The In Vivo Activity of Ime1, the Key Transcriptional Activator of ...

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Jan 19, 2004 - Ifat Rubin-Bejerano,†‡ Shira Sagee,† Osnat Friedman, Lilach Pnueli, and Yona Kassir* ...... Bowdish, K. S., H. E. Yuan, and A. P. Mitchell. 1994.

MOLECULAR AND CELLULAR BIOLOGY, Aug. 2004, p. 6967–6979 0270-7306/04/$08.00⫹0 DOI: 10.1128/MCB.24.16.6967–6979.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Vol. 24, No. 16

The In Vivo Activity of Ime1, the Key Transcriptional Activator of Meiosis-Specific Genes in Saccharomyces cerevisiae, Is Inhibited by the Cyclic AMP/Protein Kinase A Signal Pathway through the Glycogen Synthase Kinase 3-␤ Homolog Rim11 Ifat Rubin-Bejerano,†‡ Shira Sagee,† Osnat Friedman, Lilach Pnueli, and Yona Kassir* Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel Received 19 January 2004/Returned for modification 8 March 2004/Accepted 20 May 2004

Phosphorylation is the main mode by which signals are transmitted to key regulators of developmental pathways. The glycogen synthase kinase 3 family plays pivotal roles in the development and well-being of all eukaryotic organisms. Similarly, the budding yeast homolog Rim11 is essential for the exit of diploid cells from the cell cycle and for entry into the meiotic developmental pathway. In this report we show that in vivo, in cells grown in a medium promoting vegetative growth with acetate as the sole carbon source (SA medium), Rim11 phosphorylates Ime1, the master transcriptional activator required for entry into the meiotic cycle and for the transcription of early meiosis-specific genes. We demonstrate that in the presence of glucose, the kinase activity of Rim11 is inhibited. This inhibition could be due to phosphorylation on Ser-5, Ser-8, and/or Ser-12 because in the rim11S5AS8AS12A mutant, Ime1 is incorrectly phosphorylated in the presence of glucose and cells undergo sporulation. We further show that this nutrient signal is transmitted to Rim11 and consequently to Ime1 by the cyclic AMP/protein kinase A signal transduction pathway. Ime1 is phosphorylated in SA medium on at least two residues, Tyr-359 and Ser-302 and/or Ser-306. Ser-302 and Ser-306 are part of a consensus site for the mammalian homolog of Rim11, glycogen synthase kinase 3-␤. Phosphorylation on Tyr-359 but not Ser-302 or Ser-306 is essential for the transcription of early meiosis-specific genes and sporulation. We show that Tyr-359 is phosphorylated by Rim11. In the budding yeast Saccharomyces cerevisiae, the choice between meiosis and alternative developmental pathways, such as the mitotic cell cycle or pseudohyphal growth, depends on the expression and activity of a master regulator, Ime1. IME1 encodes a transcriptional activator (30, 47) that is recruited to promoters of early meiosis-specific genes by the sequencespecific DNA-binding protein Ume6 (8, 28, 36, 40). Ume6 binds to a specific element, URS1, present in the promoter of most early meiosis-specific genes (1, 49, 55). The URS1 element and Ume6 are required for the silencing of early meiosisspecific genes in vegetative growth conditions and for their transcription under meiotic conditions (4, 48, 49). The association of Ume6 with the histone deacetylase Sin3/Rpd3 complex and the ATP-dependent chromatin-remodeling complex Isw2 leads to the silencing of early meiosis-specific genes under vegetative growth conditions with glucose as the sole carbon source (17, 21). Under meiotic conditions, there is a switch in the proteins that occupy URS1 elements. Sin3 and Rpd3 are transiently removed, and Ime1, which is expressed under these conditions, is recruited by Ume6 to these promoters (36). The signals leading to meiosis (i.e., the presence of the MATa1 and MAT␣2 gene products, the absence of glucose and nitrogen, and the presence of a nonfermentable carbon source

such as acetate) converge at Ime1, regulating its availability and activity at multiple levels (for a recent review, see reference 22). First, the transcription of IME1 depends on all meiotic signals (23). In a medium promoting vegetative growth with glucose as the sole carbon source (SD medium), IME1 is silent, whereas low levels of IME1 mRNA are detected when acetate is the sole carbon source (SA medium). Upon nitrogen depletion (SPM medium), but only in MATa/MAT␣ diploid cells, the transcription of IME1 is transiently induced (23). Nitrogen depletion is also required for the efficient translation of IME1 mRNA (44) and for the localization of Ime1 in the nuclei (8, 9). Ectopic expression of Ime1 in exponentially growing cells does not promote the transcription of early meiosis-specific genes and entry into meiosis even when Ime1 is artificially localized in the nuclei (9, 40, 44). This suggests that nutrients also regulate the activity of Ime1, Ume6, or another unknown protein. These possibilities are not mutually exclusive. Glucose and nitrogen depletion regulate the transcription of early meiosis-specific genes, sporulation, and the two-hybrid interaction between Ime1(270-360) and Ume6(1-232) (40). These events depend on two protein kinases, a glycogen synthase kinase 3-␤ (GSK3) homolog, Rim11, and Rim15 (3, 8, 28, 40, 50, 52). Rim11 associates with Ime1(270-360) under all growth conditions (28, 40). In vitro, Rim11 phosphorylates one or all four tyrosine residues confined to the 20 C-terminal amino acids of Ime1 (28). This is consistent with the function of GSK3 as a dual-specificity kinase in autophosphorylating both serine/ threonine and tyrosine residues (53). Most GSK3 substrates have the S/T-X-X-X-S/T-P04 consensus, in which priming

* Corresponding author. Mailing address: Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel. Phone: 972-4-8294214. Fax: 972-4-8225153. E-mail: [email protected] .ac.il. † I.R.-B. and S.S. contributed equally to this article. ‡ Present address: Whitehead Institute for Biomedical Research, Cambridge, Mass. 6967

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phosphorylation at position P ⫹ 4 is required for GSK3 to transfer phosphate to position P0. The C-terminal 90 amino acids of Ime1 carry four such sites (see Fig. 5). Simultaneous serine-to-alanine mutations of the distal two sites have no effect on the ability of Rim11 to in vitro phosphorylate Ime1 or on the Ime1-Ume6 two-hybrid interaction (28). This suggests that Rim11 may not phosphorylate these residues. In this report we show that, in vivo, Ime1(270-360) is subject to nutrient-regulated posttranslational modifications, including tyrosine phosphorylation on a single tyrosine residue, Tyr-359. In vivo, in SA medium, Ime1 is phosphorylated on two separate residues, Tyr-359 and Ser-302 and/or Ser-306. We show that Tyr-359 is essential for sporulation. We then show that Rim11 phosphorylates Ime1 under the same conditions and provide evidence that links Rim11 to these phosphorylation events. In a rim11S5AS8AS12A triple mutant, Ime1 is aberrantly phosphorylated in the presence of glucose, and these mutant cells sporulate in the presence of glucose. This result suggests that in the presence of glucose, the activity of Rim11 is inhibited due to phosphorylation on Ser-5, Ser-8, and/or Ser-12. We further show that the cyclic AMP (cAMP)/protein kinase A (PKA) signal transduction pathway, mainly through Tpk3, transmits the glucose and nitrogen signals to Rim11 and consequently to Ime1. MATERIALS AND METHODS Media and genetic techniques. SA medium (previously designated PSP2, minimal acetate growth medium), and SPM medium (sporulation medium) have been described (24). SD medium (synthetic dextrose) has been described (45). Meiosis was induced either by shifting stationary-phase cells grown in SD medium to SPM medium at 107 cells/ml or by shifting logarithmic-phase (107 cells/ml) cells grown in SA medium to SPM medium as described previously (24). Transformation of Saccharomyces cerevisiae with lithium acetate was done as described previously (16). Standard methods for DNA cloning and bacterial transformation were used (42). Site-directed mutagenesis was performed as described previously (27). ␤-Galactosidase activity was measured as described previously (39). Results are given in Miller units (33) and are averages of at least three independent transformants. For determining ␤-galactosidase expression on plates (i.e., the formation of white or blue colonies), 100 ␮l of 5-bromo-4-chloro3-indolyl-␤-galactopyranoside (X-Gal, 20 mg/ml) was spread on top of an SA plate. Under vegetative growth conditions with glucose as the sole carbon source, Ime1 was expressed from the ADH1 promoter. Expression of Ime1 in haploid cells in either SA or SPM medium was accomplished with a truncated IME1 promoter, IME1 (⫺31 to ⫺1364), that lacks MAT control (41). The use of two different promoters was required for the detection of Ime1 in meiotic cultures because the ADH1 promoter is not sufficiently expressed in SPM medium (40). PCR mutagenesis. The following modifications were used to perform mutagenized PCR. The reaction mixture contained between 0.05 and 0.65 mM MnCl2, and the concentration of dATP was reduced to 0.2 mM. Any PCR that gave rise to about 50% efficiency was considered to carry mutagenized DNA fragments. Isolation of Ime1(270-360) mutants defective in its two-hybrid interaction with Ume6. With a PCR mutagenesis protocol, DNA fragments carrying gal4(414-441)-ime1(810-1080)-ADH1t(1088-1306) were derived with oligonucleotides ADH1t(r) and Gal4(dbd) and YEp1338 as a template. The DNA fragments produced were transformed together with a linear vector (YCp1955) into strain GGY::171. YCp1955, which carries ADH1p-GAL4(dbd)-ADHt, was linearized by digestion with EcoRI to promote in vivo recombination with the PCR products. The transformed strain carried the gal4(ad)-ume6(1-232) gene (YEp1788) and the gal-lacZ reporter. The His⫹ transformants were screened for white colonies on SA-X-Gal plates. Blue colonies arose from interaction between Gal4(ad)-Ume6 and Gal4(dbd)-Ime1, whereas white colonies were defective in this interaction. Two hundred colonies out of 1,000 transformants gave rise to white colonies (did not express gal1-lacZ). In order to examine whether these colonies carried the pADH1-gal4(dbd)-ime1(id)-ADH1t chimera, they were subjected to PCR with oligonucleotides Gal4(dbd) and ADHt(r). The expected PCR

