Table of contents

Drosophila cyclin dependent kinases

There are two Drosophila cdc2 homologs: cdc2 and cdc2c. The two Drosophila sequences are not more related to one another (56% identity) than to the yeast or human p34cdc homologs. Drosophila cdc2 provides p34cdc2 function in yeast, while cdc2c fails to do so. The inability of cdc2c to restore cell cycle progression in yeast complementation studies is not paralleled by a deficit in any sequences thought to characterize functional p34cdc2 kinases (Lehner, 1990).

In addition to the previously identified Drosophila cdc2 and cdc2c genes, four additional cdc2-related genes have been identified by using low stringency and polymerase chain reaction approaches . Sequence comparisons suggest that the four putative kinases represent the Drosophila homologs of vertebrate cdk4/6, cdk5, PCTAIRE, and PITSLRE kinases. Although the similarity between human and Drosophila homologs is extensive in the case of cdk5, PCTAIRE, and PITSLRE kinases (78%, 58%, and 65% identity in the kinase domain), only limited conservation is observed for Drosophila cdk4/6 (47% identity). However, like vertebrate cdk4 and cdk6, Drosophila cdk4/6 binds also to a D-type cyclin, according to the results of two-hybrid experiments in yeast. Northern blot analysis indicates that the four Drosophila kinases are expressed throughout embryogenesis. Expression in early embryogenesis appears to be ubiquitous according to in situ hybridization. Abundant expression is already detected at the start of embryogenesis. Both sequence conservation and expression patterns suggest that all of these kinases perform important cellular functions. This is true even for Drosophila Cyclin-dependent kinase 5 (Cdk5) protein which has been described as predominantly neuron specific in mice (Sauer, 1996).

cdc2 function in yeast

Mitosis is regulated by MPF (maturation promoting factor), the active form of Cdc2/28-cyclin B complexes. Increasing levels of cyclin B abundance and the loss of inhibitory phosphates from Cdc2/28 drives cells into mitosis, whereas cyclin B destruction inactivates MPF and drives cells out of mitosis. Cells with defective spindles are arrested in mitosis by the spindle-assembly checkpoint, which prevents the destruction of mitotic cyclins and the inactivation of MPF. The relationship between the spindle-assembly checkpoint, cyclin destruction, inhibitory phosphorylation of Cdc2/28, and exit from mitosis has been investigated. Budding yeast mad mutants lack the spindle-assembly checkpoint. Spindle depolymerization does not arrest them in mitosis because they cannot stabilize cyclin B. In contrast, a newly isolated mutant in the budding yeast CDC55 gene, which encodes a protein phosphatase 2A (PP2A) regulatory subunit, shows a different checkpoint defect. In the presence of a defective spindle, these cells separate their sister chromatids and leave mitosis without inducing cyclin B destruction. Despite the persistence of B-type cyclins, cdc55 mutant cells inactivate MPF. Two experiments show that this inactivation is due to inhibitory phosphorylation on Cdc28: phosphotyrosine accumulates on Cdc28 in cdc55 delta cells whose spindles have been depolymerized, and a cdc28 mutant that lacks inhibitory phosphorylation sites on Cdc28 allows spindle defects to arrest cdc55 mutants in mitosis with active MPF and unseparated sister chromatids. It is concluded that perturbations of protein phosphatase activity allow MPF to be inactivated by inhibitory phosphorylation instead of by cyclin destruction. Under these conditions, sister chromatid separation appears to be regulated by MPF activity rather than by protein degradation. The role of PP2A and Cdc28 phosphorylation in cell-cycle control is discussed; it is possibile that the novel mitotic exit pathway plays a role in adaptation to prolonged activation of the spindle-assembly checkpoint (Minshull, 1996).

Eukaryotic cell cycles are controlled by the activities of cyclin-dependent kinases (cdks). The major cdk in budding yeast, Saccharomyces cerevisiae, is Cdc28p. Activation of Cdc28p requires phosphorylation on threonine 169 and binding to a cyclin. Thr-169 is phosphorylated by the cdk-activating kinase (CAK), Cak1p, which was recently identified as the physiological CAK in budding yeast. Further characterization of yeast Cak1p is presented. Cak1p is dispersed throughout the cell as shown by immunofluorescence; biochemical subcellular fractionation confirms that most of the Cak1p is found in the cytoplasm. Cak1p is a monomeric enzyme in crude yeast lysates. Mutagenesis of potential sites of activating phosphorylation has little effect on the activity of Cak1p in vitro or in vivo. Furthermore, Cak1p contains no posttranslational modifications detectable by two-dimensional isoelectric focusing. Cak1p is a stable protein during exponential growth but its expression decreases considerably when cells enter stationary phase. In contrast, Cak1p levels oscillate dramatically during meiosis, reflecting regulation at both the transcriptional and post-translational level. The localization and regulation of Cak1p are in contrast to those of the known vertebrate CAK, p40(MO15) (Kaldis, 1998).

