A Novel Functional Domain of Cdc15 Kinase Is Required for Its Interaction With Tem1 GTPase in Saccharomyces cerevisiae

Corresponding author: Akio Toh-e, Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan., toh-e{at}biol.s.u-tokyo.ac.jp (E-mail)

Communicating editor: F. WINSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Exit from mitosis requires the inactivation of cyclin-dependent kinase (CDK) activity. In the budding yeast Saccharomyces cerevisiae, a number of gene products have been identified as components of the signal transduction network regulating inactivation of CDK (called the MEN, for the mitotic exit network). Cdc15, one of such components of the MEN, is an essential protein kinase. By the two-hybrid screening, we identified Cdc15 as a binding protein of Tem1 GTPase, another essential regulator of the MEN. Coprecipitation experiments revealed that Tem1 binds to Cdc15 in vivo. By deletion analysis, we found that the Tem1-binding domain resides near the conserved kinase domain of Cdc15. The cdc15-LF mutation, which was introduced into the Tem1-binding domain, reduced the interaction with Cdc15 and Tem1 and caused temperature-sensitive growth.The kinase activity of Cdc15 was not so much affected by the cdc15-LF mutation. However, Cdc15-LF failed to localize to the SPB at the restrictive temperature. Our data show that the interaction with Tem1 is important for the function of Cdc15 and that Cdc15 and Tem1 function in a complex to direct the exit from mitosis.

EUKARYOTIC cell cycle is governed by the oscillation of cyclin-dependent kinase (CDK) activity. The cell cycle progression from anaphase to G1 phase, when two new cells are generated by division, is one of the most important steps for the faithful inheritance of genetic information. While entry into mitosis is initiated by activation of the mitotic cyclin/CDK complex, exit from mitosis is promoted by inactivation of it. One way for CDK to be inactivated at mitotic exit is the destruction of mitotic cyclins. It depends on a multisubunit ubiquitin ligase called the anaphase promoting complex (APC or cyclosome), which catalyzes ubiquitination of cyclins (KING et al. 1995 ; SUDAKIN et al. 1995 ; ZACHARIAE et al. 1996 ). The resulting multiubiquitinated cyclins are recognized and degraded by the 26S proteasome. An alternative way for CDK inactivation is the attachment of the CDK inhibitor Sic1 (MENDENHALL 1993 ; SCHWOB et al. 1994 ), whose expression dramatically increases at late anaphase and persists until late G1 phase.

In budding yeast, a group of genes essential for the exit from mitosis were isolated by various genetic screens. Such genes including CDC5, CDC14, CDC15, DBF2, LTE1, MOB1, and TEM1 are components of the mitotic exit network (MEN) regulating mitotic cyclin/CDK inactivation (JOHNSTON et al. 1990 ; SCHWEITZER and PHILIPPSEN 1991 ; KITADA et al. 1993 ; SHIRAYAMA et al. 1994A , SHIRAYAMA et al. 1994B ; LUCA and WINEY 1998 ; MORGAN 1999 ). Cdc5, a member of the conserved family of Polo-kinase, is an APC regulator for mitotic cyclin destruction (CHARLES et al. 1998 ; SHIRAYAMA et al. 1998 ), consistent with the report that its mammalian homologue Plk directly phosphorylates and activates APC (KOTANI et al. 1998 ). Dbf2 functions in mitotic exit with its binding partner Mob1 (KOMARNITSKY et al. 1998 ). Cdc14 functions as a key regulator for the exit from mitosis. Cdh1, a specificity factor of APC^Cdh1, is dephosphorylated by Cdc14 at late anaphase and activates APC^Cdh1, thereby promoting the ubiquitination of mitotic cyclins (SCHWAB et al. 1997 ; VISINTIN et al. 1997 ; ZACHARIAE et al. 1998 ; JASPERSEN et al. 1999 ). The expression of the CDK inhibitor Sic1 is stimulated by the transcription factor Swi5 (KNAPP et al. 1996 ; TOYN et al. 1997 ), which is allowed to enter the nucleus by dephosphorylation of phosphorylated Swi5 by Cdc14 (VISINTIN et al. 1998 ). Cdc14 also dephosphorylates phosphorylated Sic1 to protect it from degradation (VISINTIN et al. 1998 ), while the phosphorylation of Sic1 by CDK targets Sic1 for ubiquitin-dependent degradation (FELDMAN et al. 1997 ; VERMA et al. 1997 ; NISHIZAWA et al. 1998 ).

Recent studies about the MEN components pointed out the importance of their subcelullar localization in their function in mitotic exit. During G1 phase through metaphase/anaphase transition, Cdc14 is localized and inhibited at the nucleolus by being anchored by Net1, a component of the nucleolar RENT complex (SHOU et al. 1999 ; STRAIGHT et al. 1999 ; VISINTIN et al. 1999 ). At late anaphase, Cdc14 is released from the nucleolus and dispersed to the entire nucleus and cytoplasm, where it encounters its targets, including Cdh1, Sic1, and Swi5. The release of Cdc14 from the nucleolus does not occur in the absence of the function of Cdc5, Cdc15, Dbf2, or Tem1 (SHOU et al. 1999 ; VISINTIN et al. 1999 ), suggesting that these factors function in the regulation of Cdc14 localization. Thus, the release of Cdc14 from the nucleolus is regarded as the trigger of mitotic exit. Either of Cdc5, Cdc15, Dbf2, or Tem1, at least its fraction, localized to the spindle pole body (SPB; SHIRAYAMA et al. 1998 ; CENAMOR et al. 1999 ; BARDIN et al. 2000 ; FRENZ et al. 2000 ; PEREIRA et al. 2000 ; SONG et al. 2000 ; XU et al. 2000 ), indicating that the SPB takes an important role for mitotic exit. However, the molecular mechanisms by which Cdc5, Cdc15, Dbf2, and Tem1 regulate Cdc14 localization remain elusive. The anaphase inhibitor Pds1 (COHEN-FIX et al. 1996 ; YAMAMOTO et al. 1996A , YAMAMOTO et al. 1996B ) and Esp1, which promotes sister chromatid separation (CIOSK et al. 1998 ; UHLMANN et al. 1999 ), also regulate the release of Cdc14 and CDK inactivation at mitotic exit (COHEN-FIX and KOSHLAND 1999 ; SHIRAYAMA et al. 1999 ; TINKER-KULBERG and MORGAN 1999 ).

These components of the MEN display various genetic interactions and consist of a complicated signal transduction network (summarized in JASPERSEN et al. 1998 ). To understand how a cell exits mitosis, it is very important to characterize the molecular interaction of these MEN components. CDC15 was isolated as the responsible gene of the extragenic suppressor of lte1, the wild type of which encodes a protein containing a highly conserved motif of the guanine nucleotide exchange factor (SHIRAYAMA et al. 1994B , SHIRAYAMA et al. 1996 ). TEM1 encodes a small GTPase originally isolated as a multicopy suppressor of lte1 (SHIRAYAMA et al. 1994A ). The overproduction of Cdc15 is able to suppress the lethality of the gene disruption of TEM1 (SHIRAYAMA et al. 1994A ), indicating that a cell is able to grow in the absence of Tem1 if the Cdc15 function is elevated. These results suggest that the Cdc15/Tem1 pathway control the exit from mitosis (SHIRAYAMA et al. 1994A ). Indeed, BARDIN et al. 2000 demonstrated that Tem1 and Cdc15 are in a complex during mitosis.

In this study, we isolated Cdc15 as a binding protein of Tem1 by two-hybrid screening and showed that Tem1 physically binds to Cdc15. We also determined the novel functional domain of Cdc15 that is required for the interaction with Tem1. Using various mutants of CDC15 or TEM1, we investgated the interaction between Cdc15 and Tem1. Our results indicate that Cdc15 and Tem1 function as a complex to direct the exit from mitosis.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Microbial manipulation:
The principal yeast strains used in this study are listed in Table 1. Strains derived from them were also used as described in the text. Yeast cells were grown either in rich medium (YPD) consisting of yeast extract (Difco, Detroit), peptone (Nihon Seiyaku, Tokyo), and glucose or in synthetic glucose medium (SC), which is SD containing appropriate auxotrophic supplements (SHERMAN et al. 1986 ). For synthetic galactose medium, 5% galactose and 0.2% sucrose were replaced with glucose in SC. 5-Fluoroorotic acid (5-FOA, 0.1% w/v) was added to appropriate medium for the plasmid shuffling assay. Yeast transformations were performed by the method of ITO et al. 1983 , and other standard yeast genetic manipulations were performed as described by SHERMAN et al. 1986 . The Escherichia coli strain used is DH5 [supE44 lacU169 (80lacZ m15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1].