MOL. CELL. BIOL. fragment was obtained in only 14 colonies, suggesting that in 186 of 1,000 the vector plasmid was self-ligated. Plasmids could be rescued from 6 of the 14 positive transformants. Sequence analysis was applied to reveal the nature of the mutations. Antibodies. Mouse monoclonal antibodies directed against Gal4(1-147) ([Gal4(dbd)]) were purchased from Santa Cruz Biotechnology Inc. Mouse monoclonal antibodies directed against phosphotyrosine (4G10) were purchased from Upstate. Preparation of yeast protein extracts and Western analysis. Protein extracts were prepared from either trichloroacetic acid-treated cells as described previously (12) or by the conventional method for immunoprecipitation as described previously (2). Immunoblot procedures were essentially as described previously (12, 13). In vitro kinase assay. We used the E. coli T7 S30 in vitro transcriptiontranslation system (Promega) with or without a plasmid carrying pT7-RIM11 (P2844) to express Rim11. The manufacturer’s protocol was used. GSTIme1(270-360) (P2677) or GST-Ime1(270-360)Y359F (P2843) and controlled GST (pGEX-4T-1) proteins expressed from the tac1 promoter in E. coli BL21 were immobilized on glutathione-Sepharose beads (Pharmacia), according to the manufacturer’s protocol. Following a wash with kinase buffer (50 mM Tris [pH 7.5], 10 mM MgCl2), dithiothreitol and ATP were added to final concentrations of 1 mM and 2 ␮M, respectively. We then added the E. coli S30 Rim11 lysate (or, as a control, a lysate that did not carry the RIM11 plasmid). Following 45 min of incubation at room temperature, beads were washed with PBS, and glutathionebound proteins were eluted with Laemmli sample buffer and 3 min of boiling. Tyrosine phosphorylation was detected with Western analysis. Oligonucleotides. Sequences are available upon request. Plasmids. The plasmids used and constructed in this study are listed in Table 1. A detailed description of how these plasmids were constructed is available upon request. All site-directed mutations were confirmed by sequencing. Strains. The strains used are listed in Table 2. Strains carrying the (rim11⌬ 157-370)::LEU2 allele were constructed by transforming their parental strains with a PvuII fragment carrying the rim11::LEU2 allele from YIp1611. Strains carrying the cdc25-5 allele were constructed by replacing the CDC25 allele in their parental strains following transformation with a SalI-PvuII fragment carrying cdc25-5::URA3 from P1902. Y1174 carries the rim11K68A allele from YIp2127 integrated at the HIS3 locus. Y1235-1 was constructed in three steps. First, the RIM15 allele was replaced with rim15::loxP-URA3-KAN-loxP by transforming Y1064 with YIp2211 cut the SpeI and EcoRI. Then, the URA3-KAN chimera was deleted by site-specific recombination, following transformation with pSH47 (18). Finally, the rim15K823A allele was integrated at LEU2 following transformation with YIp2670 digested with PpuMI. Y1392 was constructed by replacing the RIM11 allele with rim11K68A following transformation of Y1064 with YIp2691 digested with NruI. rim113SA was inserted at the ura3-52 locus by transforming Y1066 with YIp2804 digested with PpuMI.

RESULTS Nutrients regulate the posttranslational modification of Ime1(270-360). The following observations suggest that Ime1 needs to be phosphorylated to activate the transcription of early meiosis-specific genes: (i) in vivo, Ime1 is a phosphoprotein (8, 19, 29); (ii) in vitro, Rim11 phosphorylates Ime1 (28); (iii) two protein kinases, Rim11 and Rim15, are required for the two-hybrid interaction of Ime1 with Ume6 and the expression of early meiosis-specific genes (40, 52); and (iv) ectopic expression of Ime1 in exponentially growing cells does not promote entry into meiosis (9, 40). We hypothesized that nutrients might regulate the phosphorylation of Ime1(270-360). In the experiments reported here, Ime1(270-360) was tagged with the Gal4 DNA-binding domain [Gal4(dbd)], which by itself is not subject to any posttranslational modification (19). The pattern of Ime1(270-360) was examined in three different media: SD medium, with glucose as the sole carbon source; SA medium, with acetate as the sole carbon source; and SPM medium, with acetate as the sole carbon source and no nitrogen source. Immunoprecipitation and Western analysis with antibodies

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TABLE 1. List of plasmids Plasmid

Vector

YEp1338 YIp1611 YEp1788 YIp1902 YEp1931 YCp1955 YEp2042 YEp2112 YEp2122 YIp2127 YCp2195 YEp2196 YEp2200 YEp2206 p2211 YIp2214 YEp2215 YCp2233 YIp2234 YCp2242 YCp2302 YCp2303 YCp2306 YEp2430

HIS3 2␮m BS LEU2 2␮m URA3 TRP1 2␮m HIS3 2␮m CEN11 TRP1 2␮m TRP1 2␮m TRP1 2␮m HIS3 HIS3 2␮m CEN11 HIS3 2␮m TRP1 2␮m TRP1 2␮m T Easy LEU2 TRP1 2␮m HIS3 ARSH4 ⫹ CEN6 LEU2 HIS3 ARSH4 ⫹ CEN6 HIS3 ARSH4 ⫹ CEN6 HIS3 ARSH4 ⫹ CEN6 HIS3 ARSH4 ⫹ CEN6 TRP1 2␮m

YCp2435 YCp2436

HIS3 ARSH4 ⫹ CEN6 HIS3 ARSH4 ⫹ CEN6

YIp2670 P2677 YIp2691 YEp2713 YEp2780 YIp2804 P2843 P2844

LEU2 LEU2 TRP1 2␮m TRP1 2␮m URA3

Insert

pADH1-gal4(dbd)-ime1(270–360) rim11⌬::LEU2 pUME6-ume6(1–232)-gal4(ad)-ADHt cdc25-5::URA3 pIME1-gal4(dbd)-ime1(270–360) pAdh1-gal4(dbd) hop1-lacZ pIME1-gal4(dbd)-ime1S302A(270–360) pIME1-gal4(dbd)-ime1S306A(270–360) rim11K68A pADH1-gal4(dbd)-ime1Y359F(270–360) pIME1-gal4(dbd)-ime1Y359F(270–360) pIME1-gal4(dbd)-ime1(270–360) pIME1-gal4(dbd)-ime1S333A(270–360) rim15::loxp-URA3-kanr-loxp ime1Y359F(⫺1439 to ⫹1081)-ADHt pIME1-gal4(dbd)-ime1S337A(270–360) pADH1-gal4(dbd)-ime1S302A(270–360) ime1(⫺1439 to ⫹1081)-ADHt pADH1-gal4(dbd)-ime1S306A(270–360) pADH1-gal4(dbd)-ime1S302D(270–360) pADH1-gal4(dbd)-ime1S337A(270–360) pADH1-gal4(dbd)-ime1S333A(270–360) pIME1-gal4(dbd)-ime1S352A,S356A,S360A (270–360) pADH1-gal4(1–147)-ime1(270–360) pADH1-gal4(dbd)-ime1 S352A,S356A,S360A (270–360) Rim15K823A ptac-GST-Ime1(270–360) pRIM11-rim11K68A(1–223) pIME1-gal4(dbd)-ime1S306,Y359F (270–360) pADH1-gal4(dbd)-ime1(270–360) Rim11-3SA ptac-gal4(92–147)-ime1(270–360)(Y359F) pT7-RIM11-polyA

