We hypothesized that constitutively filamentous cdc28 mutants, which experience growth arrest at elevated temperatures would be valuable tools to identify connections between cell cycle and extracellular signals. Therefore, a cdc28 shuffle strain was created in which homozygous replacement of CDC28 with TRP1 gene was covered by a URA3 marked plasmid harboring CDC28. Transformation of this strain with a library of degenerate-PCR derived cdc28 alleles on a HIS3 marked plasmid, followed by negative selection against URA3 allowed screening colonies for mutants exhibiting enhanced filamentous growth on low nitrogen media. Five hundred colonies were selected 3. Secondary screening of these colonies for those that exhibited temperature sensitive growth at 35°C revealed 39 mutants with one to five amino acid substitutions.

Cyclin-dependent kinase is a bi-lobed protein with an amino terminal domain composed primarily of β-sheets and a larger carboxy-terminal domain containing several α-helices. Between these two domains lies the catalytic core, the opening of which is covered by the T-loop (activation loop) in the inactive monomeric state 20333435. We have previously modeled Cdc28 based on the crystal structure of the human CDK2 enzyme 336. Mapping the residues modified in the selected mutants on our model revealed that while some fell within the interior of the molecule, many were surface residues predicted to participate in binding to known essential partners (Fig. 1). Several mutations fell close to or within the ATP binding cleft (Fig. 1, green) and thus near Tyr19, the target of Swe1 mediated phosphorylation. Other mutations fell in the PSTAIRE helix involved in cyclin binding and catalytic function, or the activation loop, a domain involved in substrate recognition that includes Thr169, the target of Cak1 phosphorylation (Fig. 1, red). However, nearly one third of the alleles contained mutations that might impinge on Cdc28 interaction with Cks1 by altering contact residues, or residues underlying the Cks1 binding surface (Fig. 1, yellow and orange).

We were struck by the high number of mutations potentially affecting Cks1 binding, as this factor had yet to be directly linked to the apical-isotropic switch or filamentous differentiation. We examined if normal growth could be restored in these mutants by high copy CKS1. Indeed, 11 out of 15 alleles exhibited improved growth by CKS1 overexpression (Table 1). Of the 11 mutants suppressed by CKS1, four exhibited near wild type restoration of growth and morphology (1). The point mutants cdc28-E217V and cdc28-N238D were in this category suggesting that these alterations were less likely to directly affect additional partner associations. Although both mutations lie in the Cks1 binding region, co-crystal studies of the CDK2-Cks1 interface have demonstrated a direct contact only for the residue analogous to Cdc28 Glu217 with residues Glu63 and His94 in Cks1 37. Importantly, our bioinformatic analysis using algorithms designed by Chasman and Adams for assessment of the functional consequences of amino acid substitutions 38, indicated that mutation of residue E217 to valine should not otherwise affect the structure of monomeric Cdc28 (data not shown). E217 falls in the highly conserved GDSEID (214–219) motif, previously implicated in Cdc28 binding to Cks1 39. Mutations in residues 212, 213, and 215–218 were also identified by this screen, indicating that this domain of Cdc28 is particularly important for regulating morphological differentiation (Fig. 1).

To better study the genetic interactions of cdc28-E217V, we performed genomic replacement of endogenous CDC28 with cdc28-E217V. As expected, transformation of cdc28-E217V with high copy CKS1 similarly relieved mutant phenotypes as previously found in the cdc28-E217V shuffle strain (Fig. 2A). We also observed decreased pull-down of Cdc28-E217V compared to wild-type Cdc28 with beads coupled to the S. pombe Cks1 homolog p13^Suc1, under conditions of saturating amounts of Cdc28 and Cdc28-E217V proteins (Fig. 2B). These data strongly suggest a loss of association of Cdc28 with its essential partner Cks1 as the basis for the temperature sensitivity in this mutant. Cdc28-E217V also showed normal Cdc28 protein abundance and levels of Clb2- associated histone H1 kinase activity at permissive and restrictive temperatures (data not shown), in accordance with previous reports indicating that Cks1 does not affect mitotic CDK kinase activity 32.

However, we further questioned if the defects of this mutant were specifically attributable to its diminished interaction with Cks1. As a means to examine this possibility we tested overexpression of the CDK-activating kinase CAK1 in cdc28-E217V cells. Cak1 is an essential CDK activator that phosphorylates Cdc28 on Thr169 in the T loop to promote kinase activity, substrate accessibility and cyclin binding 4041. If Cak1 overexpression were to alleviate cdc28-E217V defects, it could indicate that cdc28-E217V harbors intrinsic defects in CDK function or in interaction with other binding partners or substrates. However, high copy CAK1 neither suppressed nor enhanced cdc28-E217V defects (Fig. 2A). Dephosphorylation of Thr169 has been shown to be likely mediated by phosphatases Ptc2 and Ptc3 42. We found that overexpression of PTC2 and PTC3 was similarly toxic to wild-type and cdc28-E217V cells (data not shown). Thus, cdc28-E217V phenotypes are not affected by alteration of activating phosphorylation of Cdc28. This further substantiates that cdc28-E217V defects are specific to interaction with Cks1.

