Modulation of the phosphorylation state of proteins is a major mechanism of cellular control. The predominant focus of studies of phosphoregulation is the substrate specificity of protein kinases. Less emphasis is placed on the ability of phosphatases to selectively remove phosphates, partially due to the difficulty of studying these enzymes but also due to a widely held view that phosphatase specificity is limited. Nevertheless, phosphatase specificity can have profound effects: more complex information processing is possible in a regulatory system if phosphatases evolve the ability to distinguish between the phosphates placed on substrates by distinct kinases.
Control of the eukaryotic cell cycle provides the best understood example of a complex phosphoregulatory system. The mitotic cell cycle is driven by oscillations in cyclin-dependent kinase (Cdk) activity, leading to large numbers of phosphorylation events that trigger chromosome duplication in S phase and segregation in M phase (Morgan, 2007
). Cdks are also the master regulators of the two meiotic divisions (Benjamin et al., 2003
; Marston and Amon, 2004
; Petronczki et al., 2003
). An understanding of how Cdk function is modulated to transform the single mitotic division into the two meiotic divisions could provide insight into the evolution of phosphoregulation.
In a mitotic cell cycle, the chromosomes are duplicated once and only once. To prevent over-replication and genome instability, replication origins must fire only a single time per cell cycle. Robust control of origin firing is achieved by dividing the DNA replication process into two steps. First, in late mitosis and G1, pre-replicative complexes (pre-RCs) are loaded onto origins, which are thereby licensed to initiate. Second, at the onset of S phase, Cdk activity initiates replication by phosphorylating initiator proteins at the origin. Cdks also trigger disassembly of the pre-RC by phosphorylating its components, thereby ensuring that origins cannot be re-licensed until Cdks are inactivated at the end of the cell cycle (Bell and Dutta, 2002
; Blow and Dutta, 2005
; Diffley, 2004
; Nguyen et al., 2001
After S phase, the duplicated sister chromatids are held together by a protein complex called cohesin (Nasmyth, 2002
). When the sister chromatids are properly bi-oriented on the spindle in metaphase, a ubiquitin ligase called the anaphase-promoting complex (APC) is activated by its Cdc20 regulatory subunit (Peters, 2006
ubiquitinates the chaperone securin to signal its destruction, releasing separase, a protease that cleaves a cohesin subunit, allowing the two sister chromatids to be pulled to opposite poles of the spindle (Nasmyth, 2002
; Uhlmann et al., 2000
also decreases Cdk1 activity by promoting partial destruction of the cyclins (Wäsch and Cross, 2002
; Yeong et al., 2000
The APC is also regulated by a second activator subunit, Cdh1, whose activity is suppressed from S phase until the end of mitosis by inhibitory phosphorylation by Cdk1 (Jaspersen et al., 1999
; Zachariae et al., 1998
). In budding yeast, separase activation at anaphase, in addition to separating the sister chromatids, activates the phosphatase, Cdc14, which dephosphorylates and thereby activates Cdh1 (Stegmeier et al., 2002
). Ubiquitination by APCCdh1
then targets cyclins for destruction. Cdc14 also dephosphorylates and activates the Cdk1 inhibitor, Sic1. The combined activation of Cdh1 and Sic1 leads to a complete loss of Cdk1 activity. Cdc14 and other phosphatases then catalyze the dephosphorylation of Cdk1 substrates (D′Amours and Amon, 2004
), thereby resetting the cell to a G1 biochemical state and allowing the re-licensing of replication origins.
Meiosis is a specialized form of nuclear division that involves a single round of DNA replication followed by two rounds of chromosome segregation. The meiotic divisions exact some unique demands on the balance of activities of Cdk1 and counteracting phosphatases. Most notably, it is necessary to uncouple the chromosome and spindle cycles between the first (MI) and second (MII) meiotic divisions, such that after MI the spindle reduplicates but DNA replication remains completely inhibited. In budding yeast, it is known that activation of the Cdc14 phosphatase at anaphase I is required for the reduplication of the spindle (Marston et al., 2003
), but it is unclear why Cdc14 does not dephosphorylate pre-RC components or reset the meiotic cell to a G1 state.
Two models can explain the uncoupling of events between the meiotic divisions. One possibility is that the role of Cdc14 in driving mitotic exit is specifically restricted between MI and MII, preventing Cdh1 and Sic1 activation and thereby allowing Cdk1 to remain partially active. The partial destruction of cyclins by APCCdc20
might reduce Cdk1 activity to levels that allow spindle disassembly but still prevent the licensing of DNA replication origins. This model is supported by studies in the fission yeast Schizosaccharomyces pombe
, where the APC inhibitor, Mes1, prevents complete cyclin destruction after MI (Izawa et al., 2005
), and in the frog Xenopus laevis
, where Cdks must remain partially active between the meiotic divisions to prevent re-initiation of DNA synthesis (Iwabuchi et al., 2000
An alternative, but not mutually exclusive, model is that an auxiliary kinase activity, present during the meiotic divisions, inhibits origin licensing even in the absence of Cdk1 activity and in the presence of Cdc14 activity. Such auxiliary mechanisms probably exist in mammalian meiosis, where Cdk1 is completely inactivated between MI and MII (Choi et al., 1991
). This auxiliary activity should be capable of phosphorylating pre-RC components while permitting the spindle cycle to continue.
In the budding yeast Saccharomyces cerevisiae
, Ime2 is a good candidate for an auxiliary factor that allows the unique aspects of the meiotic divisions to occur robustly. Ime2 is a Cdk-related kinase, expressed only in meiosis, that is required for Sic1 destruction and the meiotic G1/S transition, fulfilling a role played by G1 cyclin-Cdk1 complexes in a mitotic cycle (Dirick et al., 1998
; Stuart and Wittenberg, 1998
). Ime2 is also required for the meiotic divisions (Benjamin et al., 2003
; Schindler and Winter, 2006
). An extra round of DNA replication is observed in cells lacking Ime2 after prolonged incubation in sporulation media, perhaps indicating that Ime2 helps prevent DNA re-replication during meiosis (Foiani et al., 1996
; Guttmann-Raviv et al., 2001
). A small number of potential Ime2 substrates have been described, the majority of which (Cdh1, Sic1 and the meiotic transcription factor Ndt80) are also Cdk1 substrates (Honigberg, 2004
). It therefore seems likely that Ime2 is acting as an auxiliary kinase to Cdk1 in meiosis.
Here, we demonstrate that Ime2 is capable of phosphorylating a large number of known Cdk1 substrates in vitro, including Sic1, Cdh1 and multiple pre-RC components. We define the consensus phosphorylation site for Ime2 and demonstrate that Ime2 and Cdk1 phosphorylate distinct sites both in vitro and in vivo. Although the two kinases phosphorylate different sites, Ime2 and Cdk1 phosphates can have the same effect on substrate function: both kinases inactivate Cdh1 and inhibit licensing of chromosome replication. Most importantly, Ime2-dependent phosphates are highly resistant to Cdc14. Ime2 therefore fulfills all of the requirements for a factor that modifies the overall kinase/phosphatase balance to allow the events of the meiotic divisions to occur faithfully.