directed against the Gal4 DNA-binding domain for both immunoprecipitation and detection revealed that in SD medium, Gal4(dbd)-Ime1(270-360) appeared in a single band, whereas in SPM medium it appeared in two bands (Fig. 1A, lanes 1 and 2). Furthermore, in SPM medium, a faint band with reduced mobility was observed. This pattern of Ime1 implies that additional posttranslational modifications might occur, but during the extraction these modified molecules were either not stable or subjected to phosphatases. In order to overcome this problem, proteins were extracted in the presence of trichloroacetic acid, which is presumed to promptly inactivate any proteases and phosphatases. Indeed, when cell lysates were prepared by this method, Ime1 showed a more detailed pattern of posttranslational modifications (Fig. 1B). In SD medium, Ime1 appeared in two bands, with the level of the second band being either lower than that of the first or absent (Fig. 1B, lanes 3 and 6). In SA medium, Ime1 appeared in three bands (Fig. 1B, lanes 4 and 7). Following incubation for 6 h in SPM medium, a new, slower band appeared (Fig. 1B, lanes 5 and 8). These forms were specific and absent from a strain carrying only Gal4(dbd) (data not shown) (19). For clarity, these bands were designated 1, 2, 3, and 4. The slow-migrating forms of Ime1(270-360) result from different levels of phosphorylation of Ime1 (phosphatase treatment led to the detection of mainly the fast-migrating form; data not shown, but see below). Thus,

Construction

Ligation Ligation Ligation Gap repair (32) Ligation Ligation Ligation Site-directed mutagenesis Site-directed mutagenesis Site-directed mutagenesis Random mutagenesis Ligation Ligation Site-directed mutagenesis Ligation Ligation Site-directed mutagenesis Ligation Ligation Ligation Ligation Ligation Ligation Site-directed mutagenesis Ligation Ligation Ligation Ligation Ligation Ligation Ligation Site-directed mutagenesis Ligation Ligation

the isolation of Ime1 by conventional methods led to the elimination of the slow-migrating bands, suggesting that the residual activity of phosphatases resulted in the removal of phosphate(s) from Ime1. Therefore, in the experiments reported below, proteins for Western analysis were extracted only by the trichloroacetic acid method. As meiosis is induced only in MATa/MAT␣ diploid cells, the pattern of Ime1 modifications in the Western analysis was first performed in diploid and haploid strains. The appearance of Ime1 was similar in the haploid and diploid strains (Fig. 1B, compare lanes 3 to 5 to lanes 6 to 8), suggesting that MAT plays no role in regulating posttranslational modifications of Ime1(270-360). Therefore, in most experiments haploid strains were used to determine the pattern of Ime1 modifications. In vivo Rim11 phosphorylates Ime1 in SA medium. As both Rim11 and Rim15 are required for the two-hybrid interaction between Ime1 and Ume6, we examined if they would affect the phosphorylation pattern of Ime1. We used Western analysis to determine if Rim15 is required in vivo for phosphorylation of Ime1(270-360). In cells carrying the kinase-dead mutation rim15K823A, the pattern of Gal4(dbd)-Ime1(270-360) was identical to that observed in the wild-type strain (Fig. 1, compare lanes 9 and 10 to lanes 4 and 5). Similar results were observed for a strain carrying a RIM15 deletion allele (data not shown). We conclude that the effect of Rim15 on the transcrip-

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MOL. CELL. BIOL. TABLE 2. List of strains

Strain

GGY::171 Y422 Y424 Y556 Y568 Y1017 Y1064 Y1065 Y1066 Y1078 Y1120 Y1084 Y1089 Y1093 Y1174 Y1235–1 Y1343 Y1344 Y1345 Y1346 Y1392 Y1393 Y1456 Y1471 YPM252 HM110-3A

Relevant genotype

MAT␣ URA3::GAL1-lacZ gal80⌬ gal4⌬ his3-200 ura3-52 leu2-3,112 MATa/MAT␣ ura3-52/ura3-52 trp1⌬/trp1⌬ leu2-3,112/leu2-3,112 ade2-1/ade2-R8 his4-519/HIS4 his6-1/HIS6 gal/GAL⫹ can1/CAN1 ime1⌬::TRP1/ime1⌬::TRP1 MAT␣ ura3-52 trp1⌬ leu2-3,112 ade2-101 met can1-100 bcy1::URA3 MAT␣ URA3::GAL1-lacZ his3-200 leu2-3,112 ura3-52 rim11::LEU2 trp1⌬ MATa ura3-52 leu2,3-112 trp1⌬ his3⌬::hisG gal80⌬::hisG gal4⌬::hisG MAT␣ ura3-52 trp1(del) leu2-3,112 his3::hisG gal80::hisG gal4::hisG rim11⌬(157-370)::LEU2 cdc25-5 cdc25-5 rim11⌬(157-370)::LEU2 cdc25-5 rim11⌬(157-370)::LEU2 rim11⌬(157-370)::LEU2 cdc25-5 rim11⌬(157-370)::LEU2 his3⌬::hisG::rim11K68A rim15⌬(196-1335)::loxP leu2::LEU2-rim15K823A MATa/MAT␣ ura3/ura3 trp1/trp1 his3/his3 leu2/leu2 tpk3⌬::HIS3/tpk3⌬::HIS3 tpk1⌬::URA3/tpk1⌬::URA3 tpk2⌬::kanL/tpk2⌬::kanL tpk1⌬::URA3/tpk1⌬::URA3 tpk3⌬::HIS3/tpk3⌬::HIS3 tpk2⌬::kanL/tpk2⌬::kanL tpk3⌬::HIS3/tpk3⌬::HIS3 rim11K68A-LEU2 rim11K68A-LEU2 cdc25-5 rim11⌬(157-370)::LEU2 ura3-52::URA3-rim113SA MATa/MAT␣ rim11⌬(157-370)::LEU2/rim11⌬(157-370)::LEU2 ura3-52/ura3-52::URA3-rim113SA MAT␣ ura3-52 leu2⌬1 trp1⌬1 his3⌬200 cyr1-230ts

tion of early meiosis-specific genes is not mediated through Ime1(270-360) or that phosphorylation by Rim15 is not detectable in Western analysis. To examine whether Rim11 phosphorylates Ime1(270-360) in vivo, we performed a Western analysis in cells deleted for RIM11 and in cells carrying a kinase-dead allele, rim11K68A. In these mutant cells, Ime1(270-360) appeared in a single discrete band in SA medium and in two bands in SPM medium in comparison to the three and four bands, respectively, of wildtype strains (Fig. 1C, compare lanes 12 and 13 to lane 11 and lanes 15 and 16 to lane 14). These results demonstrate that in vivo Rim11 is required for phosphorylation of Ime1 in SA medium and that phosphorylation in SA medium is not a prerequisite for phosphorylation in SPM medium. Figure 1C shows that in the rim11 mutant strains incubated in SPM medium, the level of the slow-migrating band was lower than the level of the first band, whereas in the wild-type strains its level was higher (Fig. 1, compare lanes 15 and 16 to lanes 5, 8, and 14). This result implies that Rim11 might also be required for phosphorylating Ime1 in SPM medium, but an additional kinase may also contribute to this phosphorylation. We suggest that Rim11 directly phosphorylates Ime1, since in vivo it associates with Ime1 (28, 40) and in vitro it phosphorylates Ime1 (see Fig. 6E) (28). Activity of Rim11 in SD medium is inhibited by glucose. Rim11 and Ime1 associate in the presence of glucose (40), nevertheless, phosphorylation of Ime1 by Rim11 depends on glucose depletion. This fact suggests either that glucose inhibits the kinase activity of Rim11 or that Ime1 is not accessible to phosphorylation in the presence of glucose. Both hypotheses are compatible with the regulated function of the Rim11 homolog GSK3 (7, 20). We postulated that phosphorylation on

Remarks or reference

6 Isogenic to Y422 Isogenic to Y556

Isogenic to Y1064 Isogenic to Y1064 Isogenic to Y1065 Isogenic to Y1064 Isogenic to Y1065 Isogenic to Y1065 Isogenic to Y1064 Isogenic to Y1064 CEN.PK2 background Isogenic to Y1343 Isogenic to Y1064 Isogenic to Y1064 Isogenic to Y1064 Isogenic to Y422 H. Mitsuzawa Isogenic to YPM252

