The Bfa1/Bub2 GAP complex comprises a universal checkpoint required to prevent mitotic exit

At the end of the cell cycle, cyclin-dependent kinase (CDK) activity is inactivated to allow mitotic exit [1]. A protein phosphatase, Cdc14, plays a key role during mitotic exit in budding yeast by activating the Cdh1 component of the anaphase-promoting complex to degrade cyclin B (Clb) and inducing the CDK inhibitor Sic1 to inactivate Cdk1 [2]. To prevent mitotic exit when the cell cycle is arrested at G2/M, cells must prevent CDK inactivation. In the spindle checkpoint pathway, this is accomplished through Bfa1/Bub2, a heteromeric GTPase-activating protein (GAP) that inhibits Clb degradation by keeping the G protein Tem1 inactive [3-5]. Tem1 is required for Cdc14 activation. Here we show that in budding yeast, BUB2 and BFA1 are also required for the maintenance of G2/M arrest in response to DNA damage and to spindle misorientation. cdc13-1 bub2 and cdc13-1 bfa1 but not cdc13-1 mad2 double mutants rebud and reduplicate their DNA at the restrictive temperature. We also found that the delay in mitotic exit in mutants with misoriented spindles depended on BUB2 and BFA1, but not on MAD2. We propose that Bfa1/Bub2 checkpoint pathway functions as a universal checkpoint in G2/M that prevents CDK inactivation in response to cell-cycle delay in G2/M.     

Molecular Cloning and Characterization of a cDNA Clone That Encodes a Cdc2 Homolog from Nicotiana tabacum

We have isolated a cDNA clone (cdc2Nt1) that encodes a homologof p34^cdc2/CDC28 kinase from tobacco (Nicotiana tabacum). Thecdc2Ntl protein showed extensive similarity to other homologsof Cdc2 from plants. Complementation studies showed that thecdc2Ntl gene was able to overcome cell cycle arrest at boththe G1/S and the G2/M transitions of cdc28^ts mutants of buddingyeast, demonstrating that the cdc2Ntl protein was able to replacethe Cdc28 kinase at both the G1/S and the G2/M transitions.Analysis of gene expression demonstrated that the cdc2Ntl genewas transcribed constitutively throughout the cell cycle butthat it was preferentially expressed in actively dividing tobaccoBY-2 cells. (Received July 13, 1995; Accepted February 15, 1996)     

Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast

DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and protein phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2A^Cdc55 showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We show that chk1Δ and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of SWE1, which encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK (cdc28F19), also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, cdc55Δ showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.     

The Molecular Function of the Yeast Polo-like Kinase Cdc5 in Cdc14 Release during Early Anaphase

In the budding yeast Saccharomyces cerevisiae, Cdc14 is sequestered within the nucleolus before anaphase entry through its association with Net1/Cfi1, a nucleolar protein. Protein phosphatase PP2A^Cdc55 dephosphorylates Net1 and keeps it as a hypophosphorylated form before anaphase. Activation of the Cdc fourteen early anaphase release (FEAR) pathway after anaphase entry induces a brief Cdc14 release from the nucleolus. Some of the components in the FEAR pathway, including Esp1, Slk19, and Spo12, inactivate PP2A^Cdc55, allowing the phosphorylation of Net1 by mitotic cyclin-dependent kinase (Cdk) (Clb2-Cdk1). However, the function of another FEAR component, the Polo-like kinase Cdc5, remains elusive. Here, we show evidence indicating that Cdc5 promotes Cdc14 release primarily by stimulating the degradation of Swe1, the inhibitory kinase for mitotic Cdk. First, we found that deletion of SWE1 partially suppresses the FEAR defects in cdc5 mutants. In contrast, high levels of Swe1 impair FEAR activation. We also demonstrated that the accumulation of Swe1 in cdc5 mutants is responsible for the decreased Net1 phosphorylation. Therefore, we conclude that the down-regulation of Swe1 protein levels by Cdc5 promotes FEAR activation by relieving the inhibition on Clb2-Cdk1, the kinase for Net1 protein.     

