INTRODUCTION

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Cyclin-dependent kinases (Cdks) drive key events in the eukaryotic cell cycle. Most eukaryotes contain multiple cyclin genes, expressed at different times in the cell cycle and appearing to control distinct cell cycle events. It has been proposed that simple oscillation of a single generic Cdk activity could account for cell cycle regulation (23, 35). However, recent observations (reviewed in reference 25) suggest that such models do not take into account strong differences in the efficiency with which different cyclins carry out different biological roles (for example, see references 8, 13, and 18). The molecular basis for such cyclin specificity remains unclear. We proposed recently that a candidate substrate "docking" site characterized in cyclin A (5, 29) might be evolutionarily conserved in many cyclins (8). Here we show that this docking site is functionally conserved in the yeast B-type cyclin Clb5p, both for the ability to bind specific proteins (the mammalian inhibitors p21 and p27) and for biological function in a range of assays presumably requiring interaction with diverse Clb5p substrates. These findings suggest that evolution of this region on the cyclin surface occurred during primordial eukaryotic evolution (predating the separation of yeast and metazoans) and probably arose as a substrate-binding domain rather than as a domain for binding inhibitors. In eukaryotic systems with multiple cyclins due to gene duplication, this region could then diverge to give different binding specificity to different cyclins, resulting in sharp differences in the biological efficiency of driving specific cell cycle events.

	MATERIALS AND METHODS

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Yeast strains. A clb3,4,5,6 GAL::CLB5 strain in the W303 background (K3418 [31]; provided by K. Nasmyth) was converted to ura3 by selecting hisG-URA3-hisG popouts on 5-fluoroorotic acid (5-FOA), to make K3418-1. ×1924, a diploid clb3,4,5,6 pGAL-CLB5 strain in the BF264-15D background, was constructed by crossing KL321 (clb3,4,5,6 pGAL-CLB5/URA3 leu2 trp1 [8]), transformed with a TRP1 vector, with strain MY21-6d (clb3::LEU2 clb4,5,6 pGAL-CLB5/URA3 trp1), followed by loss of the TRP1 plasmid by growth on nonselective galactose medium. This diploid was constructed because of variability of results for clb3,4,5,6 rescue between these two strains and because phenotypes with KL321 were variable in and between some experiments. Results with ×1924 were quantitatively more consistent, perhaps because diploids are more stable to the generation of recessive modifiers. Results with all BF264-15D-derived clb3,4,5,6 strains were qualitatively consistent with each other. A clb5::ARG4 cdc28-4 pGAL-CLB5 strain (BF264-15D background) was constructed by crossing a cdc28-4 strain carrying a pGAL-CLB5 plasmid with a clb5 strain carrying a LEU2 vector and sporulating and dissecting tetrads. Two cdc28-4 clb5 pGAL-CLB5/URA3 strains from this cross were mated. As expected from the work of Segal and Reed (32), the resulting diploid was completely inviable on glucose medium, unlike the parental haploids (data not shown), and was completely rescued by the introduction of either CDC28 or CLB5 on low-copy-number plasmids (see below). A clb5::HIS3 pds1::URA3 cdc20::LEU2 ADE2::GALL-CDC20 strain was constructed in the W303 background by crossing a pds1 cdc20 GALL-CDC20 strain with a clb5::HIS3 strain (both provided by M. Shirayama), followed by tetrad analysis (the GALL promoter is an attenuated GAL promoter used in the CDC20 construct to reduce toxicity [34]). A clb1::URA3 clb2::LEU2 pCLB2/TRP1 strain was converted to clb1::ura3 by brief UV light exposure followed by FOA selection, then transformed with a CLB2/URA3 plasmid followed by loss of the pCLB2/TRP1 plasmid. The resulting strain was FOA sensitive and could be used to test rescue of clb1,2 lethality by transformation of plasmids followed by FOA selection. FOA-resistant plasmid loss segregants could then be tested for viability at various temperatures to examine the temperature sensitivity of rescue.

