Báo cáo Y học: Rum1, an inhibitor of cyclin-dependent kinase in fission yeast, is negatively regulated by mitogen-activated protein kinase-mediated phosphorylation at Ser and Thr residues pptx

Eur J Biochem 269, 3511–3521 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03033.x Rum1, an inhibitor of cyclin-dependent kinase in fission yeast, is negatively regulated by mitogen-activated protein kinase-mediated phosphorylation at Ser and Thr residues Kentaro Matsuoka1,2, Nobutaka Kiyokawa1, Tomoko Taguchi1, Jun Matsui1, Toyo Suzuki1, Kenichi Mimori1, Hideki Nakajima1, Hisami Takenouchi1, Tang Weiran1, Yohko U Katagiri1 and Junichiro Fujimoto1 Department of Pathology, National Children’s Medical Research Center, Tokyo, Japan; 2Department of Pathology, Keio University, School of Medicine, Tokyo, Japan The p25rum1 is an inhibitor of Cdc2 kinase expressed in fission yeast and plays an important role in cell-cycle control As its amino-acid sequence suggests that p25rum1 has putative phosphorylation sites for mitogen-activated protein kinase (MAPK), we investigated the ability of MAPK to phosphorylate p25rum1 Direct in vitro kinase assay using GST-fusion proteins of wild-type as well as various mutants of p25rum1 demonstrated that MAPK phosphorylates the N-terminal portion of p25rum1 and residues Thr13 and Ser19 are major phosphorylation sites for MAPK In addition, phosphorylation of p25rum1 by MAPK revealed markedly reduced Cdc2 kinase inhibitor ability of the protein Together with the fact that replacement of both Thr13 and Ser19 with Glu, which mimics the phosphorylated state of these residues, also significantly reduces the activity of p25rum1 as a Cdc2 inhibitor, it was suggested that the phosphorylation of Thr13 and Ser19 negatively regulates the function of p25rum1 Further evidence indicates that phosphorylation of Thr13 and Ser19 may retain a negative effect on the function of p25rum1 even in vivo Therefore, MAPK may regulate the function of p25rum1 via phosphorylation of its Thr and Ser residues and thus participate in cell cycle control in fission yeast The yeasts have been the favored organisms for investigation of the basic biology, genetics, and biochemistry of the cell cycle [1] Studies of the fission yeast Schizosaccharomyces pombe have played an instrumental role in the discovery of proteins that regulate the mitotic cycle S pombe appears to be able to control its cell cycle with considerably fewer components than are used by other eukaryotes, including the budding yeast Saccharomyces cerevisiae For example, S pombe relies on a single cyclin-dependent kinase (CDK), Cdc2, to coordinate its mitotic cell-cycle events Only four cyclins, Cdc13, Cig1, Cig2, and Puc1, and only one CDK inhibitor (CKI), namely p25rum1, have been identified in S pombe [1] Therefore, S pombe is considered to provide a simple model of cell-cycle regulation CDKs are undoubtedly key molecules in cell-cycle progression in virtually all eukaryotes, including fission yeast, and are required for the G1–S transition as well as for initiation of mitosis [2–4] To control the cell-cycle process, however, CDK activities must be regulated appropriately, kept low during those phases of the cell cycle when they are not required and only increase when they are needed to bring about cell-cycle progression Therefore, a mechanism tightly regulating their activities during the cell cycle must be present In S pombe, the activity of Cdc2 is regulated positively and negatively by at least three kinds of biochemical events First, Cdc2 must associate with B-type cyclin to display its kinase activity It was reported that Cdc2-Cdc13 is the mitotic kinase [5,6] while Cdc2-Cig2 promotes the G1–S transition (G1–S kinase) [7–9] The Cdc2–Cig1 complex is suggested to also contribute to the G1–S transition because a cdc13Dcig2D double mutant can still go through S phase while the cdc13Dcig2Dcig1D triple mutant cannot [9,10] It is noteworthy that these B-type cyclins are strictly regulated in terms of protein amounts during cell cycling by transcription and ubiquitin-mediated proteolysis [11] Second, the kinase activity of Cdc2 is also regulated by the phosphorylation state of specific aminoacid residues In S pombe, the phosphorylations at Tyr15 and Thr167 residues of Cdc2 regulate its kinase activity negatively and positively, respectively The phosphorylation of Tyr15 is regulated by a combination of protein kinases, Wee1 and Mik1, and protein phosphatase, Cdc25, whereas the regulatory mechanism of Thr167 is largely unknown [12–16] In addition to the above biochemical events, CKI also plays an important role in the regulation of CDK activity [1] p25rum1, the only known CKI in S pombe, was originally isolated by Moreno & Nurse in a screen for genes that, when Correspondence to N Kiyokawa, Department of Pathology, National Children’s Medical Research Center, 3-35-31, Taishido, Setagaya-ku, Tokyo 154-8567, Japan Fax/Tel.: + 81 3487 9669, E-mail: nkiyokawa@nch.go.jp Abbreviations: CDK, cyclin-dependent kinase; CKI, CDK inhibitor; MAPK, mitogen-activated protein kinase; MAP3K, MAPK kinase kinase; MAP2K, MAPK kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun amino-terminal kinase; GST, glutathioneS-transferase; EMM, Edinburgh minimal medium; YES, yeast extract + supplements; PI, propidium iodide (Received 26 February 2002, revised May 2002, accepted 31 May 2002) Keywords: cell cycle; Rum1; Cdc2; mitogen-activated protein kinase; phosphorylation Ó FEBS 2002 3512 K Matsuoka et al (Eur J Biochem 269) overproduced, would induce extra rounds of DNA replication [17] The over-replication can be extensive, with cells having