DBF2, a cell cycle-regulated protein kinase, is physically and functionally associated with the CCR4 transcriptional regulatory complex
1997; Springer Nature; Volume: 16; Issue: 17 Linguagem: Inglês
10.1093/emboj/16.17.5289
ISSN1460-2075
Autores Tópico(s)DNA Repair Mechanisms
ResumoArticle1 September 1997free access DBF2, a cell cycle-regulated protein kinase, is physically and functionally associated with the CCR4 transcriptional regulatory complex Hai-Yan Liu Hai-Yan Liu Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, NH 03824 USA Search for more papers by this author Jeremy H. Toyn Jeremy H. Toyn Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA UK Search for more papers by this author Yueh-Chin Chiang Yueh-Chin Chiang Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, NH 03824 USA Search for more papers by this author Michael P. Draper Michael P. Draper Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, NH 03824 USA Search for more papers by this author Leland H. Johnston Leland H. Johnston Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA UK Search for more papers by this author Clyde L. Denis Corresponding Author Clyde L. Denis Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, NH 03824 USA Search for more papers by this author Hai-Yan Liu Hai-Yan Liu Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, NH 03824 USA Search for more papers by this author Jeremy H. Toyn Jeremy H. Toyn Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA UK Search for more papers by this author Yueh-Chin Chiang Yueh-Chin Chiang Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, NH 03824 USA Search for more papers by this author Michael P. Draper Michael P. Draper Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, NH 03824 USA Search for more papers by this author Leland H. Johnston Leland H. Johnston Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA UK Search for more papers by this author Clyde L. Denis Corresponding Author Clyde L. Denis Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, NH 03824 USA Search for more papers by this author Author Information Hai-Yan Liu1, Jeremy H. Toyn2, Yueh-Chin Chiang1, Michael P. Draper1,3, Leland H. Johnston2 and Clyde L. Denis 1 1Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, NH 03824 USA 2Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA UK 3Department of Molecular Biology and Microbiology, Tufts University, Boston, MA, 02111 USA The EMBO Journal (1997)16:5289-5298https://doi.org/10.1093/emboj/16.17.5289 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info CCR4, a general transcriptional regulator affecting the expression of a number of genes in yeast, forms a multi-subunit complex in vivo. Using the yeast two-hybrid screen, we have identified DBF2, a cell cycle-regulated protein kinase, as a CCR4-associated protein. DBF2 is required for cell cycle progression at the telophase to G1 cell cycle transition. DBF2 co-immunoprecipitated with CCR4 and CAF1/POP2, a CCR4-associated factor, and co-purified with the CCR4 complex. Moreover, a dbf2 disruption resulted in phenotypes and transcriptional defects similar to those observed in strains deficient for CCR4 or CAF1. ccr4 and caf1 mutations, on the other hand, were found to affect cell cycle progression in a manner similar to that observed for dbf2 defects. These data indicate that DBF2 is involved in the control of gene expression and suggest that the CCR4 complex regulates transcription during the late mitotic part of the cell cycle. Introduction The CCR4 protein from Saccharoymces cerevisiae affects the expression of a number of genes and processes. CCR4 is required for full derepression of ADH2 and other non-fermentative genes under glucose-derepressed conditions (Denis, 1984; Denis and Malvar, 1990). ccr4 mutations also reduce the enhanced gene expression resulting from defects in the SPT6 or SPT10 proteins (Denis, 1984; Denis and Malvar, 1990) that appear important in maintaining a proper chromatin structure (Natsoulis et al., 1991; Dollard et al., 1994; Bortvin and Winston, 1996). CCR4 functions downstream of SPT6 and SPT10, at a