CDK-Dependent Nuclear Localization of B-Cyclin Clb1 Promotes FEAR Activation during Meiosis I in Budding Yeast

Cyclin-dependent kinases (CDK) are master regulators of the cell cycle in eukaryotes. CDK activity is regulated by the presence, post-translational modification and spatial localization of its regulatory subunit cyclin. In budding yeast, the B-cyclin Clb1 is phosphorylated and localizes to the nucleus during meiosis I. However the functional significance of Clb1''s phosphorylation and nuclear localization and their mutual dependency is unknown. In this paper, we demonstrate that meiosis-specific phosphorylation of Clb1 requires its import to the nucleus but not vice versa. While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity. Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway. We discuss the significance of our results in relation to regulation of exit from meiosis I.     

Tobacco retinoblastoma-related protein phosphorylated by a distinct cyclin-dependent kinase complex with Cdc2/cyclin D in vitro

The retinoblastoma (Rb) protein was originally identified as a product of a tumour suppressor gene that plays a pivotal role in regulating both the cell cycle and differentiation in mammals. The growth-suppressive activity of Rb is regulated by phosphorylation with cyclin-dependent kinase (CDK), and inactivation of the Rb function is one of the critical steps for transition from the G1 to the S phase. We report here the cloning of a cDNA (NtRb1) from Nicotiana tabacum which encodes a Rb-related protein, and show that this gene is expressed in all the organs examined at the mRNA level. We have demonstrated that NtRb1 interacts with tobacco cyclin D by using yeast two-hybrid and in vitro binding assays. In mammals, cyclin D can assemble with CDK4 and CDK6, but not with Cdc2, to form active complexes. Surprisingly, tobacco cyclin D and Cdc2 proteins can form a complex in insect cells, which is able to phosphorylate tobacco Rb-related protein in vitro. Using immunoprecipitation with the anti-cyclin D anti-body, cyclin D can be found in a complex with Cdc2 in suspension-cultured tobacco BY-2 cells. These results suggest that the cdc2 gene modulates the cell cycle through the phosphorylation of Rb-related protein by forming an active complex with cyclin D in plants.     

Human T-lymphotropic virus type 1 oncoprotein tax promotes unscheduled degradation of Pds1p/securin and Clb2p/cyclin B1 and causes chromosomal instability

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia. The HTLV-1 transactivator, Tax, is implicated as the viral oncoprotein. Na?ve cells expressing Tax for the first time develop severe cell cycle abnormalities that include increased DNA synthesis, mitotic arrest, appearance of convoluted nuclei with decondensed DNA, and formation of multinucleated cells. Here we report that Tax causes a drastic reduction in Pds1p/securin and Clb2p/cyclin B levels in yeast, rodent, and human cells and a loss of cell viability. With a temperature-sensitive mutant of the CDC23 subunit of the anaphase-promoting complex (APC), cdc23(ts); a temperature-sensitive mutant of cdc20; and a cdh1-null mutant, we show that the diminution of Pds1p and Clb2p brought on by Tax is mediated via the Cdc20p-associated anaphase-promoting complex, APC(Cdc20p). This loss of Pds1p/securin and Clb2p/cyclin B1 occurred before cellular entry into mitosis, caused a G(2)/M cell cycle block, and was accompanied by severe chromosome aneuploidy in both Saccharomyces cerevisiae cells and human diploid fibroblasts. Our results support the notion that Tax aberrantly targets and activates APC(Cdc20p), leading to unscheduled degradation of Pds1p/securin and Clb2p/cyclin B1, a delay or failure in mitotic entry and progression, and faulty chromosome transmission. The chromosomal instability resulting from a Tax-induced deficiency in securin and cyclin B1 provides an explanation for the highly aneuploid nature of adult T-cell leukemia cells.     