more than 16 times the normal complement of DNA, thus earning rum1 its name (replication uncoupled from mitosis) Direct in vitro assays have demonstrated that p25rum1 is an effective inhibitor of Cdc2–Cdc13 and Cdc2– Cig2, but not Cdc2–Cig1 [18–20] The p25rum1 begins to accumulate at anaphase, persists in G1 and is destroyed during S phase [20] This molecule is thought to play a central role in maintenance of the G1 phase by regulating CDK activity [17–19,21] By inhibiting G1–S kinase activity, p25rum1 determines the cell-cycle timing of the G1–S transition (Start), maintaining cells in the pre-Start state until they have attained the minimal critical mass required to initiate the cell cycle In addition, it would stabilize Cdc18 and promote S-phase initiation [22] As a consequence, this molecule determines the length of the G1 phase interval By inhibiting mitotic CDK activity, p25rum1 also prevents the premature-onset of mitosis in cells that have not initiated DNA replication [17] Although a number of studies have clarified the function and regulatory mechanism of p25rum1 [17–30], details remain unclear For example, the amino acid sequence of p25rum1 has revealed the presence of five putative phosphorylation sites for mitogen-activated protein kinase (MAPK), i.e Thr5, Thr13, Thr16, Ser19, and Thr58 [17], while the precise roles of these sites are largely unknown Whether these sites are indeed phosphorylated by MAPK, and have any functional importance in regulating p25rum1 activity, has not yet been determined MAPK is a central component of the evolutionarily conserved growth-promoting signaling pathway The MAPK cascade, which transmits extracellular signals from cell surface receptors to nuclear transcription factors [31], consists of a module of three sequentially activated protein kinases, namely, MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK The extracellular signals are mediated intracellularly as an activation of MAP3K that further activates MAP2K by phosphorylating specific Ser and Thr residues on it The activated MAP2K then phosphorylates specific Thr and Tyr residues on MAPK, leading to the activation of this kinase Once activated, MAPK not only phosphorylates its cytoplasmic targets but also promotes their nuclear translocation and subsequent modulation of transcriptional factors resulting in stimulusdependent alterations in gene expression In many mammalian cells, three distinct classes of MAPK families, including the extracellular signal-regulated kinase (ERK) family, c-Jun N-terminal kinase (JNK) family, and the p38 MAPK family, have been identified In the case of fission yeast, MAPK Sty1/Spc1 is found to be highly related to the p38 MAPKs [32,33] Although it is well documented that MAPKs are important in cell growth and proliferation of eukaryotic cells, how stimulation of MAPKs culminates in cell-cycle progression is still poorly understood In an attempt to clarify the functional correlation between MAPK and p25rum1 in cell-cycle regulation, we generated and examined a series of recombinant p25rum1 proteins containing various mutations in their putative MAPK phophsorylation sites Here, we show that MAPK can phosphorylate p25rum1 leading to suppression of the activity of this molecule as a CKI in vitro We further demonstrate the possibility that this regulatory mechanism of p25rum1 also plays a role in vivo MATERIALS AND METHODS Materials The 796-bp fragment of rum1 cDNA (nucleotides 107–902) was amplified by PCR from cDNA prepared from S pombe (Library-in-a-TubeTM S pombe, log phase, BIO 101, Inc., Vista, CA, USA) using the primers, 5¢-GTTTTTGG ATTGTCAGTTCG-3¢ (sense) and 5¢-CATGAATAAGG CAGAAGAGT-3¢ (antisense) PCR was performed using high-fidelity UlTmaTM DNA polymerase (PerkinElmer Co., Foster City, LA, USA) The PCR product was subcloned into the EcoRV site of pGEMÒ-5zf(+) vector (Promega, Madison, WI, USA) The obtained cDNA was sequenced and used as a template for PCR in the following experiments Enzymes used for molecular biological manipulation were obtained from New England Biolabs, Inc (New England Biolabs, Bevery, MA, USA) All chemical reagents were obtained from Sigma–Aldrich Fine Chemicals (St Louis, MO, USA), unless otherwise indicated Plasmid construction All plasmids generated and used in this study are listed in Table Oligonucleotides used for PCR primers were as follows: antisense primer 5¢-GTGATTGATCATTTAT ATAAACGGTAT-3¢ (carries a BclI site); DN2-sense primer, 5¢-TCGCTAGGATCCCTTCAACACCACCTA 3¢; DN13–sense primer, 5¢-GGTTGTGGATCCCATCTA CCCCAGAGTCTCCT-3¢; DN16-sense primer, 5¢-CTCC ATGGATCCCAGAGTCTCCTGGGAGTT-3¢; DN41sense primer, 5¢-TAGATGGGATCCCTGAAAGCGAT TTACC-3¢ Each sense primer carries a BamHI site The underlined nucleotides contain the mutated sequence for generating restriction sites To generate a pGEX-rum1DN2 plasmid, rum1 cDNA fragments (nucleotides 124– Table Plasmid list pGEX-rum1-WT (DN2) pGEX-rum1-DN13 pGEX-rum1-DN16 pGEX-rum1-DN41 pGEX-rum1-DN74 pGEX-rum1-DC52 pGEX-rum1-DC81 pGEX-rum1-DC102 pGEX-rum1-DC130 pGEX-rum1-5A pGEX-rum1-13A pGEX-rum1-16A pGEX-rum1-19A pGEX-rum1-58A pGEX-rum1-13A16A pGEX-rum1-13A19A pGEX-rum1-16A19A pGEX-rum1-13A16A19A pGEX-rum1-13E19E pESP-rum1-WT (DN2) pESP-rum1-13E19E Ó FEBS 2002 MAP kinase negatively regulates Rum1 (Eur J Biochem 269) 3513 877) were amplified by PCR using the antisense primer and DN2-sense primer A 741-bp BamHI and blunt-ended BclI fragment of the cDNA that corresponding to amino acids 3–230 of p25rum1 was excised and subcloned into pGEX-3X bacterial expression vector (Pharmacia Biotech, Uppsala, Sweden) at BamHI and blunt-ended EcoRI sites The consequent pGEX-rum1-D2 