post-chromatin remodeling event (Denis et al., 1994; M.Caserta, personal communication). In addition to affecting these processes, a ccr4 allele affects the expression of genes involved in cell wall integrity (A.Sakai, personal communication), in UV sensitivity (Schild, 1995) and in methionine biosynthesis (McKenzie et al., 1993). Moreover, CCR4 is required by different transactivators to function maximally (Draper et al., 1994). These observations indicate that CCR4 plays an important general transcriptional role in diverse cellular events. CCR4 is a leucine-rich repeat (LRR)-containing protein (Malvar et al., 1992). Proteins containing LRRs have often been found to be associated with other proteins through their LRR region (Kobe and Diesenhofer, 1994). CCR4 is a component of a multi-subunit complex, and the LRR region is essential for its protein-protein interactions in this complex (Draper et al., 1994, 1995). The CAF1/POP2 protein has been identified as a component of the CCR4 complex (Draper et al., 1995), and caf1 disruptions display very similar phenotypes and transcriptional defects to those of ccr4 (Sakai et al., 1992; Draper et al., 1995). We report here that DBF2 is another component of the CCR4 complex. DBF2 was identified as a temperature-sensitive mutation that causes cell cycle arrest at the end of mitosis in which the cells have a fully extended spindle and divided chromatin, a characterisitic of telophase (Johnston et al., 1990). Consistent with a mitotic role for DBF2, the gene is expressed under cell cycle control in M phase. DBF2 encodes a protein kinase and this activity is also cell cycle regulated, with a peak in late mitosis (Toyn and Johnston, 1994). Despite there being temperature-sensitive alleles of DBF2, deletions of the gene are viable (Toyn et al., 1997) due to the existence of a homolog, DBF20 (Toyn et al., 1991). However, deletion of both DBF2 and DBF20 results in strains that are non-viable, indicating that these genes encode closely related protein kinases that are essential for the ending of mitosis. The target protein substrates of the DBF2 kinase have not been identified, and, therefore, the molecular basis for its role in regulation of the cell cycle is not known. Our present work indicates a role for DBF2 in transcriptional regulation. We find that a defect in DBF2 results in phenotypes and transcriptional defects similar to those observed for a ccr4 or caf1 disruption. Conversely, ccr4 and caf1 disruptions affect cell cycle progression in late mitosis similarly to dbf2 mutations. The CCR4 complex appears, therefore, to be important to the control of specific sets of genes, including those involved in the late mitotic phase of the cell cycle. Results DBF2 associates with CCR4 and CAF1 To identify further members of the CCR4 and CAF1 complex, a yeast two-hybrid screen was carried out using the LexA-CCR4 fusion protein as the bait (Draper et al., 1995). The interaction library contained the Escherichia coli-derived B42 activator fused to yeast genomic DNA fragments under the control of a GAL1 promoter (Zervos et al., 1993). Fifty six colonies that displayed galactose-dependent activation of both the LexAop-LEU2 and the LexAop-lacZ reporters were isolated from ∼2×106 transformants. Library plasmids were isolated from the 56 colonies, and each was found to contain one of seven genes, two of which were identified previously. One of these, CAF1, encodes a protein which has been shown previously to interact with LexA-CCR4 and to be physically associated with CCR4 in vivo (Draper et al., 1995). The second gene, designated CAF2, was found to be identical to the yeast gene, DBF2. DBF2 is a cell cycle-regulated protein kinase that plays an important role in the telophase to G1 transition (Toyn and Johnston, 1994). As summarized in Table I, the B42-DBF2 fusion containing DBF2 residues 205 to its C-terminus interacted with LexA-CCR4 but failed to interact with the LexA moiety alone. DBF2 (205-561) is truncated midway in its protein kinase domain and would be expected to be inactive as a protein kinase. To confirm the interaction between CCR4 