Cdc5 influences phosphorylation of Net1 and disassembly of the RENT complex
© Shou et al; licensee BioMed Central Ltd. 2002
Received: 15 April 2002
Accepted: 17 April 2002
Published: 17 April 2002
In S. cerevisiae, the mitotic exit network (MEN) proteins, including the Polo-like protein kinase Cdc5 and the protein phosphatase Cdc14, are required for exit from mitosis. In pre-anaphase cells, Cdc14 is sequestered to the nucleolus by Net1 as a part of the RENT complex. When cells are primed to exit mitosis, the RENT complex is disassembled and Cdc14 is released from the nucleolus.
Here, we show that Cdc5 is necessary to free nucleolar Cdc14 in late mitosis, that elevated Cdc5 activity provokes ectopic release of Cdc14 in pre-anaphase cells, and that the phosphorylation state of Net1 is regulated by Cdc5 during anaphase. Furthermore, recombinant Cdc5 and Xenopus Polo-like kinase can disassemble the RENT complex in vitro by phosphorylating Net1 and thereby reducing its affinity for Cdc14. Surprisingly, although RENT complexes containing Net1 mutants (Net1(7m) and Net1(19m') lacking sites phosphorylated by Cdc5 in vitro are refractory to disassembly by Polo-like kinases in vitro, net1(7m) and net1(19m') cells grow normally and exhibit only minor defects in releasing Cdc14 during anaphase. However, net1(19m') cells exhibit a synergistic growth defect when combined with mutations in CDC5 or DBF2 (another MEN gene).
We propose that although Cdc5 potentially disassembles RENT by directly phosphorylating Net1, Cdc5 mediates exit from mitosis primarily by phosphorylating other targets. Our study suggests that Cdc5/Polo is unusually promiscuous and highlights the need to validate Cdc5/Polo in vitro phosphorylation sites by direct in vivo mapping experiments.
In the budding yeast Saccharomyces cerevisiae, the mitotic exit network (MEN) plays a pivotal role in driving cells from mitosis to G1. Identified components of the MEN include Tem1 (a GTP-binding protein), Lte1 (a putative guanine nucleotide releasing factor), Nud1 (a spindle-pole-body protein), Cdc15 (a protein kinase), Dbf2/Mob1 (a protein kinase complex), Cdc5 (a Polo-like protein kinase), and Cdc14 (a protein phosphatase). When cells harboring conditional-lethal temperature-sensitive (ts) mutations in any of these proteins are shifted to the restrictive temperature, they uniformly arrest in late anaphase as large-budded cells with segregated chromosomes, fully elongated microtubule spindles, and in all tested cases, elevated Cdc28/Clb2 protein kinase activity (Cdc28 is the major cyclin-dependent kinase Cdk1 and Clb2 the major mitosis-specific cyclin of budding yeast) (reviewed in , ).
The protein phosphatase Cdc14 is likely to be the most downstream element in the MEN because it can act directly on cell cycle regulators to promote exit from mitosis. By counteracting Cdk1-mediated phosphorylations, Cdc14 activates both Hct1/Cdh1, which targets Clb2 for degradation [3–5], and Swi5, which turns on the transcription of Cdk1 inhibitor Sic1 [6, 3]. Degradation of Clb2 and accumulation of Sic1 act in concert to eliminate mitotic Cdk activity, which is required for exit from mitosis . Thus, elucidating how Cdc14 is regulated holds the key to understanding how cells exit mitosis. In pre-anaphase cells, Cdc14 is confined to the nucleolus as a subunit of the RENT complex (which also contains Net1/Cfi1 and Sir2), and its phosphatase activity is inhibited by Net1 [8–11]. Damaged DNA and mis-oriented mitotic spindles delay exit from mitosis (reviewed in ), but after errors have been rectified or bypassed, Lte1, Nud1, and possibly other unidentified factors  promote Tem1 activation. Tem1 recruits Cdc15 to the spindle pole body, which in turn recruits and phosphorylates the Dbf2/Mob1 protein kinase complex, thereby switching it on [2, 14–17]. The Tem1-signaling pathway eventually triggers disassembly of the RENT complex, enabling the release of Cdc14 (and a portion of Sir2) from Net1. Active Cdc14 diffuses into the nucleus and cytoplasm to extinguish mitotic Cdk activity and to promote exit from mitosis.
