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The 14-3-3 Proteins Rad24 and Rad25 Negatively Regulate Byr2 by Affecting Its Localization in Schizosaccharomyces pombe

Department of Life Science and Biotechnology, Faculty of Life and Environmental Science,¹ Research Institute of Molecular Genetics, Shimane University, Matsue 690-8504,³ Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, Nagano 399-4598, Japan²

Received 5 October 2001/ Returned for modification 14 December 2001/ Accepted 5 July 2002

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

 
In Schizosaccharomyces pombe, rad24 and rad25 have been identified to be homologous to mammalian 14-3-3 genes and found to be involved in many cellular events, including checkpoint and meiosis. In the present study, we present evidences that Rad24 and Rad25 act as negative regulators of Byr2 (mitogen-activated protein kinase [MAPK] kinase kinase). Overexpression of rad24 or rad25 reduced mating and sporulation in homothallic wild-type cells. In contrast, the mating and sporulation efficiency of rad24- or rad25-null cells was higher than that of wild-type cells. Deletion of rad24 or rad25 increased sporulation efficiency in ras1-null diploid cells but not in byr2-, ste4-, byr1-, and spk1-null cells. Rad24 and Rad25 had no effect on the activity of constitutively active Byr1^S214DT218D. Rad24 and Rad25 bound to both the N-terminal and the C-terminal domains of Byr2 when these bacterially expressed proteins were examined. The formation of complexes in vivo between Byr2 and either Rad24 or Rad25 was also confirmed by immunocoprecipitation. Furthermore, we showed negative regulation of Byr2 by Rad25, by monitoring the mRNA level of mam2, which is regulated by both the Ras1/MAPK pathway and ste11, in various combinations of mutants. In addition, the cellular localization of Byr2 in living cells was observed by using fusion to green fluorescent protein. Byr2 was mainly localized in the cytoplasm during vegetative growth and then concentrated at the plasma membrane in response to nitrogen starvation. Deletion of rad24 or rad25 fastened the timing of Byr2 translocation. Our results are consistent with the hypothesis that one of the roles of 14-3-3 is to keep Byr2 in the cytoplasm and to affect the timing of Byr2 translocation in response to sexual developmental signal.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the fission yeast Schizosaccharomyces pombe, both nutrient starvation and mating pheromones lead to sexual development, which results in conjugation and sporulation (reviewed in references 12 and 70). The Byr2 protein kinase, a homolog of mammalian mitogen-activated protein kinase (MAPK) kinase kinase/MAPKKK, plays a pivotal role in S. pombe sexual development (62) and is an important downstream effector of Ras1 (64). Gpa1 (G-GTP), subunit of a receptor-coupled heterotrimeric G protein, mediates the signal from mating pheromones and affects the MAPK cascade (44, 67), which includes Byr2, Byr1, and Spk1 (42, 43, 61, 64). Another very early event in activation is the recruitment of Byr2 from the cytoplasm to the plasma membrane, depending on GTP-bound Ras1 (6). Other proteins are also involved in the activation of Byr2; these include Shk1/Pak1 (36, 46) and Ste4 (5, 45). Shk1/Pak1, an S. pombe homolog of mammalian PAK, is thought to phosphorylate Byr2. Ste4, which is a leucine zipper protein and capable of homodimerization, is also thought to dimerize Byr2 (62).

14-3-3 proteins are a family of highly conserved proteins, which are expressed in all eukaryotic cells (reviewed in reference 1). They are thought to play important roles in a wide range of signal transduction pathways, including those involved in cell cycle regulation and cell development. For example, the 14-3-3 protein binds Cdc25C phosphatase and prevents it from activating the Cdc2 kinase in mammalian cells and Xenopus laevis (11, 32, 47, 54, 71). Another member of the 14-3-3 family has been identified as a ligand for Raf1 in Drosophila melanogaster (8, 31), mammalian cells (14, 17, 18, 34, 50, 60), and yeast (23). In the budding yeast Saccharomyces cerevisiae, the 14-3-3 proteins Bmh1p and Bmh2p associate with Ste20p, a homolog of S. pombe Shk1/Pak1 and mammalian PAK, and are specifically required for RAS/MAPK cascade signaling during pseudohyphal development (49). In S. pombe, two 14-3-3 homologs, Rad24 and Rad25, regulate a DNA damage checkpoint (15). For other targets of 14-3-3 proteins in S. pombe, Cdc25 (35, 72), Plc1 (4), and Chk1 (9) have been reported. In addition, Rad24 inhibits the function of Mei2, a key regulator of meiosis, by binding with it (59).

In the present study, we found that the S. pombe 14-3-3 proteins Rad24 and Rad25 are involved in the Ras1/Byr2 signaling pathway and physically interact with Byr2 and that this interaction affects the timing of Byr2 translocation in response to sexual developmental signal.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Yeast and Escherichia coli strains, media, genetic manipulation, and analysis. All S. pombe strains used in the present study are listed in Table 1. All S. pombe strains constructed in the present study were derived from the wild-type SP870 strain. Standard yeast culture media YES and PM for S. pombe and genetic manipulation techniques were employed (3, 24, 38). Sporulation of suppressor mutants was detected by the iodine vapor staining method as described by Gutz et al. (20). The homoazygous diploid strains which lack ras1, byr2, ste4, byr1, or spk1 were generated by the protoplast fusion (3). Ploidy was confirmed by the color of colony on an agar medium containing 0.0005% Phloxin-B and by cell size and the presence of azygotic sporulation. E. coli DH5 was used as a host for all plasmid manipulations. E. coli BL21 and JM109 were used for expressing bacterial fusion proteins. Nucleotide sequences were determined by Dye Terminator Cycle Sequencing by using an ABI Prism 377 DNA sequencer (Perkin-Elmer).

Isolation of a suppressor mutant from a ras1-null strain. A homothallic h⁹⁰ ras1 null (ras1) diploid strain, SPRN1DA, was mutagenized with ethyl methanesulfonate. Among the mutagenized colonies, several mutants, which were fertile in spite of ras1, were identified, and one of these was named SRA1DA for further analysis. By fusion of the suppressor mutant haploid, SRA1 or SRA1A, with the ras1 haploid, SPRN1 or SPRN1A, to make a diploid, the mutant allele, named sra1, was shown to be recessive.