Given the well-established roles of Cln1 and Clb2 cyclins at the G1/S and the G2/M transitions respectively, as well as their opposing effects on the apical-isotropic switch, we examined modulation of cdc28-E217V phenotypes by CLB2 and CLN1. CLB2 overexpression partly restored cdc28-E217V growth at semi-permissive temperature (Fig. 2A) while cdc28-E217V clb2 double mutant segregants were inviable, germinating to form only elongated, terminal buds (Fig. 2C). In turn, CLN1 overexpression diminished proliferation at semi-permissive temperature (Fig. 2A) while deletion of CLN1 partly restored growth (data not shown). These results further support that the phenotype of cdc28-E217V is due to delayed mitotic progression.

In filamentous growth signaling, Ras2 activates both the PKA pathway and the Kss1-MAPK cascade 43. We questioned if cdc28-E217V phenotypes were dependent on Ras2 signaling. Deletion of RAS2 restored near wild-type growth and morphology to cdc28-E217V cells (Fig. 3A,B). Two transcriptional regulators, Flo8 and Sfl1, function as downstream effectors of the PKA pathway 13. Deletion of FLO8 partially alleviated both the growth and morphology defects of cdc28-E217V (Fig. 3C,D), whereas deletion of SFL1 showed no effect (Fig. 4A). Deletion of TEC1, a transcription factor required for MAPK signaling, also did not alter cdc28-E217V growth or morphology (Fig. 4B, C). These results suggested that the Ras2-activated PKA pathway specifically impinged on cdc28-E217V through Flo8. Providing further confirmation of this connection we found that a high-copy plasmid harboring the cAMP phosphodiesterase PDE2, a negative regulator of PKA, suppressed cdc28-E217V defects (data not shown) 4445.

It has also been reported that the PKA pathway, but not the Kss1-MAPK cascade, may be required for activation of filamentation during amino acid starvation by the transcription factor Gcn4 46. In accordance, we observed that deletion of GCN4 provided some suppression to cdc28-E217V cells (Fig. 3E). As a final test we reexamined each of the Cks1-binding mutants for genetic interaction with FLO8. Interestingly less that half of these mutants were suppressed by deletion of FLO8 (Table 1). These observations indicate that the interaction of PKA through Flo8 with Cdc28 may be highly specific to a particular mechanism of CDK regulation.

These results supported the Ras2-activated PKA pathway via Flo8 to be an important regulator of Cdc28. Previous genetic evidence by La Valle and Wittenberg suggested that Swe1 regulation of Cdc28 may be an effector of PKA 18. Therefore, we directly tested if Swe1 function is modulated by the PKA pathway. We found that deletions of RAS2, FLO8, or the filamentous-growth specific PKA catalytic subunit TPK2 47 in otherwise wild-type cells each diminished Cdc28 Tyr19 phosphorylation, placing the PKA signaling upstream of Swe1 and Cdc28 (Figure 5A).

Given the relationship between PKA and Swe1, we also tested if cdc28-E217V is modulated by SWE1. Strikingly, deletion of SWE1 conferred suppression of both growth and morphology defects of cdc28-E217V (Fig. 5B, C). Again, we asked if these effects were specific to Swe1 or if the absence of any negative regulator of CDK function would improve cdc28-E217V defects. Toward examining this question, we observed that deletion of the Cdc28 stoichiometic inhibitor SIC1 had only subtle effects on growth of cdc28-E217V cells 48.

One interpretation of these data is that Swe1 functions upstream of Cks1. Nonetheless, arguing against a role for Swe1 binding or Cdc28 Tyr19 phosphorylation in modulating Cks1 binding, Cdc28 was pulled down by p13^suc1 beads to a similar extent from extracts of cells lacking SWE1 or its opposing phosphatase, MIH1 (Fig. 6A). An alternative explanation is that Cks1 antagonizes Cdc28 binding to or phosphorylation by Swe1. However, GST-Swe1 beads pulled down Cdc28-E217V and wild-type Cdc28 with similar levels, and independent of expression of high copy CKS1. In turn, increased Cdc28 Tyr19 phosphorylation was not detected in the mutant cells (data not shown). Together, these results argue against Swe1 and Cks1 binding competitively to Cdc28.

