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In all eukaryotes, a series of cyclin-dependent kinasecomplexes (CDK–cyclins) drives cells through thecell-division cycle (reviewed in Refs 1–3). To main-tain the correct temporal ordering of cell-cycleevents, individual CDK–cyclin pairs must act only atspecific points in the cell cycle. Therefore, multiplemechanisms have evolved to regulate CDK–cyclinactivity. While it is well established that cyclinsynthesis and destruction, specific phosphoryl-ation–dephosphorylation of CDKs and associationof CDK–cyclin complexes with inhibitor peptides all serve to restrict the action of CDK–cyclin com-plexes, it is now recognized that controlling the subcellular localization of CDK–cyclins and theirregulators is also essential for proper cell-cycle co-ordination. Indeed, recent studies on nuclear traf-ficking of CDK–cyclins have added a new dimensionto our understanding of cell-cycle control.
At the beginning of the rideProgression through G1 and S phase is driven by
the G1 cyclins and their catalytic subunits, includ-ing Cdk2–cyclin A, Cdk2–cyclin E, Cdk4–cyclin Dand Cdk6–cyclin D4. In HeLa cells, both immuno-staining and subcellular fractionation indicate thatcyclin A is predominantly nuclear from the time ofits initial appearance in G1 through to M phase,when it is degraded5 (Fig. 1). Similarly, cyclin E isconcentrated in the nucleus in a variety of celltypes6,7. It is likely that the constitutive nuclear lo-calization of these two cyclins and their associatedCDKs reflects their roles in promoting initiation andmaintenance of DNA replication – phosphorylationof components of the replicative machinery bythese kinase complexes promotes S phase, while pre-venting re-replication. Cyclin D1 has a less staticsubcellular localization pattern than the A- and E-type cyclins – the levels of nuclear cyclin D1 kinasecomplexes increase during G1, but, as S phase be-gins, CDK–cyclin D1 complexes are exported to thecytoplasm8 (Fig. 1). Interestingly, it was reported re-cently that phosphorylation of cyclin D1 by glyco-gen synthase kinase-3b leads to the relocalization of cyclin D1 from the nucleus to the cytoplasm, potentially targeting cyclin D1 for cytoplasmic proteolysis9.
In analysing Cdk6–cyclin D3 complexes in T cells,Mahony et al. found these complexes in both nu-clear and cytoplasmic subcellular fractions. However,only complexes isolated from nuclei exhibited Rb-directed kinase activity10. It is not yet clear how thecytoplasmic Cdk6–cyclin D3 complexes are held in-active in the cytoplasm. Moreover, it is interestingto note that Cdk6 also exists in a cytoplasmic pooluncomplexed to cyclin D, but bound to Hsp90,which potentially serves as a kinase chaperone.
Rounding the G2–M bendActivation of mitotic CDK–cyclin complexes pro-
motes progression through the G2–M transition inboth mitotic and meiotic cell cycles. Analysis ofCdc2–cyclin A, Cdc2–cyclin B1 and Cdc2–cyclin B2complexes has revealed that these three major mi-totic catalysts exhibit distinct patterns of subcellular
localization through the cell cycle. As mentionedabove, immunostaining, analysis of GFP-labelledproteins and subcellular-fractionation experimentsall support the conclusion that cyclin A is constitu-tively nuclear (Ref. 5; J. Pines, pers. commun.). By con-trast, as cyclins B1 and B2 are synthesized during interphase, they accumulate almost exclusively inthe cytoplasm. As cells enter prophase, cyclin B1translocates precipitously from the cytoplasm to thenucleus (Fig. 1), whereas cyclins A and B2 continue toreside in the nucleus and cytoplasm, respectively5,11,12.These strikingly distinct localization patterns suggestthat the different mitotic CDK–cyclin complexesphosphorylate distinct substrates to promote mitoticchanges in different subcellular locales.