Clb2 and the APC/CCdh1 regulate Swe1 stability

Swe1/Wee1 regulates mitotic entry by inhibiting Clb2-Cdk1 and its accumulation is involved in stress induced G2 arrest. The APC/C^Cdh1 substrates Cdc5, Clb2 and Hsl1 regulate Swe1 degradation. We observed that clb2Dcdh1D double mutant S. cerevisiae does not express any detectable levels of Swe1, presumably due to its constitutive degradation. This effect of Cdh1 inactivation is due to stabilization of Cdc5 and Hsl1, as expression of the non-degradable Cdc5^T29A in clb2D cells prevented Swe1 accumulation. Strikingly, expression of non-degradable Hsl1^mdb/mkb prevented Swe1 accumulation even in wild type Clb2 cells. Interestingly Swe1 accumulation could be reconstituted in all these mutants by eliciting a replication fork stress with hydroxyurea. Cells expressing the Clb2^ME mutant, that cannot bind Swe1, behaved like clb2D cells, and failed to accumulate Swe1 in the absence of Cdh1 or the presence of Cdc5^T29A. This suggests that for Swe1 to accumulate it must interact with Clb2. We further show that in the absence of Clb2, Hsl1 is no longer essential for Swe1 degradation. We hypothesize that Clb2-Cdk1 protects Swe1 from premature degradation until its Hsl1 mediated de-protection, which enables its Cdc5 mediated degradation. Swe1 levels are thus regulated by monitoring the levels of three major mitotic regulators.     

Novel alleles ofcdc13 andcdc2 isolated as suppressors of mitotic catastrophe inSchizosaccharomyces pombe

Cell cycle control in the fission yeastSchizosaccharomyces pombe involves interplay amongst a number of regulatory molecules, including thecdc2, cdc13, cdc25, weel, andmik1 gene products. Cdc2, Cdc13, and Cdc25 act as positive regulators of cell cycle progression at the G2/M boundary, while Wee1 and Mik1 play a negative regulatory role. Here, we have screened for suppressors of the lethal premature entry into mitosis, termed mitotic catastrophe, which results from simultaneous loss of function of both Wee1 and Mik1. Through such a screen, we hoped to identify additional components of the cell cycle regulatory network, and/or G2/M-specific substrates of Cdc2. Although we did not identify such molecules, we isolated a number of alleles of bothcdc2 andcdc13, including a novel wee allele ofcdc2, cdc2-5w. Here, we characterizecdc2-5w and two alleles ofcdc13, which have implications for the understanding of details of the interactions amongst Cdc2, Cdc13, and Wee1.     

The cyclin-dependent kinase inhibitory domain of the yeast Sic1 protein is contained within the C-terminal 70 amino acids

By inhibiting the activity of Cdc28/Clb cyclin-dependent protein kinase (CDK) complexes, Sic1 prevents the premature initiation of S phase in the yeast Saccharomyces cerevisiae. By testing a series of Sic1 truncation mutants, we have mapped the minimal domain necessary for Cdc28/Clb inhibition in vivo to the C-terminal 70 amino acids of Sic1. Site-directed mutagenesis was used to show that a sequence that matches the zRxL motif found in mammalian CDK inhibitors is essential for Sic1 function. This motif is not found in the Schizosaccharomyces CDK inhibitor p25^rum1, which appears to be a structural and functional homolog of Sic1. Based on the mutational data and sequence comparisons, we argue that Sic1 and p25^rum1 are structurally distinct from the known mammalian CDK inhibitors, but may bind CDK complexes in a manner more closely resembling CDK substrates like the retinoblastoma and E2F proteins. Received: 3 February 1999 / Accepted: 23 April 1999     

Hyperactive Cdc2 kinase interferes with the response to broken replication forks by trapping S.pombe Crb2 in its mitotic T215 phosphorylated state

