Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase.

To identify cyclin-dependent kinase mutants with relaxed cyclin requirements, CDC28 alleles were selected that could rescue a yeast strain expressing as its only CLN G1 cyclin a mutant Cln2p (K129A,E183A) that is defective for Cdc28p binding. Rescue of this strain by mutant CDC28 was dependent upon the mutant cln2-KAEA, but additional mutagenesis and DNA shuffling yielded multiply mutant CDC28-BYC alleles (bypass of CLNs) that could support highly efficient cell cycle initiation in the complete absence of CLN genes. By gel filtration chromatography, one of the mutant Cdc28 proteins exhibited kinase activity associated with cyclin-free monomer. Thus, the mutants' CLN bypass activity might result from constitutive, cyclin-independent activity, suggesting that Cdk targeting by cyclins is not required for cell cycle initiation.     

Isolation and Characterization of New Alleles of the Cyclin-Dependent Kinase Gene CDC28 with Cyclin-Specific Functional and Biochemical Defects

The G₁ cyclin Cln2 negatively regulates the mating-factor pathway. In a genetic screen to identify factors required for this regulation, we identified an allele of CDC28 (cdc28-csr1) that blocked this function of Cln2. Cln2 immunoprecipitated from cdc28-csr1 cells was completely defective in histone H1 kinase activity, due to defects in Cdc28 binding and activation by Cln2. In contrast, Clb2-associated H1 kinase and Cdc28 binding was normal in immunoprecipitates from these cells. cdc28-csr1 was significantly deficient in other aspects of genetic interaction with Cln2. The cdc28-csr1 mutation was determined to be Q188P, in the T loop distal to most of the probable Cdk-cyclin interaction regions. We performed random mutagenesis of CDC28 to identify additional alleles incapable of causing CLN2-dependent mating-factor resistance but capable of complementing cdc28 temperature-sensitive and null alleles. Two such mutants had highly defective Cln2-associated kinase, but, surprisingly, two other mutants had levels of Cln2-associated kinase near to wild-type levels. We performed a complementary screen for CDC28 mutants that could cause efficient Cln2-dependent mating-factor resistance but not complement a cdc28 null allele. Most such mutants were found to alter residues essential for kinase activity; the proteins had little or no associated kinase activity in bulk or in association with Cln2. Several of these mutants also functioned in another assay for CLN2-dependent function not involving the mating-factor pathway, complementing the temperature sensitivity of a cln1 cln3 cdc28-csr1 strain. These results could indicate that Cln2-Cdc28 kinase activity is not directly relevant to some CLN2-mediated functions. Mutants of this sort should be useful in differentiating the function of Cdc28 complexed with different cyclin regulatory subunits.     

Molecular Evolution Allows Bypass of the Requirement for Activation Loop Phosphorylation of the Cdc28 Cyclin-Dependent Kinase

Many protein kinases are regulated by phosphorylation in the activation loop, which is required for enzymatic activity. Glutamic acid can substitute for phosphothreonine in some proteins activated by phosphorylation, but this substitution (T169E) at the site of activation loop phosphorylation in the Saccharomyces cerevisiae cyclin-dependent kinase (Cdk) Cdc28p blocks biological function and protein kinase activity. Using cycles of error-prone DNA amplification followed by selection for successively higher levels of function, we identified mutant versions of Cdc28p-T169E with high biological activity. The enzymatic and biological activity of the mutant Cdc28p was essentially normally regulated by cyclin, and the mutants supported normal cell cycle progression and regulation. Therefore, it is not a requirement for control of the yeast cell cycle that Cdc28p be cyclically phosphorylated and dephosphorylated. These CDC28 mutants allow viability in the absence of Cak1p, the essential kinase that phosphorylates Cdc28p-T169, demonstrating that T169 phosphorylation is the only essential function of Cak1p. Some growth defects remain in suppressed cak1 cdc28 strains carrying the mutant CDC28 genes, consistent with additional nonessential roles for CAK1.     