To understand the mechanisms underlying theobserved differences in cyclin A and B1 localizationpatterns, Pines and Hunter examined the subcellularlocalization of a panel of cyclin deletion mutants andchimeras between the nuclear cyclin A and cytoplas-mic cyclin B1 proteins11. These analyses identified a42-amino-acid region in the N-terminus of cyclin B1,termed the cytoplasmic-retention sequence (CRS),
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trends in CELL BIOLOGY (Vol. 9) June 1999 0962-8924/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. 207PII: S0962-8924(99)01577-9
All aboard thecyclin train:subcellular
trafficking ofcyclins and their
CDK partners
Jing Yang and Sally Kornbluth
Progression through the cell cycle is governed by the periodic
activation and inactivation of cyclin-dependent kinase complexes
(CDK–cyclins). Although the enzymatic activity of these complexes
is regulated tightly, it has recently been demonstrated that an
additional facet of cell-cycle control involves the modulation of
CDK–cyclin subcellular localization. Recent discoveries include the
identification of nuclear transport factors responsible for ferrying
some of the CDK–cyclins in and out of the nucleus, the
demonstration that phosphorylation can regulate these transport
processes and the establishment of potential links between cell-cycle
checkpoints and the control of CDK–cyclin subcellular localization.
The authors are inthe Dept ofPharmacologyand CancerBiology, DukeUniversity MedicalCenter, Box 3686,C366 LSRC,Durham, NC 27710, USA.E-mail: [email protected]
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208 trends in CELL BIOLOGY (Vol. 9) June 1999
which was both necessary and sufficient for retainingcyclin B1 in the cytoplasm during interphase. TheCRS region is well conserved between cyclins B1 andB2 (Fig. 2); indeed, deletion of this region from eithercyclin led to their constitutive nuclear localization.Based on these observations, they hypothesized thatthe CRS region might tether the cyclins to cytoplasmicconstituents, thereby keeping cyclin B1 and B2 outof the nucleus during interphase11.
A re-evaluation of CRS function was prompted bystudies in several different laboratories that demon-strated that cyclin B1 could serve as a substrate of thenuclear-export machinery13–15. Moreover, careful ex-amination of the cyclin B1 sequence revealed a hydro-phobic leucine-rich nuclear-export sequence (NES)embedded within the CRS region13–15. This NES se-quence mediates binding to the nuclear export fac-tor CRM1 and can serve as an NES when linked to aheterologous protein15. Treatment of human cultured
cells or Xenopus oocytes with lepto-mycin B, a stoichiometric inhibitor ofCRM1-mediated nuclear export, pro-moted interphase nuclear accumulationof cyclin B1, suggesting that cyclin B1can shuttle continually in and out of nu-clei13–15. Therefore, the apparent cyto-plasmic localization of cyclin B1 duringinterphase reflects a balance betweenongoing nuclear import and more rapidre-export.
The dramatic relocalization of cyclinB1 to nuclei at the G2–M transition(Fig. 3) suggested that mechanisms existto increase the nuclear import rate ofcyclin B1, decrease the nuclear exportrate, or both. Studies of Xenopus oocytematuration by Donoghue and col-leagues indicated that phosphorylationof four conserved Ser residues withinthe CRS region, which occurs duringmeiotic maturation, controls the biologi-cal activity of cyclin B116,17. Mutation ofthese four serines to alanine greatly re-duced the ability of microinjectedmRNA encoding cyclin B1 to promotemeiotic maturation, while also decreas-ing the nuclear accumulation of cy-clin B116,17. Conversely, changing thesesites to glutamic acid, to mimicphosphorylation, enhanced the abilityof cyclin B1 to induce meiotic meta-phase. At least a partial explanation forthese data was provided by experimentsdemonstrating that phosphorylation ofcyclin B1 within the CRS markedly re-duces its affinity for CRM1, thereby de-creasing its nuclear-export rate and in-creasing its nuclear concentration15.Therefore, a plausible model for thecontrol of cyclin B1 localization atG2–M proposes that phosphorylation ofcyclin B1 attenuates its nuclear export,thereby promoting cyclin B1 nuclear accumulation and entry into mitosis.