Although it is well established that Cdc2 kinase phosphorylates the DNA damage checkpoint protein Crb2^53BP1 in mitosis, the full impact of this modification is still unclear. The Tudor-BRCT domain protein Crb2 binds to modified histones at DNA lesions to mediate the activation of Chk1 by Rad3^ATR kinase. We demonstrate here that fission yeast cells harbouring a hyperactive Cdc2^CDK1 mutation (cdc2.1w) are specifically sensitive to the topoisomerase 1 inhibitor camptothecin (CPT) which breaks DNA replication forks. Unlike wild-type cells, which delay only briefly in CPT medium by activating Chk1 kinase, cdc2.1w cells bypass Chk1 to enter an extended cell-cycle arrest which depends on Cds1 kinase. Intriguingly, the ability to bypass Chk1 requires the mitotic Cdc2 phosphorylation site Crb2-T215. This implies that the presence of the mitotic phosphorylation at Crb2-T215 channels Rad3 activity towards Cds1 instead of Chk1 when forks break in S phase. We also provide evidence that hyperactive Cdc2.1w locks cells in a G1-like DNA repair mode which favours non-homologous end joining over interchromosomal recombination. Taken together, our data support a model such that elevated Cdc2 activity delays the transition of Crb2 from its G1 to its G2 mode by blocking Srs2 DNA helicase and Casein Kinase 1 (Hhp1).     

Loss of CDC5 function in Saccharomyces cerevisiae leads to defects in Swe1p regulation and Bfa1p/Bub2p-independent cytokinesis

In many organisms, polo kinases appear to play multiple roles during M-phase progression. To provide new insights into the function of budding yeast polo kinase Cdc5p, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal domain. Here we show that, at a semipermissive temperature, the cdc5-3 mutant exhibited a synergistic bud elongation and growth defect with loss of HSL1, a component important for normal G(2)/M transition. Loss of SWE1, which phosphorylates and inactivates the budding yeast Cdk1 homolog Cdc28p, suppressed the cdc5-3 hsl1Delta defect, suggesting that Cdc5p functions at a point upstream of Swe1p. In addition, the cdc5-4 and cdc5-7 mutants exhibited chained cell morphologies with shared cytoplasms between the connected cell bodies, indicating a cytokinetic defect. Close examination of these mutants revealed delayed septin assembly at the incipient bud site and loosely organized septin rings at the mother-bud neck. Components in the mitotic exit network (MEN) play important roles in normal cytokinesis. However, loss of BFA1 or BUB2, negative regulators of the MEN, failed to remedy the cytokinetic defect of these mutants, indicating that Cdc5p promotes cytokinesis independently of Bfa1p and Bub2p. Thus, Cdc5p contributes to the activation of the Swe1p-dependent Cdc28p/Clb pathway, normal septin function, and cytokinesis.     

Novel alleles ofcdc13 andcdc2 isolated as suppressors of mitotic catastrophe inSchizosaccharomyces pombe

Cell cycle control in the fission yeastSchizosaccharomyces pombe involves interplay amongst a number of regulatory molecules, including thecdc2, cdc13, cdc25, weel, andmik1 gene products. Cdc2, Cdc13, and Cdc25 act as positive regulators of cell cycle progression at the G2/M boundary, while Wee1 and Mik1 play a negative regulatory role. Here, we have screened for suppressors of the lethal premature entry into mitosis, termed mitotic catastrophe, which results from simultaneous loss of function of both Wee1 and Mik1. Through such a screen, we hoped to identify additional components of the cell cycle regulatory network, and/or G2/M-specific substrates of Cdc2. Although we did not identify such molecules, we isolated a number of alleles of bothcdc2 andcdc13, including a novel wee allele ofcdc2, cdc2-5w. Here, we characterizecdc2-5w and two alleles ofcdc13, which have implications for the understanding of details of the interactions amongst Cdc2, Cdc13, and Wee1.     