TEM1 plasmids:
Site-directed mutagenesis of TEM1 was carried out as follows. The BamHI-SalI fragment containing the open reading frame (ORF) of TEM1 was amplified by PCR using a pair of synthetic oligonucleotides TEM1#3 (5'-CCGGATCCATGGCTACACCAAGCAC-3') and TEM1#4 (5'-TGCGGTCGACTCATGTATTAACGCCCGG-3') and cloned between the BamHI site and the SalI site of pBluescript KS⁺ (Stratagene, La Jolla, CA) generating pKZ126. To construct the tem1T34A mutant gene, the 34th codon (ACA for Thr) of TEM1 was replaced with GCA (for Ala) by PCR-aided site-directed mutagenesis by using TEM1#3, TEM1#4, and a pair of complementary oligonucleotides as mutagenic primers (5'-GTAGGGAAAGCATCGCTG-3' and 5'-CAGCGATGCTTTCCCTAC-3'). pKZ126 was used as a template. The tem1T52A mutant gene (in which the 52nd codon ACG for Thr was changed to GCG for Ala) was constructed in the same way as the tem1T34A mutagenesis using a pair of oligonucleotides (5'-AGGAATACACACAGGCGCTGGGAGTGAACT-3' and 5'-AGTTCACTCCCAGCGCCTGTGTGTATTCCT-3') as mutagenic primers. To generate the expression plasmids of tem1 mutant genes, the BamHI site and the SalI site were introduced just in front of the start codon and right after the stop codon of TEM1, respectively, in pKZ134 containing the SacI^-293-PvuII⁺¹⁵²⁵ fragment (for the sequence coordinate, see below) of TEM1 in YCplac111 (GIETZ and SUGINO 1988 ). The ORF of wild-type TEM1 was replaced by either the BamHI-SalI fragment of tem1T34A or that of tem1T52A generating pKZ154 or pKZ141, respectively. Plasmids for the expression of tagged Tem1 or its derivatives were constructed as follows. The fragment containing the promoter region (300 bp) and the ORF of TEM1 or mutant tem1 was introduced into pTS903CL, pTS902CL, pTS905CL, or pTS910CU carrying the fragment encoding two hemagglutinin (HA) epitopes and six histidines tandemly (pTS903CL), two HA epitopes (pTS902CL), nine myc epitopes and six histidines tandemly (pTS905CL), or a green fluorescent protein (GFP; pTS910CU) followed by the 300-bp TDH3 terminator. The expression plasmid of TEM1-HA2, TEM1-HA2His6, tem1T34A-HA2His6, or tem1T52A-HA2His6 was designated as pKZ178, pKZ173, pKZ181, or pKZ180, respectively. The expression plasmid of TEM1-myc9His6 was designated as pKZ174. The expression plasmid of TEM1-GFP, tem1T34A-GFP, tem1T52A-GFP, or tem1-3-GFP was designated as pTGCU1, pT34G, pT52G, or pT135G, respectively. For two-hybrid assays, the BamHI-SalI fragment of the ORF of wild-type TEM1, tem1T34A, tem1T52A, or tem1-3 was introduced between the BamHI site and the SalI site of pGBDUC1 (JAMES et al. 1996 ) generating pKZ104, pKZ158, pKZ142, or pKZ131, respectively. The DNA sequence of each mutant tem1 gene (and of each mutant cdc15 gene, see below) was determined by the method of SANGER et al. 1977 using Thermo Sequenase dye terminator cycle sequencing premix kit (Amersham Life Science, Inc., Cleveland) and the ABI 373A DNA sequencer (Applied Biosystems, Foster City, CA). Number +1 indicates the position of the adenine residue of the start codon.

CDC15 plasmids:
Site-directed mutagenesis of CDC15 was carried out as follows. The PvuII^-410-PvuII⁺³⁶³⁷ fragment containing the CDC15 gene was cloned into PvuII-digested YCp lac111 generating pKZ412. To construct the cdc15-DD gene, the 298th codon (GAU for Asp) and the 300th codon (GAU for Asp) of CDC15 were replaced with GGU (for Gly) and GGC (for Gly), respectively, by PCR-aided site-directed mutagenesis using CDC15-77as (5'-GATCTGCCTCACAAAAGG-3'), CDC 15#4 (5'-TCCCCCGGGGTTTGAAATGTTGTAATGG-3'), and a pair of complementary oligonucleotides as mutagenic primers (5'-TATCATTGGGGTGCCGGCTTTCAAGAAG-3' and 5'-CTTCTTGAAAGCCGGCACCCCAATGATA-3'). pKZ412 was used as a template. For creating the cdc15-S309A gene, in which the 309th codon UCA for Ser was changed to GCA for Ala, a pair of complementary oligonucleotides (5'-CTAAATATAGCACCCTCTAAA-3' and 5'-TTTAGAGGGTGCTATATTTAG-3') was used as mutagenic primers. For creating the cdc15-LF gene, in which the 357th codon CUU for Leu and the 358th codon UUC for Phe were changed to CGU for Arg and CGC for Arg, respectively, the pair of complementary oligonucleotides (5'-CTTGCATGTGCGTCGCAGTGTTTGC-3' and 5'-GCAAACACTGCGACGCACATGCAAG-3') was used as mutagenic primers. For creating the cdc15-lyt1 gene (the 410th codon GGA for Gly was changed to GAA for Glu; JIMENEZ et al. 1998 ), a pair of complementary oligonucleotides (5'-TATGGGAGAAATTCCACTG-3' and 5'-CTGTGGAATTTCTCCCATA-3') was used as mutagenic primers. The SalI⁺⁸³⁸-XhoI⁺¹⁰⁸⁴ fragment of the CDC15 gene on pKZ412 was replaced with the corresponding SalI-XhoI fragment containing each of the mutations (cdc15-DD, cdc15-S309A, or cdc15-LF) generating pKZ434, pKZ436, or pFM420, respectively. The SalI⁺⁸³⁸-NcoI⁺¹⁵⁰⁴ fragment of the CDC15 gene on pKZ412 was replaced with the corresponding SalI-NcoI fragment containing the cdc15-lyt1 mutation generating pFM414. The cdc15d82SX was constructed by deleting the SalI⁺⁸³⁸-XhoI⁺¹⁰⁸⁴ fragment from CDC15 and religating the cohesive ends generated by the SalI and the XhoI digestion. The expression plasmid carrying CDC15-HA5 or CDC15-GFP was constructed as follows. The fragment containing the promoter region (400 bp) and the ORF of CDC15 were introduced into pTS901CU or pTS910EU, carrying the fragment encoding five HA epitopes or a GFP followed by the 300-bp TDH3 terminator, generating pKZ416 or pCFG1, respectively. The fragment encoding Cdc15-HA5 was introduced into multicopy plasmid YEplac195 (GIETZ and SUGINO 1988 ) or centromeric plasmid YCplac22 (GIETZ and SUGINO 1988 ) generating pKZ425 or pKZ438, respectively. The expression plasmids of mutant Cdc15 proteins were constructed by replacing the BglII^-95-BamHI⁺²⁵⁹² fragment of CDC15 on pKZ425, pKZ438, or pCFG1 with the corresponding BglII-BamHI fragment containing each of the cdc15 mutations. For the disruption of CDC15, the PvuII^-410-PvuII⁺³⁶³⁷ fragment containing the CDC15 gene was cloned into the PvuII-digested pBluescript KS⁺ and the part of the CDC15 fragment (from the XhoI^-57 site to the EcoRI⁺²⁷¹⁵ site) was replaced by the HIS3 gene, generating pFM418. TS902CL, pTS903CL, pTS905CL, pTS910CU, and pTS910EU were gifts from T. Sasaki (Department of Biological Sciences, Graduate School of Science, University of Tokyo).

