Can Fizzy fly solo?
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NEWS AND VIEWS 864 NATURE CELL BIOLOGY VOLUME 5 | NUMBER 10 | OCTOBER 2003 Negotiating the orderly steps through mitosis is a little like passing through immigration and customs after an international flight. If the appropriate certifications are presented at the right time to each presiding official with a min- imum of commotion, then you probably won’t be held up. But fail to satisfy any official and you may be detained at the gate. If detained for too long, you may suffer catastrophic events like missing your connecting flight. In mitosis, there are several critical gates, including the separation of sister chromatids, spindle elongation and exit from mitosis. In most eukaryotes, the APC/cyclosome directs a series of specific ubiquitin-dependent prote- olytic events that open these gates 1,2 . These include destruction of the chromosome seg- regation inhibitor securin/Pds1 (ref. 3) and the central factor sustaining mitosis, cyclin B. Cdc20 (also called Fizzy) activates the APC for destruction of securin, which allows sister chromatids to separate (see Fig. 1). This step is blocked until the mitotic spindle is fully formed by a mechanism called the ‘spindle assembly checkpoint’, ensuring that securin is not destroyed and chromosomes do not seg- regate before spindle assembly is complete 7 . In the budding yeast Saccharomyces cerevisiae, however, the mitotic spindle can form during a slow S phase while DNA replication is ongo- ing, suggesting that the completion of S phase may be the more important event triggering chromosome segregation, rather than spindle assembly itself. Now a study from Reed and colleagues on page 928 provides a detailed genetic analysis, showing that the well-known Rad53p and Mec1p components of the S-phase checkpoint also limit accumulation of the Cdc20 protein 8 . Thus, Reed and col- leagues create a unifying view that Cdc20p is a central target restraining chromosome segre- gation until both chromosome replication and spindle assembly are complete. More sur- prisingly, the authors provide genetic evidence that the ability of the S-phase checkpoint to restrain spindle elongation requires CDC20, but not components of the APC. This suggests the heterodox model that CDC20 has func- tions independent of the APC. So, can the acti- vator protein Cdc20 direct steps of mitosis on its own? The APC is an E3 ubiquitin ligase complex, an enzyme that directs the formation of ubiq- uitin chains on target proteins such as securin and cyclin B 1,2 . These ubiquitinated targets are then transported to the 26S proteasome and proteolytically destroyed. Genetic and biochemical experiments clearly support the notion that the APC requires an activating protein Cdc20 (or it close cousin Cdh1) to ubiquitinate and trigger destruction of APC target proteins. Recent biochemical experi- ments emphasize the notion that Cdc20 may work as an adaptor protein by binding to spe- cific recognition motifs or ‘destruction boxes’ on targets and recruiting those targets to the core APC 4–6 , although the precise nature of this adaptor function is not well understood. The APC activator proteins are the direct targets of specific checkpoint mechanisms. For example, in many eukaryotes the ability of Cdc20 to activate the APC is blocked by components of the spindle assembly check- point. Here, the biochemical details are com- plex and somewhat controversial, but the genetic requirements support the idea that the central target of the checkpoint is the Cdc20 activator protein itself. Once the spin- dle is properly assembled, the spindle assem- bly checkpoint is satisfied and the APC–Cdc20 complex activates destruction of securin (called Pds1 in yeast). Securin func- tions to inhibit a central enzyme, separase, which in turn causes the release of proteins holding sister chromatids together, called cohesins 13 . Despite the fact that destruction of securin is essential for chromosome segre- gation, securin itself is not strictly required for viability, although yeast deficient in securin segregate their chromosomes prema- turely. Indeed, yeast deficient in securin still fail to elongate their spindles in response to an early S-phase block imposed by addition of hydroxyurea 9,10 . Reed and colleagues perform a series of experiments describing Cdc20 as an essential target of the S-phase checkpoint. Previous studies showed that the S-phase checkpoint works in part by controlling the levels of securin 10,14,15 . Would a part of that check- point circuit work through Cdc20? The authors begin with the observation that cells blocked in S phase with hydroxyurea nor- mally arrest with a short spindle. cdc20 mutants do not affect this block. However, mutants in the S-phase checkpoint gene rad53 initiated spindle elongation early, even though DNA replication was incomplete. The surprise was that cdc20 rad53 double mutants did not elongate their spindles, indicating that Cdc20 is required for premature spindle elongation. In unperturbed