vector was designated wild type vector in this study for convenience To generate rum1 mutants containing different N-terminal deletions, rum1 cDNA fragments were amplified by PCR using the antisense primer and eitherDN13-sense (nucleotides 157–877), DN16-sense (nucleotides 166–877), or DN41-sense primer (nucleotides 244–877) The BamHI and EcoRI fragments (493 bp, 484 bp, and 406 bp, respectively) were excised from the PCR products and were subcloned into a pGEX-rum1-WT vector at BamHI and EcoRI sites The consequent plasmids were designated pGEX-rum1-DN13, -DN16, and -DN41 corresponding to amino acids 14–230, 17–230, and 42–230 of p25rum1, respectively To generate a pGEX-rum1-DN74 vector, a 310-bp blunt-ended NdeII and EcoRI fragment was excised from the rum1 cDNA and subcloned into a pGEX-rum1-WT vector at blunt-ended BamHI and EcoRI sites Consequent plasmid is correspond to amino acids 75–230 of p25rum1 To generate rum1 mutants containing different C-terminal deletions, BamHI and either EcoRI, blunt-ended NarI, XmnI, or AluI fragments (526 bp, 445 bp, 383 bp, and 298 bp, respectively) were excised from the PCR products amplified with antisense and DN2-sense primers and were subcloned into a pGEX-3X vector at BamHI and (blunt-ended) EcoRI sites The consequent plasmids were designated pGEXrum1-DC52, -DC81, -DC102, and -DC130 corresponding to amino acids 3–178, 3–149, 3–128, and 3–100 of p25rum1, respectively All plasmids described above were sequenced after the construction The pGEX-rum1-WT plasmid was used to generate different rum1 mutants by site-directed mutagenesis using the TransformerTM Site-Directed Mutagenesis kit (Clontech Laboratories, Inc., Palo Alto, CA, USA) and the following oligonucleotides: 5A, 5¢-GGTCGTGGGATCCCTTCAG CACCACCTATGCGAGGG-3¢; 13A, 5¢-GCGAGGGTT GTGTGCTCCATCTACCCCAGAGTCTCCTGGG-3¢; 16A, 5¢-GGGTTGTGTACTCCATCTGCCCCAGAGTC TCCTGGG-3¢; 19A, 5¢-CTACCCCAGAGGCTCCTGG GAG-3¢; 58 A, 5¢-GCACATTTCCACCTGCACCTGCT AAAACTCCC-3¢; 13A19A, 5¢-GCGAGGGTTGTGTG CTCCATCTACCCCAGAGGCTCCTGGG-3¢; 13E19E, 5¢-CACCACCTATGCGAGGGTTGTGTGAGCCATC TACCCCAGAGGAGCCTGGGAGTTTTAAAG-3¢ The underlined nucleotides contain the mutated sequence All mutants were sequenced after the mutagenesis To generate a glutathione S-transferase (GST)-fusion protein expression vector for fission yeast, a BamHI and blunt-ended BclI fragment was excised from either pGEXrum1-WT or -E13E19 and subcloned into pESP-1 (Stratagene Cloning Systems, La Jolla, CA, USA) at BamHI and SmaI sites To obtain an in-frame sequence between GST and rum1 genes, subsequent plasmids were digested with BamHI and re-ligated after blunt-ending with klenow DNA polymerase The consequent plasmids were designated pESP-rum1-WT and -E13E19, respectively, and were sequenced after construction Generation and purification of GST fusion proteins of p25rum1 E coli strain BL21 (Riken Gene Bank, Ibaragi, Japan) was transformed with the resulting pGEX plasmids, cultured in Luria–Bertani broth with 50 mgỈL)1 of ampicillin at 25 °C, and induced for h with 0.1 mM isopropyl thio-bD-galactoside Subsequent purification of GST fusion proteins on glutathione–Sepharose was performed as described previously [34] The GST fusion proteins bound to glutathione–SepharoseÒ4B (Pharmacia) were eluted by 10 mM of reduced form of glutathione and subsequent protein concentration was measured by Bradford method using Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) Purified GST fusion proteins were separated on a 10% SDS/polyacrylamide gel and stained with Coomassie Blue In vitro kinase assay In this study, mainly two types of in vitro kinase assay were performed First, we examined whether MAPK phosphorylates p25rum1 by in vitro kinase assay GST-p25rum1 fusion proteins were bound on glutathione–SepharoseÒ4B After intensive washing with NaCl/Pi and kinase assay buffer (50 mM Tris/HCl, pH 7.5, 10 mM MgCl2, mM dithiothreitol, mM EGTA, 100 lM ATP), the precipitates were mixed with 100 ng of either purified sea star Pisaster ochraceus oocyte MAPK, p44mpk (Seikagaku Co., Tokyo, Japan) or recombinant murine Erk2 prepared from E coli (New England Biolabs) and incubated for 15 at room temperature in 30 lL of kinase assay buffer with 10 lCi of [c-32P]ATP (specific activity > 3000 CiỈmM)1; NEN Life Science Products, Inc., Boston, MA, USA) Subsequent specific activity of ATP in the assays was 90.8 c.p.m.Ỉpmol)1 Reactions were stopped by adding lL of 6· SDSsample loading buffer [350 mM Tris/HCl, pH 6.8, 600 mM dithiothreitol, 36% glycerol, 350 mM SDS, 0.012% (w/v) Bromophenol Blue] After separation on a 10% SDS/ PAGE gel, phosphorylated proteins were visualized with autoradiography as described previously [35] As positive and negative controls for MAPK activity, Histone H1 (5 lg per test) and GST were used as substrates, respectively Second, the inhibitory effect of p25rum1 on Cdc2 kinase activity was also tested by in vitro kinase assay The kinase activity of 25 ng of either Cdc2 complex purified from sea star P ochraceus oocytes (Upstate biotechnology, Lake Placid, NY, USA) or the recombinant human Cdc2/cyclin B complex (prepared from Spondoptera frugiperda sf9 cells using baculovirus system, New England Biolabs) was examined using histone H1 as a substrate in 30 lL of kinase assay buffer with 10 lCi of [c-32P] ATP, essentially as described above To test the effects on phosphorylation activity of Cdc2 kinase against Histone H1, GST-fusion proteins of wild-type and various mutants of p25rum1 purified on glutathione–SepharoseÒ4B were added to each kinase reaction mixture As a negative control not affecting Cdc2 kinase activity, GST was also tested To test whether