The Wee1 kinase (Drosophila homolog: Wee) inhibits entry into mitosis by phosphorylation of the Cdc2 kinase. Searching for multicopy suppressors that abolish this inhibition in the fission yeast, a novel gene was identified and named wos2; it encodes a protein with significant homology to human p23, an Hsp90-associated cochaperone. The deletion mutant has a modest phenotype, being heat-shock sensitive. Using antibodies raised against bacterially produced protein, it was determined that Wos2 is very abundant, ubiquitously distributed in the yeast cell, and its expression drops drastically as cells enter into early stationary phase, indicating that its function is associated with cell proliferation. In proliferating cells, the amount of Wos2 protein is not subjected to cell cycle regulation. However, in vitro assays demonstrate that this Hsp90 cochaperone is potentially regulated by phosphorylation. In addition to suppressing Wee1 activity, overproduction of Wos2 displays synthetic lethality with Cdc2 mutant proteins, indicating that this Hsp90 cochaperone functionally interacts with Cdc2. The level of Cdc2 protein and its associated H1 kinase activity under synthetic lethal conditions suggests a regulatory role for this Wos2-Cdc2 interaction. Hsp90 complexes are required for CDK regulation; the synergy found between the excess of Wos2 and a deficiency in Hsp90 activity suggests that Wos2 could specifically interfere with the Hsp90-dependent regulation of Cdc2. In vitro analysis indicates that the above genetic interactions could take place by physical association of Wos2 with the single CDK complex of the fission yeast. Expression of the budding yeast p23 protein (encoded by the SBA1 gene) in the fission yeast indicates that Wos2 and Sba1 are functionally exchangeable and therefore that properties described for Wos2 could be of wide significance in understanding the biological function of cochaperone p23 in eukaryotic cells (Munoz, 1999).

In eukaryotes, mitosis requires the activation of cdc2 kinase via association with cyclin B and dephosphorylation of the threonine 14 and tyrosine 15 residues. It is known that in the budding yeast Saccharomyces cerevisiae, a homologous kinase, Cdc28, mediates the progression through M phase, but it is not clear what specific mitotic function is served by its activation as a result of the dephosphorylation of an equivalent tyrosine (Tyr-19). Cells expressing cdc28-E19 (in which Tyr-19 is replaced by glutamic acid) perform Start-related functions, complete DNA synthesis, and exhibit high levels of Clb2-associated kinase activity but are unable to form bipolar spindles. The failure of these cells to form mitotic spindles is due to their inability to segregate duplicated spindle pole bodies (SPBs), a phenotype strikingly similar to that exhibited by a previously reported mutant defective in both kinesin-like motor proteins Cin8 and Kip1. The overexpression of SWE1, the budding-yeast homolog of wee1, also leads to a failure to segregate SPBs. These results imply that dephosphorylation of Tyr-19 is required for the segregation of SPBs. The requirement of Tyr-19 dephosphorylation for spindle assembly is also observed under conditions in which spindle formation is independent of mitosis, suggesting that the involvement of Cdc28/Clb kinase in SPB separation is direct. On the basis of these results, it is proposed that one of the roles of Tyr-19 dephosphorylation is to promote SPB separation (Lim 1998).

Exit from mitosis requires inactivation of mitotic cyclin-dependent kinases (CDKs). A key mechanism of CDK inactivation is ubiquitin-mediated cyclin proteolysis, which is triggered by the late mitotic activation of a ubiquitin ligase known as the anaphase-promoting complex (APC). Activation of the APC requires its association with substoichiometric activating subunits, termed Cdc20 and Hct1 (also known as Cdh1). The molecular function and regulation of the APC regulatory subunit Hct1 has been examined in Saccharomyces cerevisiae. Recombinant Hct1 activates the cyclin-ubiquitin ligase activity of APC isolated from multiple cell cycle stages. APC isolated from cells arrested in G1, or in late mitosis due to the cdc14-1 mutation, is more responsive to Hct1 than APC isolated from other stages. Hct1 is phosphorylated in vivo at multiple CDK consensus sites during cell cycle stages when activity of the cyclin-dependent kinase Cdc28 is high and APC activity is low. Purified Hct1 is phosphorylated in vitro at these sites by purified Cdc28-Clb2 complexes, and phosphorylation abolishes the ability of Hct1 to activate the APC in vitro. The phosphatase Cdc14, which is known to be required for APC activation in vivo, is able to reverse the effects of Cdc28 by catalyzing Hct1 dephosphorylation and activation. It is concluded that Hct1 phosphorylation is a key regulatory mechanism in the control of cyclin destruction. Phosphorylation of Hct1 provides a mechanism by which Cdc28 blocks its own inactivation during S phase and early mitosis. Following anaphase, dephosphorylation of Hct1 by Cdc14 may help initiate cyclin destruction (Jaspersen, 1999).