Genetic interactions between Hsp90 and the Cdc2 mitotic machineryin the fission yeast Schizosaccharomyces pombe

In Schizosaccharomyces pombe, wee1 encodes a tyrosine kinase that inhibits entry into mitosis by phophorylating Cdc2, the universal cyclin-dependent kinase (Cdk) that regulates the G2/M transition in all eukaryotic cells. A search for suppressors of the G2 arrest caused by overexpression of wee1 led to the isolation of a new allele of swo1 (named swo1-w1), the gene coding for chaperone Hsp90, which is required to stabilise Wee1. The swo1-w1 allele carries a glycine to aspartic acid substitution at amino acid 155 that results in a partial loss of Hsp90 function. Cells bearing the swo1-w1 mutation in combination with the point mutation cdc2-33 or cdc2-M26 showed severe mitotic defects. Genetic interactions were not observed in combination with point mutations in other cdc genes, suggesting that Cdc2 specifically interacts with Hsp90. This synthetic lethal swo1-w1 cdc2-33 (or cdc2-M26) strain had normal levels of Cdc2 protein and histone H1 phosphorylation activity, indicating that Hsp90 is required to enable Cdc2 to interact with its mitotic substrates or regulators, rather than for its proper folding or stabilisation. In a wild-type background, swo1-w1 mutant cells were sensitive to temperature as well as to other stress agents, such as KCl, ethanol and formamide. Under these stressful growth conditions, the swo1-w1 cells displayed anaphase B arrest and aberrant septation patterns, indicating that a subset of proteins involved in mitosis and cytokinesis is highly dependent on chaperone Hsp90 for function.     

Candida albicans Cyclin Clb4 Carries S-Phase Cyclin Activity

Cyclin-dependent kinases (CDKs) are key regulators of eukaryotic cell cycle progression. The cyclin subunit activates the CDK and also imparts to the complex, at least in some cases, substrate specificity. Saccharomyces cerevisiae, an organism in which the roles of individual cyclins are best studied, contains nine cyclins (three G₁ cyclins and six B-type cyclins) capable of activating the main cell cycle CDK, Cdc28. Analysis of the genome of the pathogenic yeast Candida albicans revealed only two sequences corresponding to B-type cyclins, C. albicans Clb2 (CaClb2) and CaClb4. Notably, no homolog of the S. cerevisiae S-phase-specific cyclins, Clb5/Clb6, could be detected. Here, we performed an in vitro analysis of the activity of CaClb2 and CaClb4 and of three G₁ cyclins, as well as an analysis of the phenotype of S. cerevisiae cells expressing CaClb2 or CaClb4 instead of Clb5. Remarkably, replacement of CLB5 by CaCLB4 caused rapid diploidization of S. cerevisiae. In addition, both in vivo and in vitro analyses indicate that, in spite of the higher sequence similarity of CaClb2 to Clb5/Clb6, CaClb4 is the functional homolog of Clb5/Clb6. The activity of a CaClb2/CaClb4 cyclin hybrid suggests that the cyclin box domain of CaClb4 carries the functional specificity of the protein. These results have implications for our understanding of the evolution of specificity of the cell cycle cyclins.Cyclin-dependent kinases (CDKs) regulate many cellular processes but are best known for their role in the promotion of cell cycle progression. CDK activity depends on the binding of activatory subunits, the cyclins, which periodically appear during the cell cycle. Saccharomyces cerevisiae contains a single essential cell cycle CDK, S. cerevisiae Cdc28 (ScCdc28)/Cdk1, which in turn can be activated by nine cyclins: three G₁-type cyclins (Cln1, Cln2, and Cln3) and six B-type cyclins (S. cerevisiae Clb1 [ScCbl1] to ScCbl6) (34). Cln3 together with Cln1 and Cln2 (Cln1/2) induces a large class of cell cycle-regulated genes, including genes involved in S-phase initiation, such as the B-cyclins Clb5 and Clb6 (Clb5/6) (44, 47). Clb3 and Clb4 are expressed from early S phase to anaphase (22) and play a role in spindle orientation (Clb4) (31) and morphogenesis (Clb3 and Clb4) (25, 37), and Clb1 and Clb2 are expressed in G₂ (22) and play a role in entry into anaphase and spindle elongation (18). Genetic analysis suggests that the genes CLB1 to CLB4 have overlapping functions, as deletions of all four is lethal, but a mutant with deletion of all but CLB2 is still viable (18). Deletion of both CLB5 and CLB6 or of CLB5 alone is not lethal but results in a delay in S-phase initiation (41).The diverged yeast Schizosaccharomyces pombe contains one G₁ cyclin and three B-type cyclins. Studies indicating that a single S. pombe B-type cyclin, Cdc13, is sufficient to promote cell cycle progression led to the suggestion that the cyclin''s function is solely to periodically activate the CDK (17, 32). It is now clear, however, that the cyclin subunit imparts specificity to the CDK in at least some cases. Notably, biochemical analysis suggests that the different cellular function of the S. cerevisiae B-type cyclins may be based upon different substrate specificities: comparative analysis by in vitro phosphorylation of CDK substrates by Clb2-Cdk1 versus Clb5-Cdk1 indicates that whereas Clb2-Cdk1 carries a higher kinase activity toward most substrates, Clb5-Cdk1 is differentially much more active on a subclass of CDK substrates, including many S-phase proteins (30). A specific region of the cyclin box domain of Clb5 was identified that is essential for interaction with S-phase-specific substrates such as Orc6 (46) and Cdc6 (1).Candida albicans is a pathogenic yeast in the order Saccharomycetales, distantly related to S. cerevisiae. Given the cumbersome genetics of C. albicans, a diploid organism lacking a traditional sexual cycle, assignment of gene function in C. albicans has often been informed by sequence comparison with S. cerevisiae. However, the complete genome sequence of C. albicans, while including a Cdk1/Cdc28 homolog as well as sequence homologs of the cyclins Cln1/2, Cln3, Clb2, and Clb4—5 predicted Cdk1/Cdc28 cyclins in total—lacks an obvious homolog of Clb5/6. Here, we show by biochemical analysis and functional complementation that the homologous function of ScClb5 is carried by C. albicans Clb4 (CaClb4).     