phosphorylation of p25rum1 affects electrophoretic mobility and the activity as a Cdc2 kinase inhibitor of this protein, GST–p25rum1 proteins bound on glutathione–SepharoseÒ4B were nonisotopically phosphorylated by MAPK as described above with an exception Ó FEBS 2002 3514 K Matsuoka et al (Eur J Biochem 269) of the absence of [c-32P]ATP After intensive washing, nonisotopically prephosphorylated GST–p25rum1 were used for following SDS/PAGE and Cdc2 kinase assays Binding of GST–p25rum1 to the Cdc2–cyclin B complex Either untreated or prephosphorylated GST-p25rum1 proteins bound on glutathione–SepharoseÒ4B were incubated with 50 ng of Cdc2-cyclin B complex of human origin in 50 lL of NaCl/Pi for h at °C After intensive washing with NaCl/Pi, proteins bound on sepharose beads were separated by SDS/PAGE and were transferred to a nitrocellulose membrane Immunoblotting assay was performed as described previously [35] using monoclonal anti-Cdc2 Ig (Santa Cruz Biotechnology, Santa Cruz, CA, USA) Expression of GST–p25rum1 in S pombe The expression of GST–p25rum1 in S pombe was induced using an ESPTM Yeast Protein Expression System (Stratagene) according to the manufacturer’s protocol Briefly, p25rum1 expression vectors, either pESP-rum1-WT or -E13E19, were transformed into SP-Q01 S pombe cells using a YEASTMAKERTM Yeast Transformation System (Clontech Laboratories, Inc., Palo Alto, CA, USA) After being grown on Edinburgh minimal medium (EMM) agar supplemented with thiamine at 30 °C, positive clones were selected by PCR using specific primers for the pESP vector (Stratagene); sense, 5¢-GTACTTGAAATCCAGCAAGT ATATAGC-3¢; antisense, 5¢-CAAAATCGTAATATGCA GCTTGAATGGGCTTCC-3¢ The selected positive colony was grown in yeast extract plus supplements (YES) medium to D600 ¼ 1.0 at 30 °C After intensive washing in H2O, yeast cells were further cultured in EMM broth without thiamine at 30 °C for 24 h to induce the expression of GST fusion proteins of either the p25rum1-WT or the -E13E19 mutant To determine DNA contents, · 107 cells were harvested and washed with H2O After fixation in 70% ethanol, cells were stained with propidium iodide and analyzed by flow cytometry (EPICS XL, Beckman Coulter, Inc., Westbrook, MA, USA) To determine the expression of GST-fusion proteins, protein extracts were prepared using Y-PERTM Fig MAPK phosphorylates the N-terminal portion of p25rum1 (A) GST fusion proteins of wild-type (WT) (lane 3) and the DN74 deletion mutant, lacking 74 N-terminal amino acids (lane 2), of p25rum1 were produced in E coli After purification with glutathione– Sepharose, a 1.8-lg sample of each protein was separated by SDS/ PAGE in 10% acrylamide gel and visualized with Coomassie Bluestaining Purified GST proteins (GST, lane 4) were also examined The molecular mass standards are presented in lane (B) GST fusion proteins shown in (A) are schematically presented In the DN74 mutant, all of the putative Ser or Thr phosphorylation residues for MAPK, Thr5, Thr13, Thr16, Ser19, and Thr58, are deleted Shaded box represents GST (C) Using GST fusion proteins shown in (A) as substrates (lanes 2,3), an in vitro kinase assay was performed with MAPK purified from sea star oocytes as described in Materials and methods GST protein (lane 4) and Histone H1 (lane 5) were also examined as negative and positive control substrates, respectively In each kinase assay, the same amounts of GST and GST fusion proteins as presented in (A) were examined Ó FEBS 2002 MAP kinase negatively regulates Rum1 (Eur J Biochem 269) 3515 (Yeast Protein Extraction Reagent, PIERCE, Rockford, IL, USA) Fifty-microgram samples of each protein extract were applied for electrophoretic separation by SDS/PAGE, and Western immunoblotting was performed using rabbit polyclonal anti-GST antibody (Boeringer Manheim Biochemica, Manheim, Germany) as described previously [35] RESULTS In vitro phosphorylation of p25rum1 by MAPK As putative phosphorylation sites for MAPK were found in the amino acid sequence of p25rum1 [17], we first examined whether MAPK can phosphorylate p25rum1 in vitro by generating GST fusion proteins of p25rum1 When recombinant GST-fusion p25rum1-WT prepared from E coli (Fig 1A) was incubated with MAPK purified from sea star oocytes in the presence of 32P-labeled ATP, apparent incorporation of 32P into GST–p25rum1 was observed (Fig 1C) We also similarly used recombinant murine MAPK and obtained identical results Given that GST protein itself was not labeled with 32P under the same conditions (Fig 1C), it is obvious that MAPK phosphorylates p25rum1-WT in vitro Fig Inhibition of Cdc2 kinase activity by GST fusion proteins of p25rum1 (A) GST fusion proteins of various C-terminal deletion mutants of p25rum1, as indicated, were generated and examined by SDS/PAGE essentially as in Fig 1A (B) GST fusion proteins shown in (A) are schematically presented together with the DN74 mutant (C) Inhibitory effects of GST–p25rum1 fusion proteins on Cdc2 kinase activity were examined by in vitro kinase assay The transphosphorylation activity of Cdc2 kinase complex purified from sea star oocytes toward histone H1 was examined in the presence or absence (lane 8) of GST fusion proteins of wild-type (lane 2) and C- and N-terminal deletion mutants (lanes 1, 3–6) of p25rum1, as indicated In lane 7, GST protein was tested as a negative control In each assay, the same amounts of GST fusion proteins as presented in (A) were examined (D) Subsequent phosphorylation of histone H1 in each experiment was quantified by densitometry and expressed as a percentage of that treated with Cdc2 kinase alone (lane 8) The mean + SD numbers obtained from three independent