Disruption of CDC15:
The PvuII-PvuII fragment of pFM418 was introduced to YKZ0023 to obtain a His⁺ cell of which one of the CDC15 alleles was replaced by the HIS3 gene (YKZ0237). The gene disruption of CDC15 was confirmed by PCR. For the plasmid shuffling assay, we obtained His⁺ Ura⁺ progeny (YKZ0255) from tetrads derived from YKZ0237 harboring pKZ406, which is the centromeric plasmid carrying the CDC15 gene and the URA3 gene.

Protein extraction and immunoblotting:
Cells grown to mid-log phase in 5 ml of appropriate medium were washed with lysis buffer [100 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM EDTA, 5% glycerol, 0.5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 µg/ml antipain, 1 µg/ml aprotinin], resuspended in 30 µl of lysis buffer, and vortexed for 10 min at 4° with acid-washed glass beads ( = 0.5 mm). Lysis buffer (100 µl) was added and the extract was clarified by 10-min spins at 14,000 rpm (Sakuma M150-IV, Tokyo) at 4°. The concentration of protein in the extract was determined by BCA Protein Assay Reagent (Pierce, Rockford, IL) using bovine serum albumin (BSA) as a standard. For immunoblots, samples were boiled in SDS gel sample buffer (LAEMMLI 1970 ) and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electrophoretically transferred to nitrocellulose membrane followed by Western blotting using mouse monoclonal anti-HA antibody 16B12 (BAbCO, Richmond, CA) or mouse monoclonal anti-myc antibody 9E10 (Calbiochem, Cambridge, MA) as primary antibody. As secondary antibody, anti-mouse IgG goat antibody conjugated to alkaline phosphatase [anti-mouse IgG(H+L) AP conjugate, Promega, Madison, WI] or to horseradish peroxidase [anti-mouse IgG(H+L) HRP conjugate, Promega] was used. For detection of alkaline phosphatase, BCIP/NBT color substrate (Promega) was used. For detection of horseradish peroxidase, Western blot chemiluminescence reagent (NEN Life Science Products, Boston) was used.

ß-Galactosidase assay:
PJ69-4A cells expressing both GBD-fusion protein and GAD-fusion protein were grown in 3 ml of SC-(URA, LEU) at 25° to mid-log phase and harvested. Cells washed once with Z buffer (MILLER 1972 ) were suspended in 30 µl of Z buffer and vortexed with acid-washed glass beads for 10 min at 4°. Z buffer (100 µl) was added to the extract followed by 10-min spins at 14,000 rpm at 4°. Fifty microliters of extract was diluted to 1 ml with Z buffer. Then, 200 µl of 4 mg/ml o-nitrophenyl ß-D-galactopyranoside was added and samples were incubated at 30° until a pale yellow color developed. The reactions were stopped by adding 500 µl of 1 M Na₂CO₃. Units were calculated with the following equation: U = , where t = time of reaction (ADAMS et al. 1997 ). Amounts of protein in the extract were determined by a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) using BSA as a standard.

Coprecipitation experiments:
For His6-tagged Tem1 precipitation, cell extracts were prepared as described above. Protein (1 mg in 500 µl of lysis buffer) was incubated at 4° for 1 hr with 20 µl of 50% Ni-NTA beads (QIAGEN, Valencia, CA) that had been washed with wash buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 20 mM imidazole, 1 mM PMSF, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 µg/ml antipain, 1 µg/ml aprotinin). Beads were washed with 500 µl of wash buffer five times and then with 50 µl of elution buffer (wash buffer containing 100 mM imidazole) twice. Twenty microliters of extract before or after incubation with Ni-NTA beads and 20 µl of the eluate were boiled in SDS gel sample buffer and loaded on a 10% polyacrylamide gel. For Tem1myc9His6 precipitation, cell extracts were prepared as described above using lysis buffer containing 0.1% NP-40. Protein (1 mg in 500 µl of lysis buffer) was incubated with anti-myc antibody (1 µg of 9E10) at 4° for 1 hr followed by another 1-hr incubation with 60 µl of 50% (v/v) protein A-Sepharose (Amersham, Uppsala, Sweden) that had been swollen with lysis buffer. Protein A-Sepharose-associated immunoprecipitates were washed with lysis buffer, boiled in SDS gel sample buffer, and loaded on polyacrylamide gels. For detection of Tem1myc9His6 by Western blotting, rabbit polyclonal anti-myc antibody [c-MYC (A-14)-G, Santa Cruz Biotechnology, Inc.] was used as primary antibody and mouse monoclonal anti-rabbit IgG antibody conjugated to horseradish peroxidase (RG-16, Sigma, St. Louis) was used as secondary antibody. Preparation of cell extracts and immunoprecipitation of Cdc15-HA3 was performed as described in BARDIN et al. 2000 using 16B12 antibody. HA-tagged, myc-tagged, or GFP-tagged proteins were detected by Western blotting using 16B12, 9E10, or mouse anti-GFP monoclonal antibody (mixture of clones 7.1 and 13.1, Boehringer Mannheim, Indianapolis), respectively, as primary antibody and anti-mouse IgG goat antibody conjugated to horseradish peroxidase or anti-mouse IgG goat antibody conjugated to alkaline phosphatase as secondary antibody.

Kinase assay:
Cells (YKZ0024) expressing Cdc15-HA5 and its derivatives from multicopy plasmid were grown in 10 ml of SC-URA medium at 25° to mid-log phase and harvested. Cell extracts were prepared as described above except that LLB (50 mM HEPES-NaOH, pH 7.4, 75 mM KCl, 50 mM NaF, 50 mM ß-gylcerophosphate, 1 mM EGTA, 0.1% NP-40, 1 mM DTT, 1 mM PMSF, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 2 µg/ml aprotinin; JASPERSEN et al. 1998 ) was used instead of lysis buffer. A total of 300 µl of cell extract containing 750 µg protein was incubated at 4° for 2 hr with 3 µg of 16B12 and 60 µl 50% (v/v) protein A-Sepharose that had been swollen with LLB. The precipitants with protein A-Sepharose were divided into three equal parts, one for the kinase assay at 25°, one for the kinase assay at 37°, and one for the Western blotting. For estimation of the amount of HA-tagged Cdc15 proteins, anti-mouse IgG goat antibody conjugated to alkaline phosphatase was used as secondary antibody. AttoPhos substrate (Boehringer Mannheim, Mannheim, Germany) was used as a fluorogenic substrate of alkaline phosphatase. Fluorescence was detected by using Fluoro Image Analyzer (FLA-2000, Fuji, Tokyo). For the kinase assay, the immunoprecipitants were washed three times with 300 µl of LLB and once with 300 µl of kinase buffer (50 mM HEPES-NaOH, pH 7.4, 5 mM MgCl₂, 2.5 mM MnCl₂, 5 mM ß-gylcerophosphate, and 1 mM DTT) and incubated at either 25° or 37° for 10 min in 20 µl of kinase buffer containing 20 µM ATP, 2 µg myelin basic protein (MBP), and 5 µCi of [-³²P]ATP (3000 Ci/mmol). Reaction was stopped by boiling in SDS gel sample buffer and products were analyzed on a 12% polyacrylamide gel followed by autoradiography using Bio Imaging Analyzer (Fujix BAS1000, Fuji, Tokyo).