Isolation of the rad25 gene. SRA1DA was transformed with an S. pombe cDNA library (26) in the expression vector pAAU, in which cDNA expression is driven by the adh1 promoter and which contains ars1 and the ura4 marker. Colonies stained negative with iodine vapour were selected. Five plasmids were recovered from these colonies, and subsequent restriction mapping and sequencing analysis indicated that all isolated plasmids carried the rad25 gene.

Construction of homothallic rad24, rad25, and related mutants. A homothallic rad24 mutant was created by transforming SP870 with a rad24::ura4 DNA fragment which was amplified by PCR from the h^- rad24 mutant obtained from Antony M. Carr (15). A homothallic rad25 mutant was created by the same method with the h^- rad25 mutant, which is also obtained from Antony M. Carr. In the byr2, ste4, byr1, and spk1 deletion mutants, the ura4 gene which disrupted the indicated genes was replaced with a ura4::ADE2 fragment, resulting in SPSA (byr2::ura4::ADE2), SPFA (ste4::ura4::ADE2), SPBA (byr1::ura4::ADE2), and SPKA (spk1::ura4::ADE2) mutants. The ura4::ADE2 fragment was generated from the shk1 (shk1::ura4::ADE2)-null mutant by PCR (36). To create double-deletion mutants, SPRN1 (ras1), SPRN1A (ras1), SPSA (byr2), SPFA (ste4), SPBA (byr1), and SPKA (spk1) mutants were transformed with the rad24::ura4 fragment or the rad25::ura4 fragment. The resultant double mutants were named SP24U2 (ras1 rad24), SP24U2A (ras1 rad24), SP24U3 (byr2 rad24), SP24U4 (ste4 rad24), SP24U5 (byr1 rad24), SP24U6 (spk1 rad24), SP25U2 (ras1 rad25), SP25U2A (ras1 rad25), SP25U3 (byr2 rad25), SP25U4 (ste4 rad25), SP25U5 (byr1 rad25), and SP25U6 (spk1 rad25). Gene deletions of all strains were confirmed by Southern blot.

Plasmids used for bacterial expression studies. Plasmids expressing His₆ epitope-tagged Byr2-full length B2FL (residues 1 to 659), Byr2-N-terminal B2NT (residues 1 to 392), and C-terminal B2CT (residues 393 to 659) were constructed by inserting a PCR-generated BamHI-SmaI fragment of B2FL, a BamHI-SalI fragment of B2NT, and a SalI-PstI fragment of B2CT into the corresponding multiple cloning sites in pQE30, pQE31, and pQE32 (Qiagen), respectively. A plasmid expressing FLAG epitope-tagged Rad24 or Rad25 was constructed by inserting each PCR-generated BamHI-SalI fragment of rad24 or rad25 cDNA into a BglII-SalI site in pFLAGcts (Sigma).

Plasmids used for S. pombe studies. pREP41/42-rad24 and pREP41/42-rad25 were constructed by inserting a PCR-generated NdeI-SmaI fragment of rad24 or rad25 coding sequence into the corresponding multiple cloning site in pREP41/42 (37). An insert DNA in pREP41/42 is under the control of the moderate nmt1 promoter, whereby gene expression could be repressed by thiamine and induced in the absence of thiamine. pREP42byr2FL-HA, pREP42byr2NT-HA, and pREP42HA-byr2CT were constructed by inserting a PCR-generated NotI-NotI fragment of B2FL, a NotI-NotI fragment of B2NT, and a SalI-PstI fragment of B2CT into the corresponding multiple cloning site in the hemagglutinin (HA) epitope-tagged vectors pSLF272, pSLF272, and pSLF273, respectively (16). pREP1moc1-HA (our unpublished data) was constructed by inserting a PCR-generated SalI-SmaI fragment of moc1 coding sequence into the corresponding multiple cloning site in pSLF173 (16). pREP41-GFP^S65A, a green fluorescent protein (GFP) epitope (GFP^S65A)-tagged expression vector controlled by the moderate nmt1 promoter, was derived from pGP110 (41) and pREP1-GFP^S65A (58). pREP41rad24-GFP^S65A and pREP41rad25-GFP^S65A were constructed by inserting a PCR-generated SalI-BamHI fragment of the rad24 or rad25 coding sequence into the corresponding multiple cloning site in pREP41-GFP^S65A. The GFP and HA tags did not interfere with the normal function of each protein, since each tagged protein suppressed the mutation of the corresponding gene.

Site-specific mutagenesis of the byr1 gene and construction of chromosomal byr1^DD-3HA-tagged gene. Site-directed mutagenesis by using the Sculptor in vitro mutagenesis system (Amersham Pharmacia) was used to change residues 214 (serine) and 218 (threonine) of the byr1 gene to aspartic acid. As a template, a PCR-generated NotI-NotI fragment of the byr1 coding sequence was cloned into pBluescript SK(+) (pBS) (Stratagene). The primer 5'-GTCCCCACAAAATCTTGAGCAACATCGTTA-3' (the two aspartic acid codons are underlined) was annealed to a single-stranded pBS-byr1 DNA, and the complementary strand was synthesized in vitro. The mutated codons in pBS-byr1^DD were confirmed by sequencing.

The NotI-NotI fragment of the byr1^DD gene was cloned into the HA epitope-tagged expression vector pSLF172 (16). A 1,026-bp HindIII-SmaI fragment of byr1^DD35-3HA from pSLF172byr1^DD was cloned into a plasmid for integration, pYC11, which is the derivative of pBluescript KS(+) carrying the S. cerevisiae LEU2 gene (57). The resulting plasmid was named pFO1byr1^DDNHA. The stop codon was deleted from the restriction fragment of genomic clones. This plasmid was digested with HindIII to linearize and integrated at the genomic locus of the target gene in wild-type cells. The resulting strain was named FOB1^DD. Stable and precise integration of the tagged gene at the genomic locus was confirmed by Southern blot and the expression of Byr1^DD-HA fusion protein was also confirmed by Western blot.