FIG. 1. Rim11, glucose, and nitrogen regulate the posttranslational modifications of Ime1(270-360). Immunoprecipitation and Western (A) and Western (B to D) analysis of Gal4(dbd)-Ime1(270-360). Proteins were extracted from cells grown in SD medium (lanes 1, 3, 6, 17, 18, and 20) or SA medium (lanes 4, 7, 9, 11 to 13, and 19) or following 6 h of incubation in SPM medium (lanes 2, 5, 8, 10, 14 to 16, and 21). Ime1 was detected with antibodies directed against Gal4(dbd). The strains used were a wild-type haploid (Y1064, lanes 1 to 5, 11, 14, and 17), a wild-type diploid strain resulting from mating between Y1064 and Y1065 (lanes 6 to 8), rim15K823A (Y1235-1, lanes 9 and 10), rim11⌬ (Y1066, lanes 12 and 15), rim11K68A (Y1392, lanes 13 and 16), and rim11-3SA (Y1456, lanes 18 to 21). The gal4(dbd)-ime1(270-360) chimera was expressed from either the ADH1 promoter (YEp2780, lanes 1 and 3; YEp1338, lanes 6, 17, and 18 to 21) or the IME1 promoter (YEp1931, lanes 2, 4, 5, and 7 to 16).

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TABLE 3. The rim11-3SA mutation bypasses the glucose signal required for meiosisa Sporulation (%) Strain

Wild type rim11-3SA

SPM ⫹ 2% glucose

SPM 24 h

48 h

24 h

48 h

64.8 69.8

72.6 82.1

0 13.2

0 56.2

a Cells grown to 5 ⫻ 107 cells/ml were washed once in water and transferred to either SPM medium or SPM medium supplemented with 2% glucose at 107 cells/ml. Strains used were Y422 (wild type) and Y1471 (rim11-3SA).

serine residues at the N terminus of Rim11 might inhibit its function in SD medium, similar to the mode by which the insulin signal, through protein kinase B, inactivates GSK3 (7). We used site-directed PCR mutagenesis to mutate to alanine the three N-terminal serine residues in Rim11, Ser-5, Ser-8, and Ser-12. The phosphorylation pattern of Ime1(270-360) was examined in a strain whose only RIM11 was this allele (rim113SA). In this rim11-3SA mutant, Ime1 abnormally appeared in four bands in SD medium (Fig. 1C, compare lanes 17 and 18). We conclude that normally phosphorylation of Rim11 on Ser-5, Ser-8, and/or Ser-12 inhibits its kinase activity in the presence of glucose and nitrogen. In SA medium, the level of phosphorylation was further increased, as judged by the increase in the intensity of band 2 in comparison to band 1 (Fig. 1D, compare lanes 19 and 20). This suggests that the Rim113SA mutant protein is still subject to glucose regulation. In SPM medium, Ime1 became hyperphosphorylated (Fig. 1D, lane 21), reinforcing the conclusion that in SPM medium, Ime1 is phosphorylated by an additional kinase. Ectopic expression of Ime1 in SD medium or SA medium does not suffice for entry into meiosis and sporulation (40, 44), suggesting that nutrient-regulated phosphorylation of Ime1 might be required for sporulation. We postulated that in cells expressing the Rim11-3SA mutant protein, the constitutive phosphorylation of Ime1 might suffice to induce meiosis in the presence of glucose. Diploid cells grown in SA medium to 5 ⫻ 107 cells/ml were washed once in water and transferred to either SPM medium or SPM medium supplemented with 2% glucose at 107 cells/ml. Addition of glucose to SPM medium inhibited meiosis of wild-type diploid cells (Y422). On the other hand, isogenic rim11⌬ diploid cells carrying the rim113SA allele (Y1471) were sporulation proficient (Table 3). In the presence of glucose, sporulation was delayed (Table 3), and most of the asci formed were not mature (data not shown). Ectopic expression of Ime1 does not relieve glucose repression (44), suggesting that the rim11-3SA mutation bypassed the glucose signal that inhibits the activity of Ime1. In agreement, asci were not formed when wild-type or rim11-3SA mutant cells, which were grown in SA medium to 107 cells/ml, were transferred to SPM medium supplemented with 2% glucose. Lack of sporulation under these conditions resulted from the absence of Ime1, which is not sufficiently expressed in cells grown exponentially in SA medium (19, 23). Thus, the rim113SA mutation bypassed the glucose signal inhibiting sporulation only in cells expressing Ime1. We conclude that glucose inhibits the kinase activity of Rim11. This explains why Rim11

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does not phosphorylate Ime1 in SD medium and why meiosis is not initiated in cells that express Ime1 in SD medium. cAMP/PKA pathway inhibits the phosphorylation of Ime1. We considered the possibility that the cAMP/PKA signal transduction pathway might transmit the glucose signal that inhibits Rim11 activity. In such a case, this signal transduction pathway is expected to affect Ime1 phosphorylation and function. This assertion is based on the following. Mutations that attenuate the activity of PKA bypass the requirement for nutrient depletion for the transcription of IME1 and induction of meiosis. When cyr1-230 or cdc25-5 logarithmic-phase cells are shifted to the nonpermissive temperature, Ime1 is expressed (32, 41) and spores are formed (31, 46). CYR1 encodes adenylate cyclase (5), and CDC25 encodes the RAS GDP/GTP exchange factor, which serves as a positive regulator of adenylate cyclase (31). On the other hand, in cells deleted for BCY1, the regulatory subunit of PKA (51), IME1 is not expressed, and sporulation is not induced (32). However, as stated above, ectopic expression of Ime1 does not promote sporulation in cells grown in either SD medium or SA medium (44). We surmised, therefore, that in SD medium the cAMP/PKA pathway might inhibit phosphorylation of Ime1 and consequently the expression of meiosis-specific genes and sporulation. To test this hypothesis, we determined the pattern of Ime1 phosphorylation in the temperature-sensitive mutants cdc25-5 and cyr1-230 and in bcy1⌬ and their isogenic wild-type strains. In wild-type cells grown in SD medium at 25°C or 30°C or following a 6-h shift to 34°C, Ime1 appeared mainly in a single band (Fig. 2, lanes 1, 2, and 19). In bcy1⌬, which has high PKA activity, Ime1 appeared in a single band in both SD medium and SPM medium (Fig. 2, lanes 21 and 22). In cdc25-5 and cyr1-230 cells, which have low PKA activity, Ime1 appeared in two bands in cells grown in SD medium at 25°C (Fig. 2, lanes 3 and 9). A 6-h shift to the nonpermissive temperature of the SD medium-grown cells led to the formation of all four bands (Fig. 2, lanes 4 and 10). In SD medium, the level of the slow-migrating band was less robust than its level in SPM medium (Fig. 2, compare lane 4 to lanes 5 to 8 and lane 10 to lanes 15 to 18), suggesting that an additional signal transduction pathway is also responsible for this posttranslational modification. Thus, two mutations in the cAMP/PKA signal pathway which have impaired the activity of PKA, cdc25-5 and cyr1-230, resulted in phosphorylation of Ime1 in SD medium rather than SPM medium. We conclude, therefore, that the cAMP/PKA pathway prevents the phosphorylation events of Ime1 which normally take place in SA medium and SPM medium from happening in SD medium. Yeast cells carry three genes, TPK1, TPK2, and TPK3, each of which encodes the catalytic domain of PKA. We wished to determine the role of these genes in transmitting the glucose signal to Ime1. To this end, we used isogenic diploid strains carrying single or double mutations of these genes to determine the pattern of Ime1 by Western analysis (Fig. 3). In the double mutants tpk1⌬ tpk3⌬ and tpk2⌬ tpk3⌬, in contrast to the tpk3⌬ single mutant and the tpk1⌬ tpk2⌬ double mutant, Ime1 was hyperphosphorylated in both SD medium and SA medium. In SD medium, two bands were observed (Fig. 3, compare lanes 3 and 4 to lanes 1 and 2), whereas in SA medium, four bands were observed (Fig. 3, compare lanes 7

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FIG. 3. Glucose signal is transmitted to Ime1 mainly by Tpk3. Western analysis of Gal4(1-147)-Ime1(270-360) is shown. Proteins were extracted from 107 cells/ml grown in SD medium (lanes 1 to 4) SA medium (lanes 5 to 8) or following 6 h of incubation in SPM medium (lanes 9 to 12). Strains used were tpk3⌬ (Y1343, lanes 1, 5, and 9), tpk1⌬ tpk2⌬ (Y1344, lanes 2, 6, and 10), tpk1⌬ tpk3⌬ (Y1345, lanes 3, 7, and 11), and tpk2⌬ tpk3⌬ (strain Y1346, lanes 4, 8, and 12). The gal4(1-147)-ime1(270-360) chimera was expressed from either the ADH1 promoter (YEp1338, lanes 1 to 4) or the IME1 promoter (YEp1931, lanes 5 to 12). Immunoblot analysis was performed with antibodies directed against Gal4(dbd).