The First Green Lineage cdc25 Dual-Specificity Phosphatase

The Cdc25 protein phosphatase is a key enzyme involved in the regulation of the G2/M transition in metazoans and yeast. However, no Cdc25 ortholog has so far been identified in plants, although functional studies have shown that an activating dephosphorylation of the CDK-cyclin complex regulates the G2/M transition. In this paper, the first green lineage Cdc25 ortholog is described in the unicellular alga Ostreococcus tauri. It encodes a protein which is able to rescue the yeast S. pombe cdc25-22 conditional mutant. Furthermore, microinjection of GST-tagged O. tauri Cdc25 specifically activates prophase-arrested starfish oocytes. In vitro histone H1 kinase assays and anti-phosphotyrosine Western Blotting confirmed the in vivo activating dephosphorylation of starfish CDK1-cyclinB by recombinant O. tauri Cdc25. We propose that there has been co-evolution of the regulatory proteins involved in the control of M-phase entry in the metazoan, yeast and green lineages.

Link to supplemental material:

http://www.landesbioscience.com/journals/cc/khadarooCC3-4-sup.pdf     

Grr1 functions in the ubiquitin pathway in Saccharomyces cerevisiae through association with Skp1

Cdc34, a ubiquitin-conjugating enzyme in Saccharomyces cerevisiae, is required for cell cycle progression. Sic1, an S-phase cyclin-dependent kinase (CDK) inhibitor, is a critical target of Cdc34-mediated ubiquitination. Other essential target protein(s) could be defined since cdc34 sic1 double mutants still arrest in G2 phase. To identify proteins which function in the Cdc34-dependent ubiquitin pathway, a series of extragenic suppressors of the cdc34-1 sic1 double mutations was isolated. One of them was found to be defective in GRR1, which is involved not only in glucose repression but also in G1 cyclin destabilization. However, neither lack of glucose repression nor stabilization of G1 cyclin caused the suppression of cdc34-1 sic1. Conversely, Grr1 overproduction in cdc34-1 sic1 cells impaired colony formation, even at the permissive temperature. A multicopy suppressor, MGO1, which rescued the growth defect associated with Grr1 overproduction was isolated, and found to be identical to SKP1. Furthermore, Grr1 bound Skp1 directly in vitro. These results strongly suggest that Grr1 functions in the ubiquitin pathway through association with Skp1.     

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.