The relative position of the Polo-like kinase Cdc5 in the MEN is obscure. Cdc5 is required for full activation of the Dbf2-Mob1 protein kinase complex [15–17], and has been proposed to impinge on the MEN both upstream and downstream of Tem1 [15–18]. During exit from mitosis, Cdc5 is required for Clb2 destruction, and similar to over-expression of Cdc14 , induction of Cdc5 triggers precocious Clb2 degradation in pre-anaphase cells [19, 20]. In addition to its role in exit from mitosis, Cdc5 has other functions during mitosis in budding yeast. Depletion of Cdc5 or Polo-like kinase in cdc5▲ cells arrests a significant fraction of the cells in early anaphase with short mitotic spindles [20, 21], and phosphorylation of the cohesin subunit Scc1 by Cdc5 enhances the efficiency of Scc1 cleavage by the separase Esp1 during the metaphase to anaphase transition .
Although many proteins are known to be essential for exit from mitosis, the factor that sets Cdc14 free from the nucleolus still remains elusive. In this study, we assess the roles of Cdc5 in disassembly of the RENT complex and exit from mitosis.
Release of Cdc14 from the nucleolus in late anaphase requires Cdc5
Overexpression of Cdc5 evicts Cdc14 from the nucleolus
Cdc5 influences the phosphorylation state of Net1 in vivo
Our analysis of Cdc5's role in release of Cdc14 from the RENT complex revealed that Cdc5 is required for disassembly of RENT in late anaphase, that Cdc5 can provoke premature disassembly of RENT in pre-anaphase cells, and that Cdc5 controls (at least in part) the phosphorylation state of Net1 during anaphase. Moreover, the arrest of cdc5-1, cdc5-2, cdc5-4, and cdc5-7 temperature-sensitive mutants in late mitosis was potently suppressed by the TAB6-1 allele (Park et al., submitted), which encodes a form of Cdc14 that binds weakly to Net1 and is released from Net1 during mitosis even in the absence of MEN function . Taken together, these data suggested the hypothesis that cdc5 mutants arrest in late anaphase because inadequate Cdc5-dependent phosphorylation of Net1 results in failure to release active Cdc14 from the RENT complex.
Polo-like kinases can disrupt the RENT complex in vitro
The essential functions of Cdc5 can be performed by mammalian Polo-like kinase Plk1 , which led us to test if Cdc5 can also be substituted by other Polo-like kinases in the release assay. His6-T7-Net1N/GST-T7-Cdc14 complex was assembled on anti-GST beads, and treated with either active or N172A kinase-inactive Plx1 (Polo-like kinase from Xenopus; ) in the presence of ATP. Active but not inactive Plx1 phosphorylated both Net1N and Cdc14, and completely dislodged Net1N from bead-bound Cdc14 (Figure 4B). Thus, we conclude that like Cdc5, the protein kinase activity of Plx1 can disrupt the interaction between Net1N and Cdc14. Because recombinant Plx1 but not Cdc5 was consistently expressed at high levels in insect cells, and because release assays using Plx1 and Cdc5 yielded similar results, we used Plx1 in most of our release assays.
The Net1N/Cdc14 complex is only a crude approximation of RENT, because it does not contain all components of RENT (such as Sir2), lacks modifications which normally occur in yeast cells, and contains Net1N instead of Net1. Thus, we tested whether Polo-like kinases could disrupt a physiological RENT complex. Myc9-Net1 and the associated RENT subunits were immunopurified from extracts of myc9-NET1 cells on 9E10 beads and treated with Plx1 plus ATP. Active but not inactive Plx1 released a significant portion of Cdc14 and Sir2 from bead-bound Net1 to the supernatant fraction (Figure 4C). In a similar experiment, Cdc5 also disrupted the RENT complex (data not shown). Taken together, these data suggest that Polo-like kinases are sufficient to release Cdc14 and Sir2 from Net1 in vitro.