Microscopic analysis:
For detection of GFP fluorescence, cells expressing GFP-tagged proteins grown at 25° were harvested, washed three times with phosphate-buffered saline, and immediately subjected to microscopic analysis with an epifluorescence microscope Olympus IX70 (Olympus, Tokyo) with a lens UPlanApo100x (Olympus) and a CCD camera (SENSYS, Photometrics, Tucson, AZ). Cells grown at 37° were fixed with 3.7% formaldehyde for 10 min at 37°, washed three times with phosphate-buffered saline, and subjected to microscopic analysis as above.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of Cdc15 as a Tem1-binding protein:
To isolate Tem1-binding proteins, we performed two-hybrid screening using Tem1 as bait. To generate pKZ104, wild-type TEM1 was fused to the coding region of the Gal4-DNA binding domain on pGBDUC1 (JAMES et al. 1996 ). The library containing genomic DNA fused to the coding region of the Gal4-transcription activation domain (GAD; Y2HL, JAMES et al. 1996 ) was introduced into PJ69-4A cells (JAMES et al. 1996 ) carrying pKZ104. We isolated 94 His⁺Ade⁺ clones out of 96,000 transformants and rescued library plasmid from each of the His⁺Ade⁺ clones. DNA sequencing at the cloning junction revealed that the cloned fragments were classified into 34 genes. UETZ et al. 2000 also performed the two-hybrid screening using Tem1 as bait and isolated 24 genes. Surprisingly, none of the 34 genes that we isolated was found in the collection of UETZ et al. 2000 .

One of such isolates was found to contain a part of the CDC15 gene, the 250-bp TaqI-TaqI fragment (from 839th thymine to 1088th adenine) introduced into the ClaI site of pGADC1 (JAMES et al. 1996 ; Table 2). CDC15 encodes a protein kinase that is essential for the exit from mitosis, consisting of 974 amino acid residues (Mw 110 kD; SCHWEITZER and PHILIPPSEN 1991 ). The fact that Cdc15 and Tem1, two essential regulators for the exit from mitosis, have two-hybrid interaction suggests that they form a complex to function during mitotic exit.

To confirm the physical interaction in vivo between Cdc15 and Tem1, we performed a coprecipitation experiment. Cdc15-HA5 and Tem1-HA2His6 or Cdc15-HA5 and Tem1-HA2 were simultaneously expressed in the wild-type cell. Cell extract was incubated with Ni-NTA beads. The precipitants with the beads were washed five times with wash buffer containing 20 mM imidazole followed by elution with elution buffer containing 100 mM imidazole. Western blots of eluates showed that Tem1-HA2His6, in contrast to Tem1-HA2, efficiently precipitated with Ni-NTA beads (Fig 1). Cdc15-HA5 was detected in the eluate from Ni-NTA beads incubated with the extract containing Tem1-HA2His6 but not from those incubated with the extract containing Tem1-HA2. These data indicate that Cdc15 coprecipitated with Tem1. We concluded that Cdc15 and Tem1 form a complex in vivo.

View larger version (34K): In this window In a new window Download PPT slide   Figure 1. Cdc15 binds to Tem1 in vivo. The wild-type cells (YKZ0024) expressing Cdc15-HA5 (pKZ416) and Tem1-HA2His6 (pKZ173) or those expressing Cdc15-HA5 and Tem1-HA2 (pKZ178) were grown at 25° to mid-log phase and harvested. Extracts were prepared and incubated with Ni-NTA beads for 1 hr at 4°. Pull-down experiments with Ni-NTA beads were carried out as described in MATERIALS AND METHODS. Tem1-HA2His6 (+) or Tem1-HA2 (-) in the extract before (Ext) or after (Sup) the incubation with Ni-NTA beads and in the eluate (Elu) with 100 mM imidazole was detected by Western blotting using 16B12 as primary antibody and anti-mouse IgG goat antibody conjugated to horseradish peroxidase as secondary antibody. Cdc15-5HA in the extract or eluate was also detected. Asterisk indicates the nonspecific cross-reacting protein.

We noted that there are nucleotide sequence discrepancies between the sequence of the TaqI-TaqI fragment isolated from the library (Y2HL) in this study and that reported by SCHWEITZER and PHILIPPSEN 1991 ; the 946th cytosine, the 947th guanine, and the 961st cytosine in the results of Schweitzer and Philippsen are guanine, cytosine, and guanine in our results, respectively.

Tem1 binds to Cdc15 in a manner dependent on the properties of small GTPase:
To characterize the interaction between Cdc15 and Tem1, we investigated the interaction between Cdc15 and mutant Tem1. We introduced a novel mutation into the motif that is well conserved among small GTPases by site-directed mutagenesis (Fig 2A). One mutation substitutes the 34th threonine in the motif involved in GTP binding and hydrolysis with alanine (T34A) and the other substitutes the 52nd threonine in the effector domain with alanine (T52A).

View larger version (42K): In this window In a new window Download PPT slide   Figure 2. Tem1 binds to Cdc15 in a manner dependent on properties of small GTPase. (A) Schematic diagram of Tem1 and three mutation sites introduced by site-directed mutagenesis. The 34th threonine residue (in the motif involved in GTP binding and hydrolysis shown as solid boxes, I) and the 52nd threonine (in the effector domain, shown as III) were replaced with alanine (T34A and T52A, respectively) by site-directed PCR mutagenesis. The mutated threonine residues are highlighted. The mutation site of tem1-3 is also shown (II; SHIRAYAMA et al. 1994A ). (B) Growth of tem1 mutants. E0096 cells (tem1::HIS3 PGAL7-TEM1) carrying YCplac111 (vector), pKZ134 (TEM1), pKZ141 (T52A), and pKZ154 (T34A) were grown on a synthetic galactose (left) or a glucose (right) plate at 25° for 3 days. (C) Estimation of intracellular levels of Tem1 and its derivatives. Extracts prepared from E0096 cells expressing tagged Tem1 or its derivatives, which were grown in SG-LEU medium to mid-log phase followed by 5 hr of incubation in SC-LEU medium, were analyzed by Western blotting using anti-HA monoclonal antibody (16B12) as primary antibody and anti-mouse IgG goat antibody conjugated to horseradish peroxidase as secondary antibody. Thirty micrograms protein of each extract was loaded on a 10% polyacrylamide gel. Arrowheads point out the blots of HA-tagged Tem1. Lane no tag, E0096[YCplac111]; lane TEM1, E0096[pKZ173]; lane T52A, E0096[pKZ180]; lane T34A, E0096[pKZ181]. (D) Interactions between Cdc15 and Tem1 mutants in the two-hybrid system. Interactions between GAD-CDC15 and GBD (1), GBD-TEM1 (2), GBD-tem1T34A (3), GBD-tem1T52A (4), or GBD-tem1-3 (5) were estimated by measuring ß-galactosidase activity as described in MATERIALS AND METHODS and shown by solid bars. Open bars show the interactions between each construct and GAD. The results shown are representative of two independent trials. (E) Immunoprecipitation experiment. Extracts prepared from SLJ511 (CDC15-HA3) cells harboring indicated plasmid grown at 25° in SC-URA medium to mid-log phase were subjected to immunoprecipitation experiment as described in MATERIALS AND METHODS. Anti-HA antibody (16B12) was used to precipitate Cdc15-HA3. Lane TEM1 (right), pTGCU1 (YCpTEM1-GFP); lane tem1-3, pT135G (YCpctem1-3-GFP); lane T52A, pT52G (YCptem1T52A-GFP); lane T34A, pT34G (YCptem1T34A-GFP). The sample prepared from cells with pTGCU1 expressing untagged Cdc15 was shown in lane TEM1 (left) as a control. Amounts of Tem1-GFP and Cdc15-HA3 in extracts were shown as (ext Tem1) and (ext Cdc15), respectively. Asterisk or double asterisks indicate the nonspecific cross-reacting protein to anti-HA antibody or IgG, respectively. Arrowheads indicate the immunoprecipitated Tem1 or Cdc15.