The molecular chaperone Ydj1 is required for the p34CDC28-dependent phosphorylation of the cyclin Cln3 that signals its degradation.

The G1 cyclin Cln3 of the yeast Saccharomyces cerevisiae is rapidly degraded by the ubiquitin-proteasome pathway. This process is triggered by p34CDC28-dependent phosphorylation of Cln3. Here we demonstrate that the molecular chaperone Ydj1, a DnaJ homolog, is required for this phosphorylation. In a ydj1 mutant at the nonpermissive temperature, both phosphorylation and degradation of Cln3 were deficient. No change was seen upon inactivation of Sis1, another DnaJ homolog. The phosphorylation defect in the ydj1 mutant was specific to Cln3, because no reduction in the phosphorylation of Cln2 or histone H1, which also requires p34CDC28, was observed. Ydj1 was required for Cln3 phosphorylation and degradation rather than for the proper folding of this cyclin, since Cln3 produced in the ydj1 mutant was fully active in the stimulation of p34CDC28 histone kinase activity. Moreover, Ydj1 directly associates with Cln3 in close proximity to the segment that is phosphorylated and signals degradation. Thus, binding of Ydj1 to this domain of Cln3 seems to be essential for the phosphorylation and breakdown of this cyclin. In a cell-free system, purified Ydj1 stimulated the p34CDC28-dependent phosphorylation of the C-terminal segment of Cln3 and did not affect phosphorylation of Cln2 (as was found in vivo). The reconstitution of this process with pure components provides evidence of a direct role for the chaperone in the phosphorylation of Cln3.     

Dephosphorylation of threonine 169 of Cdc28 is not required for exit from mitosis but may be necessary for start in Saccharomyces cerevisiae.

Entry into mitosis requires activation of cdc2 kinase brought on by its association with cyclin B, phosphorylation of the conserved threonine (Thr-167 in Schizosaccharomyces pombe) in the T loop, and dephosphorylation of the tyrosine residue at position 15. Exit from mitosis, on the other hand, is induced by inactivation of cdc2 activity via cyclin destruction. It has been suggested that in addition to cyclin degradation, dephosphorylation of Thr-167 may also be required for exit from the M phase. Here we show that Saccharomyces cerevisiae cells expressing cdc28-E169 (a CDC28 allele in which the equivalent threonine, Thr-169, has been replaced by glutamic acid) are able to degrade mitotic cyclin Clb2, inactivate the Cdc28/Clb2 kinase, and disassemble the anaphase spindles, suggesting that they exit mitosis normally. The cdc28-E169 allele is active with respect to its mitotic functions, since it complements the mitosis-defective cdc28-1N allele. Whereas replacement of Thr-169 with serine affects neither Start nor the mitotic activity of Cdc28, replacement with glutamic acid or alanine renders Cdc28 inactive for Start-related functions. Coimmunoprecipitation experiments show that although Cdc28-E169 associates with mitotic cyclin Clb2, it fails to associate with the G1 cyclin Cln2. Thus, an unmodified threonine at position 169 in Cdc28 is important for interaction with G1 cyclins. We propose that in S. cerevisiae, dephosphorylation of Thr-169 is not required for exit from mitosis but may be necessary for commitment to the subsequent division cycle.     

Ubiquitination of the G1 cyclin Cln2p by a Cdc34p-dependent pathway.

Recombinant G1 cyclin Cln2p can bind to and stimulate the protein kinase activity of p34CDC28 (Cdc28p) in an extract derived from cyclin-depleted and G1-arrested Saccharomyces cerevisiae cells. Upon activating Cdc28p, Cln2p is extensively phosphorylated and conjugated with multiubiquitin chains. Ubiquitination of Cln2p in vitro requires the Cdc34p ubiquitin-conjugating enzyme, Cdc28p, protein phosphorylation and unidentified factors in yeast extract. Ubiquitination of Cln2p by Cdc34p contributes to the instability of Cln2p in vivo, as the rate of Cln2p degradation is reduced in cdc34ts cells. These results provide a molecular framework for G1 cyclin instability and suggest that a multicomponent, regulated pathway specifies the selective ubiquitination of G1 cyclins.     