The cytoplasmic-retention sequences of cyclin B1and cyclin B2 are well conserved overall, but, in thecase of Xenopus cyclin B2, several of the NES consen-sus residues found in the B1 CRS are altered in the B2sequence in a way expected to compromise NES func-tion (Fig. 2). Consistent with this, the Xenopus B2 CRSdoes not bind to CRM1 (J. Yang, J. D. Moore andS. Kornbluth, unpublished). Therefore, it seems likelythat some mechanism other than CRM1-dependentnuclear export keeps Xenopus cyclin B2 in the cyto-plasm. Elucidating this mechanism might help us tounderstand the potentially distinct roles played bycyclin B1 and cyclin B2 in promoting the events ofmitosis. Indeed, in Xenopus oocytes, overexpressionof a CRS-containing N-terminal fragment of cyclin B2leads to formation of a defective meiotic monopolarspindle, perhaps by displacing endogenous cyclin B2from cytoplasmic anchoring proteins; expression of asimilar fragment of cyclin B1 has no such effect18.
14-3-3
Cyclin B2
Cdc2
Cyclin A
Cdc2
Cyclin B1
Cdc2
Cyclin A
Cdc2Cyclin A
Cdc2
Cyclin A
Cdk2
Cyclin E
Cdk2
Cyclin B1
Cdc2
Cyclin E
Cdk2
Cyclin A
Cdk2Cyclin D3
Cdk6
Cyclin D1
Cdk4/6
Cyclin B2
Cdc2
Cyclin B2
Cdc2
Cyclin B1
Cdc2
Cdk4/6
Degradation
Cyclin D1
Cdk4/6
Cyclin D3
Cdk6
Wee 1
Cdc25C
Cytoplasm
Cytoplasm
Nucleus
Nuclear envelope
Cdc25C
Active
Active
Active
Export
Export
Import
P
Wee 1
P
PP
P
M
G1
Pro
phas
e
G2 S Cyclin D1
FIGURE 1
The subcellular localization of CDK–cyclins and their regulators during the cell cycle. During G1 phaseof the cell cycle, cyclins A, E and D are synthesized and bind to their cyclin-dependent kinase (CDK)
partners. CDK complexes containing cyclins A, E and D1 are then imported into and concentratedwithin nuclei. Cdk6–cyclin D3 has been localized to both cytoplasmic and nuclear compartments,
although only the nuclear complex is active. As cells enter S phase, cyclin A and cyclin E complexesremain within the nucleus, whereas cyclin D1 relocalizes to the cytoplasm for proteolysis at the onset
of S phase. Like Cdk2–cyclin A, Cdc2–cyclin A is nuclear and remains so until it is degraded duringmitosis. By contrast, as a result of ongoing nuclear import and more rapid re-export, cyclin B1, which
binds to Cdc2 upon synthesis during S phase, is predominantly cytoplasmic. Cdc2–cyclin B2 is alsocytoplasmic, although this might occur through anchoring of the complex to some cytoplasmic
constituent. At prophase, phosphorylation of cyclin B1 promotes accumulation of Cdc2–cyclin B1 inthe nucleus, whereas cyclin B2 remains in the cytoplasm until nuclear envelope breakdown. Also
shown are two crucial regulators of Cdc2–cyclin B – Wee1 and Cdc25C. Wee1 is a nuclear proteinthroughout the cell cycle, whereas Cdc25C binds to 14–3–3 proteins during interphase and remains
predominantly cytoplasmic (and biologically ‘inactive’). In some systems, Cdc25C, like cyclin B1,rushes precipitously into the nucleus just before entry into mitosis.
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trends in CELL BIOLOGY (Vol. 9) June 1999 209
Although the NES region of the human cyclin B2CRS also differs from that of human cyclin B1, thedeviations from an NES consensus are less signifi-cant than those in Xenopus cyclin B2. Hence, it willbe important to determine whether the human B2CRS maintains the ability to mediate nuclear exportor relies on another mechanism for cytoplasmic lo-calization, as is likely to be the case for Xenopus. Ashuman cyclin B2 appears to colocalize with theGolgi apparatus, there might, for example, be aGolgi-associated cyclin B2 anchor12.