Plasmid constructions. CLB5 plasmids were based on HAdR1: RS314-CLB5-HA, described previously (8). Mutations were introduced into this plasmid by splice overlap extension PCR and gap repair in yeast, also as described previously (8). All CLB5 mutants were completely sequenced to confirm the intended mutation and the absence of fortuitous mutations due to PCR misincorporation. In some cases, CLB5 mutants with such extraneous mutations were repaired by gap repair using appropriate restriction fragments and digestion of the target plasmid. URA3 versions of these plasmids were constructed by cotransformation of a ura3 strain with the RS314-based (TRP1) plasmids and with PvuII-digested RS416; this results in gap repair of RS416 with the insert of the RS314-based plasmid. Myc-tagged versions of the CLB5-HA plasmids were constructed by replacing the NotI fragment containing the triple hemagglutinin (HA) tag with a NotI fragment containing nine copies of the myc tag (from a plasmid provided by A. Gartner).

Protein analysis. Immunoprecipitation, Western blotting, and an immune complex histone H1 kinase assay were performed on HA-tagged Clb5p, as described previously (8).

Sequence analysis and modeling. The protein database file for the cyclin A-Cdk2-p27 trimeric complex (26) was obtained from the Brookhaven database. Residues at the site of interaction between cyclin A and p27 were first determined by inspection using the program RasMol (documentation at http://www.bernstein-plus-sons.com/software/rasmol/doc/rasmol.html). Residues conserved between cyclin A and various yeast B-type cyclins were determined using the program CLUSTAL W (37). The surface representations of the van der Waals contacts between cyclin A and Cdk2 or p27 were produced using the program GRASP (24). The surface representations of conserved residues were produced using GRASP following replacement of B factors in the PDB file with a homology score derived from a CLUSTAL W alignment. For this analysis, bovine cyclin A, human cyclin E, Clb2p, and Clb5p were aligned. This set was chosen to avoid biasing the homology in favor of B-type-cyclin-specific residues (as would occur if more B-type cyclin sequences were included in the alignment). Sequence conservation was calculated from the alignment by averaging similarity scores at each position for all possible pairs of sequences (D. Jeruzalmi, unpublished software). Equivalence of nonidentical residues was established through use of the BLOSUM62 amino acid substitution matrix (14). Final renderings of the images in Fig. 1A were performed using the programs LOOSENGRASP (D. Jeruzalmi) and RASTER3D (22). A qualitatively similar profile of surface conservation was produced using RasMol (http://www.bernstein-plus-sons.com/software/rasmol/), by coloring of side chains that are identical in cyclin A and in a majority of yeast CLB1-6 proteins and fission yeast cig1, cig2, and cdc13 proteins, based on a CLUSTAL W alignment (36), using the PDB file 1JSU for the cyclin A-Cdk2-p27 structure (24). Therefore, the restriction to cyclins A and E, CLB5 protein, and CLB2 protein in Fig. 1A does not significantly affect the results.

	RESULTS

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Conservation of Clb5p residues involved in cyclin A-p27 binding. The inhibitor p27 binds to cyclin A-Cdk2 complexes by interacting at the p27 N terminus with a hydrophobic patch on cyclin A, on a distinct face from the Cdk binding interface. p27 also traverses the "top" face of the cyclin to interact with Cdk2, finally interacting with the ATP-binding region and displacing bound ATP (26). A comparison of the p27-interacting surface of cyclin A to surface residues of cyclin A that are conserved with yeast B-type cyclins demonstrates substantial overlap in the binding interface (Fig. 1A), where cyclin A interacts with the RXLF motif in p27 (26) (Fig. 1B). The degree of conservation at this surface is comparable to conservation of the Cdk binding interface (Fig. 1A). Thus, a region of cyclin A known to be required for interaction with the p27 inhibitor (26, 29) has been conserved throughout eukaryotic evolution, despite the absence of a p27 homolog in the yeast genome sequence identifiable by BLAST search (data not shown). Aside from the p27-binding surface and the Cdk-binding surface, there is very little conservation of surface residues (Fig. 1A and data not shown), suggesting that selective pressure for the p27-binding surface has been maintained throughout eukaryotic evolution. Most other residues conserved in the cyclin superfamily are internal residues presumably required for structural stability of the folded protein (data not shown).