To examine whether the tem1 mutants thus constructed cause a phenotypic change, these mutant genes were expressed under the control of the TEM1 promoter in the tem1 disruptant carrying integrated P_GAL7-TEM1 (E0096; Fig 2B). On a glucose plate, in which the expression of the TEM1 gene from the GAL7 promoter is shut off, cells carrying the wild-type TEM1 plasmid grew well while the cells carrying the vector, the tem1T34A plasmid, or the tem1T52A plasmid failed to grow. To estimate the expression levels of these mutant proteins, we constructed plasmids encoding Tem1 or mutant Tem1 proteins with a HA2-His6 tag at the carboxy terminus (TEM1-HA2His6, tem1T34A-HA2His6, and tem1T52A-HA2His6, respectively). The tem1 disruptant harboring TEM1-HA2His6 plasmid was able to grow on the glucose plate as well as the one harboring wild-type TEM1 plasmid, indicating that Tem1-HA2His6 protein is fully functional (data not shown). Western blot revealed that mutant proteins were expressed in an amount comparable to that of wild-type Tem1 (Fig 2C), showing that Tem1 lost its function by the mutation of T34A or T52A. These data indicate that the GTP binding and hydrolysis or the interaction with its effectors are essential for the function of Tem1. The overexpression of Tem1T34A protein from multicopy plasmid under the control of the GAL7 promoter, as well as that of wild-type Tem1 protein, did not affect the growth of wild-type cells on the galactose plate (data not shown) while that of Tem1T52A protein inhibited cell growth (data not shown), indicating that the Tem1T52A protein is not simply a loss-of-function mutant but has some dominantly negative effects for cell growth.

Next, we evaluated the interaction between Cdc15 and mutant Tem1 by the two-hybrid assays. Each of the three mutant tem1 (tem1T34A, tem1T52A, and tem1-3) was fused to the DNA segment encoding the Gal4-DNA binding domain and the entire open reading frame of CDC15 was fused to the DNA segment encoding the Gal4-transcription activation domain. The two-hybrid interactions between Tem1 mutants and Cdc15 were estimated by measuring ß-galactosidase activity (Fig 2D). The interaction was abolished either by the tem1T34A mutation or by the tem1-3 mutation and was reduced by half by the tem1T52A mutation. The tem1-3 mutation is a temperature-sensitive mutation that substituted the 135th aspartate residue, in the consensus motif needed for GTP binding and hydrolysis, with alanine (D135A; Fig 2A; SHIRAYAMA et al. 1994A ). We further tested whether these tem1 mutations affect the interaction with Tem1 and Cdc15 in vivo by immunoprecipitation assays. Each tem1 mutant protein that had a GFP tag at the carboxy terminus was expressed in the cell with the chromosomal CDC15 gene tagged with three HA epitopes (CDC15-HA3). We confirmed that Tem1-GFP is functional as Tem1 (data not shown). We hardly detected either of the tem1 mutant proteins precipitated with Cdc15-HA3 (Fig 2E) whereas wild-type Tem1 coprecipitated with Cdc15-HA3. These results indicate that Tem1 binds to Cdc15 in a manner dependent on the molecular properties of small GTPase. Notably, Tem1T34A-HA2His6 was detected as a single band whereas Tem1-HA2His6 and Tem1T52A-HA2His6 were detected as a doublet (Fig 2C). This observation suggests that some forms of Tem1 are post-transcriptionally modified; however, phosphorylation was found not to be responsible for the modification (data not shown).

Determination of Tem1-binding domain of Cdc15:
Besides a report that the deletion of 32 amino acid residues of its carboxy terminus abolished the function of Cdc15 (SCHWEITZER and PHILIPPSEN 1991 ), there is no report on a functional domain except for the kinase domain. Now that we know Tem1 binds to Cdc15, it is important for understanding the function of Cdc15 to determine the domain required for the interaction with Tem1. To determine the Tem1-binding domain of Cdc15, we analyzed the interaction between Tem1 and truncated Cdc15 by two-hybrid assays. Each truncated fragment of CDC15 was fused to the coding region of the Gal4-DNA transcription activation domain and introduced into PJ69-4A cells expressing GBD-Tem1 (Fig 3A). By measuring ß-galactosidase activity, we examined the interaction between Tem1 and Cdc15 fragments.

View larger version (38K): In this window In a new window Download PPT slide   Figure 3. The Tem1-binding domain of CDC15. (A) Construction of truncated CDC15. The nucleotide sequence AGACTA (-5 to -10) upstream of CDC15 was replaced by the BglII site (AGATCT) by PCR mutagenesis. The SalI site (+838) and the XhoI site (+1084) are shown. The open bar shows the coding region of the conserved kinase domain. Truncated fragments of CDC15 were introduced into the GAD vector (JAMES et al. 1996 ). a, pKZ401 (CDC15); b, pKZ415 (cdc15d82SX); c, pKZ420 (the SalI-XhoI fragment); d, pKZ422 (the BglII-XhoI fragment); e, pKZ421 (the BglII-SalI fragment); f, pKZ402 (the fragment from the SalI site to the stop codon). (B) Interactions between Tem1 and truncated Cdc15 in the two-hybrid system. Interactions between GBD-TEM1 and truncated CDC15 fragments shown in A were estimated by measuring ß-galactosidase activity and are shown by solid bars. Open bars show the interactions between each construct and GBD. The interaction between GAD and GBD-TEM1 or GBD is shown as control. The result shown is representative of two independent experiments. (C) Functional analysis of Cdc15d82SX by the plasmid shuffling method. YKZ0255 (cdc15 [YCpCDC15-URA3]) was transformed with YCplac22 (vec), pKZ439 (CDC15), pKZ438 (CDC15-HA5), pKZ448 (d82SX-HA5 [YCp]) or pKZ449 (d82SX-HA5 [YEp]). Trp+ Ura+ transformants were streaked on a SC-TRP plate with or without 5-FOA and incubated at 25° for 3 days. (D) cdc15 [YEpcdc15d82SX-HA5] cells are temperature sensitive. cdc15 [pKZ449] (left) cells and cdc15 [pKZ438] (right) cells were streaked on a YPD plate and incubated at 37° for 3 days. (E) Western blot of Cdc15d82SX-HA5. Extracts from YKZ0255 cells harboring pKZ438 (1, 2; Cdc15-HA5), pKZ448 (3, 4; Cdc15d82SX-HA5 expressed from centromeric plasmid), or pKZ449 (5, 6; Cdc15d82SX-HA5 expressed from multicopy plasmid) grown in SC-(URA, TRP) medium to mid-log phase were prepared and analyzed by Western blotting using anti-HA monoclonal antibody (16B12) as primary antibody and anti-mouse IgG goat antibody conjugated to alkaline phosphatase as secondary antibody. The samples of cells grown at 25° or 37° were loaded in odd number lanes or even number lanes, respectively. Thirty micrograms protein of each extract was loaded on a 7.5% polyacrylamide gel. Asterisk indicates the nonspecific cross-reacting protein.

Knowing that the segment consisting of 82 amino acid residues encoded by the SalI-XhoI fragment of CDC15 (corresponding to the TaqI-TaqI fragment isolated by the two-hybrid screening; Table 2) alone was able to interact with Tem1 (Fig 3B, Fig C), we constructed the mutant gene encoding Cdc15 without the 82-amino-acid residues (cdc15d82SX; see MATERIALS AND METHODS). As expected, Cdc15d82SX showed a reduced interaction with Tem1 (Fig 3B, Fig B), indicating that the 82-amino-acid domain plays a major role for the interaction. The construct containing the kinase domain and the 82-amino-acid domain (Fig 3B, Fig D) interacted with Tem1 as strongly as the full-length Cdc15, while the kinase domain alone hardly interacted with Tem1 (Fig 3B, Fig E). The Western blot showed that the GAD fusion of kinase domain (e) was expressed more than that containing both the kinase domain and the 82-amino-acid domain (d, data not shown), suggesting that the difference of the ß-galactosidase activity was not due to the difference of the expression levels of the GAD fusions but to the intensity of the interaction. The fragment without the kinase domain showed the interaction at a level comparable to that of the 82-amino-acid domain (Fig 3B, Fig C and Fig F) showing that the carboxy terminus of Cdc15 has less significance for the interaction. These results indicate that the region consisting of the kinase domain and the following 82-amino-acid residues is important for the interaction between Tem1 and Cdc15. We tentatively designated the segment consisting of the 82-amino-acid residues the Tem1-binding domain.