How do they get through the tunnel?As cyclins A, D, E and B1 all spend some portion of
the cell cycle within the nucleus, either they, or theirCDK partners, must contain nuclear-targeting infor-mation. Proteins containing classical basic nuclear-localization sequences (NLSs) bind to a soluble NLSreceptor, importin-a, which then interacts with thecytoplasmic nuclear transport factor, importin-b,for transport through the nuclear pore (for review,see Refs 19–22). However, as no obvious NLS can befound upon scanning the primary sequences of vertebrate cyclins or their CDK partners, several hypothetical mechanisms have been proposed to account for CDK–cyclin nuclear import: CDK–cyclincomplexes might utilize novel import receptors; aclassical NLS might be reconstituted by binding ofthe CDKs to the cyclins; and CDK–cyclin complexesmight ‘piggyback’ into the nucleus on karyophilicproteins that do contain a consensus NLS.
However, in the case of cyclins B1 and E, recentstudies have demonstrated that nuclear targeting in-formation is contained entirely within the cyclinsubunit23. Moreover, cyclin E seems to contain acryptic (or as-yet-unidentified) ‘classical’ NLS sinceit binds to and can be imported into nuclei by theimportin a–b heterodimer. Surprisingly, cyclin B1enters nuclei through direct binding to importin-bin the absence of an importin-a adaptor. Indeed, cyclinB1 binds to a site on importin-b distinct from thebinding site used by importin-a, suggesting that anovel transport mechanism is used for cyclin B123.Indeed, cyclin B1 nuclear import has been observedin the absence of the small GTPase Ran, which wouldbe highly unusual for a classical NLS-dependent nuclear-import pathway (C. Takizawa and D. Morgan,pers. commun.).
Although different cyclins share significant hom-ology, they appear to use diverse mechanisms fornuclear import. As reported by Moore et al., both cy-clin B1 and cyclin E, which use distinct transport re-ceptors, do not appear to require CDKs for nuclearimport23. However, in accordance with an earlierdeletion analysis of cyclin A performed by the Nigglaboratory24, cyclin A nuclear import requires the activity of an associated CDK (J. Pines, pers. com-mun.). Moreover, cyclin A appears to enter nucleithrough a mechanism distinct from that identifiedfor cyclins B1 and E (J. Pines, pers. commun.). Tocomplicate matters further, a point mutant of cyclinD1, which results in inappropriate cytoplasmic re-tention of Cdk4–cyclin D1 can be partially inducedto enter nuclei through overexpression of the
CDK inhibitor p21, suggesting that Cdk4–cyclin D1 might be able to use the classical NLS on p21 fornuclear import9.
Why do all of the cyclins examined appear to usedifferent mechanisms for nuclear import? It is likelythat their different cell-cycle roles necessitate differ-ent nuclear-import efficiencies or different opportu-nities for transport regulation. For example, main-taining a low nuclear-import rate for cyclin B1during interphase is essential if nuclear export is tokeep it in the cytoplasm efficiently. As appending aclassical NLS onto cyclin B1 results in its constitu-tive nuclear localization, it is likely that direct bind-ing to importin-b offers a less efficient nuclear-import mechanism than binding to importin-b viathe importin-a adaptor. The rate of nuclear relocal-ization of cyclin B1 at the G2–M transition appearsto exceed the rate expected by inhibiting nuclear export alone, so it is quite possible that phosphoryl-ation of cyclin B1 also boosts its rate of nuclear import. Whether cyclin B1 employs a novel trans-port mechanism at the G2–M transition or utilizesthe importin-b-dependent pathway more efficientlyremains to be determined.
CDK–cyclin trafficking and keeping the cell cycleon track
How does control of CDK–cyclin localization con-tribute to cell-cycle regulation? Perhaps the most ex-tensively studied connection is that between cyclin B1nuclear translocation and proper coordination ofthe G2–M transition. In diverse organisms, cyclin B1nuclear accumulation directly precedes the onset ofmitosis. Heald et al. showed that high levels of cyto-plasmic Cdc2–cyclin B kinase activity alone couldnot induce mitosis in BHK cells25. In addition, as described above, reduced nuclear accumulation ofcyclin B1 in Xenopus oocytes correlates with a reducedability to promote the G2–M transition16. Hence,nuclear accumulation of Cdc2–cyclin B kinaseseems to be required for mitosis to proceed.