Cell cycle-specific proteolysis is critical for proper execution of mitosis in all eukaryotes. Ubiquitination and subsequent proteolysis of the mitotic regulators Clb2 and Pds1 depend on the cyclosome/APC and the 26S proteasome. Components of the cell cycle machinery in yeast, specifically the cell cycle regulatory cyclin-dependent kinase Cdc28 and a conserved associated protein Cks1/Suc1, interact genetically, physically, and functionally with components of the 26S proteasome. A mutation in Cdc28 (cdc28-1N) that interferes with Cks1 binding, or inactivation of Cks1 itself, confers stabilization of Clb2, the principal mitotic B-type cyclin in budding yeast. Surprisingly, Clb2-ubiquitination in vivo and in vitro is not affected by mutations in cks1, indicating that Cks1 is not essential for cyclosome/APC activity. However, mutant Cks1 proteins no longer physically interact with the proteasome, suggesting that Cks1 is required for some aspect of proteasome function during M-phase-specific proteolysis. Evidence is provided that Cks1 function is required for degradation of the anaphase inhibitor Pds1. Stabilization of Pds1 is partially responsible for the metaphase arrest phenotype of cks1 mutants because deletion of PDS1 partially relieves the metaphase block in these mutants (Kaiser, 1999).

Perhaps a clue to Cks1 function lies in the observation that more cdc28-1N is found in immunocomplexes with proteasome subunits than wild-type Cdc28. Similar observations have also been made in in vitro experiments, in which portions of the 19S proteasome cap have been reconstituted. Taking into account both differential binding of Cdc28-1N and Cdc28 to the proteasome and the independent association of Cks1 with the proteasome, it is suggested that Cks1 may function as a recycling factor for Cdc28. After proteolysis of Clb2, Cks1 binding to Cdc28 would release the kinase subunit from a putative receptor on the proteasome and allow new Cdc28/Clb2 complexes to bind. A defect in Cdc28/Cks1 complex formation would disrupt the recycling cycle and result in a blocked receptor leading to defects in Clb2 proteolysis. Consistent with this idea, Cdc28-1N confers a partial dominant-negative phenotype when overexpressed. This model is largely hypothetical and a more direct mechanism of regulation of proteasome activity by Cdc28/Cks1 is equally plausible (Kaiser, 1999).

In the budding yeast Saccharomyces cerevisiae, the cyclin-dependent kinases of the Clb/Cdc28 family restrict the initiation of DNA replication to once per cell cycle by preventing the re-assembly of pre-replicative complexes (pre-RCs) at replication origins that have already initiated replication. This assembly involves the Cdc6-dependent loading of six minichromosome maintenance (Mcm) proteins, Mcm2-7, onto origins. How Clb/Cdc28 kinases prevent pre-RC assembly is not understood. In living cells, the Mcm proteins are found to colocalize in a cell-cycle-regulated manner. Mcm2-4, 6 and 7 are concentrated in the nucleus in G1 phase, gradually exported to the cytoplasm during S phase, and excluded from the nucleus by G2 and M phase. Tagging any single Mcm protein with the SV40 nuclear localization signal makes all Mcm proteins constitutively nuclear. In the absence of functional Cdc6 (Drosophila homolog: CG5971), Clb/Cdc28 kinases are necessary and sufficient for efficient net nuclear export of a fusion protein between Mcm7 and the green fluorescent protein (Mcm7-GFP), whereas inactivation of these kinases at the end of mitosis coincides with the net nuclear import of Mcm7-GFP. In contrast, in the presence of functional Cdc6, which loads Mcm proteins onto chromatin, S-phase progression as well as Clb/Cdc28 kinases are required for Mcm-GFP export. It is proposed that Clb/Cdc28 kinases prevent pre-RC reassembly in part by promoting the net nuclear export of Mcm proteins. It is further proposed that Mcm proteins become refractory to this regulation when they load onto chromatin and must be dislodged by DNA replication before they can be exported. Such an arrangement could ensure that Mcm proteins complete their replication function before they are removed from the nucleus (Nguyen, 2000).

The activity of the cyclin-dependent kinase 1 (Cdk1), Cdc28, inhibits the transition from anaphase to G1 in budding yeast. CDC28-T18V, Y19F (CDC28-VF), a mutant that lacks inhibitory phosphorylation sites, delays the exit from mitosis and is hypersensitive to perturbations that arrest cells in mitosis. Surprisingly, this behavior is not due to a lack of inhibitory phosphorylation or increased kinase activity, but reflects reduced activity of the anaphase-promoting complex (APC), a defect shared with other mutants that lower Cdc28/Clb activity in mitosis. The activity of the APC depends on its interaction with the WD-40 protein Cdc20. CDC28-VF has reduced Cdc20-dependent APC activity in mitosis, but normal Hct1-dependent APC activity in the G1 phase of the cell cycle. The defect in Cdc20-dependent APC activity in CDC28-VF correlates with reduced association of Cdc20 with the APC. The defects of CDC28-VF suggest that Cdc28 activity is required to induce the metaphase to anaphase transition and initiate the transition from anaphase to G1 in budding yeast. Indeed, APC is phosphorylated by Cdc28 in budding yeast, and that a defect in this phosphorylation causes reduced Cdc20-dependent APC activity and contributes to the CDC28-VF phenotype (Rudner, 2000a).