View larger version (85K): [in this window] [in a new window]   FIG. 1.   (A) Interaction surfaces and conserved surface residues in cyclin A. Images of the cyclin A molecular surface are shown. The van der Waals interaction surfaces on cyclin A for p27 (top row) and Cdk2 (bottom row) are indicated in blue (left), using the cyclin A-Cdk2-p27 trimeric crystal structure (26) (PDB file 1JSU). The perspective for the Cdk2 interface is obtained by an approximately 90° rotation of the top set of images from left to right. Degrees of evolutionary conservation on these same faces of cyclin A between mammalian cyclins A and E and yeast cyclins Clb5 and Clb2 are indicated (middle) in shades of yellow to green (green is highest conservation; dark green is 100% identity). The images on the right are overlays of the left and middle images, merged using Photoshop software, and the positions of side chains mutated in this study are shown. For ease of understanding the mutagenic experiments described here, the numbering is for Clb5p. Sequence alignments from cyclin A to Clb5p are as follows: D220-D207; M210-M197; L214-L201; W217-W204; Q254-Q241; K266-K253; E295-E282; and F304-F291 (3). (B) Schematic of interaction between residues in cyclin A and the conserved RXLF motif in proteins that interact with cyclin A (1, 5, 26). The numbering is for Clb5p (see above for translation to cyclin A).

View larger version (79K): [in this window] [in a new window]   FIG. 2.   Clb5p interacts with p27 and p21 dependent on a conserved surface. Clb5-Gal4 DBD plasmids, wild type or mutant in conserved residues involved in p27-cyclin A binding (Fig. 1), and a vector control (all URA3 plasmids) were transformed into PJ469- (16). PJ469- (16) was transformed with Gal4-AD vector and Gal4-AD-Cdc28 fusion plasmid and with VP16-AD fusions with p27 and p21 (all LEU2 plasmids). (The VP16-AD vector was as negative for activation as was the GAL4-AD vector; data not shown). Diploids between the DBD and AD transformants were selected by mixing and growth on yeast extract-peptone-dextrose (YEPD), followed by replica plating to medium lacking leucine and containing uracil, selecting for both plasmids. Activation was scored by growth on medium lacking histidine, on medium lacking histidine but containing 10 mM 3-aminotriazole (AT) (both scoring the same GAL1 promoter-HIS3 fusion, with differing stringency [16]), and on medium lacking adenine (scoring a GAL2 promoter fused to ADE2 [16]).

The p27-binding domain in Clb5p is largely dispensable for Sic1p binding. The conservation of the p27-binding domain is surprising, given that S. cerevisiae does not contain a p27 homolog detectable by BLAST search. The Clb-Cdc28p kinase inhibitor Sic1p has been proposed to bind to Clb5p via a C-terminal sequence with some distant similarity to the cyclin A-interacting regions of p27 and other cyclin A targets (15, 39). We tested the ability of Sic1p to bind to Clb5p with and without a functional p27-binding domain. Sic1p (HA-tagged and overexpressed from the GAL1 promoter) could bind both wild-type and hpm mutant Clb5p, assayed by immunoprecipitating Sic1p-HA and assaying for myc-tagged Clb5p in the immunoprecipitates, although a moderate reduction in binding of the mutant protein was reproducibly observed (Fig. 3A). Similarly, when untagged Sic1p was expressed from the GAL1 promoter in cells expressing wild-type or hpm Clb5p-HA, immunoprecipitated Clb5p-associated kinase activity was strongly inhibited in both cases, although a low level of residual kinase activity was detected with the hpm mutant but not with the wild type (Fig. 3B). Since both of these assays require stable Sic1p-Clb5p binding to persist through multiple washes of the immunoprecipitates, Sic1p-Clb5p-hpm binding must be reasonably tight.