FIG. 2. In SD medium, the cAMP/PKA signal transduction pathway prevents phosphorylation of Ime1(270-360). Western analysis of Gal4(1-147)-Ime1(270-360) is shown. Proteins were extracted from 107 cells/ml grown in SD medium at 30°C (lanes 19 and 21) or 25°C (lanes 1, 3, and 9) or following 6 h of incubation at 34°C (lanes 2, 4, and 10). Cells grown in SD medium at 25°C to the stationary phase were shifted to SPM medium and incubated for 6 h at either 25°C (lanes 5 and 7) or 34°C (lanes 6 and 8). Proteins were also extracted from 107 cells/ml grown in SA medium at 30°C (lane 20) or 25°C (lanes 11 and 13) or following 6 h of incubation at 34°C (lanes 12 and 14). In addition, these cells were shifted to SPM medium and incubated for 6 h at 30°C (lane 22), 25°C (lanes 15 and 17), or 34°C (lanes 16 and 18). Strains used were wild-type Y1064 (lanes 1, 2, 5, and 6), the cdc25-5 mutant (Y1078, lanes 3, 4, 7, and 8), wild-type isogenic to the cyr1-230 mutant (HM1103A, lanes 9, 10, 13, 14, 17, and 18), the cyr1-230 mutant (YPM252, lanes 11, 12, 15, and 16), wild-type isogenic to the bcy1⌬ mutant (Y556, lanes 19 and 20), and the bcy1⌬ mutant (Y568, lanes 21 and 22). The gal4(1-147)-ime1(270-360) chimera was expressed from either the ADH1 promoter (YEp1338, lanes 1 to 4, 9 to 10; YEp2780, lanes 19 to 22) or the IME1 promoter (YEp1931, lanes 5 to 8 and 11 to 18). Immunoblot analysis was performed with antibodies directed against Gal4(dbd).

and 8 to lanes 5 and 6). These results imply that Tpk3 is the main kinase required for transmitting the nutrient signals to Ime1. In its absence, the homologs of Tpk3, Tpk1, and Tpk2 probably take over its function. The correlation between phosphorylation of Ime1(270-360) and expression of early meiosis-specific genes was examined with the early meiosis-specific gene HOP1, whose promoter was fused to the lacZ reporter. In wild-type cells grown at 25°C, hop1-lacZ was silent in SD medium and SA medium, whereas high expression (an increase greater than 1,000-fold) was detected in SPM medium (Table 4). Interestingly, a shift of these cells to 34°C for 6 h led to nine- and twofold increases in expression in SA medium and SPM medium, respectively (Table 4). This elevated expression might result from the increase in the expression of IME1 in response to heat shock treatment (44). However, in SD medium, such an increase in Ime1 was not sufficient to induce hop1-lacZ. When cdc25-5 diploid cells

grown in SD medium were shifted to the nonpermissive temperature for 6 h, expression of hop1-lacZ was increased 30-fold (Table 4), suggesting that Cdc25 transmits a glucose signal that prevents hop1-lacZ expression. Moreover, cdc25-5 diploid cells bypassed the glucose signal that prevents sporulation in the presence of glucose (data not shown). In SA medium, a shift to the nonpermissive temperature led to an additional 15-fold increase in the expression of hop1-lacZ (Table 4). This increase might result from heat shock treatment or a decrease in Cdc25 activity. We prefer the latter explanation because in the wild-type strain, the level of hop1lacZ expression following incubation for 6 h in SA medium at 34°C was about 1% of that observed in SPM medium, whereas in the cdc25-5 strain, that level reached 30% (Table 4). These results indicate that Cdc25 also transmits a nitrogen signal. The expression of hop1-lacZ correlates well with the four bands observed for Ime1 in SD medium in cdc25-5 cells incubated at the nonpermissive temperature (Fig. 2, lane 4). In SPM medium, expression of hop1-lacZ was further increased threefold in cdc25-5 cells (Table 4). This result and the phosphorylation pattern of Ime1(270-360) (Fig. 2) suggest that an additional signal pathway might transmit the nitrogen signal to Ime1(270-360). cAMP/PKA signal transduction pathway transmits the glucose and nitrogen signals to Rim11. We hypothesized that

TABLE 4. Mitotic and meiotic levels of hopI-lacZ expression ␤-Galactosidase activity (Miller units) Strain

a

Wild type cdc25-5 rim11⌬ rim11K68A rim11⌬cdc25-5

SD

SA

SPM

25°C

34°C

25°C

34°C

25°C

34°C

0.10 0.39 0.06 NT 0.05

0.04 12.23 0.42 NT 0.14

0.97 8.85 0.06 0.01 0.98

8.98 129.14 1.57 0.45 11.11

435.68 599.15 0.02 0.02 0.02

948.05 422.40 0.30 0.03 0.36

a The strains used were diploids constructed by the following matings: wild type, Y1064 ⫻ Y1065; cdc25-5, Y1078 ⫻ Y1120; rim11⌬, Y1066 ⫻ Y1089; rim11⌬ cdc25-5, Y1084 ⫻ Y1093; and rim11K68A, Y1174 ⫻ Y1089. These cells carry the hop1-lacZ chimera on a 2␮m TRP1 vector (YEp2042). Cells grown at 25°C to 0.5 ⫻ 107 cells/ml were shifted to 34°C for 6 h. Cells grown at 25°C in SA were shifted to SPM and incubated at 25 or 34°C for 6 h. NT, not tested.

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FIG. 4. PKA inhibits phosphorylation of Ime1 by Rim11. Proteins were extracted from 107 cells/ml that were grown in SD medium and shifted to 37°C for 3 h (lanes 1 to 3) or from cells that were grown in SA medium, transferred to SPM medium for 6 h, and incubated at 37°C. Strains used were cdc25-5 (Y1078, lanes 1 and 4), cdc25-5 rim11⌬ (Y1084, lanes 2 and 5), and cdc25-5 rim11K68A (Y1393, lanes 3 and 6). The gal4(1-147)-ime1(270-360) chimera was expressed from the IME1 promoter (YEp1931). Immunoblot analysis was performed with antibodies directed against Gal4(dbd).

PKA negatively regulates the function of Rim11. This hypothesis predicts that cdc25-5 will not suppress the meiotic defect of rim11⌬ cells in Ime1 phosphorylation, hop1-lacZ expression, or sporulation. In the double mutants cdc25-5 rim11⌬ and cdc25-5 rim11K68A, Ime1 appeared in two bands in SD medium and SPM medium (Fig. 4, lanes 2, 3, 5, and 6). In contrast, in the cdc25-5 single mutant, Ime1 appeared in four bands in both SD medium and SPM medium (Fig. 4, lanes 1 and 4). Accordingly, diploid cells homozygous for rim11⌬ cdc25-5 were sporulation deficient and did not express hop1lacZ when incubated in SPM medium at either the permissive or restrictive temperature (Table 4). However, an 11-fold increase in the expression of hop1-lacZ was observed when rim11⌬ cdc25-5 cells grown in SA medium were shifted to the nonpermissive temperature. This increase is similar to that observed for the wild-type strain transferred to 34°C (Table 4). Why the level of expression was reduced in SPM medium (0.36 units in comparison to 11.11 units) is not clear. Ime1 Ser-302 and Ser-306 are phosphorylated. The Rim11 homolog GSK3 phosphorylates serine or threonine residues in an S/T X X X S/T consensus sequence (20). Figure 5 shows that Ime1(270-360) carries four such sites, designated sites I, II, III, and IV. In order to determine whether Ime1 is phosphorylated on any of these sites, we created serine-to-alanine mutations in these sites and examined their effect on the phos-

FIG. 5. Amino acid sequence of Ime1(270-360). The putative amino acid sequence of Ime1(270-360), which corresponds to the domain that is sufficient for interaction with Ume6, is shown. Marked in large boxes are the four consensus sites for phosphorylation by the Rim11 homolog GSK3. Amino acid residues whose role in phosphorylation and activity was studied in this report are marked by stars.