experiments were presented The amino acid sequence data suggested that putative phosphorylation sites of p25rum1, Thr5, Thr13, Thr16, Ser19, and Thr58, are clustered in the N-terminal portion We then tested whether an actual phosphorylation site(s) by MAPK is present in the N-terminal portion of p25rum1 To test this, we generated a GST-fusion deletion mutant of p25rum1 lacking 74 amino acids from the N-terminus (DN74, Fig 1B) As shown in Fig 1C, in vitro kinase assay revealed that MAPK cannot translocate 32P into DN74 mutants, indicating that the MAPK phosphorylation site(s) is indeed located in the N-terminal portion of p25rum1 protein within amino acids 3–74 N-Terminal deletion does not influence the activity of p25rum1 as a Cdc2 kinase inhibitor The p25rum1 protein is known to inhibit the kinase activity of a complex of Cdc2 and B type cyclin and thereby regulate the cell cycle of fission yeast Consistently, GST fusion p25rum1-WT effectively inhibited the phosphorylation of histone H1 mediated by a Cdc2 kinase complex purified from sea star oocytes in vitro (Fig 2, lane 2) and 32P incorporation into histone H1 was reduced to less than 30% of that in the absence of p25rum1-WT, as assessed by 3516 K Matsuoka et al (Eur J Biochem 269) Ó FEBS 2002 densitometry We also similarly used recombinant human Cdc2–cyclin B complex and obtained identical results When GST–p25rum1-DN74 protein was similarly examined, inhibition of Cdc2 kinase activity comparable to that of p25rum1-WT was observed (Fig 2C,D, compare lanes and 2) In contrast, a C-terminal deletion induced apparent loss of the activity of p25rum1 as a Cdc2 kinase inhibitor As shown in Fig 2C,D, functional reduction of p25rum1 depends on the length of C-terminal deletion and longer deletions yielded greater recovery of the phosphorylation of histone H1 mediated by Cdc2 kinase in an in vitro kinase assay These data indicate that the catalytic domain of p25rum1 is located in its C-terminal portion and deletion of 74 N-terminal amino acids does not affect the activity of p25rum1 as an inhibitor of Cdc2 kinase Effect of N-terminal phosphorylation on p25rum1 function Although an N-terminal deletion does not directly affect the Cdc2 kinase inhibitor function of p25rum1, it is possible that the N-terminal portion contributes to functional regulation of this protein upon phosphorylation Therefore, we next examined whether N-terminal phosphorylation of p25rum1 mediated by MAPK affects the activity of this protein as a Cdc2 kinase inhibitor We thus prepared the phosphorylated GST–p25rum1-WT by combining MAPK and nonisotopic ATP In contrast to the untreated case (Fig 3A, lane 1), the same amount of prephosphorylated p25rum1-WT failed to inhibit the Cdc2 kinase activity (Fig 3A, lane 2) and incorporation of 32P into histone H1 comparable to that achieved in the absence of p25rum1-WT (Fig 3A, lane 4) was observed As prephosphorylated GST–p25rum1 itself did not show apparent transphosphorylation activity towards histone H1 (Fig 3A, lane 3), recovery of the 32P translocation into histone H1 being mediated by MAPK, possibly contaminating in the process of p25rum1 prephosphorylation, could be ruled out In contrast, pretreatment with a combination of MAPK and nonisotopic ATP did not affect the activity of GST–p25rum1-DN74 proteins as a Cdc2 kinase inhibitor (Fig 3B) Therefore, the data indicate that N-terminal phosphorylation of p25rum1 indeed reduces the Cdc2 kinase inhibitor activity of this protein Determination of the MAPK-mediated phosphorylation sites in p25rum1 In the experiment presented in Fig 1,  60 pmol phosphate were incorporated in 35 pmols p25rum1, suggesting the presence of two phosphorylation sites for MAPK in the molecule Next we determined which Ser and Thr residues in p25rum1 are responsible for MAPK-mediated phosphorylation To test this, we first generated various N-terminal deletion mutants of p25rum1 as indicated in Table 1, and examined 32P incorporation into these mutants mediated by MAPK The DN41 mutant showed almost no 32P incorporation, whereas the DN16 mutant showed an  50% reduction of phosphorylation (data not shown), suggesting that Ser19 is a MAPK phosphorylation site In contrast, the DN13 mutant showed almost equivalent 32P incorporation in comparison with that of DN16 (data not shown), suggesting that another MAPK phosphorylation site is Thr5 or Thr13 We further determined the MAPK-medi- Fig Functional modulation of p25rum1 by MAPK-mediated phosphorylation (A) The same amounts of GST–p25rum1-WT proteins were nonisotopically phosphorylated (P, lanes and 3) or not phosphorylated (lane 1) by MAPK prior to the assay Cdc2 kinase activity against histone H1 was examined by in vitro kinase assay in the presence or absence (lane 4) of either untreated (lane 1) or prephosphorylated (lane 2) GST–p25rum1proteins and consequent inhibition of Cdc2 kinase activity was compared In lane 3, the background phosphorylation level in the absence of Cdc2 kinase of prephosphorylated GST-p25rum1 added to lane was examined (B) GST fusion proteins of the DN74 mutant were similarly examined as in (A) ated phosphorylation sites in p25rum1 by generating aminoacid point mutants of wild-type in which each Ser and Thr residue was replaced with Ala Consistent with the data obtained using the N-terminal deletion mutants, A16 or A58 mutants in which Thr16 or Thr58 were replaced with Ala showed no reduction in 32P incorporation after kinase assay with MAPK (data not shown) In addition, there was no significant reduction in 32P incorporation mediated by MAPK with the A5 mutant in which Thr5 were replaced with Ala as compared with