Prior observations coupled with those reported in this study lead us to propose the following sequence of events in exit from mitosis. Accumulation of GTP-bound Tem1 triggered by division of the nucleus along the mother–bud axis leads to sequential activation of the protein kinases Cdc15 and Dbf2–Mob1 (Visintin and Amon, 2001
). The activated Dbf2–Mob1 translocates to the nucleus where it comes into contact with protein phosphatase Cdc14 (Stoepel et al., 2005
). Dbf2–Mob1 phosphorylates an unknown protein to dislodge Cdc14 from Net1, resulting in diffusion of Cdc14 throughout the nucleus. Dbf2–Mob1 also phosphorylates Cdc14 on several sites that flank C-terminal NLS sequences, thereby inhibiting the NLS. Because of this, phosphorylated Cdc14 molecules that escape to the cytoplasm cannot efficiently return to the nucleus and thus linger in the cytoplasm where they can dephosphorylate substrates such as Cdh1 and Swi5. Although phosphorylated Cdh1 and Swi5 are retained in the cytoplasm (Jans et al., 1995
; Knapp et al., 1996
; Jaquenoud et al., 2002
), the molecules dephosphorylated by cytoplasmic Cdc14 can now gain access to the nucleus, where they activate APC and expression of Sic1, respectively (Visintin et al., 1998
; Jaspersen et al., 1999
). Cdh1-APC and Sic1 extinguish cyclin B–Cdk activity, thereby enabling a return to G1 phase.
Several key pieces of evidence reported in this study support this model. First, Cdc14 contains a C-terminal NLS that is sufficient to direct localization of a GFP reporter (). Second, the activity of this NLS is transiently inhibited in late mitosis in a DBF2
-dependent manner (). Third, the NLS sequences of Cdc14 are flanked by multiple consensus phosphorylation sites for protein kinase Dbf2–Mob1 ( and ). We can confirm by direct MS analysis that at least some of these sites are phosphorylated in vivo (Fig. S3) and are phosphorylated by purified Dbf2–Mob1 in vitro (; and Fig. S1). Fourth, mutation (to Ala) of consensus and validated Dbf2 phosphorylation sites flanking the NLS leads to a loss of DBF2
-dependent regulation, such that NLS activity is no longer inhibited during late mitosis (). Fifth, this key regulatory control is not confined to the overexpressed NLS reporter, as it can also be observed with full-length Cdc14 expressed at subendogenous levels. Although wild-type Cdc14 is dispersed throughout the cell during late anaphase in a MEN-dependent manner (Shou et al., 1999
; Visintin et al., 1999
), a mutant with a nonphosphorylatable C-terminal NLS is not (). The role of regulated nuclear transport in control of mitotic exit described in this study adds to a long list of processes that are known to be controlled by phosphorylation-dependent inhibition of NLS activity, dating back to the observation that phosphorylation of consensus Cdk phosphorylation sites adjacent to NLS of Swi5 blocks its uptake into the nucleus during mitosis (Moll et al., 1991
The behavior of Cdc14 NLS mutants suggests that MEN promotes exit from mitosis by at least two distinct mechanisms. This interpretation is based on the observation that cells can be sustained by an allele of Cdc14 in which the Dbf2–Mob1 phosphorylation sites have been changed to Ala (Fig. S4). Further support is provided by the observation that a phosphomimetic mutant (CDC14-PS1,2E) can bypass cdc15Δ () when expressed from a CEN plasmid. If Cdc14 is the only target (or the only essential target) of Dbf2–Mob1, a nonphosphorylatable allele of Cdc14 should mimic a dbf2 loss of function mutant. This is clearly not the case, which suggests the existence of a second activity for Dbf2–Mob1 that can promote exit from mitosis even if Dbf2–Mob1 cannot phosphorylate the C-terminal NLS of Cdc14 and is required for exit from mitosis even if the C-terminal NLS is inactivated by mutation. However, it is clear that phosphorylation of Cdc14’s NLS contributes to mitotic exit because a mutant lacking these phosphorylation sites is unable to recover efficiently from a cdc15ts block and resume exit from mitosis ().