Preparation of His fusion protein and FLAG fusion proteins and in vitro interaction assay. His fusion plasmids and FLAG fusion plasmids were transformed into E. coli JM109 and BL21, respectively. Cells were grown at 37°C and induced to express His-Byr2 full-length, His-Byr2 N-terminal, His-Byr2 C-terminal, Rad24-FLAG, and Rad25-FLAG forms by the addition of 1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) for 4 h at 37°C. Cells expressing His fusions or FLAG fusions were harvested and lysed in phosphate buffer or Tris buffer, respectively, by sonication. The phosphate buffer contained 50 mM NaH₂PO₄ (pH 8.0), 300 mM NaCl, 1 mM imidazole and protease inhibitors, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail tablets (Complete; Boehringer Mannheim). Tris buffer contained 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and the protease inhibitors described above. After centrifugation, each supernatant was incubated with Ni²⁺-nitrilotriacetic acid (Ni-NTA) agarose beads (Qiagen) against His fusions or anti-FLAG M2 affinity gel (Sigma) against FLAG fusions for 2 h at 4°C. FLAG fusions were eluted from anti-FLAG M2 affinity gel into elution buffer containing 0.1 M glycine (pH 3.5) in 20 mM Tris-HCl (pH 8.0). His fusion proteins immobilized on Ni-NTA-agarose beads were incubated with FLAG fusions for 2 h at 4°C and washed with wash buffer containing 50 mM NaH₂PO₄ (pH 8.0), 300 mM NaCl, and 5 mM imidazole. Complexes bound with Ni-NTA-agarose beads were detected by anti-His antibody (Qiagen) or anti-FLAG antibody (Sigma) Western blot analysis.

Yeast cell extracts. Cells were grown in the synthetic medium PM with appropriate supplements to mid-logarithmic phase (ca. 2 x 10⁷ cells/ml, total of 2 x 10⁸ cells), harvested by centrifugation, and washed once with ice-cold stop buffer (150 µM NaCl, 50 µM NaF, 10 µM EDTA, 1 µM NaN₃ [pH 8]) (38). The cells were lysed in 100 µl of ice-cold lysis buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 0.8% Nonidet-P40, 5 µM EDTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail tablets [Complete; Boehringer Mannheim]) (33) by vigorous vortexing with four 15-s pulses with 0.5-mm-diameter zirconia/silica beads (Biospec Products, Inc.). After centrifugation (14,000 rpm for 15 min at 4°C), the protein concentration in the supernatant was estimated by measuring the A₂₈₀ and adjusted to 10 mg/ml with lysis buffer.

Coimmunoprecipitation of endogenous Rad24 with exogenously expressed Rad24-GFP or Rad25-GFP. Wild-type cells expressing either GFP (control vector), Rad24-GFP, or Rad25-GFP were induced by growth at 30°C for 18 h in PM lacking thiamine. Immunoprecipitation analysis was performed with anti-GFP antibody by the method described below. The endogenous Rad24 protein was detected on immunoblots with anti-Rad24 antibody, which was kindly provided by Akio Toh-e (4).

Coimmunoprecipitation of Byr2FL-3HA, Byr2NT-3HA, or 3HA-Byr2CT with Rad24-GFP or Rad25-GFP. Byr2FL-3HA, Byr2NT-3HA, 3HA-Byr2CT, or Moc1-3HA expression combined with Rad24-GFP, Rad25-GFP, or GFP (vector) was induced by growth at 30°C for 18 h in PM lacking thiamine. As negative controls expressing HA fusion and GFP fusion, a nonrelevant gene moc1 (27) and GFP (vector alone) were used, respectively. The moc1 gene is under the control of the strongest nmt1 promoter (37). Anti-HA monoclonal antibody sc-7392 (Santa Cruz Biotechnology) against HA and anti-GFP polyclonal antibody A-6455 (Molecular Probes) against GFP were used in the immunoprecipitation of the HA fusions and GFP fusions, respectively. Then, 1 mg of cell extract was incubated with 1 µg of anti-HA antibody or 1 µg of anti-GFP antibody for 4 h at 4°C, and the mixture was added to 60 µl of protein A-Sepharose beads (50% slurry; Amersham-Pharmacia) and incubated again for 4 h at 4°C. The beads were washed six times with 0.5 ml of lysis buffer in a vortex mixer and resuspended in 30 µl of lysis buffer.

Samples of the immunoprecipitants were suspended in sodium dodecyl sulfate loading buffer, immediately boiled for 5 min, and resolved by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis. Proteins were then transferred to a polyvinylidene difluoride membrane (Immobilon; Millipore). Standard procedures for immunoblotting were followed as described previously (22). Primary antibodies raised from different animals, anti-HA polyclonal antibody sc-805 (Santa Cruz Biotechnology) and anti-GFP monoclonal antibody M048-3 (MBL), were used to avoid overlapping signals associated with immunoglobulin G (IgG) antibodies, which were used for immunoprecipitation. A horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) and goat anti-mouse IgG (Santa Cruz Biotechnology) were used as secondary antibodies to detect HA fusions and GFP fusions, respectively. The immunoreaction was visualized by using the ECL kit (Amersham Pharmacia).

Northern blot analysis. Total RNA was extracted from S. pombe cells as follows. Cells were cultured in 50 ml of PM to mid-log phase at 5 x 10⁶ cells/ml at 30°C; 2.5 x 10⁷ cells were washed with water and then with PM lacking ammonium chloride and finally resuspended in 50 ml of PM lacking ammonium chloride and grown for 5 h at 25°C. Cells for time course experiments were cultured as described above for 9 h with cultures being scaled up by 10-fold. Cells were resuspended in 0.5 ml of Isogen RNA isolation reagent (Nippon Gene) and vigorously vortexed for 6 min with 0.5-mm-diameter zirconia/silica beads (Biospec). After centrifugation (14,000 rpm for 15 min at 4°C), nucleic acid in the supernatant was precipitated with isopropanol. Approximately 15 µg of total RNA was resolved on 1.0% denaturing formaldehyde-agarose gels and transferred to a hybridization membrane (Hybond-N+; Amersham Pharmacia) in alkali transfer buffer (0.05 M NaOH) for 4 h or overnight. Standard procedures for hybridization were followed (53). Probes used for RNA-DNA hybridization were a 1.3-kb PvuII-PvuII restriction fragment of the ste11 gene from pSX1 (56), a 1.0-kb BamHI-BamHI restriction fragment of the mam2 gene from pCMVLX-mam2 (obtained from K. Kitamura), and a 1.0-kb EcoRV-EcoRV restriction fragment of the leu1 gene from pJK148 (28). The probe was labeled with [-³²P]dCTP (Amersham Pharmacia) by using the BcaBEST labeling kit (Takara Biomedicals). The transcription on the blot was analyzed by autoradiography with the image analyzer, BAS1500-Mac (Fuji Film Co.). The relative intensity of each band was then calculated with the amount of leu1 by using the NIH Image program (version 1.61).