that of wild-type (data not shown) Therefore, it was suggested that Thr5, Thr16, and Thr58 are not responsible for MAPK-mediated phosphorylation In contrast, the A13 and A19 mutants in which Thr13 and Ser19 were replaced with Ala, respectively, both showed an  50% reduction of 32P incorporation as compared with that of wild-type (Fig 4A,B), indicating that these two amino-acid residues are major phosphorylation sites in p25rum1 mediated by MAPK To confirm this, we generated an A13A19 mutant (Fig 4C) in which both Thr13 and Ser19 were replaced with Ala (Fig 4D), and found that this mutant showed only a faint incorporation of 32 P mediated by MAPK (Fig 4E) Based on these data, Thr13 and Ser19 were assumed to be major MAPKmediated phosphorylation sites in p25rum1 Ó FEBS 2002 MAP kinase negatively regulates Rum1 (Eur J Biochem 269) 3517 Fig Phosphorylation of wild-type and A13A19 mutant of p25rum1 by MAPK (A) GST fusion proteins of wild-type (WT) (lanes and 3), the A13 deletion mutant (lane 2), and the A19 mutant (lane 4) of p25rum1 were examined as in Fig 1A,C (upper panels and lower panels, respectively) (B) Subsequent phosphorylation of GST fusion proteins in each experiment was quantified and expressed as a percentage of wild-type proteins as in Fig 2D (C) GST fusion proteins of wild-type (lane 1) and the A13A19 mutant (lane 2) of p25rum1 were generated and examined by SDS/PAGE, essentially as in Fig 1A (D) The GST fusion proteins are schematically presented In the A13A19 mutant of p25rum1 (lane 2), both the Thr13 and the Ser19 residues of the WT were replaced with Ala (E) Phosphorylation activities of MAPK against GST-fusion proteins of either the wild-type (lane 1) or the A13A19 mutant of p25rum1 (lane 2) were examined as in Fig 1(C) Effect of Glu13/Glu19 mutation on the function of p25rum1 It has been well documented in some proteins that replacement of Ser and Thr residues with Glu mimics the conformational and functional changes of the proteins mediated by phosphorylation at these residues To test whether phosphorylation of Thr13/Ser19 does indeed lead to inactivation of p25rum1 function, we generated a GST– E13E19 mutant of p25rum1 in which both Thr13 and Ser19 are replaced with Glu When the GST–E13E19 mutant was examined by SDS/PAGE, it exhibited retardation of electrophoretic mobility as compared with that of GST– p25rum1-WT (Fig 5A) It was reported that phosphorylation of protein leads to an electrophoretic mobility change in many cases [35] Indeed, GST–p25rum1-WT prephosphorylated by MAPK had a mobility shift on SDS/PAGE gel similar to that observed for the GST–E13E19 mutant (Fig 5A) In contrast, pretreatment with a combination of MAPK and nonisotopic ATP did not affect the electrophoretic mobility of GST–p25rum1-D74 that lacks N-terminal portion (Fig 5C) Considering the above, it is most likely that replacement of Thr13 and Ser19 with Glu mimics the change in electrophoretic mobility of p25rum1 mediated by phosphorylation at Thr13/Ser19 Next, we examined whether Glu13/Glu19 mutation alters the function of p25rum1 as a Cdc2 kinase inhibitor using an in vitro kinase assay In comparison with the significant inhibition achieved by wild-type, the E13E19 mutant showed only a limited inhibition of the 32P translocation in histone H1 mediated by Cdc2 kinase (Fig 5D) As shown in Fig 5D, wild-type was sufficient to inhibit Cdc2 kinase activity even at lower concentration, whereas E13E19 mutant presented a weak inhibition of Cdc2 kinase activity only at higher concentration, indicating that replacement of Thr13/Ser19 with Glu inactivates the function of p25rum1 The data strongly suggest that phosphorylation of Thr13/ Ser19 blocks the activity of p25rum1 as a Cdc2 inhibitor As it was reported previously, p25rum1 interacts with the complex of Cdc2 and B type cyclin and thus inhibits the Cdc2 kinase activity Therefore we tested whether MAPKmediated phosphorylation of p25rum1 prevents this interaction with Cdc2 kinase complex and hence abolishes the activity of p25rum1 as a Cdc2 kinase inhibitor As shown in Fig 5E, untreated GST–p25rum1 protein could bind with Cdc2–cyclin B complex However, when phosphorylated Ó FEBS 2002 3518 K Matsuoka et al (Eur J Biochem 269) Fig Effect of E13E19 mutant of p25rum1 on Cdc2 kinase activity (A) GST fusion proteins of wild-type (WT) (lane 1) and the E13E19 mutant (lane 3) of p25rum1 were generated and examined by SDS/PAGE, essentially as in Fig 1A Simultaneously, WT nonisotopically prephosphorylated by MAPK was also examined (lane 2) (B) The GST fusion proteins are schematically presented In lane 2, wild-type was prephosphorylated at both Thr13 and Ser19 by MAPK In the E13E19 mutant (lane 3), both Thr13 and Ser19 were replaced with Glu (C) Both untreated (lane 1) and prephosphorylated (lane 2) GST fusion proteins of the DN74 mutant were examined as in (A) (D) Cdc2 kinase activity against Histone H1 was examined by in vitro kinase assay in the presence or absence (lane 7) of different concentrations (lanes and 4, 0.45 lg; lanes and 5, 0.9 lg; lanes and 6, 1.8 lg) of wild-type (lanes 1–3) and the E13E19 mutant (lanes 4–6) proteins (E) Either untreated (lane 4) or prephosphorylated (lane 5) GST–p25rum1 immobilized on the glutathion–Sepharose beads were incubated with Cdc2-cyclin B complex The Cdc2 proteins bound to GST p25rum1 were detected by immunoblotting as described in Materials and methods As negative controls for coprecipitation, GST proteins were similarly examined (lanes and 3) As a positive control for immunoblotting, Cdc2–cyclin B complex was applied in lane (CNT) GST–p25rum1 was similarly tested, no reduction of the binding