Properties of Saccharomyces cerevisiae wee1 and its differential regulation of p34CDC28 in response to G1 and G2 cyclins.

Wee1 is a protein kinase that negatively regulates p34cdc2 kinase activity. We have identified a Saccharomyces cerevisiae wee1 homolog encoded by the SWE1 gene. SWE1 overexpression arrests cells in G2 with short spindles whereas deletion of SWE1 did not alter the cell cycle but did eliminate the G2 delay observed in mih1- mutants. Swe1 immunoprecipitates were capable of tyrosine phosphorylating and inactivating p34CDC28 complexed with Clb2, a G2-type cyclin, but not p34CDC28 complexed with Cln2, a G1-type cyclin, consistent with the inability of Swe1 overexpression to inhibit the G1/S transition. These results suggest that specific cyclin subunits target p34CDC28 for distinct regulatory controls which may be important for ensuring proper p34CDC28 function during the cell cycle.     

A Cdc28 mutant uncouples G1 cyclin phosphorylation and ubiquitination from G1 cyclin proteolysis

Proteolysis of the yeast G(1) cyclins is triggered by their Cdc28-dependent phosphorylation. Phosphorylated Cln1 and Cln2 are ubiquitinated by the SCF-Grr1 complex and then degraded by the 26 S proteasome. In this study, we identified a cak1 allele in a genetic screen for mutants that stabilize the yeast G(1) cyclins. Further characterization showed that Cln2HA was hypophosphorylated, unable to bind Cdc28, and stabilized in cak1 mutants at the restrictive temperature. Hypophosphorylation of Cln2HA could thus explain its stabilization. To test this possibility, we expressed a Cak1-independent mutant of Cdc28 (Cdc28-43244) in cak1 mutants and found that Cln2HA phosphorylation was restored, but surprisingly, the phospho-Cln2HA was stabilized. When bound to Cdc28-43244, Cln2HA was recognized and polyubiquitinated by SCF-Grr1. The Cdc28-43244 mutant thus reveals an unexpected complexity in the degradation of polyubiquitinated Cln2HA by the proteasome.     

The oncogenic activity of cyclin E is not confined to Cdk2 activation alone but relies on several other,distinct functions of the protein

We have previously shown that cyclin E can malignantly transform primary rat embryo fibroblasts in cooperation with constitutively active Ha-Ras. In addition, we demonstrated that high level cyclin E expression potentiates the development of methyl-nitroso-urea-induced T-cell lymphomas in mice. To further investigate the mechanism underlying cyclin E-mediated malignant transformation, we have performed a mutational analysis of cyclin E function. Here we show that cyclin E mutants defective to form an active kinase complex with Cdk2 are unable to drive cells from G(1) into S phase but can still malignantly transform rat embryo fibroblasts in cooperation with Ha-Ras. In addition, Cdk2 activation is not a prerequisite for the ability of cyclin E to rescue yeast triple cln mutations. We also find that the oncogenic properties of cyclin E did not entirely correspond with its ability to interact with the negative cell cycle regulator p27(Kip1) or the pocket protein p130. These findings suggest that the oncogenic activity of cyclin E does not exclusively rely on its ability as a positive regulator of G(1) progression. Rather, we propose that cyclin E harbors other functions, independent of Cdk2 activation and p27(Kip1) binding, that contribute significantly to its oncogenic activity.     

p34Cdc28-mediated control of Cln3 cyclin degradation.