Polo-like kinase abolishes Cdc14-binding activity of Net1 in vitro
RENT complexes comprised of Net1 phospho-site mutants are resistant to disassembly by Polo-like kinase, but exhibit only minor phenotypes in vivo
If Cdc5 disassembled the RENT complex through phosphorylating Net1, then a mutant Net1 lacking Cdc5 phosphorylation sites should bind Cdc14 even in the presence of active Cdc5/Polo-like kinase. To test this hypothesis, we prepared Net1N phosphorylated by Cdc5 in vitro, and mapped 12 definitive and more than 20 potential phosphorylation sites using mass spectrometry, which is described in detail elsewhere .
We constructed an extensive set of phospho-site mutants of Net1N, which were tested for their ability to remain bound to Cdc14 beads in the presence of Plx1 . By successively combining individual mutations in Cdc5 phosphorylation sites, we created the phosphosite mutants Net1N(7m) (with S30, S31, S48, S60, S64, S242, and S335 all changed to A) and Net1N(19m') (with T16, S30, S31, S48, S60, S64, T194, T195, T196, T197, S223, S224, S228, S231, S242, S295, S301, T302, S335 all changed to A). Net1N(7m) contained mutations required to strongly inhibit Plx1-dependent disruption of Net1N-Cdc14 complexes, whereas Net1N(19m') contained additional mutations without compromising the affinity of Net1N to Cdc14 . Both of these mutant proteins, when expressed in E. coli and assembled with recombinant Cdc14, were found to be severely defective in releasing Cdc14 upon treatment with Plx1 (data not shown).
To evaluate the effects of the Net1(7m) and Net1(19m') mutants in vivo, DNA fragments encoding either the wild type or the mutant NET1N alleles were subcloned into a TRP1-marked integrating plasmid to generate full-length NET1-myc9 [894L1], net1(7m)-myc9 [894L2], and net1(19m')-myc9 [894L3] plasmids, respectively, which were then integrated into the TRP1 locus of net1▲/+ diploid yeast cells. After sporulation, net1▲ NET1-myc9, net1▲ net1(7m)-myc9, and net1▲ net1(19m')-myc9 strains were obtained and analyzed.
First, we compared in vivo phosphorylation states of Net1 and Net1(7m). Net1 and Net1(7m) from asynchronously growing cells migrated identically in an SDS-polyacrylamide gel (Figure 3, Lanes 1 and 5). Net1(7m) from cdc14-1 cells arrested at 37°C appeared to migrate slightly faster than the corresponding Net1 (Figure 3, compare Lanes 8 and 4), and the reduced mobility of Net1(7m) in cdc14 cells was no longer dependent upon CDC5 (compare lanes 7 and 8). Net1(19m') behaved identically to Net1(7m) in this assay (data not shown). We conclude that there is diminished Cdc5-dependent phosphorylation of Net1(7m) in cdc14 cells arrested in anaphase.
Despite the striking defects of the phosphosite mutant proteins in vitro, neither net1(7m)-myc9 nor net1(19m')-myc9 cells expressed a dramatic phenotype. We observed that ~40% of net1(7m)-myc9 and net1(19m')-myc9 cells with long mitotic spindles showed focal (i.e. nucleolar) Cdc14 localization, compared to ~6% of wild type cells (Figure 6B). However, this result indicates that the majority (~ 60%) of net1 phosphosite-mutant cells released Cdc14 from the nucleolus during anaphase with normal kinetics. Indeed, the kinetics of cell cycle progression (monitored by either elongation of the mitotic spindle or delocalization of Cdc14 from the nucleolus) in the wild-type and the net1(7m) mutant strains were almost identical (Figure 6C).
While this manuscript was under consideration, Cdc5 was shown to be involved in promoting 'Cdc14 early anaphase release' (FEAR, ). FEAR is a transient phenomenon that depends on Cdc5, but does not depend upon other components of the MEN pathway. It was reported that in early anaphase, Cdc14 is transiently released from the nucleolus by the action of the FEAR pathway, but quickly returns to the nucleolus unless the MEN is activated to promote sustained release of Cdc14 and subsequent exit from mitosis. To evaluate whether the net1(7m) and net1(19m') phosphosite mutants have an impact on FEAR, we analyzed Cdc14 localization in synchronous populations of dbf2-1, net1(7m) dbf2-1, and net1(19m') dbf2-1 cells following release from an α-factor block in G1 phase at 25°C to α-factor-free medium prewarmed to 37°C (nonpermissive temperature for dbf2-1). As shown in Figure 6D, the percentage of cells that completely released Cdc14 during early anaphase (spindle length 0–8 μm) was reduced by ~50% in net1(7m) and ~25% in net1(19m') cells compared with wild-type cells.