To test if the Tem1-binding domain is important for the function of Cdc15, we expressed Cdc15d82SX in the cdc15 disruptant. The centromeric plasmid carrying cdc15d82SX was introduced into the cdc15 disruptant, which was kept alive by expressing the wild-type CDC15 from plasmid harboring the URA3 gene. On medium containing 5-FOA, the cdc15 disruptant with Cdc15d82SX no longer survived (Fig 3C). Western blots showed that Cdc15d82SX was expressed from centromeric plasmid at a level lower than that of the wild-type Cdc15 (Fig 3E). However, when Cdc15d82SX was expressed from multicopy plasmid, the cdc15 disruptant restored growth at 25° but displayed the temperature-sensitive growth (Fig 3C and Fig D). The amount of Cdc15d82SX expressed from multicopy plasmid was more than that of the wild-type Cdc15 (Fig 3E), suggesting that the deletion of the Tem1-binding domain impaired the function of Cdc15.

Mutagenesis analysis of Tem1-binding domain:
In contrast to the high conservation of the sequence of the kinase domain of Cdc15, any obvious sequence similar to the Tem1-binding domain is not found near the kinase domain of other protein kinases including the Cdc15 homologue in fission yeast, Cdc7 kinase (FANKHAUSER and SIMANIS 1994 ). The Tem1-binding domain is rich in charged amino acid residues in the amino-terminal side and has one putative Cdc28-phosphorylation site. Further, we found an amino acid sequence that is slightly similar to the carboxy-terminal region of the fission yeast Cdc7 (LxxLFxVCxL) in the Tem1-binding domain. To characterize the function of the Tem1-binding domain more precisely, we introduced the point mutations into the indicated sites by PCR-aided site-directed mutagenesis (Fig 4A).

View larger version (53K): In this window In a new window Download PPT slide   Figure 4. Mutagenesis analysis of Tem1-binding domain. (A) Amino acid sequence of the Tem1-binding domain and the mutation sites introduced by site-directed mutagenesis. Construction of each mutant was described in MATERIALS AND METHODS. The region rich in charged amino acid residues was underlined. (B) Functional analysis of Tem1-binding domain mutants by the plasmid shuffling method. YKZ0255 (cdc15 [YCpCDC15-URA3]) was transformed with YCplac111 (vec), pKZ412 (CDC15), pKZ434 (DD), pKZ436 (S309A), or pFM420 (LF). Transformants were streaked on each of the SC-LEU plates with or without 5-FOA and incubated at 25° for 3 days. (C) The cdc15-LF cells displayed the temperature-sensitive growth that is suppressed by overproduction of Tem1. YKZ0376 (cdc15-LF) was transformed with YCplac33 (vec), pKZ406 (CDC15), or L11 (TEM1[YEp], SHIRAYAMA et al. 1994A ). Transformants were streaked on a SC-URA plate and incubated at 37° for 3 days. (D) Western blot of mutant Cdc15 proteins. Extracts prepared from YKZ0255 cells harboring indicated plasmid grown at 25° or 37° in SC-(URA, TRP) medium to mid-log phase were analyzed by Western blotting using anti-HA monoclonal antibody (16B12) as primary antibody and anti-mouse IgG goat antibody conjugated to horseradish peroxidase as secondary antibody. Asterisk indicates the nonspecific cross-reacting protein. Lane 1, pKZ438 (YCpCDC15-HA5) at 25°; lane 2, pKZ455 (YCpcdc15-DD-HA5) at 25°; lane 3, pKZ456 (YCpcdc15-S309A-HA5) at 25°; lane 4, pKZ442 (YCpcdc15-LF-HA5) at 25°; lane 5, pKZ442 (YCpcdc15-LF-HA5) at 37°. (E) Coimmunoprecipitation experiment between Tem1 and Cdc15 mutants. Extracts prepared from YKZ0454 (tem1::URA3 [YCpTEM1-myc9His6]) cells harboring indicated plasmid grown at 25° in SC-(LEU, TRP) medium to mid-log phase were subjected to immunoprecipitation experiment as described in MATERIALS AND METHODS. Lane CDC15 (right), pKZ453 (YEpCDC15-HA5); lane d82SX, pKZ449 (YEpcdc15d82SX-HA5), lane DD; pKZ465 (YEpcdc15-DD-HA5), lane LF; pKZ464 (YEpcdc15-LF-HA5). The sample prepared from cells with pKZ453 expressing Tem1 without a myc9His6-tag is shown in lane CDC15 (left) as a control. Amounts of Cdc15-HA5 in extracts are shown as (ext Cdc15). (F) Overexpression of cdc15 mutants complements the tem1 disruption. E0096 cells (tem1::HIS3 PGAL7-TEM1) carrying indicated plasmid were grown on a synthetic galactose (left) or a glucose (right) plate at 25° for 3 days. Section vec, YCplac22; section TEM1, CP (YCpTEM1); section CDC15, pKZ453 (YEpCDC15); section d82SX, pKZ449 (YEpcdc15d82SX); section DD, pKZ455 (YEpcdc15DD); section S309A, pKZ456 (YEpcdc15S309A); section LF, pKZ454 (YEpcdc15LF).

To analyze the effects of these mutations, centromeric TRP1 plasmid carrying each of the mutant genes under the CDC15 promoter was introduced into the cdc15 disruptant carrying pKZ416 followed by the plasmid shuffling on the FOA plate. Western blots showed that all the mutant proteins tested were produced at a comparable level (Fig 4D). The cdc15-DD strain was not able to grow in the presence of 5-FOA, indicating that the cdc15-DD mutation is lethal (Fig 4B). The cdc15-LF strain displayed temperature-sensitive growth (Fig 4B and Fig C) that is not due to the instability of Cdc15-LF protein at a high temperature (Fig 4D and Fig 6E).

View larger version (33K): In this window In a new window Download PPT slide   Figure 5. Cdc15 kinase activity of Tem1-binding domain mutants. (A) Wild-type cells (YKZ0024) carrying the multicopy plasmid encoding Cdc15-HA5 (pKZ425), Cdc15d82SX-HA5 (pKZ419), Cdc15-DD-HA5 (pKZ435), Cdc15-S309A-HA5 (pKZ454), Cdc15-LF-HA5 (pKZ431), or Cdc15-2-HA5 (pKZ429) were grown at 25°. Extract was prepared for Cdc15-HA5 or its derivatives and was immunoprecipitated with monoclonal antibody (16B12). The kinase activity associated with the precipitate was assayed by using MBP as substrate (top) or by measuring the autophosphorylation activity (bottom). (B) Cdc15-HA5 or its derivatives were detected by Western blotting using anti-HA monoclonal antibody (16B12) as primary antibody and anti-mouse IgG goat antibody conjugated to horseradish peroxidase as secondary antibody. For the estimation of Cdc15-HA5 and mutant derivatives, anti-mouse IgG goat antibody conjugated to alkaline phosphatase as secondary antibody was used. AttoPhos substrate was used as a fluorogenic substrate of alkaline phosphatase (data not shown, see MATERIALS AND METHODS). (C) Specific activity of MBP phosphorylation. The intensity of the signal of autoradiography was normalized by the amount of immunoprecipitated Cdc15-HA5 or its mutant derivatives. The results shown are representative of two independent trials.

View larger version (42K): In this window In a new window Download PPT slide   Figure 6. Cdc15 kinase activity of temperature-sensitive mutants. (A) Schematic diagram of Cdc15 and the temperature-sensitive mutation sites. The open box and the solid box represent the kinase domain and the Tem1-binding domain, respectively. Arrowheads indicate the temperature-sensitive mutation sites. Wild-type cells (YKZ0024) carrying the multicopy plasmid encoding Cdc15-HA5 (pKZ425), Cdc15-LF-HA5 (pKZ431), Cdc15-lyt1-HA5 (pKZ430), or Cdc15-rlt1-HA5 (pKZ428) were grown at 25°. Extract of each culture was prepared and Cdc15-HA5 or its derivatives was immunoprecipitated with monoclonal antibody (16B12). The kinase activity associated with the precipitate was assayed at 25° or 37° by using MBP as substrate (B) or by measuring the autophosphorylaion activity (C). (D) The amount of the Cdc15-HA5 or its derivatives was estimated by Western blotting using anti-HA monoclonal antibody (16B12) as primary antibody and anti-mouse IgG goat antibody conjugated to alkaline phosphatase as secondary antibody. AttoPhos substrate was used as a fluorogenic substrate of alkaline phosphatase. (E) Specific activity of MBP phosphorylation. The intensity of the signal of autoradiography was normalized by the amount of immunoprecipitated Cdc15-HA5 or its mutant derivatives. Solid boxes or open boxes show the specific activities at 25° or 37°, respectively. The numbers above the open boxes represent the ratio of the specific activity at 37° to that at 25°. The result shown is representative of two independent trials.