The work presented in this study represents a significant step forward in our understanding of exit from mitosis, in which we identify Cdc14 as the first known substrate of Dbf2–Mob1. Our findings thereby yield the first mechanistic insights into how this protein kinase acts directly to trigger mitotic exit, which is a topic that has remained an enigma since it was first discovered nearly a decade ago that Dbf2–Mob1 underlies the release of Cdc14 from the nucleolus during late anaphase (Shou et al., 1999
; Visintin et al., 1999
). However, a complete description of the molecular basis for the exit from mitosis awaits identification of the additional mechanisms mobilized by Dbf2–Mob1. What is the best candidate for this mechanism? Our data indicate that even though Cdc14 that lacks Dbf2–Mob1 phosphorylation sites is not efficiently localized to the cytoplasm during late mitosis, it is nevertheless released from the nucleolar tether protein Net1 (). This implies that the other way in which Dbf2–Mob1 acts is to disrupt the Cdc14–Net1 complex. A Cdc14 mutant lacking the C-terminal phosphorylation sites is essentially not a substrate for Dbf2–Mob1 in vitro (), but the corresponding phosphomimetic mutant still localizes primarily to the nucleolus in interphase cells (not depicted), suggesting that these phosphorylations do not disrupt the Cdc14–Net1 complex. If Dbf2–Mob1 activates parallel mechanisms to secure mitotic exit, then its second target need not be essential. Multiple proteins implicated in regulator of nucleolar silencing and telophase formation or function are plausible targets, including RNA Pol I, Sir2, Spo12, and Net1. Indeed, Net1 was identified in an in vitro proteomic screen for Dbf2–Mob1 substrates (Mah et al., 2005
). Evaluation of this possibility will require analysis of Net1 phosphorylation during exit from mitosis and phenotypic characterization of PS mutants.
A question posed by our results that remains unresolved is why is cytoplasmic retention of Cdc14 not essential for exit from mitosis? We consider two potential explanations. First, the CDC14-PS1,2A mutant may not be a complete PS null. We have attempted to address this by eliminating all three PS clusters, but mutations in PS3 have the paradoxical effect of suppressing cdc15ts (unpublished data), pointing to either a nonspecific effect of the mutations or further complexity in the regulation of Cdc14. Our second explanation relies on the notion that Cdc14 is constantly being exported to the cytoplasm during mitotic exit. Even though Cdc14-PS1,2A molecules are quickly resequestered in the nucleus as a result of action of the uninhibitable mutant C-terminal NLS, they may nonetheless be capable of exerting activity on their substrates during their brief sojourn in the cytoplasm. According to this viewpoint, the chief function of the regulatory circuit we have uncovered in this study is to delay the return of Cdc14 to the nucleus, thereby allowing it to achieve a higher concentration in the cytoplasm. Although this increase would not be as robust in cdc14-PS1,2A cells, it could be sufficient to exceed a threshold level of cytoplasmic Cdc14 activity to enable exit from mitosis unless the other activities of the Cdc15–Dbf2–Mob1 module have been circumscribed.
While this manuscript was in revision, a paper appeared from Chen et al. (2008)
on the phosphorylation of Clp1 by Sid2. Clp1 is the S. pombe
homologue of Cdc14, and Sid2 is a homologue of Dbf2. Although S. pombe
contains a pathway very similar to MEN, in fission yeast, this pathway controls the initiation of septation and not exit from mitosis. Chen et al. (2008)
show that Sid2 phosphorylates Clp1 on a cluster of sites in vitro and in vivo. The 14-3-3 protein Rad24 binds to phospho-Clp1, which retains Clp1 in the cytoplasm throughout cytokinesis. A nonphosphorylatable mutant, Clp1, returns to the nucleus prematurely during cytokinesis, and this renders the cells sensitive to perturbations of the cytokinetic machinery. There are some interesting differences in the phosphorylation sites of Cdc14 and Clp1. Although the target region of Clp1 spans ~125 residues and has a significant content of basic (10 Lys and Arg) and acidic (11 Glu and Asp) residues, the target region of Cdc14 is compact, with all sites clustered within a stretch of 25 amino acids (lacking Asp or Glu) and tightly interdigitated with four pairs of Lys or Arg that have NLS activity. NLS for Clp1 has not been mapped. It could be that both yeasts use different strategies to retain Cdc14/Clp1 in the cytoplasm during exit from mitosis/cytokinesis or that a combination of phosphorylation and binding of 14-3-3 protein inactivates a C-terminal NLS in both proteins.