To test for the possibility that the proteins encoded by net1(7m) and net1(19m') might be hypomorphic with respect to their resistance toward phosphorylation by Cdc5 (i.e., not all Cdc5 phosphorylation sites on Net1 were mapped and mutated), we evaluated whether the net1 phosphosite-mutants exhibited a synthetic growth phenotype when combined with msd2-1 (a mutant allele of CDC5). This experiment was based on the idea that a reduction in phosphorylation sites on Net1 coupled with a reduction in Cdc5 activity might synergize to prevent efficient release of Cdc14 during anaphase, resulting in failure to exit mitosis. The net1(19m') but not net1(7m) allele showed a synthetic growth defect when combined with msd2-1 (Figure 6E). The net1(19m') allele also showed a synthetic growth defect when combined with dbf2-1. These observations imply that some of the residues mutated in net1(19m') represent in vivo phosphorylation sites that influence the MEN-dependent release of Cdc14 during anaphase.
The 'RENT control' hypothesis posits that disassembly of the RENT complex and subsequent release of Cdc14 from the nucleolus drive cells from mitosis to interphase. This study analyzes how the Polo-like kinase Cdc5 participates in this key step of the mitotic exit program.
Cdc5 participates in disassembly of the RENT complex during anaphase
CDC5 was originally discovered as a gene essential for exit from mitosis . We found that Cdc5 was required to release Cdc14 from the nucleolus: cdc5-1 cells in late anaphase displayed focal nucleolar Cdc14 staining, whether grown at the permissive or restrictive temperature. By contrast, Cdc14 was released from the nucleolus in the great majority of wild type cells by late anaphase. Surprisingly, cdc5-1 cells grown at 25°C could exit mitosis despite persistent nucleolar localization of Cdc14, which may have been due to release of a small fraction of Cdc14 that was nevertheless sufficient to drive exit from mitosis in these cells. Alternatively, it is possible that complete release of Cdc14 from the nucleolus in cdc5-1 cells was so brief that it escaped our detection. A ramification of this observation is that indirect immunofluorescence assays based on Cdc14 localization may not correctly predict whether a cell can exit mitosis.
Not only was Cdc5 required for Cdc14 release, but a stabilized mutant lacking the destruction box (Cdc5(DB▲)) triggered inappropriate Cdc14 release in pre-anaphase cells when overexpressed from the GAL1,10 promoter. Ectopic release of Cdc14 might contribute to the proliferation block and Clb2 degradation caused by induction of GAL-CDC5(DB▲) [19, 20]. Overexpression of Cdc5(DB▲) also altered nucleolar structure in nocodazole-arrested cells (Figure 2B), the reason for which is not understood. In contrast, GAL-mediated overexpression of Tem1 (R. Li, personal communication), Cdc15 , or Dbf2/GST-Mob1 (RA, unpublished data) had little or no detectable effect on either exit from mitosis or cell proliferation, suggesting that the Tem1/Cdc15/Dbf2/Mob1 branch of the MEN plays a more restricted role in promoting the exit from mitosis.
The observations that Cdc5 was both necessary and sufficient for Cdc14 release and that Cdc5 influenced phosphorylation state of Net1 in vivo prompted us to examine whether Cdc5 directly catalyzes disassembly of the RENT complex. Indeed, Cdc5 and the Xenopus polo-like kinase Plx1 reduced the binding affinity between recombinant Cdc14 and Net1 by phosphorylating Net1, and dislodged both Cdc14 and Sir2 from the RENT complex in vitro.