cells, Cdc20p is not synthe- sized until soon before anaphase and would not be present in S phase arrested cells to trig- ger spindle elongation. Satisfyingly, the authors observe that the rad53 mutation allows Cdc20 to accumulate in early S phase. Furthermore, overexpression of Cdc20 in S-phase cells treated with hydroxyurea causes spindle elongation to occur prematurely, with timing similar to the rad53 mutants. Thus, overexpression of Cdc20p is sufficient to deliver spindle elongation signal and bypass the normal RAD53-dependent checkpoint. The obvious possibility may be that Securin/Pds1 is indeed the critical target of Cdc20 for spindle elongation, but several results argue against this. Notably, rad53-1 cells show premature spindle elongation in early S phase, whereas pds1 mutants only show elongation in late S phase 10 . Wild-type cells only elongate their spindles after replica- tion is complete. So how does RAD53 delay Cdc20p accumu- lation during an S-phase block? Here, the authors show that both mRNA and protein lev- els are affected. In the rad53 mutant, CDC20 mRNA did accumulate, but the Cdc20 protein did not, suggesting that Rad53p functions post- transcriptionally. Mutants in mec1, another mediator of the S-phase checkpoint, showed a delay in CDC20 mRNA accumulation. Thus, Can Fizzy fly solo? Peter K. Jackson To ensure genome stability, the S-phase checkpoint blocks spindle elongation during S phase. Later, the spindle assembly checkpoint blocks chromosome segregation by restraining CDC20/Fizzy-dependent activation of the anaphase-promoting complex (APC). Now, a study suggests that the S-phase checkpoint also limits accumulation of Cdc20p and, unexpectedly, an APC-independent CDC20 function for spindle elongation. Peter K. Jackson is in the Programs in Chemical Biology, Biophysics and Cancer Biology, and the Department of Pathology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5324, USA e-mail: [email protected] ©2003 Nature Publishing Group
Transcript of Can Fizzy fly solo?
N E W S A N D V I E W S
864 NATURE CELL BIOLOGY VOLUME 5 | NUMBER 10 | OCTOBER 2003
Negotiating the orderly steps through mitosis isa little like passing through immigration andcustoms after an international flight. If theappropriate certifications are presented at theright time to each presiding official with a min-imum of commotion, then you probably won’tbe held up. But fail to satisfy any official andyou may be detained at the gate. If detained fortoo long, you may suffer catastrophic eventslike missing your connecting flight.
In mitosis, there are several critical gates,including the separation of sister chromatids,spindle elongation and exit from mitosis. Inmost eukaryotes, the APC/cyclosome directs aseries of specific ubiquitin-dependent prote-olytic events that open these gates1,2. Theseinclude destruction of the chromosome seg-regation inhibitor securin/Pds1 (ref. 3) andthe central factor sustaining mitosis, cyclin B.Cdc20 (also called Fizzy) activates the APC fordestruction of securin, which allows sisterchromatids to separate (see Fig. 1). This stepis blocked until the mitotic spindle is fullyformed by a mechanism called the ‘spindleassembly checkpoint’, ensuring that securin isnot destroyed and chromosomes do not seg-regate before spindle assembly is complete7.In the budding yeast Saccharomyces cerevisiae,however, the mitotic spindle can form duringa slow S phase while DNA replication is ongo-ing, suggesting that the completion of S phasemay be the more important event triggeringchromosome segregation, rather than spindleassembly itself. Now a study from Reed andcolleagues on page 928 provides a detailedgenetic analysis, showing that the well-knownRad53p and Mec1p components of the S-phase checkpoint also limit accumulationof the Cdc20 protein8. Thus, Reed and col-leagues create a unifying view that Cdc20p is acentral target restraining chromosome segre-gation until both chromosome replication
and spindle assembly are complete. More sur-prisingly, the authors provide genetic evidencethat the ability of the S-phase checkpoint torestrain spindle elongation requires CDC20,but not components of the APC. This suggeststhe heterodox model that CDC20 has func-tions independent of the APC. So, can the acti-vator protein Cdc20 direct steps of mitosis onits own?
The APC is an E3 ubiquitin ligase complex,an enzyme that directs the formation of ubiq-uitin chains on target proteins such as securinand cyclin B1,2. These ubiquitinated targetsare then transported to the 26S proteasomeand proteolytically destroyed. Genetic andbiochemical experiments clearly support thenotion that the APC requires an activatingprotein Cdc20 (or it close cousin Cdh1) toubiquitinate and trigger destruction of APCtarget proteins. Recent biochemical experi-ments emphasize the notion that Cdc20 maywork as an adaptor protein by binding to spe-cific recognition motifs or ‘destruction boxes’on targets and recruiting those targets to thecore APC4–6, although the precise nature ofthis adaptor function is not well understood.