Construction of chromosomal byr2-GFP tagged gene. A BamHI-SmaI fragment of GFP^S65A (58) was amplified by PCR. To construct pYC11GFP^S65A, the BamHI-SmaI fragment of GFP^S65A was first subcloned into pYC11 (57). A 1.7-kb XhoI-BamHI restriction fragment of genomic 80-byr2 was inserted into pYC11GFP^S65A at the N terminus of GFP. The resulting plasmid was named pFO1byr2NGFP^S65A. The stop codon was deleted from the restriction fragment of the genomic clone. This plasmid was digested with XhoI to linearize it and integrated at the genomic locus of the target gene in wild-type cells, rad24 cells, and rad25 cells. These integrant strains were named FOWB2G, FO24, and FO25, respectively. These GFP-tagged strains harbor one copy of the target gene with a GFP tag and one copy of the target gene with a deletion of the 5' end. Stable and precise integration of the tagged gene at the genomic locus was confirmed by Southern blot, and the expression of Byr2-GFP fusion protein was also confirmed by Western blot.

Byr2-GFP localization in living cells by fluorescence microscopy. Strains (FOWB2G, FO24, and FO25) were cultured in an appropriate volume of PM to mid-log phase at 5 x 10⁶ cells/ml at 30°C, washed with water and then with PM lacking ammonium chloride, and finally resuspended in PM lacking ammonium chloride and grown for 5 to 6 h at 25°C. Byr2-GFPs in living cells were monitored at an appropriate interval by fluorescence microscopy as described previously (13). Microscopic images were obtained on a charge-coupled device by using an Olympus oil-immersion objective lens 60x/NA1.4 on the DeltaVision microscope system (Applied Precision, Inc., Seattle, Wash.); details of the microscope system are provided by Haraguchi et al. (21).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Screening of chromosomal mutants of ras1-null (ras1) cells. S. pombe cells that lack ras1 fail to conjugate and sporulate (19). We mutagenized ras1 diploid cells (SPRN1DA) with ethyl methanesulfonate and selected the colonies of cells that did sporulate, as determined by iodine staining. A method of screening similar to the one used in the present study has been described elsewhere (25). We selected several ras1 mutants carrying chromosomal suppressors that rescue their sporulation defect. We obtained more than 16 suppressor mutants from the ras1-null mutant that rescued their sporulation defect. The mutations in these mutants were classified as either recessive or dominant by fusion of ras1 cells. One mutant was found to be recessive, and the others were all dominant. This recessive mutant was named SRA1DA, and its mutation allele was named sra1^- for further study. The mutation in SRA1DA (ras1sra1^-/ras1sra1^-) restores the sporulation defect in ras1 diploid cells.

Isolation of the rad25 gene. To identify the mutation allele sra1^-, we screened a high-copy cDNA library, constructed in the vector pAAU (26), for suppressors of SRA1DA. We then isolated five clones, which reduced sporulation efficiency in SRA1DA. After sequencing, all of these clones were found to encode the rad25 gene. rad25, as well as rad24, has been previously identified to be homologous to mammalian 14-3-3 genes (15). Thus, we constructed a rad25 deletion mutant of SRA1DA, named SRA25UDA (ras1sra1^-rad25/ras1sra1^-rad25), and tested its sporulation efficiency, but unexpectedly it was higher than that of SRA1DA, 19.7% versus 9.7%. Simultaneously, we amplified the rad25 gene from SRA1 (ras1sra1^-), including both the open reading frame region and the intron region by PCR, with Pfu polymerase to ensure high fidelity, and sequenced several clones of the rad25 gene. As a result, the nucleotide sequence of rad25 in SRA1 was normal. In addition, we detected the normal level of rad25 transcript from SRA1DA by Northern analysis (data not shown). Considering these results, we concluded that rad25 is not in the same complementation group with sra1. We also determined the nucleotide sequence of the rad24 gene from SRA1 to know the possible gene alteration, but it was normal. We also detected the normal level of rad24 transcript from SRA1DA by Northern analysis (data not shown). Therefore, we concluded that the sra1 mutant allele is not located at either rad24 or rad25 locus.

Even though rad24 or rad25 was not the originally mutated sra1 allele we had sought, we thought it would be meaningful to explore the role of rad24 and rad25 in the sexual development process in S. pombe.

Rad24 and Rad25 act in the same pathway in sexual differentiation, with the forming of a complex. To test whether Rad24 and Rad25 act in the sexual differentiation, especially in the Ras1/MAPK cascade, we examined their effects on mating and sporulation efficiency of a homothallic wild-type strain by overexpressing rad24 or rad25. Two plasmids, pREP41rad24 and pREP41rad25, were constructed to express rad24 and rad25 under the control of the moderate nmt1 promoter (37). Overexpression of rad24 or rad25 reduced mating and sporulation efficiency in wild-type cells (Fig. 1A). Rad24 was twofold more effective than Rad25, and simultaneous expression of rad24 and rad25 in wild-type cells caused the same effect on mating and sporulation as did expression of rad24 alone (data not shown). Next, we tested the phenotype of rad24 cells or rad25 cells, since a rad24 rad25 double mutant had been reported to be lethal (15). The mating and sporulation efficiency of rad24 cells was increased compared with that of wild-type cells, but that of rad25 cells showed no difference from that of wild-type cells (Table 2). However, rad25 cells, which were grown on agar plates, showed considerably increased mating and sporulation efficiency compared to wild-type cells. A complementation test in rad24 or rad25 cells by overexpressing rad24 or rad25 was performed (Fig. 1B and C). The activity of Rad24 exceeded that of Rad25 in either case. Together, these data indicate that both Rad24 and Rad25 negatively regulate sexual development and that their function is redundant, although Rad24 preferentially acts compared to Rad25 in the context of mating and sporulation.