between p25rum1 and Cdc2 was observed (Fig 5E) We also tested the E13E19 mutant and observed that its binding to Cdc2–cyclin B complex is comparable to that of wild-type (data not shown) All of the above data were obtained from in vitro studies Therefore, we next examined whether the Glu13/Glu19 mutation inactivated p25rum1 function in yeast cells It was reported that overexpression of p25rum1 induces massive over-replication in S pombe, thereby leading to elongation of the cells as well as an increase in DNA content [17] Consistent with the previous report, GST–p25rum1 induced a massive increase in DNA content and cell elongation upon expression in S pombe, as assessed by flow cytometry and microscopic observation, respectively (Fig 6A) The data indicate that p25rum1 retains its function in yeast cells even in the form of GST-fusion protein When the GST-E13E19 mutant was expressed in S pombe, however, no significant change in either DNA content or cell size was observed (Fig 6A) As immunoblot analysis revealed a level of protein expression of the GST–E13E19 mutant comparable to that of GST–p25rum1, it is suggested that the GST– E13E19 mutant did not function as a Cdc2 kinase inhibitor in yeast cells DISCUSSION Our data clearly indicate that p25rum1 is a potent substrate for MAPK In vitro experiments also revealed that MAPK mediated phosphorylation negatively regulates the activity of p25rum1 as a Cdc2 inhibitor Direct in vitro kinase assay using GST fusion proteins of wild type as well as various mutants of p25rum1 demonstrated that residues Thr13/Ser19 are major phosphorylation sites for MAPK Since the weak but visible phosphorylation of A13A19 mutant by MAPK was observed (Fig 4E), the other Ser or Thr residue(s) might also be involved in the phosphorylation by MAPK However, Glu13/Glu19 mutation (E13E19 mutant), which mimics the phosphorylated state of Thr13/ Ser19, significantly abolishes p25rum1 function in in vitro, it is suggested that residues Thr13/Ser19 are essential for phosphorylation-mediated inactivation of the protein by MAPK Given that E13E19 mutant also abolishes p25rum1 function in yeast cells, it is most likely that the Thr13/Ser19 phosphorylation negatively regulates p25rum1 activity even in vivo We also found that C-terminal deletion inhibits the p25rum1 activity of Cdc2 kinase inhibitor, whereas N-terminal deletion, including Thr13/Ser19, does not Therefore, we speculate that the C-terminal portion of Ó FEBS 2002 MAP kinase negatively regulates Rum1 (Eur J Biochem 269) 3519 Fig Effect of E13E19 mutant of p25rum1 on mitosis of S pombe (A) The pESP expression vectors of GST fusion proteins of either the wild-type (WT) (mid panels) or the E13E19 mutant (lower panels) of p25rum1, as well as vector only (upper panels) were introduced into S pombe as described in Materials and methods After a 24-h cultivation, the cells were stained with PI and nuclear DNA contents were examined by flow cytometry (left panels) At the same time, the morphology of S pombe in each transformation was also examined by light microscopy (right panels) (B) Cell extracts were prepared from S pombes which had been transformed with wild-type (lane 1) and E13E19 mutant (lane 2) expression vectors Fifty micrograms of each lysate were examined by immunoblot analysis using anti-GST Ig p25rum1 is a catalytic domain of the protein while the N-terminal portion is a regulatory domain mediating a reduction in activity upon phosphorylation of Thr13/Ser19 by MAPK At this moment, the precise mechanism of negative regulation of the function of p25rum1 that induced by Thr13/ Ser19 phosphorylation is unclear As p25rum1 interacts with the complex of Cdc2 and B type cyclin and thus inhibits the Cdc2 kinase activity, it is possible that phosphorylation of p25rum1 at Thr13/Ser19 prevent its interaction with the Cdc2-B type cyclin complex However, our data in the present study indicated that MAPK-mediated phosphorylation does not affect interaction between p25rum1 and Cdc2–cyclin B complex Further experiments to clarify the mechanism of phosphorylation-mediated functional regulation of p25rum1 are now underway The CKI p25rum1 plays an important role in the regulation of G1 phase in the fission yeast cell cycle [17– 24] By preventing Cdc2 kinase activity, p25rum1 has two essential roles First, it determines the length of the pre-Start G1 period Secondly, it prevents mitosis from occurring in early G1 cells [17] As the regulation of CDK activity must be accurate, the function of p25rum1 must also be regulated tightly in the cell-cycle process The regulatory mechanism of p25rum1 function has been well characterized by CorreaBordes et al and Benito et al [20,24] According to their observations, p25rum1 protein levels are regulated sharply and periodically during the cell cycle and can hence contribute to appropriate control of the cell cycle The p25rum1 begins to accumulate in anaphase, persisting in G1 and disappearing during S phase As p25rum1 is stabilized and polyubiquitinated in a mutant with a defective 26S proteosome, it is suggested that degradation normally occurs via the ubiquitin-dependent 26S proteosome pathway Interestingly, these authors also observed that p25rum1 is phosphorylated by the Cdc2–B type cyclin complex at residues Thr58 and Thr62, the distinct Thr residues from MAPK-mediated phosphorylation residues which we identified Among three Cdc2-cyclin complexes in fission yeast, p25rum1 inhibits the activities of Cdc2–Cdc13 and Cdc2– Cig2 complexes but not the Cdc2–Cig1 complex [19,24] In contrast, Cdc2–Cig1, but neither Cdc2–Cdc13 nor Cdc2– Cig2, is a Cdc2 kinase responsible for the