and DBF2, we constructed a full-length DBF2 fused to B42 and expressed it along with LexA-CCR4. The B42-DBF2 (full-length) fusion, which is capable of complementing phenotypes associated with a dbf2 disruption (data not shown), also interacted with LexA-CCR4 and did so to the same extent as the truncated B42-DBF2 fusion (Table I). A LexA-CCR4 derivative containing a deletion in the LRR region (Draper et al., 1994) failed to interact with DBF2 (Table I), suggesting that the interaction observed between CCR4 and DBF2 was dependent on the presence of the LRR region in the CCR4 protein. Table 1. Two-hybrid interactions of DBF2 with CCR4 and CAF1 LexA fusion B42 fusion β-Gal (U/mg) LexA-CCR4 B42 30 LexA-CCR4 B42-DBF2 (205-561) 150 LexA-CCR4 B42-DBF2 (1-561) 170 LexA-CCR4 B42-DBF2 (K195T) 110 LexA B42-DBF2 (205-561) 1.3 LexA-CCR4 (ΔLRR) B42-DBF2 (205-561) <1 LexA-CAF1 B42 110 LexA-CAF1 B42-DBF2 (205-561) 210 LexA-CAF1 B42-DBF2 (1-561) 1700 LexA-CAF1 B42-DBF2 (K195T) 1500 We also analyzed the ability of B42-DBF2 to interact with LexA-CAF1. The full-length B42-DBF2 fusion displayed an interaction with either LexA-CAF1 (residues 124-441) (Table I) or with LexA-CAF1 (full-length) (data not shown). In contrast, truncated B42-DBF2 (205-561) did not interact with either LexA-CAF1 construct (Table I; data not shown). Also, the interactions between CCR4 and DBF2, CCR4 and CAF1, and CAF1 and DBF2 essentially were unaffected by deletion of the gene encoding the other factor, CAF1, DBF2 and CCR4, respectively (data not shown). Other factors, therefore, appear to mediate or stabilize the association of these three proteins. DBF2 is physically associated with the CCR4 complex The physical association of B42-DBF2 with CCR4 was examined by co-immunoprecipitation. Whole-cell extract expressing the B42-DBF2 (full-length) fusion protein containing an HA1 tag was incubated with CCR4 antibody. The immunoprecipitated samples were analyzed by Western blotting using antibody directed against CCR4 or HA1 (Figure 1). The B42-DBF2 protein was co-immunoprecipitated specifically with the CCR4 antibody (Figure 1A, lane 4) but was not immunoprecipitated by an antibody raised against the LexA protein (lane 5). In addition, B42-DBF2 was found not to be immunoprecipitated from extracts prepared from a strain lacking CCR4 protein (Figure 1B, compare lanes 4 and 5). In a control experiment, a B42-SIP1 fusion protein [SIP1 is a protein associated with the SNF1 protein kinase (Yang et al., 1992) and has been shown not to be associated with the CCR4 complex, data not shown] did not co-immunoprecipitate with CCR4 (Figure 1A, lane 3). These data indicate that B42-DBF2 interacts with CCR4 specifically via the DBF2 moiety. We also showed that B42-DBF2 was also co-immunoprecipitated with CAF1 using an anti-CAF1 antibody (Figure 1B, lane 7) and was not immunoprecipitated from extracts prepared from a strain lacking CAF1 protein (lane 6). We were not able, however, to co-immunoprecipitate CCR4 or CAF1 with B42-DBF2 in strains deleted for caf1 or ccr4, respectively. Figure 1.Co-immunoprecipation of B42-DBF2 with CCR4 and CAF1. (A) Extracts from strain EGY188 containing either B42-DBF2 or B42-SIP1 were incubated with either anti-CCR4 or anti-LexA antibodies and the resulting immunoprecipitates were subjected to electrophoresis on a 10% SDS-PAGE gel. CCR4- and HA1-containing proteins were detected by Western analysis as described (Draper et al., 1995). Lanes 1 and 2, crude extracts containing B42-SIP1 and B42-DBF2, respectively; lane 3, B42-SIP1-containing extracts treated with anti-CCR4 antibody; lane 4, same as lane 3 except B42-DBF2; lane 5, same as lane 4 except extracts were treated with anti-LexA antibody. (B) Extracts containing B42-DBF2 were immunoprecipitated and analyzed as described in (A) above. Lane 1, crude extract from EGY188-1 (ccr4); lane 2, crude extract from EGY188-c1 (caf1); lane 3, crude extract from EGY188 (wt); lanes 4 and 5, CCR4 immunoprecipitations using strains EGY188-1 and EGY188, respectively; lanes 6 and 7, CAF1 immunoprecipitations using strains EGY188-c1 and EGY188, respectively. Lanes 