View larger version (35K): [in this window] [in a new window]   FIG. 1. Expression of rad24 or rad25 inhibits mating and sporulation efficiency. The host wild-type (SP870) (A), rad24 (SP24U1) (B), rad25 (SP25U1) (C), ras1 rad24 diploid (SP24U2DA) (D), and ras1 rad25 diploid (SP25U2DA) (E) strains were transformed with control vector pREP41, pREP41rad24, or pREP41rad25. Each transformant cell was grown in PM+N with thiamine to early log phase at 30°C and subsequently inoculated into PM-N with or without thiamine for 48 h at 25°C. The mating and sporulation efficiency was scored microscopically as the prevalence of zygotic or azygotic asci relative to haploid or diploid cells, respectively, in a given population. About 500 to 1,000 cells were observed. Inhibition of mating and sporulation is reported as a percentage of that seen for cells induced with thiamine. Each error bar represents the standard deviation of three independent experiments.

View larger version (39K): [in this window] [in a new window]   FIG. 2. Homo- and heterodimerization are observed for Rad24 and Rad25. A wild-type strain (SP870) was transformed with either pREP41-GFP, pREP41rad24-GFP, or pREP41rad25-GFP. GFP fusions in each transformant were induced by growth at 30°C for 18 h in PM lacking thiamine. The protein extracts prepared from each transformant cell were subjected to immunoprecipitation (IP) with anti-GFP antibody.

ras1

rad24

rad25

rad24

rad25

ras1 rad24

ras1 rad25

ras1

byr2 rad24

byr2 rad25

byr2

ras1 rad24

ras1 rad25

ras1

ras1 rad24

ras1 rad25

ras1

View larger version (79K): [in this window] [in a new window]   FIG. 3. Photomicrographs of S. pombe homothallic wild-type cells, rad24-null mutants, and rad25-null mutants, which were combined with mutants lacking the Ras1/MAPK cascade function. Strains wild-type (SP870), ras1 diploid (SPRN1DA), byr2 diploid (SPSAD), ste4 diploid (SPFAD), byr1 diploid (SPBAD), spk1 diploid (SPKAD), rad24 (SP24U1), ras1 rad24 diploid (SP24U2DA), byr2 rad24 diploid (SP24U3D), ste4 rad24 diploid (SP24U4D), byr1 rad24 diploid (SP24U5D), spk1 rad24 diploid (SP24U6D), rad25 (SP25U1), ras1 rad25 diploid (SP25U2DA), byr2 rad25 diploid (SP25U3D), ste4 rad25 diploid (SP25U4D), byr1 rad25 diploid (SP25U5D), and spk1 rad25 diploid (SP25U6D) are shown in panels A to R, respectively. Wild-type, rad24, and rad25 strains were grown on PM+N plates, and the other diploid strains were grown on PM+N plates containing 0.0005% Phloxin-B to check the ploidy at 25°C for 4 days. The ascospores in panels A, G, M, H, and N are indicated with arrowheads. Scale bar, 10 µm.

rad24

ras1

rad25

Activated Byr1 is independent of Rad24 and Rad25. We performed further genetic analysis to determine whether Rad24 and Rad25 directly modulate Byr2 function. MAPKK family kinases, including those from mammalian to yeast sources, are activated by phosphorylation of two conserved Ser/Thr residues (74), which correspond to serine-214 and threonine-218 in Byr1. We generated constitutively active Byr1 (designated Byr1^DD) by substituting aspartic acid for serine-214 and threonine-218. Since overexpression of byr1^DD in byr1 cells resulted in growth inhibition (data not shown), we constructed a strain named FOB1^DD, in which the wild-type byr1 allele was replaced with the byr1^DD allele. The byr1^DD cells show weak growth inhibition, much aggregation (data not shown), and elongated conjugation tubes (Fig. 4A). In byr1^DD cells, expression of rad24 or rad25 in liquid minimal medium without nitrogen (PM-N) in the absence of thiamine did not show any alternation in morphology compared to expression of control vector (Fig. 4A). We next quantitatively analyzed the effect of Rad24 or Rad25 in byr1^DD cells by counting abnormally shaped cells (i.e., that had elongated conjugation tubes) in a given population (Fig. 4B). The ratio of the prevalence of abnormally shaped cells under nitrogen starvation was not altered by overexpressing rad24 or rad25. These observations suggest that neither Rad24 nor Rad25 acts as a modulator of components downstream of Byr1. It therefore appeared that the target of Rad24 and Rad25 might be Byr2.

View larger version (66K): [in this window] [in a new window]   FIG. 4. Rad24 and Rad25 have no effect on the activity of constitutively active Byr1DD. Strain byr1DD (FOB1DD) was transformed with either pREP41 (control), pREP41rad24, or pREP41rad25. Transformants were cultured after being starved in nitrogen-free minimal liquid medium (PM-N) with or without thiamine for 48 h at 25°C, following vegetative growth in minimal liquid medium (PM+N). (A) Photomicrographs of byr1DD cells. byr1DD cells carried the control vector pREP42 (left panel), pREP42-rad24 (middle panel), or pREP42-rad25 (right panel) without thiamine under conditions of nitrogen starvation. The arrows indicate abnormally shaped cells, which had elongated conjugation tubes. Scale bar, 10 µm. (B) The prevalence ratio of abnormally shaped cells. Abnormally shaped cells were microscopically scored in byr1DD cells in each condition as indicated. Each error bar represents the standard deviation of three independent experiments.

