All dividing cells must replicate their chromosomes and deliver a complement of genetic material to each daughter cell with extreme fidelity. During the latter stages of cell division it is of particular importance that chromosome segregation and spindle positioning are properly coordinated temporally and spatially with cytokinesis.
Much of our understanding of how late mitotic events are regulated has come from studies in budding and fission yeast. In the budding yeast S. cerevisiae
, a signaling pathway called the mitotic exit network (MEN) initiates mitotic exit only after correct positioning of the spindle in the mother-neck bud [1
]. The MEN is a GTPase-driven signaling network regulated by the small Ras-like molecule Tem1p that becomes activated upon entry of the yeast spindle pole body (SPB) into the bud [4
]. The downstream effector of the mitotic exit network is the Cdc14p dual-specificity phosphatase, which promotes Cdk inactivation by dephosphorylating specific substrates including the Cdk inhibitor Sic1p, the APC activator Cdh1p, and the transcription factor Swi5 [5
Cdc14p activity in S. cerevisiae
appears to be regulated primarily through its subcellular localization. During interphase of the cell cycle, Cdc14p is sequestered in the nucleolus by its stoichiometric inhibitor Net1p [8
] and is released from the nucleolus in two phases during mitosis [11
]. The first phase occurs at the metaphase-anaphase transition, when APCCdc20
-directed destruction of the anaphase inhibitor securin Pds1 activates the separase Esp1 to initiate sister chromatid separation. Esp1, Slk19p, Spo12p and Cdc5p, collectively known as the FEAR network for Cdc f
elease), promote the release of Cdc14 from the nucleolus in early anaphase in a manner that is not well understood [11
]. During this first phase, only a subset of Cdc14p is released and transiently localizes to the SPB. It has been postulated that the SPB localization of Cdc14 primes the activity of the MEN, perhaps by dephosphorylating and inactivating the Tem1p GAP inhibitor Bfa1p [11
]. The requirement of Esp1 for the first stage of Cdc14 release provides an elegant mechanism to ensure that mitotic exit proceeds only after prior passage through the metaphase to anaphase transition. The second phase of Cdc14 release occurs upon proper spindle orientation and activation of Tem1p, when Cdc14p becomes fully released from the nucleolus and localizes throughout the cell in an activated form. This second phase requires the activity of all gene products of the MEN, although the mechanism by which the MEN promotes Cdc14p release from Net1p is not well understood [8
]. No homolog of budding yeast Net1p has been identified in any other species, suggesting that Net1p inhibition of Cdc14p may be unique to budding yeast.
In the fission yeast S. pombe
, easily identifiable homologues of S. cerevisiae
MEN pathway genes regulate the initiation of septum formation during cytokinesis and has been termed the septation initiation network, or SIN [2
]. Similar to the MEN, the SIN plays a major role in coupling a late mitotic event (here, cytokinesis) to the prior completion of sister chromatid separation [13
]. However, the wiring of the two pathways is distinct. Whereas in S. cerevisiae
Cdc14p is the downstream effector of the MEN, in S. pombe the downstream effector appears to be the Sid2p kinase bound to its coactivator Mob1p [14
]. Characterization of the S. pombe
Cdc14 orthologue, clp1/flp1, led to the surprising conclusion that clp1/flp1 was not required for septum formation since only ~3% of clp1/flp1 null cells displayed characteristic cytokinesis defects, but instead appeared to regulate a cytokinesis checkpoint by arresting cells at the G2/M transition in the event of defective cytokinesis [16
]. In addition, clp1/flp1 differed from S. cerevisiae
Cdc14p in its localization pattern and substrate specificity. This divergence of Cdc14 function between budding and fission yeast was unexpected and suggested that Cdc14, although highly conserved through evolution, would be likely be co-opted for other functions and perhaps modes of regulation in higher organisms.
The lone Cdc14 homologue present in C. elegans was recently shown to be required for central spindle formation and cytokinesis [18
]. CeCdc14 localized to the central spindle in anaphase and midbody in telophase, but notably did not localize to the nucleolus or centrosome [18
]. This pattern of localization contrasted with that of fission yeast, budding yeast and human Cdc14 homologs (see below), which localize to the nucleolus and spindle pole body at different times in the cell cycle. Several C elegans MEN/SIN homologs were identified based on sequence homology, including BUB2, DBF2, MOB1, and sid1+
, but it is not presently understood how they relate to CeCdc14.
Humans contain two highly related Cdc14 paralogs (50% identity) that have diverged in function through evolution. Despite their high sequence similarity, the localization patterns of hCdc14A and B differ dramatically, with hCdc14A localizing to the centrosome and hCdc14B to the nucleolus [19
]. A 54 amino acid peptide present at the very N-terminus of hCdc14B is necessary and sufficient to direct hCdc14B to the nucleolus [20
], whereas hCdc14A contains a nuclear export sequence (amino acids 352–367) required for centrosomal localization [21
]. Both hCdc14A and B appear to be dynamic in their subcellular distributions, which is likely a central aspect of their regulation. Disruption of hCdc14A in tissue culture cells by overexpression or depletion using siRNA provides strong evidence that hCdc14A regulates the centrosome duplication cycle and cytokinesis [20
]. The function of hCdc14B has yet to be elucidated.
The three dimensional structure of the core domain of hCdc14B (residues 44–378) was recently solved by x-ray crystallography both by itself and complexed to a peptide substrate [22
]. The most striking feature of the structure is that Cdc14 contains two domains in its N-terminus that are highly related structurally (rmsd of 2.6 Å) despite very limited amino acid conservation (11% identity). Both domains adopt a dual-specificity phosphatase (DSP) signature fold, but only the more C-terminal "B" domain contains the active site Cys and Arg residues. However, the more N-terminal "A" contributes important contact residues from two loops that facilitate peptide substrate specificity.
and genetic analyses and suggest that the Cdc14 family dephosphorylates substrates of cyclin-dependent kinases (Cdk's) [7
], whose canonical recognition sequence is (S/T)PXK. However, budding yeast Cdc14p is also able to recognize tyrosine-phosphorylated peptides as substrates [24
]. The three dimensional structure of Cdc14 in complex with a peptide substrate provides strong evidence that Cdc14 has a strong preference for phosphoserine/threonine residues located immediately N-terminal to a proline residue, consistent with Cdc14 dephosphorylating Cdk substrates [22
Despite high sequence conservation, studies in budding and fission yeast, human tissue culture cells and C. elegans have demonstrated that Cdc14 homologs have clearly adopted different functions and mechanisms of regulation. To better understand the role of metazoan Cdc14 and its regulation, we have begun to characterize homologs of Cdc14 from the South African clawed frog Xenopus laevis. We find that XCdc14 proteins localize to both centrosomes and nucleoli and are mitotically phosphorylated. Furthermore, antibody microinjection into Xenopus embryos demonstrates that XCdc14 proteins are essential for cell division.