Cln3 cyclin of the budding yeast Saccharomyces cerevisiae is a key regulator of Start, a cell cycle event in G1 phase at which cells become committed to division. The time of Start is sensitive to Cln3 levels, which in turn depend on the balance between synthesis and rapid degradation. Here we report that the breakdown of Cln3 is ubiquitin dependent and involves the ubiquitin-conjugating enzyme Cdc34 (Ubc3). The C-terminal tail of Cln3 functions as a transferable signal for degradation. Sequences important for Cln3 degradation are spread throughout the tail and consist largely of PEST elements, which have been previously suggested to target certain proteins for rapid turnover. The Cln3 tail also appears to contain multiple phosphorylation sites, and both phosphorylation and degradation of Cln3 are deficient in a cdc28ts mutant at the nonpermissive temperature. A point mutation at Ser-468, which lies within a Cdc28 kinase consensus site, causes approximately fivefold stabilization of a Cln3-beta-galactosidase fusion protein that contains a portion of the Cln3 tail and strongly reduces the phosphorylation of this protein. These data indicate that the degradation of Cln3 involves CDC28-dependent phosphorylation events.     

G1/S cyclin-dependent kinase regulates small GTPase Rho1p through phosphorylation of RhoGEF Tus1p in Saccharomyces cerevisiae

Rho1p is an essential small GTPase that plays a key role in the morphogenesis of Saccharomyces cerevisiae. We show here that the activation of Rho1p is regulated by a cyclin-dependent kinase (CDK). Rho1p is activated at the G1/S transition at the incipient-bud sites by the Cln2p (G1 cyclin) and Cdc28p (CDK) complex, in a process mediated by Tus1p, a guanine nucleotide exchange factor for Rho1p. Tus1p interacts physically with Cln2p/Cdc28p and is phosphorylated in a Cln2p/Cdc28p-dependent manner. CDK phosphorylation consensus sites in Tus1p are required for both Cln2p-dependent activation of Rho1p and polarized organization of the actin cytoskeleton. We propose that Cln2p/Cdc28p-dependent phosphorylation of Tus1p is required for appropriate temporal and spatial activation of Rho1p at the G1/S transition.     

Ras Signals to the Cell Cycle Machinery via Multiple Pathways To Induce Anchorage-Independent Growth

Several specific cell cycle activities are dependent on cell-substratum adhesion in nontransformed cells, and the ability of the Ras oncoprotein to induce anchorage-independent growth is linked to its ability to abrogate this adhesion requirement. Ras signals via multiple downstream effector proteins, a synergistic combination of which may be required for the highly altered phenotype of fully transformed cells. We describe here studies on cell cycle regulation of anchorage-independent growth that utilize Ras effector loop mutants in NIH 3T3 and Rat 6 cells. Stable expression of activated H-Ras (12V) induced soft agar colony formation by both cell types, but each of three effector loop mutants (12V,35S, 12V,37G, and 12V,40C) was defective in producing this response. Expression of all three possible pairwise combinations of these mutants synergized to induce anchorage-independent growth of NIH 3T3 cells, but only the 12V,35S-12V,37G and 12V,37G-12V,40C combinations were complementary in Rat 6 cells. Each individual effector loop mutant partially relieved adhesion dependence of pRB phosphorylation, cyclin E-dependent kinase activity, and expression of cyclin A in NIH 3T3, but not Rat 6, cells. The pairwise combinations of effector loop mutants that were synergistic in producing anchorage-independent growth in Rat 6 cells also led to synergistic abrogation of the adhesion requirement for these cell cycle activities. The relationship between complementation in producing anchorage-independent growth and enhancement of cell cycle activities was not as clear in NIH 3T3 cells that expressed pairs of mutants, implying the existence of either thresholds for these activities or additional requirements in the induction of anchorage-independent growth. Ectopic expression of cyclin D1, E, or A synergized with individual effector loop mutants to induce soft agar colony formation in NIH 3T3 cells, cyclin A being particularly effective. Taken together, these data indicate that Ras utilizes multiple pathways to signal to the cell cycle machinery and that these pathways synergize to supplant the adhesion requirements of specific cell cycle events, leading to anchorage-independent growth.     