To examine whether Cdc15d82SX, Cdc15-DD, or Cdc15-LF is defective in binding to Tem1 in vivo, we performed immunoprecipitation assay. Consistent with the result obtained from the two-hybrid assay (see Fig 3B), Cdc15d82SX-HA5 was not precipitated with Tem1-myc9His6. We hardly detected Cdc15-LF-HA5 coprecipitated with Tem1-myc9His6 (Fig 4E), indicating that the cdc15-LF mutation affects the interaction between Tem1 and Cdc15. Interestingly, some amount of Cdc15-DD-HA5 was precipitated with Tem1-myc9His6, indicating that the cdc15-DD mutation does not abolish the ability to form the Cdc15/Tem1 complex (see DISCUSSION). We found that the temperature-sensitive growth of the cdc15-LF cell was efficiently suppressed by the overexpression of Tem1 (Fig 4C). This result suggests that the primary defect of the cdc15-LF mutation is the reduction in the interaction with Tem1 and, therefore, the interaction with Tem1 is required for the Cdc15 function under physiological conditions. We also found that the overproduction of Cdc15d82SX, Cdc15-DD, or Cdc15-LF suppressed the lethality of tem1 disruption (Fig 4F). These results are consistent with the notion that the point mutation or the deletion mutation of the Tem1-binding domain affects only the interaction between Tem1 and Cdc15. We failed to detect an effect of the cdc15-S309A mutation at least under the normal growth conditions. The phosphorylation at the 309th serine, if it occurs, alone does not affect the cell growth (JASPERSEN and MORGAN 2000 ).

Effects of mutations in the Tem1-binding domain on kinase activity:
Analysis of the point mutations (cdc15-DD and cdc15-LF) in addition to the deletion mutation (cdc15d82SX) confirmed the importance of the Tem1-binding domain for the function of Cdc15. Next, we tested the possibility that these mutations in the Tem1-binding domain affect the kinase activity of Cdc15. To measure the kinase activity, Cdc15-HA5 and its mutant derivatives expressed from multicopy plasmid in the wild-type cells were immunoprecipitated by anti-HA antibody and the immunocomplex was subjected to the kinase assay using MBP as an artificial substrate (Fig 5A, top; JASPERSEN et al. 1998 ). The autoradiogram of the autophosphorylation of Cdc15 and its mutant derivatives was also shown in Fig 5A (bottom). We found that Cdc15-DD-HA5 or Cdc15-LF-HA5 retains, if not full, kinase activity toward MBP (Fig 5C). These results indicate that a defect caused by either of the two mutations is not due to a simple reduction in kinase activity.

We estimated kinase activity of temperature-sensitive Cdc15, other than Cdc15-LF (Fig 6A). The cdc15-rlt1 mutation resides in the kinase domain (SHIRAYAMA et al. 1996 ) and the cdc15-lyt1 mutation in the middle region of the Cdc15 (JIMENEZ et al. 1998 ). Either the cdc15-lyt1 mutation or the cdc15-rlt1 mutation caused a reduction in kinase activity, although the cdc15-rlt1 mutation much more severely impaired the activity (Fig 6B and Fig C). We performed the kinase assay at a high temperature to determine how the temperature shift affects the kinase activity. At 37°, the kinase activity toward MBP of the wild-type Cdc15-HA5 was higher than that at 25° by 1.6-fold (Fig 6D). Cdc15-LF-HA5 and Cdc15-lyt1-HA5 each displayed the increase in kinase activity at a high temperature as did the wild-type Cdc15-HA5. However, Cdc15-rlt1-HA5 showed a decrease in the activity at a high temperature. These results indicate that the Cdc15-rlt1 enzyme itself is temperature sensitive; in contrast, Cdc15-LF or Cdc15-lyt1 has a reduced enzymatic activity that is not temperature sensitive.

Effects of the tem1 mutations or the cdc15 mutations on their localization to the spindle pole body:
Tem1 and Cdc15 localize to the spindle pole body (CENAMOR et al. 1999 ; BARDIN et al. 2000 ; PEREIRA et al. 2000 ; XU et al. 2000 ). We examined whether the mutation that reduced the function of Tem1 or Cdc15 might cause any effect on their SPB localization. Carboxy-terminally GFP-tagged Tem1 or its mutant derivative was expressed from the centromeric plasmid in the wild-type cell. Wild-type Tem1-GFP was detected at the SPB as one or two dots associated with DNA (Fig 7A, a and b). We hardly detected Tem1T34A-GFP localized to the SPB (Fig 7A, Fig C and Fig D). In turn, we observed dispersed GFP fluorescence throughout the cytosol. Tem1-3-GFP weakly localized to the SPB at 25° but not at 37° (Fig 7A, Fig G and Fig I). The cytosolic GFP signal of Tem1-3-GFP at 37° was much brighter than that at 25°. These results suggest that the SPB localization of Tem1 is regulated by nucleotide bound to it because the tem1T34A mutation or the tem1-3 mutation resides in the motif involved in GTP binding and hydrolysis (Fig 2A). We detected Tem1T52A-GFP localized to the SPB (Fig 7A, Fig E and Fig F), suggesting that the functional defect of Tem1 by the tem1T52A mutation is not due to its inability to localize properly to the SPB but due to its inability to interact with Cdc15.

View larger version (92K): In this window In a new window Download PPT slide   Figure 7. Localization analysis of GFP-tagged Tem1 proteins or Cdc15 proteins. (A) Localization of GFP-tagged Tem1 and its mutant derivatives. The wild-type cell (YKZ0024) expressing indicated plasmind was grown to mid-log phase at 25° (a, c, e, or g) or to mid-log phase at 25° followed by 3-hr incubation at 37°. The GFP fluorescence was detected as described in MATERIALS AND METHODS. a, pTGCU1 (YCpTEM1-GFP); c, pT52G (YCptem1T52A-GFP); e, pT34G (YCptem1T34A-GFP); g and i, pT135G (YCptem1-3-GFP). DNA was visualized by DAPI staining and is shown in b, d, f, h, or j. (B) Localization of GFP-tagged Cdc15 and its mutant derivatives. The cdc15-2 (YKZ0200) cell expressing indicated plasmid was grown to mid-log phase at 25° followed by a 3-hr incubation at 37°. The GFP fluorescence was detected as described in MATERIALS AND METHODS. a, pCFG1 (YEpCDC15-GFP); c, pRLTG (YEpcdc15-rlt1-GFP); e, pLFG (YEpcdc15-LF-GFP); g, pLYTG (YEpcdc15-lyt-GFP); i, pDDG (YEpcdc15-DD-GFP). Arrowheads indicate the highly fluorescent bodies that probably were artifacts resulting from overexpression of GFP-tagged protein. DNA was visualized by DAPI staining and is shown in b, d, f, h, or j.