View larger version (24K): [in this window] [in a new window]   FIG. 3.   The p27-binding domain makes only a marginal contribution to Sic1p-Clb5p interaction. (A) 1255-5C (wild type, BF264-15D background) and 1255-5C-3 (GAL1::SIC1-HA, multiple-copy integrant) were transformed with TRP1 vector or CLB5-myc/TRP1 plasmids (wild type or hpm [M197A L201A W204A], expressed from the CLB5 promoter). Fresh stationary-phase cultures of transformants grown in medium lacking tryptophan were diluted in YEP-Gal medium and incubated for 6 h at 30°C, by which time most cells in the culture had adopted the long-budded morphology characteristic of Sic1p-mediated cell cycle arrest (30). Sic1p-HA was immunoprecipitated, and the immunoprecipitates were assayed for their content of Sic1p-HA and Clb5p-myc by Western blotting. An aliquot of the total extract was also assayed for Clb5p-myc. (B) 1255-5C and 1255-5C-6 (GAL1::SIC1, multiple-copy integrant) were transformed with TRP1 vector or CLB5-HA/TRP1 plasmids (wild type or hpm, expressed from the CLB5 promoter). GAL1::SIC1 was induced as for panel A. Following anti-HA immunoprecipitation, Clb5p-HA, associated Cdc28p, and histone H1 kinase activity were assayed.

Effects of mutations on Clb5p-associated kinase activity. Mutations affecting the putative p27-binding domain (hpm or Q241A) did not reduce Clb5p-associated kinase activity (8) (Fig. 4). This was expected, because these mutations are predicted to affect surface residues distant from the Cdk interface, based on the cyclin A-Cdk2 structure.

View larger version (36K): [in this window] [in a new window]   FIG. 4.   Clb5p-associated kinase activity. Transformants of a wild-type strain (W303-background strain ×1907-19C) with vector or with the indicated CLB5-HA constructs, expressed from the CLB5 promoter in RS316-based plasmids, were grown in medium lacking uracil to log phase, extracted, and immunoprecipitated with anti-HA antibody. Clb5-HAp in the immunoprecipitates was assayed by Western blotting (top). Associated histone H1 kinase activity was assayed by immune complex kinase assay (bottom). Most of these mutants were also tested in a wild-type BF264-15D strain (1255-5C) with similar results (data not shown).

Mutations in the Clb5p p27-binding region interfere with biological activity. The p27-binding domain of cyclin A might also be important for the interaction of cyclin A with substrates and other regulators, providing targeting and specificity to cyclin A-Cdk2 kinase activity by allowing interaction with proteins containing the "RXL" motif (1, 28, 29, 40). Indeed, recent structural analysis shows that the p107 substrate binds to cyclin A in a manner structurally very similar to the binding of p27 (5), including involvement of the hydrophobic patch residues and Q254 (equivalent to Clb5p Q241). If the p27-binding domain in Clb5p plays a similar role, then loss of the ability to interact with targets due to mutations in this domain should reduce or eliminate Clb5p biological activity without significantly reducing associated kinase activity.

View larger version (51K): [in this window] [in a new window]   FIG. 5.   Ability of CLB5 mutants to rescue clb3,4,5,6 inviability. (A) Transformants of K3418-1 (W303 background, a ura3 derivative of K3418, clb3,4,5,6 GAL-CLB5 [31]) with RS316-based plasmids containing the indicated CLB5-HA allele expressed from the CLB5 promoter were selected on galactose-containing medium lacking uracil. Individual transformants were grown on this medium to stationary phase and suspended in water, and 10-fold serial dilutions were plated on yeast extract-peptone-Gal (YEPGal) (GAL-CLB5 on) and yeast extract-peptone-dextrose (YEPD) (GAL-CLB5 off). Plates were incubated for 3 days at 30°C. (B) Transformants of ×1924 (BF264-15D diploid, clb3,4,5,6 pGAL-CLB5/URA3) with RS314-based plasmids containing the indicated CLB5-HA allele expressed from the CLB5 promoter were selected on ScGal-Trp. Individual transformants were grown on this medium to stationary phase and suspended in water, and 10-fold serial dilutions were plated on YEPGal (GAL-CLB5 on) and YEPD (GAL-CLB5 off). Plates were incubated for 3 days at 30°C or 35°C.