1-3 were developed with anti-HA1 antibody whereas lanes 4-7 were developed with both anti-CCR4 and anti-HA1 antibodies. For clarity, lanes 4-7 were not treated with anti-CAF1 antibody since other experiments have shown that CCR4 and CAF1 always co-immunoprecipitate (Draper et al., 1995; data not shown). Download figure Download PowerPoint We further examined the physical interaction between DBF2 and CCR4 using a second approach. We have found that CCR4 and a CAF1-6His-tagged protein co-purify following two chromatographic stages: Ni2+-NTA agarose and Mono-Q chromatography (Figure 2A). The CAF1-6His gene, containing one copy of a CAF1 gene fused at its 3′ end to a six histidine tag and integrated into the yeast genome at the TRP1 locus, was able to complement phenotypes associated with a caf1 null allele (data not shown). We subsequently used a LexA-DBF2 construct that was capable of complementing a dbf2 null allele (data not shown) to analyze the co-purification of DBF2 with CAF1. Whole-cell extract prepared from a strain expressing both LexA-DBF2 and CAF1-6His was first passed over an Ni2+-NTA-agarose column and the bound proteins were eluted with imidazole. The resulting eluant was then passed over an FPLC Mono-Q column, and the bound proteins were eluted with a linear salt gradient. The resulting Mono-Q fractions were subjected to Western blot analyses (Figure 2B). The LexA-DBF2 fusion protein co-purified with CAF1 through the Ni2+-NTA and Mono-Q columns (Figure 2B). In a control experiment, LexA alone was not retained on the Ni2+-agarose column (Figure 2C, lane 3, LexA, compared with lane 4, LexA-DBF2), indicating that it is the DBF2 moiety of LexA-DBF2 which is co-purifying with CAF1-6His. The co-purification experiment together with the co-immunoprecipitation experiments clearly indicate that DBF2 is associated with the CCR4 complex in vivo. Figure 2.Co-purification of Lex-DBF2 with CAF1 and CCR4. (A) Ten ml of extracts from strain MLF6-3 (CAF1-6His) were bound to Ni2+-NTA-agarose beads and, after imidazole elution, the 3 ml of eluent were subjected to Mono-Q chromatography. The resultant 1 ml chromatographic fractions (5 ml for flowthrough) were subjected to Western analysis following 10% SDS-PAGE. CCR4 and CAF1-6His proteins were detected as indicated with anti-CCR4 and anti-CAF1 antibodies. It should be noted that with this anti-CAF1 antibody, CAF1 protein cannot be detected in crude extracts. Also, in crude extracts, the CCR4 antibody recognizes the non-specific protein that is larger than CCR4. CrEx, 15 μl of crude extract; Ni-eluant, 50 μl of Ni2+-NTA-agarose eluant with imidazole; flow-through, 20 μl of flowthrough of Ni2+ eluant subjected to Mono-Q chromatography; Mono-Q fractions, 20 μl of fractions obtained by salt elution of Ni2+ eluant proteins subjected to Mono-Q chromatography. (B) Yeast extracts were prepared from MLF6 (CAF1-6His) containing the LexA-DBF2 plasmid. Lane designations are the same as in (A), except that 30 μl of the different samples were analyzed. The Western was developed with antibody directed against CAF1 and LexA. We estimate that ∼30% of the LexA-DBF2 bound to the Ni2+-NTA-agarose column co-migrated with CAF1-6His following Mono-Q chromatography. (C) Extracts were prepared from strain MLF6 containing LexA-202-3 (LexA) or LexA-DBF2. Lane designations are the same as in (A). The Western was developed with antibody specific to LexA. Download figure Download PowerPoint The CCR4 complex displays DBF2-dependent protein kinase activity Since DBF2 encodes a protein kinase, we addressed the question as to whether the CCR4 complex contained a protein kinase by using an in vitro kinase assay. Galactose-grown extracts prepared from a strain expressing the B42-DBF2 fusion (containing the HA1 epitope and under the control of the galactose-inducible GAL1 promoter) were immunoprecipitated with HA1, CCR4 or LexA control antibody, and the resulting immunoprecipitates were then analyzed for the ability to phosphorylate H1 histone (Figure 3A). Both anti-HA1 and anti-CCR4 immunoprecipitates displayed protein kinase activity (lanes 1 and 3). No protein