View larger version (37K): [in this window] [in a new window]   FIG. 5. Direct interaction among Byr2FL, Byr2NT, or Byr2CT with Rad24 or Rad25. (A) Expression of His6 fusion proteins and FLAG fusion proteins. His6-Byr2 full-length (Byr2FL), His6-Byr2 N-terminal (Byr2NT), and His6-Byr2 C-terminal (Byr2CT) proteins were expressed in E. coli and immobilized on Ni-NTA-agarose beads. The FLAG fusion proteins, Rad24-FLAG and Rad25-FLAG, were expressed in E. coli and purified with anti-FLAG M2 affinity Sepharose beads. The His6 fusion proteins and the FLAG fusion proteins were detected by immunoblotting. The asterisk indicates degradation products of His6-Byr2FL and His6-Byr2NT. (B) Binding of either Rad24 or Rad25 to Byr2FL, Byr2NT, and Byr2CT. The His6 fusion proteins immobilized on Ni-NTA-agarose beads were incubated with FLAG fusion proteins. Bound fraction was analyzed by Western blotting with anti-His antibody (upper panel) or anti-FLAG antibody (lower panel). The asterisk indicates the degradation products of His6-Byr2FL and His6-Byr2NT.

byr2

View larger version (78K): [in this window] [in a new window]   FIG. 6. Byr2FL, Byr2NT, and Byr2CT physically interact with Rad24 and Rad25 in S. pombe cells. A byr2 strain (SPSA) was cotransformed with a pair of fusion constructs as indicated. Protein extracts prepared from each transformant cell were subjected to immunoprecipitation (IP) with either anti-HA antibody (A and C) or anti-GFP antibody (B and D). The immunoprecipitants were analyzed by Western blotting with either anti-GFP antibody (A and D) or anti-HA antibody (B and C). The arrows in panels A and D indicate GFP, Rad24-GFP, and Rad25-GFP. The arrows in panels B and C indicate Moc1-HA, Byr2FL-HA, Byr2NT-HA, and HA-Byr2CT. Asterisks indicate the degradation products of Byr2FL-HA, and double asterisks indicate the degradation products of Rad24-GFP and Rad25-GFP. Triple asterisks indicate nonspecific bands.

Expression of mam2 and ste11 in rad24 and rad25 derivatives.

rad24

rad25

rad24

rad25

rad25

rad24

rad24

rad25

mam2

ste11

ste11

mam2

rad24

rad25

rad24

rad24

rad24

rad24

rad24

ras1 rad25

ras1

byr2

ste4

byr1

spk1

View larger version (59K): [in this window] [in a new window]   FIG. 7. Expression of mam2 and ste11 mRNA in rad24 cells, rad25 cells, and the relevant cells. (A) Time course of transcription of mam2 and ste11 in wild-type (SP870), rad24 (SP24U1), and rad25 (SP25U1) cells. Cells were harvested at the indicated times for 9 h after nitrogen starvation. Total RNA was prepared from each strain and analyzed by Northern blot. The transcription of leu1 in each strain was also analyzed as an inner loading control. (B) Dependency on the Ras1/MAPK cascade in expression of mam2 and ste11. The level of mam2 and ste11 transcript was examined 5 h after nitrogen starvation in wild-type (SP870), ras1 (SPRN1), byr2 (SPSA), ste4 (SPFA), byr1 (SPBA), spk1 (SPKA), ras1V17 (SP593), byr1DD (FOB1 DD), ste11 (KJ33-1A), and mam2 (C523-10) cells. The relative intensities of mam2 and ste11 transcript are presented under the panels. Asterisk in the middle panel shows a nonrelevant band. (C) Level of mam2 transcript in the double mutants among mutants in the Ras1/MAPK cascade and rad24 or rad25 cells. The level of mam2 transcript was examined at 5 h after nitrogen starvation in rad24 (SP24U1), ras1 rad24 (SP24U2), byr2 rad24 (SP24U3), ste4 rad24 (SP24U4), byr1 rad24 (SP24U5), spk1 rad24 (SP24U6), rad25 (SP25U1), ras1 rad25 (SP25U2), byr2 rad25 (SP25U3), ste4 rad25 (SP25U4), byr1 rad25 (SP25U5), and spk1 rad25 (SP25U6) cells. The relative intensity of mam2 transcription is presented under the panel. (D) Quantitative comparison in the levels of mam2 transcript.

rad24

rad25

View larger version (91K): [in this window] [in a new window]   FIG. 8. Localization of Byr2. (A) Byr2 translocation in response to nitrogen starvation. A byr2-GFP-tagged strain (FOWB2G) was grown in PM+N to mid-log phase and subsequently inoculated into PM-N. Localization of Byr2 in living cells was monitored with a fluorescence microscope soon after nitrogen starvation (left) and at 5.5 h (right) at 25°C. The arrows indicate Byr2-GFP, which translocated to the plasma membrane. (B) Byr2 translocation occurred earlier in rad24 or rad25 cells than in wild-type cells. The byr2-GFP-tagged strains with deletion of rad24 or rad25 (FO24 and FO25) were grown as for panel A. Localization of Byr2-GFP in each living cell was monitored at 1-h intervals. The arrows indicate Byr2-GFP, which was concentrated at the plasma membrane. Scale bar, 15 µm.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

ras1 rad24

ras1 rad25

ras1

Since deletion of rad24 or rad25 can bypass the phenotype associated with ras1 but not that associated with byr2, ste4, byr1, or spk1 and since the overexpression of either Rad24 or Rad25 does not affect the phenotype of cells expressing Byr1^DD (constitutively active Byr1), it appears that Byr2 is the target of Rad24- and Rad25-mediated regulation. The direct binding of Byr2 to either Rad24 or Rad25 was confirmed by using a bacterially expressed protein system and coimmunoprecipitation (Fig. 5 and 6). Ste4, a leucine zipper protein capable of homodimerization, is thought to dimerize Byr2 near the plasma membrane (62). Our genetic results also suggest that Ste4 is not necessary for the regulation of Byr2 by Rad24 and Rad25.

Deletion of the rad24 gene or the rad25 gene caused different changes in morphology. In the context of cell morphology, Rad24 and Rad25 might be involved in different pathways. The morphological involvement of rad24 and rad25 cannot be explained by the known targets of 14-3-3 proteins in S. pombe. It therefore appears that Rad24 and Rad25 have multiple targets other than Cdc25, Chk1, Plc1, Mei2, Ste11, and Byr2. We recently identified CAP (adenylyl cyclase-associated protein), which is involved in cell morphology, as another example of the target of 14-3-3 proteins (75).