phosphorylation of p25rum1 [20] The mutation of one or both Thr58/Thr62 residues to Ala stabilizes p25rum1 protein and induces persistent expression of the protein, resulting in a cell-cycle delay in G1 and polyploidization due to occasional re-initiation of DNA replication before mitosis Therefore, this phosphorylation might be the signal that targets p25rum1 for degradation by ubiquitination Based on their observations, earlier investigators concluded that periodic accumulation and phosphorylation-initiated ubiquitinationation of p25rum1 in G1 phase play a role in setting a threshold of cyclin levels important in determining the length of the pre-Start G1 phase and in ensuring the correct order of cell-cycle events [20] In contrast to their observation, we found MAPKphosphorylated p25rum1 to show reduced activity as a Cdc2 kinase inhibitor in vitro, indicating that negative regulation of the p25rum1 function mediated by Thr13/Ser19 phosphorylation is independent of ubiquitination, rather being induced by some conformational change of the protein Considering all of the above evidence together, we speculate that p25rum1 has, at a minimum, two distinct and independent mechanisms of functional regulation, both of which are mediated by phosphorylation The Ó FEBS 2002 3520 K Matsuoka et al (Eur J Biochem 269) function of p25rum1 in the cell cycle may be regulated mainly by ubiquitination-based control of protein levels initiated by Cdc2–Cig1-mediated Thr58/Thr62 phosphorylation However, Thr13/Ser19 phosphorylation by MAPK should be an alternate or additional mechanism that directly reduces p25rum1 activity We have demonstrated the possibility that MAPK negatively regulates p25rum1 via phosphorylation of its Ser and Thr residues, though it is still unclear whether this regulatory mechanism is indeed utilized in vivo In the case of S pombe, cells with deletion of sty1/spc1, a gene encoding S pombe MAPK, are highly elongated as a consequence of a delay in the timing of mitotic initiation, which is exacerbated in response to stresses such as high osmolarity and nutritional limitation [32] Although such mutants still undergo cell-cycle arrest in response to stress, they are unable to resume proliferation and die [32,33] These lines of evidence suggest that the Sty1/Spc1 MAPK pathway is required for recovery from stress-induced cellcycle arrest in S pombe Activation of Sty1/Spc1 induces nuclear translocation and subsequent phosphorylation of the bZIP transcription factor Atf1, a homologue of ATF2 that is targeted by the mammalian SAPKs, whereas it is currently unclear exactly how Sty1/Spc1 influences basal cell-cycle machinery However, several possibilities have been raised [31] One possibility is that Sty1/Spc1 promotes the expression of B-type cyclin Cdc13 Alternatively, Sty1/Spc1 may be required for the assembly or stability of mitotic cyclin–CDK complexes or other cellcycle components In relation to the second possibility, it is plausible that Sty1/Spc1 phosphorylates p25rum1, inhibiting its activity as a negative regulator of Cdc2 kinase and thereby stabilizes cyclin–CDK complexes Interestingly, it was observed in mammalian cells that MAPK ERK is able to phosphorylate p27KIP1, a member of the p21CIP1/WAF1 CKI family, in vitro, preventing the CKI from interacting with and inhibiting CDK2, a mammalian G1–S CDK [36] Moreover, expression of a dominant negative Ras mutant or pharmacological inhibition of MEK, both of which lead to inhibition of the MAPK cascade, results in G1/S arrest, suggesting that ERK modulates the timing of Start in vivo [37] The above evidence supports our hypothesis In conclusion, our data indicate that amino-acid residues Thr13/Ser19 are responsible for phosphorylation mediated by MAPK and that these residues negatively affect the activity of p25rum1 as a Cdc2 kinase inhibitor Although further studies are clearly necessary, investigation of MAPK-mediated regulation of p25rum1 may provide new insights into the biochemical basis of cell-cycle control ACKNOWLEDGEMENTS We thank M Sone and S Yamauchi for their excellent secretarial work This work was supported in part by a Grant for Pediatric Research (12C-01) from the Ministry of Health and Welfare of Japan This work was also supported by a grant from the Japan Health Sciences Foundation for Research on 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(1997) Mitosis-specific negative regulation of epidermal growth factor receptor, triggered by a decrease in ligand binding and dimerization, can be overcome by overexpression of receptor J Biol Chem 272, 18656–18665 Kawada, M., Yamagoe, S., Murakami, Y., Suzuki, K., Mizuno, S & Uehara, Y (1997) Induction of p27Kip1 degradation and anchorage independence by Ras through the MAP kinase signaling pathway Oncogene 15, 629–637 Takuwa, N & Takuwa, Y (1997) Ras activity late in G1 phase required for p27kip1 downregulation, passage through the restriction point, and entry into S phase in growth factorstimulated NIH 3T3 fibroblasts Mol Cell Biol 17, 5348–5358  ... function of p25rum1 in the cell cycle may be regulated mainly by ubiquitination-based control of protein levels initiated by Cdc2–Cig1-mediated Thr5 8 /Thr6 2 phosphorylation However, Thr1 3 /Ser1 9 phosphorylation. .. domain of the protein while the N-terminal portion is a regulatory domain mediating a reduction in activity upon phosphorylation of Thr1 3 /Ser1 9 by MAPK At this moment, the precise mechanism of. .. GST fusion proteins of the DN74 mutant were similarly examined as in (A) ated phosphorylation sites in p25rum1 by generating aminoacid point mutants of wild-type in which each Ser and Thr residue