corresponding to H1 histone was phosphorylated when H1 histone was left out of the reaction (lane 2). In control experiments, both the immunoprecipitates obtained with LexA antibody (lane 4) and extracts incubated with protein A-agarose alone (data not shown) contained much less kinase activity than observed with the anti-CCR4 antibody immunoprecipitates (Figure 3A, compare lane 4 with lane 3). These results indicate that the CCR4 immunoprecipitates contain protein kinase activity. To examine whether the kinase activity in the CCR4 complex is due to the presence of DBF2, we repeated the kinase experiment by immunoprecipitating the CCR4 complex from extracts of either glucose-grown or galactose-grown cultures. Protein kinase activity in anti-CCR4 antibody immunoprecipitates observed in the galactose-inducing condition (Figure 3B, lane 4) was much greater than that in the glucose-repressing condition (lane 2). Under galactose growth conditions with LexA immunoprecipitates (lane 3), protein kinase activity was much less than observed for the CCR4 immunoprecipitates (Figure 3B, lane 4). This background level of protein kinase activity was also observed in anti-CCR4 antibody immunoprecipitates of extracts from a ccr4-deleted strain (data not shown). Moreover, under galactose growth conditions, increased protein kinase activity was observed for B42-DBF2-containing strains in both the CCR4 (Figure 3C, lane 2) and HA1 (lane 4) immunoprecipitates as compared with comparable immunoprecipitates obtained from B42-only expressing strains (see lanes 1 and 3, respectively). These results indicate that the increased protein kinase activity observed under galactose conditions from CCR4 immunoprecipitates was indeed due to the expression of the B42-DBF2 fusion protein. Figure 3.CCR4 immunoprecipitates contain DBF2 protein kinase activity. (A) Extracts from strain MLF6 containing B42-DBF2 (lanes 1-4) grown under galactose growth conditions were first treated with antibody and then the immunoprecipitates were analyzed for H1 histone protein kinase activity, as described, following SDS-PAGE and fluorography (Toyn and Johnston, 1994). The presence of added H1 histone is indicated above the autoradiograms. Lane 1, immunoprecipitation was conducted with HA1 epitope antibody; lanes 2 and 3, immunoprecipitations were conducted with anti-CCR4 antibody; lane 4, same as lane 3 except LexA antibody. (B) Protein kinase assays were conducted as described in (A) except that strain MLF6 containing B42-DBF2 was grown under glucose or galactose growth conditions as indicated. Immunoprecipitations as indicated were conducted as described in (A). (C) Extracts from strain MFL6 containing B42-DBF2 (lanes 2 and 4) or B42 (lanes 1 and 3) grown under galactose growth conditions were assayed for kinase activity as described in (A) above. Lanes 1 and 2, immunoprecipitations were conducted with anti-CCR4 antibody; lanes 3 and 4, immunoprecipitations were conducted with HA1 epitope antibody. (D) Re-immunoprecipitation of extracts following the first immunoprecipitation protein kinase assay was conducted as described in Materials and methods. Lane 1, the first immunoprecipitation was conducted with anti-CCR4 antibody and the second with CCR4 antibody; lane 2, same as lane 1 except that the second immunoprecipitation was conducted with CAF1 antibody; lane 3, same as lane 1 except the second immunoprecipitation was conducted with HA1 antibody; lane 4, same as lane 3 except HA1 antibody was used for both immunoprecipitations. Download figure Download PowerPoint To test if any of the known components in the CCR4 immunoprecipitates can be phosphorylated by DBF2, we re-immunoprecipitated CCR4, CAF1 or B42-DBF2 following the kinase assay. First, the CCR4 complex was immunoprecipitated with the CCR4 antibody and the B42-DBF2 fusion protein was immunoprecipitated with the HA1 antibody. The resulting immunoprecipitates were then subjected to the in vitro kinase assay. CCR4, CAF1 and B42-DBF2 subsequently were re-immunoprecipitated out of the CCR4 immunoprecipitates by adding, respectively, the CCR4, CAF1 and HA1 