Next, we tested the localization of Cdc15 mutants. Carboxy-terminally GFP-tagged Cdc15 or its mutant derivative was expressed from multicopy plasmid in cdc15-2 cells. The SPB localization of Cdc15-GFP was detected in 20% of the cells at 37° (Fig 7B, a) or at 25° (data not shown) and SPB localization was cell cycle independent (data not shown). In some cases, we also detected highly fluorescent bodies (indicated by arrowheads in Fig 7B, Fig E and Fig G) in the cell that probably were artifacts resulting from overexpression of GFP-tagged protein and were easily distinguished from the GFP signal associated with the SPB by their separation from the 4',6-diamidino-2-phenylindole (DAPI) signal. By examining the localization of GFP-tagged Cdc15 mutant proteins, we detected Cdc15-DD-GFP at the SPB either at 25° or at 37° as a spot brighter than that of the wild-type Cdc15-GFP (data not shown and Fig 7B, Fig I) and we detected Cdc15-LF-GFP at the SPB at 25° (data not shown) but not at 37° (Fig 7B, Fig E). These results suggest that the Tem1-binding domain has some role in the proper SPB localization of Cdc15. We further tested the localization of Cdc15-rlt1-GFP and Cdc15-lyt1-GFP. Cdc15-rlt1-GFP was mainly cytosolic and was hardly detected at the SPB at 25° or 37° (data not shown and Fig 7B, Fig C), suggesting that the kinase activity of Cdc15 is required for its SPB localization. We detected Cdc15-lyt1-GFP at the SPB at 25° or 37° (data not shown and Fig 7B, Fig G), suggesting that the cdc15-lyt1 mutation has little effect on the SPB localization of Cdc15.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Cdc15/Tem1 complex:
The MEN, which eventually causes the inactivation of CDK at mitotic exit, includes protein kinases (Cdc5, Cdc15, and Dbf2), a protein phosphatase (Cdc14), and a small GTPase (Tem1), suggesting that one aspect of the MEN is a complicated phosphorylation/dephosphorylation network. Various genetic interactions among the MEN genes reported so far (JASPERSEN et al. 1998 ) reflect this complexity and the characterization of the molecular interaction between the MEN components is very important to deepen our understanding of this feature of the MEN.

In this study, we showed that Tem1 bound to Cdc15 (Fig 1). While we were preparing this article, BARDIN et al. 2000 showed that Tem1 formed a complex with Cdc15 during mitosis. We also provided evidence that each of the mutations that are believed to reduce the function of Tem1 (tem1T34A, tem1T52A, or tem1-3) also weakened the interaction with Cdc15 (Fig 2D and Fig E), suggesting that the activation of Tem1 is necessary for the formation of the Cdc15/Tem1 complex in vivo. The result that the mutation in the putative effector domain (tem1T52A) reduced the interaction suggests that the Cdc15 is likely to be an effector of Tem1.

Domain analysis of Cdc15:
Cdc15 is a protein kinase that has a kinase domain at its amino-terminal region (Fig 6A). In this study, we determined a novel functional domain of Cdc15, the Tem1-binding domain. The deletion analysis showed that Tem1 binds to the region consisting of the 82-amino-acid residues that resides next to the kinase domain of Cdc15 (Fig 3B and Fig 4E). Determination of the Tem1-binding domain provided us a clue to characterize the domain function of Cdc15.

The properties of Cdc15d82SX, which has been constructed by deleting the Tem1-binding domain, are of particular interest. Cdc15d82SX retained low kinase activity toward MBP (Fig 5D) and, when overexpressed, restored the growth of the cdc15 disruptant at least at a permissive temperature (Fig 3C). These results led us to assume that the deletion of the Tem1-binding domain does not abolish the entire function of Cdc15 and that the interaction between Cdc15 and Tem1 is required for only a limited step of the action of Cdc15. Supporting this notion, the results of the mutagenesis analysis showed that the Tem1-binding domain is not essential for the intrinsic kinase activity of Cdc15 because Cdc15-LF or Cdc15-DD still retained the kinase activity (Fig 5C and Fig 6E) even though each of the mutations caused a defect in cell growth (Fig 4B and Fig C). The results that the cdc15-DD mutation enhanced the SPB localization of Cdc15-DD while the cdc15-LF mutation diminished it (Fig 7B, Fig E and Fig I) suggest that the Tem1-binding domain is involved in the regulation of the subcellular localization of Cdc15. The cdc15-DD mutation does not completely abolish the interaction with Cdc15-DD and Tem1 as we slightly detected the Cdc15-DD coprecipitated with Tem1 (Fig 4E). This suggests that there exists a subdomain in the amino-terminal region of the Tem1-binding domain that is less important for the interaction between Cdc15 and Tem1 but is important for an essential step(s) of the action of Cdc15. It remains to be examined whether the enhanced localization of Cdc15-DD to the SPB is toxic or not.

From previous reports and this study, the amino-terminal one-third region of Cdc15 is required for phosphorylation reaction and the interaction with Tem1. However, the function of the middle or the carboxy-terminal region of Cdc15 is still largely unknown. The cdc15-lyt1 mutation resides in the middle region of Cdc15 (Fig 6A; JIMENEZ et al. 1998 ). The kinase activity of Cdc15-lyt1 was lower than that of the wild-type Cdc15 (Fig 6D) but was not temperature sensitive, suggesting that the temperature sensitivity of the cdc15-lyt1 mutant should be explained by a mechanism other than thermolabile enzyme activity. It has been suggested that the region containing the cdc15-lyt1 site might be involved in the function of Cdc15 in cytokinesis (JIMENEZ et al. 1998 ). Whether or not the region where the cdc15-lyt1 mutation resides is directly involved in the interaction with Tem1 is elusive. There are seven putative phosphorylation sites in Cdc15, one in the Tem1-binding domain and the rest in the carboxy-terminal two-thirds of Cdc15. The mutations in all of them to nonphosphorylated form stimulated the Cdc15 function probably by changing the subcellular localization of Cdc15 (JASPERSEN and MORGAN 2000 ).

Possible mechanisms of the regulation of Cdc15 by Tem1:
An interesting question is how Tem1 regulates Cdc15. Cdc15 is known to be localized to the SPB (CENAMOR et al. 1999 ; XU et al. 2000 ) and its phosphorylation activity to artificial substrate MBP is constant during the cell cycle (JASPERSEN et al. 1998 ). It is important to determine whether Tem1 is involved in these properties of Cdc15. A paradigm of the interaction between Cdc15 and Tem1 was reported in fission yeast. Cdc7 (the fission yeast Cdc15 homologue) and Spg1 (the fission yeast Tem1 homologue) form a complex that regulates septation (SCHMIDT et al. 1997 ). Cdc7 localizes to the SPB during mitosis and this SPB localization is dependent on Spg1 function (SOHRMANN et al. 1998 ). On the other hand, the kinase activity of Cdc7 is not regulated by Spg1 (SOHRMANN et al. 1998 ). We estimated the kinase activity toward MBP of Cdc15-HA5 immunoprecipitated from the extract of the temperature-sensitive tem1-3 cells grown under the restrictive temperature or from the extract overexpressing the dominant negative Tem1-T52A protein (Fig 2A) and found no effect of the tem1 mutation on the kinase activity of Cdc15 (data not shown). Together with our observations that Cdc15-LF defective in binding to Tem1 showed no significant defect in the kinase activity, these results may suggest that Tem1 is not required for the kinase activity of Cdc15, at least, toward a nonphysiological substrate. We found that Cdc15-LF failed to localize to the SPB at the restrictive temperature (Fig 7B, Fig E), suggesting that the proper interaction with Cdc15 and Tem1 may be required for the SPB localization of Cdc15. This interpretation appears inconsistent with the previous report that overproduced Cdc15 was able to localize to the SPB in the absence of Tem1 function (CENAMOR et al. 1999 ; our unpublished result). However, localization of a protein under its overproduction should be evaluated with caution. Because of a limited amount of Cdc15 in vivo, it is very hard to detect it by microscopic analysis under the physiological conditions (XU et al. 2000 ; data not shown). Furthermore, the physiological substrates of Cdc15 are not yet identified. Analysis of the physiological level of Cdc15 using physiological substrates is necessary to characterize precisely the regulatory mechanism of Cdc15 by Tem1.

	ACKNOWLEDGMENTS

We thank Viesturs Simanis for the spg1 plasmids, Philip James for PJ69-4A and plasmids used for two-hybrid screening, David Morgan for the Cdc15-HA3 strain, Yukihumi Uesono for the CDC15 plasmid, and Takeshi Sasaki for the plasmids for protein tagging. A part of this work was supported by a grant for scientific research from Monbusho.

Manuscript received June 26, 2000; Accepted for publication December 15, 2000.

	LITERATURE CITED

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