The APC activator proteins are the directtargets of specific checkpoint mechanisms.For example, in many eukaryotes the abilityof Cdc20 to activate the APC is blocked bycomponents of the spindle assembly check-point. Here, the biochemical details are com-plex and somewhat controversial, but thegenetic requirements support the idea thatthe central target of the checkpoint is theCdc20 activator protein itself. Once the spin-dle is properly assembled, the spindle assem-bly checkpoint is satisfied and theAPC–Cdc20 complex activates destruction ofsecurin (called Pds1 in yeast). Securin func-tions to inhibit a central enzyme, separase,which in turn causes the release of proteinsholding sister chromatids together, calledcohesins13. Despite the fact that destructionof securin is essential for chromosome segre-gation, securin itself is not strictly requiredfor viability, although yeast deficient insecurin segregate their chromosomes prema-turely. Indeed, yeast deficient in securin stillfail to elongate their spindles in response to
an early S-phase block imposed by additionof hydroxyurea9,10.
Reed and colleagues perform a series ofexperiments describing Cdc20 as an essentialtarget of the S-phase checkpoint. Previousstudies showed that the S-phase checkpointworks in part by controlling the levels ofsecurin10,14,15. Would a part of that check-point circuit work through Cdc20?
The authors begin with the observation thatcells blocked in S phase with hydroxyurea nor-mally arrest with a short spindle. cdc20mutants do not affect this block. However,mutants in the S-phase checkpoint gene rad53initiated spindle elongation early, even thoughDNA replication was incomplete. The surprisewas that cdc20 rad53 double mutants did notelongate their spindles, indicating that Cdc20is required for premature spindle elongation.
In unperturbed cells, Cdc20p is not synthe-sized until soon before anaphase and wouldnot be present in S phase arrested cells to trig-ger spindle elongation. Satisfyingly, theauthors observe that the rad53 mutationallows Cdc20 to accumulate in early S phase.Furthermore, overexpression of Cdc20 in S-phase cells treated with hydroxyurea causesspindle elongation to occur prematurely, withtiming similar to the rad53 mutants. Thus,overexpression of Cdc20p is sufficient todeliver spindle elongation signal and bypassthe normal RAD53-dependent checkpoint.
The obvious possibility may be thatSecurin/Pds1 is indeed the critical target ofCdc20 for spindle elongation, but severalresults argue against this. Notably, rad53-1cells show premature spindle elongation inearly S phase, whereas pds1 mutants onlyshow elongation in late S phase10. Wild-typecells only elongate their spindles after replica-tion is complete.
So how does RAD53 delay Cdc20p accumu-lation during an S-phase block? Here, theauthors show that both mRNA and protein lev-els are affected. In the rad53 mutant, CDC20mRNA did accumulate, but the Cdc20 proteindid not, suggesting that Rad53p functions post-transcriptionally. Mutants in mec1, anothermediator of the S-phase checkpoint, showed adelay in CDC20 mRNA accumulation. Thus,
Can Fizzy fly solo?Peter K. Jackson
To ensure genome stability, the S-phase checkpoint blocks spindle elongation during S phase. Later, the spindleassembly checkpoint blocks chromosome segregation by restraining CDC20/Fizzy-dependent activation of theanaphase-promoting complex (APC). Now, a study suggests that the S-phase checkpoint also limits accumulation ofCdc20p and, unexpectedly, an APC-independent CDC20 function for spindle elongation.
Peter K. Jackson is in the Programs in ChemicalBiology, Biophysics and Cancer Biology, and theDepartment of Pathology, Stanford UniversitySchool of Medicine, 300 Pasteur Drive, Stanford,CA 94305-5324, USAe-mail: [email protected]
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the two S-phase checkpoint pathways conspireto regulate Cdc20p accumulation at bothmRNA and protein levels. Levels of Cdc20p areprobably very important, because overexpres-sion of CDC20 causes mitotic catastrophe.
As Securin/Pds1p is unlikely to be the targetrestraining spindle elongation in early S phase, the authors sought other potentialtargets of Cdc20p. However, they did not seedestruction of mitotic cyclins or Pds1p in therad53 mutants, suggesting that most APC tar-gets are stable. So, if there is no obvious effecton APC substrate stability, maybe Cdc20p hasanother function. To test this idea, the authorsreturned to the rad53 mutant, which lacks theS-phase checkpoint and displays CDC20-dependent, premature spindle elongation.Next, they tested whether subunits of the APCare also required for premature spindle elonga-tion. To their amazement, spindle elongationproceeds even when temperature-sensitivemutants in three different APC subunits were
inactivated by shifting to the non-permissivetemperature. The textbook genetic interpreta-tion is that Cdc20 has a function in spindleelongation independent of the APC. Anunconventional idea indeed!