Mutations in the putative targeting domain of the mitotic cyclin CLB2 reduce Clb2-specific biological activity. Hydrophobic residues in helix 1 of the cyclin box are conserved in the cyclin superfamily (Fig. 1) (3). If this region forms a substrate-binding cleft, as proposed for cyclin A (5, 29), then the biological specificity of different cyclins might be due to interaction of the region with distinct targets based on the identity of these residues or flanking sequences. We mutated the three residues aligned with the cyclin A hydrophobic patch residues in the mitotic cyclin CLB2, N260, L264, and W267, to alanine. This CLB2-hpm mutation (expressed from the CLB5 promoter) was tested for its ability to rescue clb1,3,4 clb2-ts strain inviability and was found to be severely defective (Fig. 6A). In contrast to these results with clb1,3,4 clb2-ts lethality, the CLB2-hpm mutant had no significant defect in rescuing clb1,2 lethality (in the presence of CLB3,4), although CLB5 was completely unable to rescue (data not shown). We speculate that in the presence of CLB3,4, Clb2p may need to interact with a narrower range of substrates to provide rescue.

View larger version (72K): [in this window] [in a new window]   FIG. 6.   The hydrophobic patch mutation (N260A L264A W267A) alters the biological activity and specificity of Clb2p. (A) Transformants of K3080-1A (clb1,3,4 clb2-ts; a trp1::URA3 derivative of K3080 [2]) with the indicated plasmids (RS314 derivatives; all CLB coding sequences under control of the CLB5 promoter) were selected on medium lacking tryptophan at 23°C. Individual transformants were grown on this medium to stationary phase and suspended in water, and 10-fold serial dilutions were plated on yeast extract-peptone-dextrose (YEPD). Plates were incubated for 2 days at 35°C (semipermissive) or 37°C (nonpermissive) and for 3 days at 23°C (permissive). (B) Transformants of ×1924 (BF264-15D diploid, clb3,4,5,6 pGAL-CLB5/URA3) with RS314-based plasmids containing the indicated CLB coding sequences expressed from the CLB5 promoter were selected on galactose-containing medium lacking tryptophan. Individual transformants were grown on this medium to stationary phase and suspended in water, and 10-fold serial dilutions were plated on yeast extract-peptone-Gal (YEPGal) (GAL-CLB5 on) and YEPD (GAL-CLB5 off). Plates were incubated for 3 days at 30°C. (Note that for unknown reasons, the defect in rescue by CLB5::CLB2 seen in this strain background is not observed in the clb3,4,5,6 W303 background strain K3418-1; see the text.)

clb1,3,4 clb2-ts

clb3,4,5,6 pGAL-CLB5

Additional assays for CLB5 function. The results presented so far indicate that the essential functions shared by Clb3,4,5,6p require a substrate targeting domain in Clb5p for efficient performance. Lack of these four cyclins results in a defect in spindle assembly, as well as some defect in performance of DNA replication (31). We showed previously that efficient performance of the DNA replication function of Clb5p (9, 10, 31) requires a functional targeting domain (8). Other assays for Clb5p function were needed to determine if this requirement was general.

View larger version (57K): [in this window] [in a new window]   FIG. 7.   Cyclin- and targeting domain-restricted performance of Clb5p function in the cdc28-4 background. A diploid strain of genotype cdc28-4 clb5::ARG4 pGAL-CLB5/URA3 (×1922) was transformed with RS314-based plasmids containing the indicated CLB coding sequence under control of the CLB5 promoter. SF19, an RS314-based plasmid containing CDC28, was included as a control. Transformants were selected on galactose-containing medium lacking tryptophan at 23°C. Individual transformants were grown on this medium to stationary phase and suspended in water, and 10-fold serial dilutions were plated on yeast extract-peptone-Gal (YEPGal) (GAL-CLB5 on) and yeast extract-peptone-dextrose (YEPD) (GAL-CLB5 off). Plates were incubated for 5 days at 23°C.