Many proteins that interact with 14-3-3 proteins contain the recognition motifs RSXpSXP and RXY/FXpSXP (pS indicates phosphoserine) (40, 68). Raf1 from higher eukaryotes has these motifs and interacts with 14-3-3 proteins in a phosphorylation-dependent manner (39, 40). Although Byr2 does not contain the typical recognition motifs, it interacts with Rad24 or Rad25 through at least two binding sites, one in the N-terminal and a second in the C-terminal domain. Similarly, S. pombe Chk1 does not contain a motif matching the RSXpSXP, but it interacts with Rad24 and Rad25 (9). It has been reported that 14-3-3 proteins can bind different peptide motifs via a conserved amphipathic groove (48, 65), so the binding of Byr2 to 14-3-3 proteins may involve such an atypical motif. According to another report, some of the 14-3-3-binding motifs have deviated from typical motifs, and phosphorylation for binding is not always required (69). Since the interaction between Byr2 and either Rad24 or Rad25 was observed in an E. coli expression binding assay and a two-hybrid system assay, this interaction is thought to be unaffected by phosphorylation. We are now trying to define the 14-3-3 proteins binding sites on Byr2.

Negative effect of Rad24 and Rad25 on Byr2 translocation. Rad24 and Rad25 in S. pombe appear to act as negative regulators of Byr2. We draw this conclusion from a biochemical method in addition to genetic studies. A different situation has been reported in S. cerevisiae, in which 14-3-3 proteins act as positive regulators of Ste20 (49). However, there are two opposite observations concerned with the roles of 14-3-3 proteins for Raf in mammals, D. melanogaster, and X. laevis (10, 31, 51, 52, 63).

The Ras1/MAPK pathway can stimulate the expression of mam2 and ste11, and the expression of mam2 is totally dependent on ste11. This regulatory system on mam2 made it difficult to assess the role of Rad24 and Rad25 for Byr2. We initially observed that the ste11 transcript in rad24 cells was increased and that the timing of induction in rad24 cells was earlier than that of wild-type cells. This observation could not be explained previously, but the recent finding that Rad24 directly inhibits Ste11 by binding and inhibits the expression of ste11 in an autoregulatory way (30) explained our results well. Since Rad25 has little effect on Ste11, the role of Rad25 for Byr2 was easier to monitor by mam2 expression than Rad24. By monitoring the mam2 and ste11 transcript, we showed the involvement of Rad25 in the Ras1/MAPK pathway. It would be difficult to determine the role of Rad24 for Byr2 by Northern blot analysis unless we discover the reporter gene solely dependent upon the Ras1/MAPK cascade.

In addition, we examined the cellular localization of Byr2 tagged with GFP. Byr2 translocation from the cytoplasm to the plasma membrane was observed in response to nitrogen starvation. In contrast, deletion of rad24 or rad25 induced earlier translocation of Byr2.

In summary, our results and knowledge led us to propose the model depicted in Fig. 9. Inactive Byr2 is retained in the cytoplasm by binding to the dimerized form(s) of Rad24 and Rad25. In the presence of an extracellular signal, Byr2 is recruited to the plasma membrane, being dependent on GTP-bound Ras1, and is modified by other components necessary for its activation. This model includes a role for Rad24 and Rad25 in retaining target proteins in the cytoplasm, as is the case in the cytoplasmic anchoring of Cdc25 by Rad24 (35, 73).

View larger version (27K): [in this window] [in a new window]   FIG. 9. Model for the roles of Rad24 and Rad25 in regulation of Byr2 activity (see Discussion).

	ACKNOWLEDGMENTS

 
We thank Yasushi Hiraoka and Takaharu Yamamoto for providing fluorescence microscopic photographs of Byr2-GFP. We also thank Antony M. Carr for the rad24 and rad25 mutants, Michael Wigler and Stevan Marcus for the ras1^val mutant and the shk1 mutant, Susan L. Forsburg for HA-tagged plasmids, Mitsuhiro Yanagida for the GFP_S65A plasmid, Masayuki Yamamoto for the ste11 plasmid, Chikashi Shimoda for the mam2 mutant, Kenji Kitamura for the ste11 mutant and the mam2 plasmid, and Akio Toh-e and Tomoko Andoh for the anti-Rad24 antibody. This work was technically assisted by Yasue Ishikura, Miho Omae, Tohoko Furuta, and Ayano Era.

This work was supported by Grants-in-Aid from the Kato Memorial Foundation and the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan. Phone: 81-852-32-6587. Fax: 81-852-32-6092. E-mail: kawamuka{at}life.shimane-u.ac.jp.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Aitken, A. 1995. 14-3-3 proteins on the MAP. Trends Biochem. Sci. 20:95-97.[CrossRef][Medline]

2. Aitken, A. 1996. 14-3-3 and its possible role in co-ordinating multiple signaling pathways. Trends Cell Biol. 6:341-347.[CrossRef][Medline]

3. Alfa, C., P. Fantes, J. Hyams, M. McLeod, and E. Warbrick. 1993. Experiments with fission yeast. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

4. Andoh, T., T. J. Kato, Y. Matsui, and A. Toh-e. 1998. Phosphoinositide-specific phospholipase C forms a complex with 14-3-3 proteins and is involved in expression of UV resistance in fission yeast. Mol. Gen. Genet. 258:139-147.[CrossRef][Medline]

5. Barr, M. M., H. Tu, L. Van Aelst, and M. Wigler. 1996. Identification of Ste4 as a potential regulator of Byr2 in the sexual response pathway of Schizosaccharomyces pombe. Mol. Cell. Biol. 16:5597-5603.[Abstract]

6. Bauman, P., Q. Cheng, and C. F. Albright. 1998. The Byr2 kinase translocates to the plasma membrane in a Ras1-dependent manner. Biochem. Biophys. Res. Commun. 244:468-474.[CrossRef][Medline]

7. Braselmann, S., and F. McCormick. 1995. BCR and RAF form a complex in vivo via 14-3-3 proteins. EMBO J. 14:4839-4848.[Medline]

8. Chang, H. C., and G. M. Rubin. 1997. 14-3-3 positively regulates Ras-mediated signaling in Drosophila. Genes Dev. 11:1132-1139.[Abstract/Free Full Text]

9. Chen, L., T.-H. Liu, and N. C. Walworth. 1999. Association of Chk1 with 14-3-3 proteins is stimulated by DNA damage. Genes Dev. 13:675-685.[Abstract/Free Full Text]

10. Clark, G. J., J. K. Drugan, K. L. Rossman, J. W. Carpenter, K. Rogers-Graham, H. Fu, C. J. Der, and S. L. Campbell. 1997. 14-3-3 negatively regulates Raf-1 activity by interactions with the Raf-1 cysteine-rich domain. J. Biol. Chem. 272:20990-20993.[Abstract/Free Full Text]