Net1 mutants lacking critical Cdc5 phosphorylation sites support normal progression through mitosis
If Cdc5 directly promotes disassembly of RENT in vivo by a mechanism similar to that observed in vitro, then a Net1 mutant that cannot be phosphorylated by Cdc5 should not allow release of Cdc14 from the nucleolus in anaphase cells. To address this prediction, we mapped in vitro Cdc5-phosphorylation sites in the N-terminal part of Net1 which is sufficient to bind and inhibit Cdc14 . The details of this study are described elsewhere . Among the thirty or so mapped sites (with some being more efficiently phosphorylated than others), no strict consensus sequence for Cdc5 phosphorylation could be derived. However, the data from this and other [22, 29] studies suggest a clear preference for phenyl or aliphatic side chains (F, Y, L, I, V, M) in the +1 position.
Mutating all mapped phosphorylation sites was ruled out as being uninformative, because a Net1 mutant lacking 22 sites [Net1(22m)] already suffered significantly reduced binding affinity for Cdc14 (data not shown). Thus, we prioritized phospho-sites based on their stoichiometry of phosphorylation and on homology to sites whose phosphorylation was demonstrated to be important in earlier release assays. In total, we tested more than 20 phospho-site mutants of Net1, and identified two – Net1(7m) and Net1(19m') – that still bound Cdc14 normally, but displayed a strong defect in releasing Cdc14 upon treatment with Plx1. Full length net1(7m)-myc9 and net1(19m')-myc9 alleles were constructed and expressed in net1▲ background. Mutant RENT complexes retrieved from these cells were significantly more resistant to the Polo-like kinase Plx1 in release assays (Fig. 6A and data not shown). However, when net1(7m) and net1(19m') cells were evaluated by immunofluorescence, the majority released Cdc14 in late anaphase, and the subset that exhibited a modest delay in Cdc14 mobilization probably released enough Cdc14 to exit mitosis in a timely manner such that essentially no cell cycle progression defect was evident (Fig. 6C). In addition, Cdc14 remained nucleolar in cdc5-1 net1▲ net1(7m)-myc9 cells (data not shown), suggesting that the release event was still regulated properly.
How does Cdc5 promote disassembly of the RENT complex and exit from mitosis?
There are two extreme interpretations of our analysis of the Net1(7m) and Net1 (19m') mutants: First, Cdc5 is not directly involved in disrupting the RENT complex, and second, Cdc5 directly disassembles the RENT complex via phosphorylation of Net1, but because it is not feasible to evaluate all possible 109 (=230) combinations of phospho-site mutants or because the sites phosphorylated by Cdc5 in vivo are different from those in vitro due to the presence of factors such as Sir2, Pol I, and rDNA [10, 23], we missed key phosphorylation sites that would have given a more definitive phenotype when mutated. A variation on this theme is that it might never be possible to construct a phosphosite mutant of Net1 that binds Cdc14 but fails to release it during anaphase, if the hydroxyl groups that are phosphorylated normally form critical hydrogen bonds that tether Cdc14 to Net1.
A third hypothesis that stakes middle ground between these polar extremes is that Cdc5 affects RENT stability both directly and indirectly, with the former role being less significant than the latter. Several lines of evidence are consistent with this view. First, net1(7m) and net1(19m') mutants exerted a modest effect on release of Cdc14 from the nucleolus, in that ~40% of cells with long mitotic spindles retained focal localization of Cdc14 and that both net1 phospho-site mutants displayed a modest defect in 'Cdc14 early anaphase release' (FEAR; ). Second, net1(19m') showed a synthetic growth defect when combined with dbf2-1 or the msd2-1 allele of CDC5. Third, phosphorylation site mapping of Net1 purified from arrested cdc14-1 cells (WS and SC, unpublished data) revealed that at least one (S231) of the five in vivo phosphorylation sites in the first 341 amino acids was directly phosphorylated by Cdc5 in vitro. Nevertheless, because expression of neither Net1(7m) nor Net1(19m') had a major impact on exit from mitosis, any direct effect of Cdc5 on Net1 appears to be minor, and the requirement for Cdc5 in the release of Cdc14 might arise principally from phosphorylation of other release-promoting proteins.