An alternative link to Cks1 is that Swe1 protein might accumulate and/or persist abnormally in cdc28-E217V cells. Western blot analysis of the cdc28-E217V cells revealed accumulation of Myc epitope-tagged Swe1, which appeared as a range of apparent molecular weights, a pattern previously ascribed to Swe1 phosphorylation 49 (Fig. 6B). To further pursue a role for Cks1 in Swe1 function, we examined cells lacking CDC55, a protein phosphatase 2A (PP2A) regulatory subunit that promotes Swe1 degradation. cdc55 mutants contain elevated levels of Swe1 and show morphological defects including slow growth, elongated morphology and increased Cdc28 Tyr19 hyperphosphorylation at 16°C, which are relieved by deletion of SWE1 50. Strikingly, high copy CKS1 also strongly suppressed the growth and morphology defects of cdc55 mutants and reduced Cdc28 Tyr19 phosphorylation (Fig. 7A, B, C). These results suggest that Cks1 opposes Swe1 accumulation and inhibitory phosphorylation.

Gene replacement, allele integration, yeast mating, and yeast transformation were as described 368. For genetic analyses, multiple tetratypes were examined. Yeast strains were constructed in the Sigma 1278b background unless indicated. Strains and plasmids were derived from the following:

A degenerate-PCR derived library of cdc28 mutants cloned into pRS413 was used to transform SKY750 3. The transformants were then replica-plated onto 5-Fluoroorotic acid (5-FOA)-containing media to evict the CEN URA3 CDC28 plasmid. About 50,000 5-FOA^R colonies were plated on synthetic low ammonia dextrose (SLAD) media. Five hundred colonies exhibiting enhanced filamentous growth were isolated and from these, 64 showed dramatically reduced growth at 35°C. Plasmids were rescued and re-introduced in SKY750. After 5-FOA treatment, 39 were found to confer a strong phenotype and were sequenced. Genomic integration of the E217V mutation into SKY2256 was as described 68. Tetrad analysis and DNA sequencing were used to confirm the integration.

The effect of polymorphism at E217 was studied as described 38. Molecular modeling was performed as described 3 using Swiss-Model and Swiss-PDB-Viewer 7071.

Yeast cultures were grown to saturation in rich or selective liquid media at 22°C. Saturated cultures were diluted serially at a ratio of 1/5 and spotted onto rich or selective agar medium. We have found the cdc28 mutants, possibly in part of because of the essential nature of many Cdc28 functions, have a tendency to acquire genomic and intragenic suppressor mutations. Because of this, we repeated spot testing at least four times for each experiment and a representative assay is shown. Each of the experimental groups shown within a given experiment were performed together on the same plate at each temperature. Images were acquired with a Pixera Professional camera on a Zeiss inverted microscope using brightfield illumination and contrast-enhanced in Adobe Photoshop. Magnification for detail of growth and morphology was performed in accord with previously published assessments of filamentation 7273.

Yeast extracts were prepared from log phase cultures by bead beating in buffer containing 50 mM Tris pH 7.6, 10% glycerol, 0.5% NP-40, 2 mM EDTA, 250 mM NaCl, 1 mM phenylmethylsulphonyl fluoride, and protease inhibitor cocktail (Roche). Protein content was determined by Bradford assay. For Western blot analysis, proteins separated by SDS-PAGE were transferred to nitrocellulose membranes, and blocked in TBS-Tween-20 containing 5% milk, or 5% BSA for anti-phospho-Tyr15 Cdc2 antibody (Cell Signaling Technologies). Membranes were probed with polyclonal anti-Myc (A14, Santa Cruz) at 1/1000 dilution, anti-PSTAIRE (Santa Cruz) at 1/500, anti-phospho-Tyr15 Cdc2 (Cell Signaling Technologies) at 1/1000, or anti-PGK (Molecular Probes) at 1/2500. Membranes were washed 3× with TBS-Tween-20 and probed with HRP-linked anti-rabbit or anti-mouse IgG (Amersham) at 1/2500. For p13^Suc1 pull-down reactions, 1 mg of protein extract was incubated overnight at 4°C with 50 μ l of p13^Suc1 Sepharose bead slurry (Upstate). Beads were washed five times with extract buffer, boiled in sample buffer and the supernatants subjected to SDS-PAGE. For binding assays using GST-Swe1 coated glutathione beads, 1 mg of yeast extract was incubated overnight with 100 μ l of 50% glutathione bead slurry and treated as for p13^Suc1 Sepharose beads. GST-Swe1 was isolated from E. coli BL21(DE3) plysS harboring pGEX-GST-SWE1 69, a kind gift from Jeremy Thorner. Bacteria were grown to an O.D.₆₀₀ of 0.6, induced for 3 h at 37°C with 0.2 mM IPTG, and briefly sonicated in PBS buffer containing protease inhibitor cocktail, 1 mM PMSF, 1 mM Orthovanadate, 0.5 mM DTT, and 1% Triton X-100. Glutathione beads were incubated with 1–2 mg of protein extract overnight at 4°C. GST-Swe1 coated beads were washed five times with PBS and incubated with extracts.