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phorylation pattern of Ime1(270-360). Western analysis revealed that, in SPM medium, the phosphorylation pattern of Ime1(270-360) carrying the single mutation S333A or S337A or the triple mutation S352AS356AS360A was similar to that of the wild-type protein (four bands were observed, with bands 1 and 3 being less intense) (Fig. 6A, compare lanes 4, 5, and 7 to lanes 1 and 6). By contrast, unlike the wild-type situation, serine-to-alanine mutations in either Ser-302 or Ser-306 resulted in the appearance of only three rather than four bands in SPM medium (Fig. 6, lanes 2, 3, 9, and 21). In SA medium, these mutants were detected in two bands rather than three bands (Fig. 6, compare lanes 8, 11, and 17 to lanes 10 and 16), suggesting that both Ser-302 and Ser-306 are phosphorylated in SA medium. Since Rim11 is required for phosphorylation of Ime1(270-360) in SA medium, phosphorylation on Ser-302 and/or Ser-306 might depend on Rim11. Furthermore, phosphorylation on Ser-302 and/or Ser-306 is not a prerequisite for additional phosphorylation events taking place in SA medium and SPM medium. Rim11 is required for the two-hybrid interaction of Ime1(270-360) with Ume6(1-232) (8, 29, 40) (Fig. 7). In wildtype cells that express both Gal4(dbd)-Ime1(270-360) and Gal4(ad)-Ume6(1-232), the gal1-lacZ reporter gene is highly expressed in SPM medium, giving rise to 78.2 Miller units of ␤-galactosidase activity. In contrast, in isogenic rim11⌬ cells, only 1.2 Miller units are observed, similar to the control (1.4 Miller units). We therefore used this two-hybrid assay to determine whether mutations in the putative Rim11 phosphorylation sites will impair the interaction of Ime1 with Ume6. Gal4(dbd)-Ime1(270-360) carrying the single mutation S302A, S302D, S306A, S333A, or S337A or the triple mutation S352AS356AS360A interacted with Ume6 as well as the wildtype Gal4(dbd)-Ime1(270-360) protein (Fig. 7), suggesting that these residues in Ime1 are not essential for interacting with Ume6. The observation that alanine substitutions of Ser-302 and Ser-306 did not impair the two-hybrid interaction of Ime1with Ume6 is inconsistent with the requirement of Rim11 for the activity of Ime1 and with the notion that Rim11 mediates its function through phosphorylation on Ser-302 and/or Ser306. Nevertheless, it is possible that Rim11 phosphorylates an additional residues(s) that is not necessarily within the GSK3 consensus sites. Rim11 phosphorylates Ime1 on Tyr-359. We used random PCR mutagenesis to create mutations in Ime1(270-360) that might affect its activity. We used the two-hybrid assay to screen for mutations in Ime1 that show the same phenotype as the one observed in rim11⌬ cells, namely, low expression of the gal1-lacZ reporter gene in cells expressing Gal4(ad)-Ume6(1232) and Gal4(dbd)-Ime1(270-360) (see Materials and Methods). Sequence analysis revealed the nature of the various Ime1 mutants. A detailed description of the mutants will be given elsewhere. In this report, we focus on only one mutant that was impaired in the phosphorylation pattern of Ime1(270360). This mutant carried an A-to-T transversion at position 1076. The predicted amino acid sequence suggests that this mutation resulted in a Tyr-359 mutation to phenylalanine (designated Y359F). Figure 7 shows that the Gal4(dbd)-Ime1(270-360) that carried the Y359F mutation was defective in the two-hybrid in-

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FIG. 6. In SA medium, Ime1 is phosphorylated on Ser-302, Ser-306, and Tyr-359. (A to C) Immunoblot analysis of various Ime1(270-360) mutant proteins with antibodies directed against Gal4(dbd). (D) Proteins were immunoprecipitated with antibodies directed against Gal4(dbd), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and detected by antibodies directed against either Gal4(dbd) (upper panel) or phosphotyrosine (lower panel). Proteins were extracted from cells grown in SA medium to 107 cells/ml (lanes 8, 10 to 13, and 16 to 19) or following 6 h of incubation in SPM medium (lanes 1 to 7, 9, 14 and 15, and 20 to 26). The strain used was Y1064 carrying the following pIME1-gal4(dbd)-ime1(270-360) mutants on a 2␮m plasmid: wild-type (YEp2200, lanes 1, 6, 10, 12, 14, 16, 20, 24, and 26), S302A (YEp2112, lanes 2, 8, and 9), S306A (YEpP2122, lanes 3, 11, 17, and 21), S333A (YEp2206, lane 4), S337A (YEp2215, lane 5), S352A S356A S360A (YEp2430, lane 7), Y359F (YEp2196, lanes 13, 15, 18, 22, and 25), and S306A Y359F (YEp2713, lanes 19 and 23). (E) Rim11 phosphorylates Imel on Tyr-359. E. coli S30 transcription-translation system carrying pT7-RIM11 on P2844 (lanes 30 to 32) or the control vector plasmid (lanes 27 to 29) incubated with E. coli made Gst-Ime1(270-360) (lanes 28 and 39), Gst-Ime1(270-360)Y359F (lanes 29 and 32), and GST (lanes 27 and 30) proteins immobilized on glutathione-Sepharose beads. Proteins were then subjected to Western analysis using antibodies directed against phosphotyrosine.

teraction with Gal4(ad)-Ume6(1-232), giving rise to only 2.5 Miller units of ␤-galactosidase in SPM medium. To determine if an Ime1(1-360)Y359F can complement ime1⌬ in promoting sporulation, we constructed ime1(⫺1081 to ⫹ 1200) genes that carry either the Y359F mutation or the wild-type allele. These alleles (on plasmids YIp2214 and YIp2234, respectively) were integrated at the leu2 locus of a MATa/MAT␣ ime1⌬/ime1⌬ diploid (strain Y424). Asci were observed in the strain carrying the wild-type IME1 allele, while the strain carrying the ime1Y359F allele was sporulation deficient (0% asci).

In order to determine whether the Y359F mutation affects the phosphorylation pattern of Ime1, the pIME1-gal4(dbd)-ime1(270360)Y359F chimera was placed on a 2␮m plasmid (Western analysis could not efficiently detect the protein when the chimeric gene was present on a CEN plasmid). Figure 6 shows that in SA medium, Ime1Y359F appeared in two bands rather than three (Fig. 6, compare lanes 13 and 18 to lanes 12 and 16), and in SPM medium three bands were observed rather than four (Fig. 6, compare lanes 15 and 22 to lanes 14 and 20). We conclude that in SA medium one of the phosphorylated residues is Tyr-359.

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FIG. 7. Two-hybrid interaction between Ime1 and Ume6 depends on Tyr-359 in Ime1 and on Rim11. The level of expression of gal1-lacZ (␤-galactosidase activity in Miller units) was compared in cells expressing different Gal4(dbd)-Ime1(270-360) mutant proteins and the Gal4(ad) plasmid pGAD2F (6) (control) or Gal4(ad)-Ume6(1-232) plasmid YEp1788. The level of interaction is given relative to that of the wild-type control (set at 1). The strains used were GGY::171 and Y1017 (an isogenic rim11⌬ strain). The following plasmids carrying gal4(dbd)-ime1(270-360) mutant genes were used: wild-type (YCp2435), S302A (YCp2233), S302D (YCp2302), S306A (YCp2242), S333A (YCp2306), S337A (YCp2303), S352A S356A S360A (YCp2436), and Y359F (YCp2195).