Mutants that are impaired in mitotic Cdc28 function have difficulty leaving mitosis. This defect can be explained by a defect in APC phosphorylation, which depends on mitotic Cdc28 activity in vivo and can be catalyzed by purified Cdc28 in vitro. Mutating putative Cdc28 phosphorylation sites in three components of the APC (Cdc16, Cdc23, and Cdc27) makes the APC resistant to phosphorylation both in vivo and in vitro. The nonphosphorylatable APC has normal activity in G1, but its mitotic, Cdc20-dependent activity is compromised. These results show that Cdc28 activates the APC in budding yeast to trigger anaphase. Previous reports have shown that the budding yeast Cdc5 homolog, Plk, can also phosphorylate and activate the APC in vitro. Like cdc28 mutants, cdc5 mutants affect APC phosphorylation in vivo. However, although Cdc5 can phosphorylate Cdc16 and Cdc27 in vitro, this in vitro phosphorylation does not occur on in vivo sites of phosphorylation (Rudner, 2000b).

Organelles called centrosomes in metazoans or spindle pole bodies (SPBs) in yeast direct the assembly of a bipolar spindle that is essential for faithful segregation of chromosomes during mitosis. Abnormal accumulation of multiple centrosomes leads to genome instability, and has been observed in both tumor cells and cells with targeted mutations in tumor-suppressor genes. The defects that lead to centrosome amplification are not understood. The multiple-centrosome phenotype has been recapitulated in budding yeast by disrupting the activity of specific cyclin-dependent kinase (CDK) complexes. These observations are reminiscent of mechanisms that govern DNA replication, and show that specific cyclin/CDK activities function both to promote SPB duplication and to prevent SPB reduplication (Haase, 2001).

Evidence is presented that the major Schizosaccharomyces pombe CDK, Cdc2-cyclin B, influences recombinational repair of radiation-induced DNA double-strand breaks at two distinct stages during the G2 phase. At an early stage in homologous recombination, a defect in Cdc2 kinase activity caused by a single amino acid change in cyclin B affects the formation of Rhp51 (Rad51sp) foci in response to ionizing radiation in a process that is redundant with the function of Rad50. At a late stage in homologous recombination, low Cdc2-cyclin B activity prevents the proper regulation of topoisomerase III (Top3) function, disrupting a recombination step that occurs after the assembly of Rhp51 foci. This effect of Cdc2-cyclin B kinase on Top3 function is mediated by the BRCT-domain-containing checkpoint protein Crb2, thus linking checkpoint proteins directly with recombinational repair in G2. These data suggest a model in which CDK activity links processing of recombination intermediates to cell cycle progression via checkpoint proteins (Caspari, 2002).

As in many polarized cells, spindle alignment in yeast is essential and cell cycle regulated. A key step that governs spindle alignment is the selective binding of the Kar9 protein to only one of the two spindle pole bodies (SPBs). It has been suggested that cyclin-dependent kinase Cdc28, in complex with cyclin Clb4, associates only with the SPB in the mother cell and so prevents Kar9 binding to this SPB. However, the nonoverexpressed Clb4 associates with the budward-directed SPB through Kar9. Cdc28-Clb4 then uses Kar9 as a carrier to move from this SPB to the plus ends of astral microtubules, where Cdc28-Clb4 regulates the interactions between microtubule ends and subdomains of the bud cortex. In the absence of Cdc28-Clb4 activity (G1/S phase), astral microtubules interact with the bud tip in a manner dependent on actin, Myo2, and Kar9. Coincidentally with reaching the bud cortex in S phase, Cdc28-Clb4 facilitates the dissociation of the microtubule bud tip interaction and their capture by the bud neck. This transition prevents the preanaphase spindle from becoming prematurely pulled into the bud. Thus, Cdc28-Clb4 facilitates spindle alignment by regulating the interaction of astral microtubules with subdomains of the bud cortex (Maekawa, 2004).

The budding yeast Saccharomyces cerevisiae is a highly polarized model organism that divides perpendicular to the mother-bud axis at the predetermined site of bud emergence. This mode of division makes the alignment of the mitotic spindle along the mother-bud axis essential for survival. A precise temporal program of microtubule (MT)-cortex interactions ensures the correct positioning of the spindle. In G1/S phase of the cell cycle, astral MTs, which are organized by the yeast spindle pole body (SPB), are directed into the growing bud (the daughter cell). Astral MT interactions with the bud tip position the nucleus in the mother cell body close to the bud neck (Maekawa, 2004 and references therein).