Enhanced Cell Polarity in Mutants of the Budding Yeast Cyclin-dependent Kinase Cdc28p

The yeast cyclin-dependent kinase Cdc28p regulates bud morphogenesis and cell cycle progression via the antagonistic activities of Cln and Clb cyclins. Cln G1 cyclins direct polarized growth and bud emergence, whereas Clb G2 cyclins promote isotropic growth of the bud and chromosome segregation. Using colony morphology as a screen to dissect regulation of polarity by Cdc28p, we identified nine point mutations that block the apical-isotropic switch while maintaining other functions. Like a clb2 Delta mutation, each confers tubular bud shape, apically polarized actin distribution, unipolar budding, and delayed anaphase. The mutations are all suppressed by CLB2 overexpression and are synthetically lethal with a CLB2 deletion. However, defects in multiple independent pathways may underlie their common phenotype, because the mutations are scattered throughout the CDC28 sequence, complement each other, and confer diverse biochemical properties. Glu12Gly, a mutation that alters a residue involved in Swe1p inhibition of Cdc28p, was unique in being suppressed by deficiency of SWE1 or CLN1. With wild-type CDC28, filament formation induced by CLN1 overexpression was markedly decreased in a SWE1 deletion. These results suggest that Swe1p, via inhibition of Clb2p/Cdc28p, may mediate much of the effect of Cln1p on filamentous morphogenesis.     

Mutations in CDC14 result in high sensitivity to cyclin gene dosage in Saccharomyces cerevisiae

We screened for mutations that resulted in lethality when the G1 cyclin Cln2p was overexpressed throughout the cell cycle in Saccharomyces cerevisiae. Mutations in five complementation groups were found to give this phenotype, and three of the mutated genes were identified as MEC1, NUP170, and CDC14. Mutations in CDC14 may have been recovered in the screen because Cdc14p may reduce the cyclin B (Clb)-associated Cdc28 kinase activity in late mitosis, and Cln2p may normally activate Clb-Cdc28 kinase activity by related mechanisms. In agreement with the idea that cdc14 mutations elevate Clb-Cdc28 kinase activity, deletion of the gene for the Clb-Cdc28 inhibitor Sic1 caused synthetic lethality with cdc14-1, as did the deletion of HCT1, which is required for proteolysis of Clb2p. Surprisingly, deletion of the gene for the major B-type cyclin, CLB2, also caused synthetic lethality with the cdc14-1 mutation. The clb2 cdc14 strains arrested with replicated but unseparated DNA and unseparated spindle pole bodies; this phenotype is distinct from the late mitotic arrest of the sic1::TRP1 cdc14-1 and the cdc14-1 hct1::LEU2 double mutants and of the cdc14 CLN2 overexpressor. We found genetic interactions between CDC14 and the replication initiator gene CDC6, extending previous observations of interactions between the late mitotic function of Cdc14p and control of DNA replication. We also describe genetic interactions between CDC28 and CDC14. Received: 24 May 1999 / Accepted: 19 October 1999     

A Highlights from MBoC Selection: PP1 promotes cyclin B destruction and the metaphase–anaphase transition by dephosphorylating CDC20

Ubiquitin-dependent proteolysis of cyclin B and securin initiates sister chromatid segregation and anaphase. The anaphase-promoting complex/cyclosome and its coactivator CDC20 (APC/C^CDC20) form the main ubiquitin E3 ligase for these two proteins. APC/C^CDC20 is regulated by CDK1-cyclin B and counteracting PP1 and PP2A family phosphatases through modulation of both activating and inhibitory phosphorylation. Here, we report that PP1 promotes cyclin B destruction at the onset of anaphase by removing specific inhibitory phosphorylation in the N-terminus of CDC20. Depletion or chemical inhibition of PP1 stabilizes cyclin B and results in a pronounced delay at the metaphase-to-anaphase transition after chromosome alignment. This requirement for PP1 is lost in cells expressing CDK1 phosphorylation–defective CDC20^6A mutants. These CDC20^6A cells show a normal spindle checkpoint response and rapidly destroy cyclin B once all chromosomes have aligned and enter into anaphase in the absence of PP1 activity. PP1 therefore facilitates the metaphase-to-anaphase transition by promoting APC/C^CDC20-dependent destruction of cyclin B in human cells.