antibodies, and B42-DBF2 was re-immunoprecipitated from the HA1 immunoprecipitates by adding HA1 antibody again. The resulting immunoprecipitates were subjected to SDS-PAGE and fluorography (Figure 3D). Phosphorylated B42-DBF2 was identified in the HA1 double immunoprecipitation (lane 4) and from the CCR4 immunoprecipitate that was re-treated with HA1 antibody (lane 3). Phosphorylation of CCR4 or CAF1 was not observed from the CCR4/CCR4 and CCR4/CAF1 double immunoprecipitates (lanes 1 and 2, respectively). Separate experiments showed that CCR4 or CAF1 could be detected by Western analysis from these re-immunoprecipitated extracts (data not shown). These data suggest that neither CCR4 nor CAF1 is an in vitro target for DBF2. The initial two-hybrid interaction between CCR4 and DBF2 indicated that the DBF2 kinase domain was not required for interaction with CCR4, although it was required for interaction with CAF1 (Table I). To determine specifically if DBF2 kinase function was required for its interaction with CCR4 or CAF1, a DBF2 allele containing a mutation (K195T) in the conserved lysine residue of the ATP-binding site catalytic domain of DBF2 subsequently was analyzed (Toyn and Johnston, 1993). This dbf2-K195T allele does not complement the dbf2 null allele, is lethal in the absence of the wild-type DBF2 gene and lacks kinase activity (Toyn and Johnston, 1994). B42-DBF2-K195T interacted with both CCR4 and CAF1 to nearly the same extent as did the wild-type B42-DBF2 protein (Table I). These experiments indicate that DBF2 protein kinase activity is not important for DBF2 association with CCR4 or CAF1, and that CCR4 and CAF1 are not in vitro substrates for DBF2 under the conditions utilized. A dbf2 disruption causes transcriptional defects similar to those observed with disruption of ccr4 and caf1 Disruption of CCR4 or CAF1 results in a number of transcriptional phenotypes. We investigated the putative role of DBF2 in several of these transcriptional processes. ccr4 mutations were identified originally as specific suppressors of the enhanced ADH2 expression under glucose growth conditions caused by an spt10 defect (Denis, 1984; see Table II). Only ccr4 and caf1 alleles display this phenotype (Draper et al., 1995). A dbf2 disruption, which grows almost as well as the wild-type, similarly suppressed the enhanced ADH II enzyme levels caused by an spt10 defect (Table II). The dbf2 effect was specific to suppressing spt10-enhanced ADH2 expression, since dbf2 was incapable of suppressing an ADR1-5c allele which displays high ADH II enzyme levels under repressed conditions similar to those observed in an spt10-containing strain. In contrast, a deletion of DBF20, a non-cell cycle-controlled homolog of DBF2, had no effect on spt10-enhanced ADH2 expression (Table II). Table 2. dbf2 suppresses spt10-enhanced expression ADH II (mU/mg) spt10a 87 dbf2spt10a 9.0 ADR1-5Cb 100 dbf2 ADR1-5Cb 120 spt10c 210 dbf20 spt10c 200 spt10d 175 ccr4 spt10d 23 a Segregants from crosses 994-2×991-2-6d and 1013-2a×1013-6a. b Segregants from cross 787-6b×1007-1-4a. c Segregants from cross 808-5c×1044-5a. d Data from Denis (1984). ccr4 and caf1 mutations also cause a defect in non-fermentative growth at 37°C, a phenotype we observed to be shared by dbf2 (Table III, see glycerol column). This arrest of growth on glycerol by dbf2 was not a mitotic arrest as is observed with temperature-sensitive alleles of DBF2 (data not shown). While a ccr4 disruption affects the non-fermentative expression of ADH2 by 5-fold (Denis and Malvar, 1990) and caf1 by ∼2-fold (Draper et al., 1995), a dbf2 effect on ADH2 expression under non-fermentative growth conditions was not observed (data not shown). caf1 and ccr4 defects also cause increased sensitivity to staurosporine, a protein kinase inhibitor (A.Sakai, personal communicaation) and to caffeine, two compounds linked to effects on cell wall integrity and other processes (Posas et al., 1993; Costigan et al., 1994) (Table III). These effects are similar to those observed with defects in the protein kinase C and MAP kinase pathway (Lee et al., 1993; Costigan et al., 1994) and