The SPB then duplicates in early S by a conservative mechanism creating a newly formed and a pre-existing ('old') SPB. At this stage of the cell cycle, the Bim1-Kar9 complex has a key role in the alignment of the assembling spindle along the mother-bud axis. Bim1 is an EB1-like MT-binding protein and Kar9 shows weak similarities to the adenomatous polyposis coli (APC) tumor suppressor. The Bim1-Kar9 complex binds to only the old, but not the new, SPB and becomes transported from the old SPB to MT plus ends, where Kar9 ensures the association of MTs with the bud cortex. Because only the astral MTs organized by the old SPB have Bim1-Kar9 attached to it, and Kar9 interacts with the budward-directed actin motor Myo2, the astral MTs of the old SPB will be pulled into the bud, whereas the MTs of the new SPB fail to show persistent bud cortex interactions. This positions the old SPB close to the mother-bud junction (bud neck), whereas the new SPB becomes centered in the mother cell body (Maekawa, 2004 and references therein).

Coincident with the assembly of the bipolar spindle in S phase, astral MTs establish new interactions with the bud neck. These interactions require a functional septin ring and depend upon Kar9 function. During this phase of the cell cycle, astral MTs also depolymerize; this, in turn, further assists in the orientation of the preanaphase spindle along the mother-bud axis and positions the nucleus adjacent to the bud neck (Maekawa, 2004 and references therein).

In budding yeast the actin-dependent spindle orientation occurs in a restricted period that extends from G1 phase to shortly before anaphase. With anaphase onset, when the spindle extends into the bud, a second dynein-dependent pathway provides the force for nuclear movement. Genetic data indicate that the Kar9 and dynein pathways partially compensate for each other in spindle orientation. Failure of one pathway leads to defects in spindle alignment and chromosome instability, but mutants are viable. However, simultaneous loss of both pathways is lethal (Maekawa, 2004 and references therein).

Although these data indicate that the astral MT-cortex interactions are highly coordinated throughout the cell cycle, little is known about their regulation. However, it is clear that yeast cyclin-dependent kinase, Cdc28, is associated with SPBs and MTs. In early S phase, a fraction of Cdc28 is transported, together with Kar9, from the old SPB to MT ends directed into the bud. In contrast, Cdc28 is not associated with MTs organized by the new SPB. The role of Cdc28 in the regulation of the localization of the Bim1-Kar9 complex with the SPB and MTs is still a matter of debate. Although one study suggests that Cdc28 regulates the transport of Bim1 and Kar9, another report, in which overexpressed Clb4-GFP was used to define the localization of Clb4 to the new SPB, concludes that Cdc28-Clb4 prevents Bim1-Kar9 from binding to the new SPB. The current study has found that nonoverexpressed Clb4 selectively associates with the old SPB in a Kar9-dependent manner. The Cdc28-Clb4 complex becomes transported from the SPB to astral MT plus ends, where, in S phase, Clb4 facilitates the dissociation of MT ends from the bud tip region and their capture by the bud neck. Thus, Clb4 controls the interaction of astral MT plus ends with subdomains of the bud cortex (Maekawa, 2004).

Duplication of the Saccharomyces cerevisiae spindle pole body (SPB) once per cell cycle is essential for bipolar spindle formation and accurate chromosome segregation during mitosis. The role that the major yeast cyclin-dependent kinase Cdc28/Cdk1 plays in assembly of a core SPB component, Spc42, was investigated to better understand how SPB duplication is coordinated with cell cycle progression. Cdc28 is required for SPB duplication and Spc42 assembly, and Cdc28 was found to directly phosphorylate Spc42 to promote its assembly into the SPB. The Mps1 kinase, previously shown to regulate Spc42 phosphorylation and assembly, is also a Cdc28 substrate, and Cdc28 phosphorylation of Mps1 is needed to maintain wild-type levels of Mps1 in cells. Analysis of nonphosphorylatable mutants in SPC42 and MPS1 indicates that direct Spc42 phosphorylation and indirect regulation of Spc42 through Mps1 are two overlapping pathways by which Cdc28 regulates Spc42 assembly and SPB duplication during the cell cycle (Jaspersen, 2004).

A crystalline array of roughly 1000 copies of the coiled-coil protein Spc42 forms the SPB core layer, or central plaque, that spans the nuclear envelope. Spc42 is also a component of the satellite, the amorphous SPB precursor that forms adjacent to the existing SPB early in the cell cycle. Following activation of Cdc28-Cln in G1, the satellite expands into a structure known as the duplication plaque, which resembles the cytoplasmic half of a mature SPB. Spc42 plays a critical role in driving satellite expansion by virtue of its ability to self-assemble and to form a scaffold for assembly of other SPB components. Mutants that fail to properly assemble Spc42 into the SPB are also defective in SPB duplication, indicating that Spc42 assembly is a key event during SPB duplication (Jaspersen, 2004).