We used immunoprecipitation and Western analysis to determine if, in vivo, Ime1(270-360) is phosphorylated on Tyr359. Western analysis with antibodies directed against the Gal4 DNA-binding domain for both immunoprecipitation and detection revealed that in the wild-type strain incubated in SPM medium, Ime1 appeared in three bands, with the level of the third band lower. The third band was absent from wild-type cells that expressed Gal4(dbd)-Ime1(270-360)Y359F (Fig. 6D, compare lane 24 to lane 25), suggesting that the third band was the result of phosphorylation on Tyr-359. The third band was also missing in rim11⌬ cells expressing the wild-type Gal4(dbd)-Ime1(270-360) protein (Fig. 6D, compare lane 24 to lane 26), suggesting that the third band was the result of phosphorylation by Rim11. Antibodies directed against phosphotyrosine detected a single band, corresponding to the slowmigrating band, but only in wild-type cells expressing the wildtype Ime1(270-360) protein. In rim11⌬ cells and in wild-type cells expressing the Ime1(270-360)Y359F mutant protein, Ime1 was not recognized. We conclude, therefore, that Ime1(270-360) is phosphorylated on a single tyrosine residue, Tyr-359, and that Rim11 is required for this phosphorylation. In order to provide direct evidence that Rim11 phosphorylates Ime1 on Y359F, we used an in vitro kinase assay. Rim11 made by the E. coli T7 S30 transcription-translation system was incubated with E. coli GST-Ime1(270-360), and GST proteins were immobilized on glutathione-Sepharose beads. Following 45 min of incubation, proteins were subjected to Western analysis with antibodies directed against phosphotyrosine. Figure 6E shows that GST-Ime1 was indeed detected by this antibody

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(lane 31). GST-Ime1 detection depended on Rim11 and on the Tyr-359 residue in Ime1. GST-Ime1 was not detected in cell extracts lacking the T7-RIM11 gene (Fig. 6E, lane 28) or when Ime1 carried the Y359F mutation (Fig. 6E, lane 32). Furthermore, without Ime1, the GST peptide was not subject to tyrosine phosphorylation (Fig. 6E, lanes 27 and 30). We conclude, therefore, that Rim11 phosphorylates Tyr-359 in Ime1. Thus, phosphorylation on a single residue, Tyr-359, is required for the two-hybrid interaction of Ime1 with Ume6. Moreover, cdc25-5 did not suppress sporulation in cells expressing the ime1Y359F allele, indicating that PKA activity inhibits the activity of Rim11 and the phosphorylation of Ime1 on Tyr-359. In order to prove that, in SA medium, the two phosphorylating bands are due to phosphorylation on Ser-302, Ser-306, and Tyr-359, we constructed the double mutant Gal4(dbd)Ime1(id) S306AY359F. This mutant protein appeared in a single band in SA medium and in two bands in SPM medium (Fig. 6C, compare lane 19 to lane 16 and lane 23 to lane 20). As the pattern of Ime1(270-360) in rim11 was also one band in SA medium and two bands in SPM medium, we suggest that both Ser-302/Ser-306 and Tyr-359 are targets for Rim11 phosphorylation. Thus, by combining random and site-directed mutagenesis, we identified two residues, Tyr-359 and a combination of Ser-302 and Ser-306, that are subject to phosphorylation on Ime1(270-360) in SA medium. DISCUSSION Ime1 is subject to nutrient-regulated phosphorylation. In S. cerevisiae, entry into meiosis depends on glucose and nitrogen depletion and the presence of a nonfermentable carbon source, such as acetate. These meiotic signals that open the transcriptional program of all meiosis-specific genes converge at IME1, regulating its availability (for a review, see reference 22) and activity (this report). IME1 encodes a potent transcriptional activator that lacks known DNA-binding motifs and is recruited to the URS1 element present in the promoters of all early meiosis-specific genes by the DNA-binding protein Ume6 (8, 29, 36, 40). In this report we show that, in vivo, the domain of Ime1 that suffices for interaction with Ume6, 270 to 360, is subject to nutrient-regulated posttranslational modifications. In a medium that promotes vegetative growth with glucose as the sole carbon source (SD medium), Ime1(270-360) appeared mainly in a single band (Fig. 1). In a medium that promotes vegetative growth with acetate as the sole carbon source (SA medium), Ime1 appeared in three bands. Under sporulation conditions (SPM medium), Ime1 was subject to further posttranslational modifications. The following observations suggest that the nutrient-regulated modifications of Ime1 are the result of phosphorylation events and that, in vivo, Rim11 is the main kinase that phosphorylates Ime1(270-360). (i) One of the modified forms of Ime1(270-360) was detected by antiphosphotyrosine antibodies (Fig. 6D). (ii) In rim11, Ime1(270-360) was not subject to tyrosine phosphorylation and was not detected by antibodies directed against antiphosphotyrosine (Fig. 6D). (iii) In cells expressing a constitutively active Rim11-3SA allele, Ime1 abnormally appeared in four bands in SD medium rather than SPM medium (Fig. 1C). (iv) Ime1(270-360) carrying any of the conservative substitutions Y359F, S302A, or S306A was

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defective in posttranslational modifications (Fig. 6). (v) Finally, as in rim11 strains, in the Ime1(270-360)Y359F S306A double mutant, Ime1 appeared in SA medium in a single band and in SPM medium in two bands rather than three and four bands, respectively (Fig. 1C). Our results are in agreement with the physical association of Ime1(270-360) with Rim11 (28, 40) and the phosphorylation of Tyr-359 in Ime1 by the in vitro transcribed and translated Rim11 (Fig. 6E). The following observations suggest that phosphorylation of Ime1(270-360) is required for its activity. (i) Deletion and kinase-dead mutations in RIM11 resulted in impaired phosphorylation of Ime1, defective two-hybrid interaction between Ime1 and Ume6, and absent early meiosis-specific genes transcription and spore formation (Fig. 1C and 7, Table 4) (3, 8, 28, 40, 50, 52). (ii) A constitutively active mutation, rim11-3SA, bypassed the glucose signal that prevented phosphorylation of Ime1 and sporulation in the presence of glucose (Fig. 1 and Table 3). (iii) Ime1 carrying a nonsense mutation at Q340 or alanine substitutions of the eight serine, threonine, and tyrosine residues beyond Q340 associates with Rim11 but is impaired in both Rim11 in vitro phosphorylation, and meiosis (28). (iv) Ime1(270-360) carrying the single mutation Y359F was defective in the in vivo phosphorylation, two-hybrid interaction with Ume6, and sporulation (Fig. 7). These results demonstrate a direct role for phosphorylation on a single amino acid residue, Tyr-359, in the ability of Ime1 to promote transcription and entry into meiosis. cAMP/PKA pathway transmits the glucose and nitrogen signals that inhibit complete phosphorylation of Ime1. The cAMP/PKA pathway transmits both glucose and nitrogen signals that inhibit the transcription of IME1 (22, 32, 41, 43). Consequently, inactivation of this signal pathway leads to the transcription of early meiosis-specific genes and sporulation in a medium promoting vegetative growth (Table 4) (31, 46). Ectopic expression of Ime1 does not suffice for entry into meiosis (40, 44), suggesting that Ime1 needs to be modified to be active. We postulated that the cAMP/PKA pathway might negatively regulate the phosphorylation and consequently the activity of Ime1. Indeed, in this report we demonstrate that temperature-sensitive mutations in adenylate cyclase, Cyr1, or the RAS exchange factor, Cdc25, bypass both the glucose and nitrogen signals that prevent the complete phosphorylation of Ime1 (Fig. 2). We further show that constitutively high activity of PKA, which results from deletion of its regulatory subunit BCY1, prevents phosphorylation of Ime1. Thus, inactivation of the cAMP/PKA pathway promotes the expression and phosphorylation of Ime1 in the presence of glucose. Nevertheless, meiosis and sporulation are induced only in the absence of glucose and the presence of acetate (31, 46). We suggest that this parity reflects the complex regulation of Ime1 expression. The glucose signal is transmitted to at least four discrete elements in the promoter of IME1; the function of only one element, IREu, is modulated by PKA (22, 41, 43). Therefore, inactivation of the PKA pathway does not lead to complete expression of Ime1 in glucose-grown cells. On the other hand, in acetate-grown cells, inhibition of PKA activity leads to increased levels of Ime1 that can bypass additional nutrient controls, for instance, the sequestering of Ime1 from the nucleus (8) or Ime2 activation (see below). Yeast cells carry three genes, TPK1 to TPK3, encoding PKA