appear to result from defects in expression of genes involved in cell wall integrity (Roemer et al., 1994; Shimizu et al., 1994). A dbf2 disruption resulted in similar increased sensitivity to caffeine and staurosporine (Table III). The staurosporine sensitivity and temperature sensitivity of dbf2, caf1 and ccr4 deletions were reversed by 1 M sorbitol as were the cold sensitivity and temperature sensitivity phenotypes of these disrupted alleles (Table III) (A.Sakai, personal communication). We also observed that ccr4- (B.Anderson, personal communication), caf1- and dbf2-containing strains were hypersensitive to elevated levels of the Li+ ion (Table III). It should be noted that combining the dbf2 disruption with either a ccr4 or caf1 disruption resulted in no increased sensitivity of any of the above phenotypes, suggesting that DBF2 functions in the same pathway as CCR4 and CAF1. Table 3. dbf2, caf1 and ccr4 phenotypes Strain Relevant genotype 12°C 12°C + sorb. 37°C 37°C + sorb. Gly 37°C Stauro Stauro + sorb. Caffeine LiCl 188 wt + + + + + + + + + 188-1 ccr4 − + w + − − + − − 188-c1 caf1 − w − + − − + − w S7-4A dbf2 w + w + − − + − − In addition, we examined the role of DBF2 in affecting LexA transactivator function. Both ccr4 and caf1 defects reduce the ability of several different LexA activators to activate a LexA-lacZ reporter ∼2- to 3-fold (Draper et al., 1994, 1995). A dbf2 disruption affected the function of LexA-B42, LexA-ADR1-TADIV and LexA-CCR4-1-345 by ∼2- to 3-fold, although it had little or less effect on LexA-CAF1 or LexA-ADR1-full length activation (data not shown). These results are similar to those observed for caf1 and ccr4 defects, but the effects are not as general as for the defects in the CAF1 and CCR4 genes. The above results indicate that DBF2 is required for processes similar to those that require CCR4 and CAF1. More importantly, they indicate that DBF2 affects several different transcriptional processes and behaves as a functional component of the CCR4 complex. ccr4 and caf1 strains have cell cycle-related phenotypes The finding that DBF2 is physically associated with both CCR4 and CAF1 suggested that these proteins cooperate in cell regulation, and would therefore have related phenotypes when mutated. Because DBF2 functions at the end of mitosis (Johnston et al., 1990; Toyn and Johnston, 1994), we investigated whether deletion of CCR4 or CAF1 caused similar cell cycle defects. Two different tests indicated that ccr4 and caf1 deletion strains were partially defective for cell cycle progression at the end of mitosis: firstly, they displayed an increased proportion of late mitotic cells in log phase cultures, and, secondly, they were hypersensitive to CLB2 overexpression. Log phase ccr4, caf1 and isogenic control cultures were stained with 4′,6′-diamidino-2-phenylindole (DAPI), and the percentage of cells with buds and with divided chromatin was counted, as an indicator of the number of telophase cells (Table IV). Both mutants showed an increase in the budded population, consistent with a mitotic delay, but, more importantly, the ccr4 and caf1 cultures had ∼3-fold and 2-fold increases in the percentage of total cells with divided chromatin, respectively. This suggests that the ccr4 and caf1 genotypes result in a delayed exit from mitosis, at least in terms of causing some of the cells to spend more time in telophase. Table 4. Cells with divided chromatin in caf1 and ccr4 cultures Yeast strains Buds (%) Divided chromatin (%) Budded cells with divided chromatin (%) wta 62 15 24 ccr4b 75 44 57 wtc 45 10 22 caf1d 54 20 37 a wt, strain 612-1d; b ccr4, strain 612-1d-2A; c wt, strain 935-2; d caf1, strain 935-2-3. Deactivation of the B cyclin kinase activity is necessary for the exit from mitosis, and it has been shown that overexpression of CLB2, or expression of non-degradable CLB2, prevents B cyclin kinase deactivation, causing a stage-specific arrest of the cell cycle in telophase (Surana et al., 1993). Furthermore, yeast mutants that are partially defective for B cyclin kinase deactivation, a
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