Spc42 protein levels do not change during the cell cycle, but SPC42 expression and Spc42 phosphorylation both increase during G1. While the overall importance of each in timing SPB duplication is not understood, synthesis of Spc42 has been proposed to result in a soluble pool of the protein that can assemble into the SPB in G1. Analysis of Spc42 assembly suggests that phosphorylation also promotes SPB duplication, and Spc42 is highly phosphorylated in vivo. The phosphorylation state of Spc42 is affected by mutations in CDC28, suggesting that regulation of Spc42 phosphorylation by Cdc28 could link SPB duplication with cell cycle progression (Jaspersen, 2004).

The timing of SPB/centrosome duplication during cell division is also regulated by the conserved Mps1 kinase, although there has been disagreement about whether the requirement for Mps1 during centrosome duplication extends to human cells. In yeast, Mps1 is required for multiple steps in SPB duplication. The role of Mps1 in satellite expansion into a duplication plaque is likely due to direct phosphorylation of Spc42 by Mps1. Alterations in Mps1 levels by overexpression or by expression of mutant forms of Mps1 disrupt SPB/centrosome duplication and function, highlighting the importance of proper Mps1 regulation. In mouse cells, Mps1 appears to be regulated by cyclin-dependent kinases since inhibition of Cdk2 activity leads to a dramatic decrease in Mps1 protein levels. The mechanism by which Cdk2 phosphorylation affects Mps1 stability is unclear, but the fact that Mps1 levels are low in G1-arrested yeast cells lacking Cdc28 activity suggests that a similar pathway might exist in yeast. Regulation of Mps1 by Cdc28 might be an indirect mechanism by which Cdc28 controls Spc42 phosphorylation and SPB duplication (Jaspersen, 2004).

To elucidate the function of Cdc28 during SPB duplication, its role in phosphorylation of Spc42 was analyzed. Cdc28 was found to directly phosphorylate Spc42 and is required for Spc42 assembly. In addition, Cdc28 indirectly regulates Spc42 through phosphorylation of Mps1, which stabilizes Mps1. Neither pathway is essential for cell viability, but disruption of both impairs SPB duplication. Thus, Cdc28 regulates Spc42 phosphorylation and SPB duplication by multiple overlapping mechanisms (Jaspersen, 2004).

Cdk1-dependent phosphorylation of Cdc13 coordinates telomere elongation during cell-cycle progression

Elongation of telomeres by telomerase replenishes the loss of terminal telomeric DNA repeats during each cell cycle. In budding yeast, Cdc13 plays an essential role in telomere length homeostasis, partly through its interactions with both the telomerase complex and the competing Stn1-Ten1 complex. Previous studies in yeast have shown that telomere elongation by telomerase is cell cycle dependent, but the mechanism underlying this dependence is unclear. In S. cerevisiae, a single cyclin-dependent kinase Cdk1 (Cdc28) coordinates the serial events required for the cell division cycle, but no Cdk1 substrate has been identified among telomerase and telomere-associated factors. This study shows that Cdk1-dependent phosphorylation of Cdc13 is essential for efficient recruitment of the yeast telomerase complex to telomeres by favoring the interaction of Cdc13 with Est1 rather than the competing Stn1-Ten1 complex. These results provide a direct mechanistic link between coordination of telomere elongation and cell-cycle progression in vivo (Li, 2009).

In most eukaryotes, the ends of linear chromosomes are capped by telomeres. Telomeres are essential for both the stability of linear chromosomes and the complete replication of genomic information. Telomeres are maintained by telomerase, whose activity is highly regulated. In yeast, more than 150 genes affect telomere length maintenance. In humans, haploinsufficiency for telomerase RNA in dyskeratosis congenita patients leads to progressive bone marrow failure and premature aging. Hence even a reduction in gene dosage has severe clinical consequences, highlighting the importance of telomerase regulation (Li, 2009).

In the budding yeast S. cerevisiae, the telomeric DNA consists of 250-350 base pairs of double-stranded C1-3A/TG1-3 repeats with a short single-stranded TG1-3 3' overhang. Sequence-specific telomeric DNA-binding proteins and their associated factors form a dynamic structure at telomeres. This dynamic nucleoprotein structure is essential for telomere silencing, telomere end protection, and telomere length regulation. In S. cerevisiae, two major telomeric DNA-binding proteins are Rap1 and Cdc13. While Rap1 binds duplex C1-3A/TG1-3 DNA repeats, Cdc13 binds the single-stranded TG1-3 3' overhang. Cdc13 has been implicated as the major regulator of telomere access by the telomerase complex. Cdc13 can interact with two distinct complexes that either positively or negatively regulate telomere elongation. Association of Cdc13 with Est1 is essential for recruitment of the telomerase holoenzyme, which contains the protein catalytic subunit Est2 and the integral RNA template TLC1, as well as Est1 and Est3. Cdc13 also interacts with the Stn1-Ten1 protein complex, which negatively regulates telomere elongation and plays an essential role in telomere end protection. Hence, Cdc13 tightly regulates telomere elongation through its interactions with both the Est1-Est2-Est3-TLC1 complex and the competing Stn1-Ten1 complex. However, the mechanism by which Cdc13 coordinates the binding of these two complexes during telomere elongation had not previously been analyzed (Li, 2009).

