ORF of the stress-independent pol1 gene was used as an additional background control. Primers used relative to the transcription start codon corresponded to –214 to –193 and –168 to –149 for the cdc2 promoter; –402 to –380 and –360 to –340 for the gpd1 promoter; –264 to –245 and –220 to –199 for the hsp9 promoter; –113 to –85 and –61 to –34 for the act1 promoter; and +2520 to +2543 and +2567 to +2588 for the pol1 ORF. For real-time PCR we set up 12.5-μl reactions containing 0.5 μl of purified DNA from a particular immunoprecipitate, 1 μl of 5 μm 5′-oligonucleotide primer, 1 μl of 5 μm 3′-oligonucleotide primer, 6.25 μl of SYBR Green PCR master mix (Applied Biosystems), and 3.75 μlof dH20. Reactions were analyzed using an ABI 7900 thermal cycler according to the manufacturer’s instructions. We performed three independent PCR reactions and calculated the mean “threshold cycle number” (or Ct value). The -fold enrichment of the stress-inducible promoters relative to stress-independent promoters for a particular sample was calculated using the following formula: -fold enrichment = 2CtINPUT – CtIP/2CtINPUT – Ctbackground, where Ct IP is the Ct value for the immunoprecipitate (gpd1, hsp9, act1, and pol1), and Ct background is the Ct value for the background control (cdc2). Atf1 Protein Accumulates following Stress—The Atf1 protein possesses eleven potential MAPK sites, namely a serine or threonine residue immediately followed by a proline (Fig. 1A). We first examined the levels of Atf1 protein before and after two different types of stress, namely oxidative stress (H2O2) and osmotic stress (sorbitol) to determine whether, by analogy to the mammalian ATF2 factor, phosphorylation modulated Atf1 stability (24Firestein R. Feuerstein N. J. Biol. Chem. 1998; 273: 5892-5902Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 25Fuchs S.Y. Ronai Z. Mol. Cell. Biol. 1999; 19: 3289-3298Crossref PubMed Scopus (52) Google Scholar, 26Fuchs S.Y. Tappin I. Ronai Z. J. Biol. Chem. 2000; 275: 12560-12564Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In wild-type cells the level of Atf1 was significantly increased following stress imposition (Fig. 1B). In addition to increased levels, the mobility of the Atf1 protein was retarded; this has been shown previously to be due to phosphorylation (12Shiozaki K. Russell P. Genes Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (367) Google Scholar, 16Watanabe Y. Yamamoto M. Mol. Cell. Biol. 1996; 16: 704-711Crossref PubMed Google Scholar). These results are consistent with the possibility that stability of Atf1 is linked to its phosphorylation state. Interestingly, the extent and kinetics of phosphorylation appear to be dependent on the particular type of stress applied. To directly test whether phosphorylation affects stability, we constructed a mutant atf1 gene containing single point mutations in all potential MAPK phosphoacceptor sites (atf1-11M). This gene was integrated into the atf1 locus so that atf1 mRNA expression was under the control of the endogenous promoter. Following the imposition of stress, neither retardation nor significant accumulation of the mutant atf1-11M protein was observed (Fig. 1B). The results are consistent with a lack of phosphorylation of the mutant protein and a dependence on direct phosphorylation for Atf1 accumulation. There was a slight reduction in the amount of mRNA produced for the atf1-11M transcript compared with wild-type (Fig. 1C), but the levels did not seem to drop sufficiently enough to explain the dramatic decrease that we observed in the level of atf1-11M protein. A slight increase in the amount of atf1-11M protein over time was observed (Fig. 1B, compare levels at t = 0 to t = 30) and was probably due to a modest increase in the atf1 mRNA levels that we observed upon stress (Fig. 1C) (12Shiozaki K. Russell P. Genes Dev. 1996; 10: 2276-2288Crossref PubMed Scopus (367) Google Scholar, 33Rodriguez-Gabriel M.A. Burns G. McDonald W.H. Martin V. Yates 3rd, J.R. Baöhler J. Russell P. EMBO J. 2003; 22: 6256-6266Crossref PubMed Scopus (59) Google Scholar). Previously, we have analyzed the global changes in gene expression in response to stress and found that, in H2O2 stress, the levels of atf1 mRNA are induced 2.3- and 2.7-fold at 15 and 60 min of stress, respectively; in response to sorbitol stress the levels of atf1 mRNA are induced 2.6-fold at 15 min and have returned to basal levels by 60 min (34Chen D. Toone W.M. Mata J. Lyne R. Burns G. Kivinen K. Brazma A. Jones N. Baöhler J. Mol. Biol. Cell. 2003; 14: 214-229Crossref PubMed Scopus (628) Google Scholar). These modest increases are in agreement with our Northern blot analysis (Fig. 1C). To support the above conclusions, we examined Atf1 protein levels in different mutant backgrounds defective, or containing a constitutively active, Sty1 kinase pathway. Wis1 is an MAPK kinase that phosphorylates and thus activates Sty1. In both sty1Δ and wis1Δ cells, the level of Atf1 in the absence of stress was lower than in wild-type cells (Fig. 1D). Furthermore, no increase in levels and no retardation of mobility were observed following H2O2 stress. In contrast, however, in wis1-DD cells, which have a constitutively active allele of the MAPK kinase wis1 (35Shiozaki K. Shiozaki M. Russell P. Mol. Biol. Cell. 1998; 9: 1339-1349Crossref PubMed Scopus (99) Google Scholar), Atf1 levels were high in the absence of stress and a significant proportion had a retarded mobility. The levels did not increase further following stress. Thus all the results described above show a tight correlation between the phosphorylation of Atf1 and its level in the cell. Atf1 Is Phosphorylated under Basal and Stress Conditions but Not When Its MAPK Sites Are Mutated—Although all the potential MAPK sites in Atf1 had been mutated, it was important to examine the phosphorylation status of atf1-11M, because it was possible that Sty1 could still phosphorylate a site that did not conform to the MAPK consensus motif. A previous study has shown that the mammalian homologue of Sty1, p38α, can phosphorylate one of its targets, TAB1, at a serine-alanine site that does not fit the MAPK consensus motif (36Cheung P.C. Campbell D.G. Nebreda A.R. Cohen P. EMBO J. 2003; 22: 5793-5805Crossref PubMed Scopus (242) Google Scholar). Firstly, we stressed atf1-HA and atf1-11M-HA cells and analyzed the Atf1 protein from these and unstressed control samples. The immunoprecipitated protein fractions were split into two, and one-half of each sample was treated with λ-phosphatase to remove the phosphate groups and analyzed by Western blotting (Fig. 2A). Interestingly, the Atf1 protein growing in rich media without stress appeared to be basally phosphorylated. Upon stress, as expected, Atf1 became hyperphosphorylated. In contrast, the atf1-11M protein did not appear to be phosphorylated, eithe" @default.
• W1986301417 created "2016-06-24" @default.
• W1986301417 creator A5030900887 @default.
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