View larger version (43K): [in this window] [in a new window]   FIG. 8.   Structural requirements for a cell cycle-inhibitory function of Clb5p. A cdc20 pds1 clb5 GALL-CDC20 strain (1896-1; W303 background) was transformed with the RS314-based plasmids containing the indicated CLB5 alleles. Transformants were selected on galactose-containing medium lacking tryptophan (Gal-trp) at 23°C. Individual transformants were grown on this medium to stationary phase and suspended in water, and 10-fold serial dilutions were plated on Gal-trp (GAL-CLB5 on) and dextrose-containing medium lacking tryptophan (Dex-trp) (GAL-CLB5 off). Plates were incubated for 6 days at 23°C.

	DISCUSSION

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Specific cyclin requirements for the budding yeast cell cycle. Of the nine budding yeast cyclins (three CLN and six CLB cyclins) known to activate the Cdc28p protein kinase, no single one is essential, and most double (and some triple and quadruple) mutants are viable as well (7, 21, 23), indicating substantial functional overlap among cyclins. In addition, Haase and Reed recently described a strain in which clb1,2,3,4,5,6 were deleted and replaced with CLB1 under the control of the strong GAL promoter (11). The viability of this strain is prima facie evidence that multiple B-type cyclins are not essential, provided that a single B-type cyclin is sufficiently overexpressed. However, this result does not imply that any B-type cyclin will similarly substitute for CLB1-6, and it appears likely from previous work that CLB5, at least, will not (8, 31). We showed that at comparable levels of expression, Clb2p was inefficient at substituting for Clb5p in the activation of DNA replication (8). We also recently found that the ability of Clb2p to block mitotic exit when overexpressed without its destruction box (36) could not be provided by Clb5-dbp at comparable levels of overexpression (15a). Thus, it appears likely that the apparent degeneracy of the cyclin system may hide a significant level of cyclin-specific targeting, which is detectable only at appropriate expression levels and in specific assays.

A conserved region of cyclins that may provide interaction with targets. Although many residues in cyclins exhibit significant conservation across the cyclin superfamily, most of these residues do not contribute significantly to the cyclin molecular surface (Fig. 1 and data not shown). These residues generally contribute to the buried hydrophobic core of the cyclin and are presumably required for adoption of the double 5-helix bundles that are characteristic of the entire cyclin superfamily (3, 4, 17). Conserved surface residues are mainly in the Cdk binding interface and in a portion of the surface distinct from the Cdk binding interface that contributes part of the p27 binding interface in cyclin A (Fig. 1). These residues are conserved across eukaryotic evolution, although interestingly they are frequently not as well conserved in distantly related cyclins with distinct functions such as the yeast CLN G1 cyclins (3). Budding yeast lacks an identifiable p27 homolog in the genome, but despite this, the ability of this region to bind to p27 has been conserved since divergence of metazoans and yeast, even though p27 may not have existed at the time of this divergence. Therefore, the "p27-binding" domain may have had interaction with RXL motif cyclin-Cdk targets (1) as its original and conserved role, and p27 may have evolved to fit this region. A dual role for this region in p27 binding and substrate interaction in the case of cyclin A was proposed by Schulman et al. (29), and interaction of this region with the p107 substrate was indeed demonstrated by recent structural analysis (5). The budding yeast Cdk inhibitor Sic1p has little or no homology to p27, and a functional p27-binding domain is not required for Clb5p-Sic1p binding to Sic1p and kinase inhibition (Fig. 4). We cannot rule out the possibility, though, that p27 homologs were present in a primordial eukaryote and then lost in the yeast lineage. Also, our data do not rule out the idea that Sic1p may occupy the hydrophobic patch region in Clb5p. Indeed, the slight reduction in binding that we detected (Fig. 3) is consistent with this idea, although such an interaction is clearly not required for binding and kinase inhibition. Candidate RXL-like sequences in an important C-terminal region of Sic1p have been described (15, 39).