Telomere length homeostasis is maintained through a dynamic process. Telomerase extends the telomeric TG1-3 single-stranded DNA overhang by copying the intrinsic telomerase RNA template, while lagging strand synthesis by DNA polymerases α and δ is inferred to fill the terminal 5' gap during each cell cycle. Previous studies have shown that telomere elongation by telomerase is restricted to late S to G2 phases in vivo. The timing of telomere elongation in late S and G2 phases correlates with the binding of protein factors involved in telomere elongation, including Est1, Est2, and Cdc13. These data have suggested that the assembly of a functional telomerase complex at the telomeres is restricted to late S to G2 phases of the cell cycle (Li, 2009).

In budding yeast, the G-rich overhang is very short (about 13 bases) throughout most of the cell cycle but becomes longer around late S to G2 phases. Available data have suggested that Cdk1 activity is required for the generation of this extended 3' single-strand overhang, although the details of the mechanism were unknown. Increased binding of Cdc13 to such an extended 3' single-strand overhang could serve to subsequently recruit telomerase through the interaction of Cdc13 with Est1. Of the four telomerase components, Est1, Est2, TLC1, and Est3, only the expression of Est1 is cell cycle regulated, peaking at late S and G2 phases. Hence, expression of Est1 at late S and G2 phases likely restricts the assembly of functional telomerase complex to late S and G2 phases. How cells coordinate cell-cycle progression and the recruitment of telomerase complex to telomere has been an open question (Li, 2009).

In budding yeast, the regulation of cell-cycle progression depends on a single cyclin-dependent kinase, Cdk1 (Cdc28). Cdk1 regulates cell-cycle progression by phosphorylating hundreds of different protein substrates. The association with various, periodically expressed cyclins regulates the substrate specificity of Cdk1. While it is known that telomere elongation is cell cycle dependent, no Cdk1 substrates that regulate telomere elongation have been identified. This study shows that Cdk1-dependent phosphorylation of Cdc13 at threonine 308 plays an important role in the efficient recruitment of the telomerase complex to telomeres in late S to G2 phases of the cell cycle. Both the telomerase complex and the Stn1-Ten1 complex are recruited to telomeres during late S and G2 phases of cell-cycle progression. Therefore, since these two complexes counteract each other in terms of telomere length regulation, it is necessary to coordinate their binding to telomeres in order to ensure active telomerase function. The data show that phosphorylation of Cdc13 by Cdk1 plays such a key regulatory role, by coordinating the subsequent recruitment of these two complexes to telomeres to ensure proper telomere elongation and telomere protection (Li, 2009).

cdc2 function in invertebrates

Six protein kinases that belong to the family of cdc2-related kinases have been identified in Caenorhabditis elegans. Results from RNA interference experiments indicate that at least one of these kinases is required for cell-cycle progression during meiosis and mitosis. This kinase, encoded by the ncc-1 gene, is closely related to human Cdk1/Cdc2, Cdk2 and Cdk3 and yeast CDC28/cdc2+. It was asked whether ncc-1 acts to promote passage through a single transition or multiple transitions in the cell cycle, analogous to Cdks in vertebrates or yeasts. Five recessive ncc-1 mutations were isolated in a genetic screen for mutants that resemble larval arrested ncc-1(RNAi) animals. The results indicate that maternal ncc-1 product is sufficient for embryogenesis, and that zygotic expression is required for cell divisions during larval development. Cells that form the postembryonic lineages in wild-type animals do not enter mitosis in ncc-1 mutants, as indicated by lack of chromosome condensation and nuclear envelope breakdown. However, progression through G1 and S phase appears unaffected, as revealed by expression of ribonucleotide reductase, incorporation of BrdU and DNA quantitation. These results indicate that C. elegans uses multiple Cdks to regulate cell-cycle transitions and that ncc-1 is the C. elegans ortholog of Cdk1/Cdc2 in other metazoans, required for M phase in meiotic as well as mitotic cell cycles (Boxem, 1999).