Cyclin B1
NH2
Destruction boxCytoplasmic
retention sequence
Human: LCQAFSDVILXenopus: LCQAFSDVLI
Cyclin box
COOH
Cyclin B2
NH2
Destruction boxCytoplasmic
retention sequence
Human: LCQAFSDALLXenopus: LCQAFSDALT
Cyclin box
COOH
FIGURE 2
Alignment of the nuclear-export sequences (NESs) of Xenopus and human cyclins B1and the corresponding region of cyclin B2. Both Xenopus and human cyclin B1contain a consensus CRM1-binding hydrophobic NES. However, the correspondingamino acids in cyclin B2 diverge from the NES consensus, although the deviationfrom the consensus is less pronounced in human cyclin B2 than in Xenopus cyclin B2.The amino acids comprising the NES consensus sequence are shown in bold.
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210 trends in CELL BIOLOGY (Vol. 9) June 1999
Several studies have implicated nuclear exclusionof Cdc2–cyclin B1 in the operation of checkpointsthat prevent entry into mitosis in the presence ofDNA damage. It has been reported that Cdc2–cyclinB1 is cytoplasmic in G2-arrested cells bearing dam-aged DNA14. Indeed, forcibly localizing Cdc2–cy-clin B1 to the nucleus by appending a classical NLScan trigger premature mitotic events in DNA-dam-age-arrested cells, particularly if Cdc2 is mutated torender it insensitive to inhibitory phosphorylation(Cdc2AF)26. Similarly, Toyoshima et al. found that anuclear-export-defective mutant of cyclin B1 wasmore effective than wild-type cyclin B1 at cooperat-ing with the drug caffeine to override a DNA-damage-induced G2 arrest14. Although these datademonstrate that inappropriate nuclear localizationof Cdc2–cyclin B1 can compromise a DNA-damage-induced cell-cycle arrest, it is not yet knownwhether regulation of cyclin B1 localization is a di-rect locus of checkpoint control. It will be interest-ing to determine if and how such regulation occurs.It is an intriguing possibility that cyclin-B-directedkinases regulating Cdc2–cyclin B1 localization aremodulated by checkpoint pathways.
Where is this train headed?Although recent progress has been made towards
understanding the regulation of CDK–cyclin nu-clear trafficking, many questions remain. Some ofthe receptors responsible for CDK–cyclin nuclearimport have been defined, but in no cases have thenuclear-targeting sequences been identified. Thiswill be crucial if we are to understand how interac-tions between cyclins and import receptors are modulated and to determine how nuclear import ofthe CDK–cyclin complexes impinges upon cell-cycleregulation.
Since phosphorylation controls the localization ofCdc2–cyclin B1 and possibly other CDK–cyclincomplexes, identification of kinase/phosphatasesacting on these complexes will be of great interest.The means by which cell-cycle checkpoints mightcontrol CDK–cyclin localization also remains to bedetermined. An additional facet of cell-cycle regu-lation concerns the mechanisms that coordinate thelocalization of CDK–cyclin complexes with that oftheir regulators. For example, prior to the demon-stration that Cdc2–cyclin B1 can shuttle in and outof nuclei, it was not understood how Wee1, a nega-tive regulator of Cdc2, might gain access to thesecomplexes. It has been demonstrated recently thatthe Cdc25 phosphatase that activates Cdc2–cyclinB, can shuttle in and out of nuclei. Moreover, Cdc25nuclear trafficking appears to be controlled both byG2–M checkpoints and by cellular factors that helpto maintain a G2 arrest in oocytes27,28. Under-standing how subcellular trafficking of these cell-cycle regulators helps to coordinate downstream nuclear and cytoplasmic events of the cell cycle iscrucial if we are to obtain a complete picture of howthe cell-cycle locomotive runs.
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G2 phase
Prophase
(a) (b) (c)
(d) (e) (f)
FIGURE 3
Translocation of cyclin B1 to the nucleus at mitosis. Cyclin B1–green-fluorescent protein in a living HeLa cell is cytoplasmic in G2 phase (a–c) because
it is rapidly exported from the nucleus. At mitosis, the cyclin B1–GFP rapidlytranslocates into the nucleus (d–f) in a phosphorylation-dependent manner.
[(a,d) fluorescence; (b,e) differential interference contrast; (c,f) merged image.Images kindly provided by P. Clute, Wellcome/CRC Institute, Cambridge, UK.]