However, there may be a number of molec-ular explanations that could allow us to escapethis heterodoxy. First, the specific alleles of theAPC could have only reduced function, andthus at the non-permissive temperature thereis sufficient APC activity for the normalamount of Cdc20. Indeed, in some biochemi-cal experiments, the amount of Cdc20required to activate the APC is almost certain-ly substoichiometric, offering the possibilitythat the APC is generally in excess11. In addi-tion, the APC is a complex with over a dozensubunits and we still do not well understandthe diversity of its functions1,2. However, theauthors did test alleles of several differentAPC components, both components contain-ing possible structural subunits with TPR
repeats (cdc16, cdc23) and an allele of the corecatalytic E3 ubiquitin ligase (apc2). It could bethat the APC function is less severely reducedfor premature spindle elongation than for itsnormal role in mitosis (where these mutantswere first identified). In this case, it is possiblethat premature activation of the APC causesdeficiencies in spindle assembly by allowingdestruction of some spindle protein that nor-mally accumulates in S phase. In this case, thespindle elongation the authors observe maybypass some of its normal requirements.Obviously further experiments knocking outother APC components or finding alleles ofCdc20 that clearly discriminate between itsAPC-dependent and -independent functionswould serve to definitively answer if this effectis independent of the APC.
But if there were a critical APC-independentfunction, what might it be? The authors sug-gest a more conventional idea. Maybe Cdc20binds to its critical target(s) for spindle elon-gation and this binding is a primary form ofinactivation before a second proteolytic eventrequiring both Cdc20 and the APC. Such a pri-mary inactivating event might even requirethe destruction box sequences on the targetand could work through a variety of mecha-nisms. An interesting possibility is suggestedby the recent discovery that Cdc20 binds toand requires the CCT chaperone12 for APC-dependent destruction. Could Cdc20p-boundtargets undergo a primary unfolding (andthus inactivating) step on their pathway toAPC-dependent ubiquitination and destruc-tion? Or could Cdc20p sequester targets regu-lating spindle elongation by another means?Identifying the critical Cdc20 target limitingspindle elongation will certainly be impor-tant. Obviously genetics alone will not answerthese questions and further biochemical stud-ies are needed before we understand the bestway to reliably make the connecting flightfrom chromosome segregation to the comple-tion of mitosis.1. Peters, J. M. Mol. Cell 9, 931–943 (2002).2. Harper, J. W. et al. Genes Dev. 17, 2179–2206
(2002).3. Yamamoto, A. et al. J. Cell Biol. 133, 99–110 (1996).4. Pfleger, C. M. et al. Genes Dev. 15, 2396–2407
(2001).5. Hilioti, Z., et al. Curr. Biol. 11, 1347–1352 (2001).6. Burton, J. L. & Solomon, M. J. Genes Dev. 15,
2381–2395 (2001).7. Hwang, L. H. et al. Science 279, 1041–1044 (1998).8. Clarke, D. J. et al. Nature Cell Biol. 5, 928–935
(2003).9. Clarke, D. J. et al. Curr. Biol. 9, 365–368 (1999).10. Clarke, D. J. et al. Nature Cell Biol. 3, 619–627
(2001).11. Kramer, E. R. et al. Mol. Biol. Cell 11, 1555–1569
(2000).12. Camasses, A. et al. Mol. Cell 12, 87–100.13. Nasmyth K. Annu. Rev. Genet. 35, 673–745 (2001).14. Wang H, et al. Genes Dev. 15, 1361–1372 (2001).15. Sanchez Y, et al. Science 286, 1166–1171 (1999).
Incomplete DNA replication
Rad53p
CDC20 gene
CDC20
CDC20
Mec1p
Transcription
APCCDC20
Securin destruction
Sister chromatid separation
Regulation/inhibition of spindle protein
Block spindle elongation
?
S phase
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Spindle assemblycheckpoint
Figure 1 The S-phase checkpoint restrains the function of Cdc20. In early S phase, signalsgenerated by incomplete DNA replication structures signal to the S-phase checkpoint proteinsRad53p and Mec1p. Mec1p limits the transcription of the CDC20 gene. Rad53p blocks theaccumulation of Cdc20p, which restrains the activity of unknown proteins regulating spindleelongation. This inhibitory function may be independent of the APC. Once DNA replication iscomplete, Cdc20p accumulates to activate the APC, but is restrained by the spindle assemblycheckpoint. Once spindle assembly is complete, Cdc20 can activate destruction of securin totrigger sister chromatid separation, spindle elongation and later events of anaphase.
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© 2003 Nature Publishing Group