If Cdc5 plays a minor role in directly disassembling RENT, then how does it promote exit from mitosis? Cdc15, Dbf2, and Mob1 are not properly localized to the spindle pole body in a significant fraction of cdc5-1 cells [30, 31, 17], and Dbf2/Mob1 protein kinase activity is diminished in cdc5 cells [15–17], suggesting that at least one function of Cdc5 is to generate maximally active Dbf2/Mob1, which can then trigger Cdc14 release. This explanation is unlikely to account fully for Cdc5's role in exit from mitosis, however, because (1) overexpression of Cdc5, but not Dbf2/Mob1, provoked ectopic release of Cdc14 from the nucleolus; and (2) the Dbf2/Mob1 kinase complex is at least partially activated and is correctly localized to the spindle pole body in 50% of cdc5 cells at the nonpermissive temperature [16, 17].
A word of caution on phosphosite mapping
Cdc5 can phosphorylate up to 30 residues in the amino terminal domain of Net1 in vitro. Remarkably, only five sites in this same segment were confirmed to be phosphorylated in vivo in cdc14 cells, and only one of these corresponded to a site phosphorylated by Cdc5 in vitro. Whereas it is possible that Cdc5 protein kinase activity is qualitatively or quantitatively altered in cells held at the cdc14 block, resulting in reduced phosphorylation of Cdc5 target sites on Net1, our data suggest that Cdc5 is a promiscuous protein kinase. This raises significant concerns about the interpretation of numerous studies in the literature that report effects of Cdc5/Polo on in vitro processes without mapping and genetic confirmation of phosphorylation sites. Moreover, several recent studies have reported direct mapping of sites phosphorylated by Cdc5/Polo in vitro [22, 29, 32], but in two of these cases the authors relied exclusively on phosphoshift analysis of mutant proteins to confirm that the same sites are phosphorylated in vivo. Given the significant possibility that multiply mutated proteins are not properly phosphorylated in vivo for indirect reasons, or migrate aberrantly upon SDS-PAGE, it may be prudent to perform direct mapping experiments to confirm unambiguously the physiological significance of sites phosphorylated by Cdc5/Polo in vitro.
Although Cdc5 activity (1) is necessary to free nucleolar Cdc14 in late mitosis, (2) can elicit ectopic release of Cdc14 in pre-anaphase cells when overproduced, (3) regulates the phosphorylation state of Net1 during anaphase, and (4) can disassemble native and recombinant RENT complexes in vitro by phosphorylating Net1, a combination of in vivo and in vitro phosphosite mapping and mutagenesis experiments suggest that Cdc5 mediates exit from mitosis primarily by phosphorylating targets other than Net1. The unusual promiscuity of Cdc5/Polo highlights the need to validate Cdc5/Polo in vitro phosphorylation sites by direct in vivo mapping experiments.
Materials and Methods
The SDS-boiling method for yeast extract preparation to visualize Net1 phosphorylation state in vivo and methods for protein detection were described previously . For indirect immunofluorescence, we used 1:1000 rabbit α-A190, 1:2000 rabbit α-tubulin, 1:200 rabbit α-Cdc14, 1:1000 rat YL1/2 (against tubulin), and 1:1000 mouse monoclonal HA.11 and 9E10 antibodies (Babco/CRP). For Western blots, we used 1:2000 9E10, 1:2500 α-Sir2, 1:1000 rabbit α-Cdc14 (crude serum), 1:6000 α-GST, and 1:5000 α-T7 (Novagen).
Recombinant protein expression
GST-T7-Cdc14 (RDB 872) and GST-Cdc14 (RDB 1183) were expressed in E. coli BL21(DE3) pLysS cells, and purified on glutathione-agarose beads (Sigma) as described previously . His6-T7-Net1(1-341) (Net1N, RDB1153)  was expressed in E. coli BL21(DE3) pLysS cells, and purified on Ni-NTA agarose beads (Qiagen) as described previously for p28Kix1. Purification of His6-Plx1, His6-Plx1(N172), and His6-HA-Cdc5 from baculovirus-infected insect cells followed an earlier protocol . The concentrations of purified proteins were in the ranges of: Net1N~0.4 mg/ml; GST-Cdc14~0.1–0.2 mg/ml; Plx1~0.3 mg/ml; Plx1(N172)~0.1 mg/ml; and Cdc5~1 mg/ml (although the level of Cdc5 expression varied drastically from one batch to another).