11. Dalal, S., C. Schweitzer, J. Gan, and J. DeCaprio. 1999. Cytoplasmic localization of human cdc25C during interphase requires an intact 14-3-3 binding site. Mol. Cell. Biol. 19:4465-4479.[Abstract/Free Full Text]

12. Davey, J. 1998. Fusion of a fission yeast. Yeast 14:1529-1566.[CrossRef][Medline]

13. Ding, D.-Q., Y. Chikashige, T. Haraguchi, and Y. Hiraoka. 1998. Oscillatory nuclear movement in fission yeast meiotic prophase is driven by astral microtubules as revealed by continuous observation of chromosomes and microtubules in living cells. J. Cell Sci. 111:701-712.[Abstract]

14. Fantl, W. J., A. J. Muslin, A. Kikuchi, J. A. Martin, A. M. MacNicol, R. W. Gross, and L. T. Williams. 1994. Activation of Raf-1 by 14-3-3 protein. Nature 371:612-613.[CrossRef][Medline]

15. Ford, J. C., F. Al-Khodairy, E. Fotou, K. S. Sheldrick, D. J. F. Griffiths, and A. M. Carr. 1994. 14-3-3 protein homologs required for the DNA damage checkpoint in fission yeast. Science 265:533-535.[Abstract/Free Full Text]

16. Forsburg, S. L., and D. A. Sherman. 1997. General purpose tagging vectors for fission yeast. Gene 191:191-195.[CrossRef][Medline]

17. Freed, E., M. Symons, S. G. Macdonald, F. McCormick, and R. Ruggieri. 1994. Binding of 14-3-3 proteins to the protein kinase Raf and effects on its activation. Science 265:1713-1716.[Abstract/Free Full Text]

18. Fu, H., K. Xia, D. C. Pallas, C. Cui, K. Conroy, R. P. Narsimhan, H. Mamon, R. J. Collier, and T. M. Roberts. 1994. Interaction of the protein kinase Raf-1 with 14-3-3 proteins. Science 266:126-129.[Abstract/Free Full Text]

19. Fukui, Y., T. Kozasa, Y. Kaziro, T. Takeda, and M. Yamamoto. 1986. Role of a Ras homolog in the life cycle of Schizosaccharomyces pombe. Cell 44:329-336.[CrossRef][Medline]

20. Gutz, H., H. Heslot, U. Leupold, and N. Loprieno. 1974. Handbook of genetics. Plenum Press, Inc., New York, N.Y.

21. Haraguchi, T., D.-Q. Ding, A. Yamamoto, T. Kaneda, T. Koujin, and Y. Hiraoka. 1999. Multiple-color fluorescence imaging of chromosomes and microtubules in living cells. Cell Struct. Funct. 24:291-298.[CrossRef][Medline]

22. Harlow, E., and D. Lane. 1988. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

23. Irie, K., Y. Gotoh, B. M. Yashar, B. Errede, E. Nishida, and K. Matsumoto. 1994. Stimulatory effects of yeast and mammalian 14-3-3 proteins on the Raf protein kinase. Science 265:1716-1719.[Abstract/Free Full Text]

24. Kaiser, C., S. Michaelis, and A. Mitchell. 1994. Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

25. Katayama, S., F. Ozoe, R. Kurokawa, K. Tanaka, T. Nakagawa, H. Matsuda, and M. Kawamukai. 1996. Genetic analysis of the sam mutations, which induce sexual development with no requirement for nutritional starvation in fission yeast. Biosci. Biotechnol. Biochem. 60:994-999.[Medline]

26. Kawamukai, M., J. Gerst, J. Field, M. Riggs, L. Rodgers, M. Wigler, and D. Young. 1992. Genetic and biochemical analysis of the adenylyl cyclase-associated protein, cap, in Schizosaccharomyces pombe. Mol. Biol. Cell 3:167-180.[Abstract]

27. Kawamukai, M. 1999. Isolation of a novel gene, moc2, encoding a putative RNA helicase as a suppressor of sterile strains in Schizosaccharomyces pombe. Biochim. Biophys. Acta 1446:93-101.[Medline]

28. Keeney, J. B., and J. D. Boeke. 1994. Efficient targeted integration at leu1-32 and ura4-294 in Schizosaccharomyces pombe. Genetics 136:849-856.[Abstract]

29. Kitamura, K., and C. Shimoda. 1991. The Schizosaccharomyces pombe mam2 gene encodes a putative pheromone receptor which has a significant homology with the Saccharomyces cerevisiae Ste2 protein. EMBO J. 10:3743-3751.[Medline]

30. Kitamura, K., S. Katayama, S. Dhut, M. Sato, Y. Watanabe, M. Yamamoto, and T. Toda. 2001. Phosphorylation of Mei2 and Ste11 by Pat1 kinase inhibits sexual differentiation via ubiquitin proteolysis and 14-3-3 protein in fission yeast. Dev. Cell 1:389-399.[CrossRef][Medline]

31. Kockel, L., G. Vorbruggen, H. Jackle, M. Mlodzik, and D. Bohmann. 1997. Requirement for Drosophila 14-3-3 in Raf-dependent photoreceptor development. Genes Dev. 11:1140-1147.[Abstract/Free Full Text]

32. Kumagai, A., and W. G. Dunphy. 1999. Binding of 14-3-3 proteins and nuclear export control the intracellular localization of the mitotic inducer Cdc25. Genes Dev. 13:1067-1072.[Abstract/Free Full Text]

33. Leatherwood, J., A. Lopez-Girona, and P. Russell. 1996. Interaction of Cdc2 and Cdc18 with a fission yeast ORC2-like protein. Nature 379:360-363.[CrossRef][Medline]

34. Li, S., P. Janosch, M. Tanji, G. C. Rosenfeld, J. C. Waymire, H. Mischak, W. Kolch, and J. M. Sedivy. 1995. Regulation of Raf-1 kinase activity by the 14-3-3 family of proteins. EMBO J. 14:685-696.[Medline]

35. Lopez-Girona, A., B. Furnari, O. Mondesert, and P. Russell. 1999.