Following ncc-1 RNAi treatment, fertilized oocytes fail to complete meiotic maturation. At the time of injection, a large number of germ precursor cells are in pachytene of meiosis I. These nuclei progress through meiotic prophase I but do not initiate metaphase, indicating that ncc-1 is required to promote the transition from prophase to metaphase in meiosis. A role in meiosis is consistent with the function of Cdk1/Cdc2 in other eukaryotes. In fact, Xenopus p34cdc2 was discovered by the biochemical characterization of maturation promoting factor (MPF), a cytoplasmic factor that induces meiotic maturation when injected into immature oocytes. Completion of meiosis I and II is a two step process in amphibians. Progesterone, or injection of MPF, triggers oocytes that are arrested in diplotene of prophase I to progress through meiosis I. Mature oocytes will subsequently arrest in metaphase of meiosis II and can be triggered by fertilization to complete meiosis. Meiotic maturation in C. elegans is induced by a factor in sperm that is independent from the sperm’s function at fertilization. In the absence of sperm, C. elegans oocytes arrest for prolonged periods in diakinesis. Future studies may reveal whether progesterone and the ‘sperm factor’ activate similar pathways that induce p34cdc2 and ncc-1, respectively, and trigger progression to meiotic metaphase I (Boxem, 1999 and references).

Does progression through meiotic prophase require ncc-1 function? In the RNA-injected animals, oocyte development appears normal and chromosomes become fully condensed. It cannot be exclude that inactivation of ncc-1 by RNAi is incomplete. However, several observations support effective inactivation. If inactivation was complete, entry into meiotic development, meiotic prophase progression, condensation of chromosomes to diakinesis bivalents and oocyte development can all occur independent of ncc-1. If these processes do require ncc-1 activity, this must be less activity than required for germ cell proliferation and for the transition from diakinesis to meiosis I. As yet, formation of diakinesis bivalents of normal morphology in the absence of Cdk1 activity has not been described in any eukaryote. During spermatogenesis in Drosophila, DNA condensation can occur in the apparent absence of Dmcdc2 activity. However, male meiosis in Drosophila does not involve meiotic recombination and attachment of bivalents by chiasmata. Nuclear envelope breakdown occurs slowly in mature ncc-1( RNAi) oocytes; by the time it is accomplished, meiosis would normally have been completed. The fact that degradation still occurs could indicate incomplete inactivation of ncc-1, as Cdk1 is believed to phosphorylate nuclear lamins and to trigger nuclear envelope breakdown in mitosis. However, other kinases have also been implicated in nuclear lamin phosphorylation, including S6 kinase II and protein kinase C. Such kinases may trigger nuclear envelope breakdown in the absence of ncc-1 activity (Boxem, 1999).

In budding yeast, vacuole inheritance is tightly coordinated with the cell cycle. The movement of vacuoles and several other organelles is actin-based and is mediated by interaction between the yeast myosin V motor Myo2 and organelle-specific adaptors. Myo2 binds to vacuoles via the adaptor protein Vac17, which binds to the vacuole membrane protein Vac8. This study shows that the yeast cyclin-dependent kinase Cdk1 phosphorylates Vac17 and that phosphorylation of Vac17 parallels cell cycle-dependent movement of the vacuole. Substitution of the Cdk1 sites in Vac17 decreases its interaction with Myo2 and causes a partial defect in vacuole inheritance. This defect is enhanced in the presence of Myo2 with mutated phosphorylation sites. Thus, Cdk1 appears to control the timing of vacuole movement. The presence of multiple predicted Cdk1 sites in other organelle-specific myosin V adaptors suggests that the inheritance of other cytoplasmic organelles may be regulated by a similar mechanism (Peng, 2008).

The C. elegans germline provides an excellent model for analyzing the regulation of stem cell activity and the decision to differentiate and undergo meiotic development. The distal end of the adult hermaphrodite germline contains the proliferative zone, which includes a population of mitotically cycling cells and cells in meiotic S phase, followed by entry into meiotic prophase. The proliferative fate is specified by somatic distal tip cell (DTC) niche-germline GLP-1 Notch signaling through repression of the redundant GLD-1 and GLD-2 pathways that promote entry into meiosis. This study describes characteristics of the proliferative zone, including cell cycle kinetics and population dynamics, as well as the role of specific cell cycle factors in both cell cycle progression and the decision between the proliferative and meiotic cell fate. Mitotic cell cycle progression occurs rapidly, continuously, with little or no time spent in G1, and with cyclin E (CYE-1) levels and activity high throughout the cell cycle. In addition to driving mitotic cell cycle progression, CYE-1 and CDK-2 also play an important role in proliferative fate specification. Genetic analysis indicates that CYE-1/CDK-2 promotes the proliferative fate downstream or in parallel to the GLD-1 and GLD-2 pathways, and is important under conditions of reduced GLP-1 signaling, possibly corresponding to mitotically cycling proliferative zone cells that are displaced from the DTC niche. Furthermore, GLP-1 signaling was found to regulate a third pathway, in addition to the GLD-1 and GLD-2 pathways and also independent of CYE-1/CDK-2, to promote the proliferative fate/inhibit meiotic entry (Fox, 2011).

Table of contents

cdc2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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