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catalytic activity. These genes seem to have a redundant function in relation to growth, because the deletion of all three genes causes cell cycle arrest. However, these kinases have specific roles in regulating pseudohyphal growth, where Tpk2 is a positive regulator and Tpk3 is a negative regulator (35, 38). Similarly, in this report, we show that Tpk3 plays the major role in transmitting the nutrient signals that inhibit phosphorylation on Ime1(270-360) (Fig. 3). Regulation and targeting of Rim11, a GSK3 homolog. Rim11 is homologous to GSK3 family members that are conserved in all eukaryotes (37). This family of protein kinases have key roles in transmitting signals involved with various cellular and developmental processes, in tumorigenesis, in apoptosis, and in the development of Alzheimer’s disease (for a recent review, see references 7, 14, 15, 20, and 26). Similarly, in budding yeast, Rim11 possesses a pivotal developmental role in promoting entry into meiosis. It is necessary for the twohybrid interaction of Ime1 with Ume6, the transcription of early meiosis-specific genes, and sporulation (3, 8, 28, 29, 40). In this report we demonstrate that, in vivo, in SA medium, Ime1(270-360) was phosphorylated on Tyr-359 and Ser-302/ Ser-306. We link Tyr-359 to Rim11 and suggest that Ser302/ Ser-306 might also be phosphorylated by Rim11. This suggests that Rim11 is a dual-specificity kinase that phosphorylates both tyrosine and serine residues. This conclusion agrees with the observation that both GSK3 and Rim11 are autophosphorylated on a tyrosine residue (54, 58). However, to our knowledge, this is the first report demonstrating in vivo tyrosine phosphorylation of an external substrate of a GSK3 homolog. How is Rim11 targeted to phosphorylate these tyrosine and serine residues in Ime1(270-360)? GSK3 is targeted to the canonical S/TXXXS/T consensus site. Following phosphorylation on the C-terminal S/T residue (by another kinase), it phosphorylates the N-terminal S/T residue (for a recent review, see references 7, 14, 15, 20, and 26). The predicted amino acid sequence of Ime1(270-360) reveals four such sites (Fig. 5), one of which, site I, was subject to phosphorylation. Serine-toalanine mutations at either Ser-302 or Ser-306 eliminated a specific phosphorylation band in SA medium (Fig. 6). We assume that Western analysis could not differentiate between phosphorylation on Ser-302 and Ser-306 and phosphorylation on Ser-306 only. We suggest, therefore, that in S. cerevisiae, substrate targeting by phosphate priming (10) is regulated in the same manner as in other members of the GSK3 family. Serine-to-alanine mutations in the other three GSK3 canonical sites did not affect the phosphorylation pattern of Ime1(270360) (Fig. 6). This suggests that in the absence of these sites, additional, unrelated sites became phosphorylated, that phosphorylation on these sites was not detected by Western analysis, or that these sites were not phosphorylated by Rim11. We incline towards the last possibility, because we were able to detect mutants that did affect the phosphorylation pattern of Ime1. Moreover, site I differs from the additional sites by the presence of proline residues (SPPPS). We speculate that proline residues might target Rim11 specifically to this site. This suggestion is based on the report that phosphorylation of the microtubule-associated protein tau, which is implicated in the progression of Alzheimer’s disease, is targeted by prolines. How Rim11 is targeted to phosphorylate Tyr-359 is not known. Because this tyrosine is present in a canonical GSK3 site, it is

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intriguing to speculate that this SKTYS site might direct Rim11 to this region. Interestingly, Tyr-199 in Rim11, which is subject to autophosphorylation (58), is also present within such a canonical site (SYICS). In this report we show that the in vivo phosphorylation of Ime1(270-360) by Rim11 occurs in SA medium (Fig. 1), although Rim11 associates with Ime1(270-360) under all growth conditions (3, 28, 40). Similarly, the in vitro kinase activity of Rim11 is increased when extracted from acetate- rather than glucose-grown cells (29, 58). These results imply either that the activity of Rim11 is inhibited by glucose or that it is activated by acetate. These hypotheses are not exclusive. The latter hypothesis is based on the observation that in Dictyostelium discoideum, the activation of GSK3 requires tyrosine phosphorylation by the tyrosine kinase Zak1 (25). In agreement, in S. cerevisiae, Rim11 carrying the Y199F mutation is defective in phosphorylation of exogenous substrates (29, 58). The first hypothesis is based on the observation that GSK3 kinases are subject to negative regulation. For instance, phosphorylation on Ser-9 in GSK3 by protein kinase B inhibits its activity by the formation of a pseudosubstrate template that competes for binding with the priming phosphate (10). In this case the signal, i.e., addition of insulin, increases the activity of protein kinase B (10). In the case of Wnt signaling, inhibition of GSK3 is not mediated by phosphorylation but by sequestering the kinase into a different protein complex (for a review, see reference 7). We postulated that in S. cerevisiae, glucose inhibits the activity of Rim11 by phosphorylation on a specific serine in its N terminus. Sequence alignment could not identify the specific serine residue that might be analogous to Ser-9 in GSK3. Therefore, by site-directed PCR mutagenesis, we replaced three putative serine residues in the N-terminal domain of Rim11 with alanine. We show that this rim11-3SA allele resulted in a constitutively active Rim11 that is not inhibited by glucose. The Rim11-3SA protein bypassed the glucose and part of the nitrogen signals inhibiting phosphorylation of Ime1(270-360). Moreover, this mutant allele also bypassed the glucose inhibition of sporulation. We conclude, therefore, that in the presence of glucose, phosphorylation on Ser-5, Ser-8, and/or Ser-12 in Rim11 inhibits its kinase activity. We further show that the glucose signal is transmitted to Rim11 by the cAMP/PKA signal transduction pathway. cdc25-5, cyr1-1, and rim11-3SA cells showed the same pattern of Ime1(270-360) phosphorylation in SD medium, four bands, with the levels of bands 3 and 4 being almost identical (Fig. 1 and 2). Moreover, under these conditions, the early meiosisspecific gene HOP1 was induced (Table 4) and cells sporulated in the presence of nutrients (Table 3). By contrast, in rim11 mutant cells grown in SA medium, phosphorylation of Ime1(270-360) was impaired (Fig. 1), HOP1 was not expressed (Table 4), and cells were sporulation deficient. Moreover, the double mutant rim11⌬ cdc25-5 was defective in the phosphorylation of Ime1(Fig. 4) and in the expression of HOP1 (Table 4). The predicted 14 N-terminal amino acids of Rim11, MNIQSNNPNLSNN, which contain the rim11-3SA mutations, do not carry a consensus site for PKA phosphorylation. This observation suggests that Rim11 might be phosphorylated by a different kinase whose activity is regulated by PKA. Rim11 is also subject to autophosphorylation on Tyr-199

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(58). Interestingly, this phosphorylation is increased in SA medium in comparison to SD medium (58). Moreover, a tyrosine-to-phenylalanine mutation in this residue results in impaired expression of ime2-lacZ in vivo and in defective phosphorylation of Ume6 in vitro, suggesting that this phosphorylation is required for the kinase activity of Rim11 (58). Further work will elucidate whether phosphorylation of Rim11 Ser-5, Ser-8, and/or Ser-12 inhibits Tyr-199 phosphorylation or whether this phosphorylation is subject to distinct regulation. Glucose inhibits the expression of early meiosis-specific genes through multiple mechanisms. Cell fate in diploid yeast cells is controlled by nutrient signals. In the presence of glucose, diploid cells propagate in the yeast form when high levels of nitrogen are available and in a filamentous form in the presence of low levels of nitrogen. A different optional developmental decision is made in the absence of glucose and the presence of a nonfermentable carbon source such as acetate. In the presence of nitrogen, cells propagate in the yeast form, while in the absence of nitrogen they arrest proliferation and enter a meiotic cycle, which is concluded by the formation of four haploid spores (for a recent review, see reference 22 and references therein). The effect of glucose on the transcription of early meiosisspecific genes occurs at multiple levels. It represses the transcription of IME1 (23) and the posttranslational modifications of Ime1(270-360). We show that in the absence of glucose, phosphorylation of Ime1 on a single tyrosine residue, Tyr-359, is essential for its activity. Thus, cells expressing the Ime1Y359F mutant protein are sporulation deficient, whereas cells in which this residue is abnormally phosphorylated, cdc25-5 or rim11-3SA cells, sporulate even in the presence of glucose. These results demonstrate at least one mechanism by which glucose prevents entry into the meiotic cycle even in the presence of Ime1. These results do not exclude the possibility that the availability and/or activity of Ume6 or an additional regulator is also modulated by glucose. On the contrary, the glucose signal is apparently transmitted to additional proteins. Ume6 is subject to nutrient-regulated phosphorylation. The in vivo phosphorylation of Ume6 is increased in SA medium in comparison to SD medium, and upon nitrogen depletion, Ume6 becomes hyperphosphorylated (56). Rim11 phosphorylates specific serine and threonine residues in the N-terminal domain of Ume6. Alanine substitution of these five residues in Ume6 causes a defect in phosphorylation, the Ime1-Ume6 two-hybrid interaction, transcription of the early meiosis-specific gene IME2, and sporulation (29). IME2 encodes a serine/ threonine kinase required for high-level transcription of early meiosis-specific genes (34, 57). The activity of Ime2 is negatively regulated by glucose through the interaction of its Cterminal domain with the small G-protein Gpa2 (11). Exit from the cell cycle and entry into a specific developmental program, such as meiosis, is a crucial decision that must take place only under the appropriate conditions. It is not surprising, therefore, that the glucose signal is transmitted to several target proteins. In this way, cells ensure that meiosis will be initiated only when appropriate conditions exist.

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