The substrates for release assays were RENT (isolated from yeast cells) or Cdc14/Net1N (assembled in vitro) complexes anchored on resins through tags on either Net1 (Figures 4A, 4C, and 6A) or Cdc14 (Figures 4B and 5), and were prepared at 4°C. For experiments in Figures 4C and 6A, immunoprecipitation of RENT through Myc9-Net1 was carried out as described earlier , except that EDTA (final 2 mM) was included in the lysis buffer. Each release reaction contained RENT from ~10 OD600 units (1 unit equals 1 ml of culture at OD600 = 1.0) of cells bound to 15 μl of 9E10 beads. To assemble Cdc14 beads with Net1N for two assays (Figure 4B, detection method: Western blotting), 2 μl of rabbit-anti-GST antibodies were absorbed to 40 μl of protein A beads (Sigma) in 100 μl of HBS [20 mM HEPES (pH 7.2), 150 mM NaCl] + 2 mM EDTA + 0.1 mg/ml BSA + 1 mM DTT (HEBD) buffer for 1 hr. The beads were washed twice with HBS + 2 mM DTT + 0.2% Triton and once with HBS + 1 mM DTT. The beads were incubated with 250 ng of GST-T7-Cdc14 in 100 μl of HEBD for 1 hr, followed by the same sequence of washes. Finally, the beads were incubated with Net1N (450 ng) for 1 hr. To assemble Net1N beads with Cdc14 for six reactions (Figure 4A, detection method: Coomassie Blue staining), 105 μl of anti-T7 beads (Novagen) were incubated with 24 μg of His6-T7-Net1N in 100 μl of HEBD for 1 hr, washed four times with HBS + 0.2% n-octylglucoside + 2 mM DTT, and incubated with 6 μg of GST-Cdc14 in 100 μl of HEBD for 1 hr. After the final incubation step of the protein complete assembly process, beads were extensively washed, followed by a final wash with Kinase Buffer [5 mM Tris·HCl (pH 8.0), 25 mM NaCl, 10 mM MgCl2, 1 mM DTT, 0.1 mg/ml BSA, 1 mM ATP, 10 μg/ml pepstatin + chymostatin + leupeptin (PCL)]. The beads were then distributed into equal aliquots for release assays. Kinase reactions were carried out in 30 μl Kinase Buffer at room temperature for 0.5–1 hr, and the supernatant was transferred to new tubes at the end of assays. The beads were washed once with 70 μl of HEBD, and the wash was pooled with the supernatant, and precipitated with TCA (10% final) on ice for 30 min. After centrifugation in a microfuge for 10 min, the pellet was saved as "sup". Proteins present on the beads or in the sup were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
For experiments in Figure 5, all four reactions consisted of 125 ng GST-T7-Cdc14 adsorbed to 20 μl anti-GST beads, 200 ng His6-T7-Net1N, 1 μl Plx1, 20 μl kinase buffer containing ATP, and the ATP depletion system (see below).
Kinase reaction was terminated by depleting ATP with 1 μl of hexokinase (1 U/μl) and 2 μl of 10 × G+ I (1 μl 10 mg/ml PCL+ 20 μl 50% glucose + 79 μl H2O) for 20 min at room temperature.
We thank R. Verma for advice on phospho-site mapping experiments; R. Verma, B. Hay, and D. Chan for discussions; and W. Seufert and E. Traverso for sharing unpublished data. We thank G. Alexandru, D. Chan, R. Lipford, E. Traverso, and R. Verma for critically reading the manuscript. We also thank J. Charles (Morgan lab), A. Kumagai (Dunphy lab), and M. Shirayama (Nasmyth lab) for the Cdc5 baculoviruses, Plx1 baculoviruses, and GAL-CDC5(DB▲) strain, respectively; M. Nomura and F. Soloman labs provided antibodies against A190 and tubulin, respectively. WS was an HHMI predoctoral fellow. This research was supported by an NIH grant to RJD. WS dedicates this paper to Maste R. Brain, Rubbe R. Stamp, and their invaluable yet dispensable lessons.
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