2. Lefebvre, S. et al. Cell 80, 155–165 (1995).

3. Lorson, C. L., Hahnen, E., Androphy, E. J. & Wirth, B. Proc.

Natl Acad. Sci. USA 96, 6307–6311 (1999).

4. Frugier, T. et al. Hum. Mol. Genet. 9, 849–858 (2000).

5. Gangwani, L., Mikrut, M., Galcheva-Gargova, Z. & Davis, R. J. J

Cell Biol 143, 1471–1484 (1998).

6. Galcheva-Gargova, Z. et al. Science 272, 1797–1802 (1996).

7. Schlessinger, J. Cell 103, 211–225 (2000).

8. Galcheva-Gargova, Z. et al. Mol. Biol. Cell 9, 2963–2971 (1998).

9. Gangwani, L., Mikrut, M., Theroux, S., Sharma, M. & Davis, R.

J. Nature Cell Biol. 3, 376–383 (2001).

10. Matera, A. G. Trends Cell Biol. 9, 302–309 (1999).

11. Fischer, U., Liu, Q. & Dreyfuss, G. Cell 90, 1023–1029 (1997).

12. Pellizzoni, L., Charroux, B., Rappsilber, J., Mann, M. &

Dreyfuss, G. J. Cell. Biol. 152, 75–86 (2001).

13. Friesen, W. J. & Dreyfuss, G. J. Biol. Chem. 275, 26370–26375

(2000).

14. Charroux, B. et al. J. Cell Biol. 148, 1177–1186 (2000).

15. Gall, J. G. Annu. Rev. Cell Dev. Biol. 16, 273–300 (2000).

16. Francis, J. W., Sandrock, A. W., Bhide, P. G., Vonsattel, J. P. &

Brown, R. H. J. Proc. Natl Acad. Sci. USA 95, 6492–6497 (1998).

The cell’s ability to self-reproduce is afundamental feature of life, essentialfor species continuation and embryon-

ic development, as well as tissue renewal inadult organisms, including humans.Cellular self-reproduction reflects the work-ings of the cell division cycle machinery,whose two major driving forces, the activi-

ties of cyclin-dependent kinases (CDKs)and ubiquitin-mediated, proteasome-dependent proteolysis, are carefully orches-trated at multiple levels. Failure to controlthe cell cycle adequately can lead to diverselife-threatening diseases such as cancer.

This aspect has motivated the rapid paceof discovery in this dynamic field of cell

biology. Given the impressive progress ofcell cycle research over the past decade orso, it is surprising that a group of essential,phylogenetically conserved cell cycle regu-lators known as the Cks/Suc1 proteins havenot been linked with a specific molecularfunction for some 15 years. The work oftwo research teams1,2 using differentapproaches has recently converged to revealan unexpected role for Cks1, one of the twomammalian Cks orthologues. Cks1 seemsto be an essential accessory factor forSCFSkp2, the ubiquitin ligase that targets thep27Kip1 CDK inhibitor for proteasome-dependent destruction, thereby allowingCDK activity to drive cells into S phase.This discovery is intriguing from severalpoints of view and it has important impli-cations for biomedicine.

In the first report, Ganoth et al.1 identifiedCks1 as the missing factor that complement-ed the otherwise inefficient ubiquitinylationof p27Kip1. This was done by reconstitutingthe SCFSkp2 complex purified from insectcells using baculovirus expression vectorscomplemented with factors biochemically

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NATURE CELL BIOLOGY VOL 3 APRIL 2001 http://cellbio.nature.com E95

p27 destruction: Cks1pulls the trigger

Jiri Bartek and Jiri Lukas

Recent work has discovered the role of the cyclin-dependent kinase(CDK)-binding protein Cks1 in degrading the CDK inhibitor p27Kip1. Thisprocess is essential for DNA replication and is aberrantly enhanced incancer. Surprisingly, new work indicates that this function of Cks1 isindependent of CDKs and links Cks1 with the Skp2 subunit of the SCFubiquitin ligase.

Wiskott–Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT)are disorders resulting in variable immunodeficiency. The protein mutat-ed in these diseases, WASP, is expressed in cells of a haematopoeticlineage and is known to regulate the actin cytoskeleton by linking sig-nalling via Rho-family GTPases and activation of the Arp2/3 complex,which promotes actin nucleation and crosslinking.

A recent paper in Nature Genetics by Devriendt and colleagues(Nature Genetics 27, 313–317; 2001) describes a third disorder aris-ing from a defect in WASP: X-linked severe congenital neutropenia(XLN). Linkage analysis of a family whose male members displayedimmunodeficiency and neutropenia revealed the presence of a singlebase-pair mutation (resulting in an L270P substitution) in WASP. Thisfinding was surprising because both WAS and XLT patients have normalneutrophil counts, and, although no WASP was detectable in an XLT cellline, WASP was present in almost normal amounts in XLN lines.

WASP activity is regulated by an intramolecular interaction betweenits GBD and VCA domains. Binding of Cdc42 to WASP disrupts this inter-action and allows WASP to interact with and activate the Arp2/3 com-plex. L270 (green residue in figure) is located within an α helix that ispart of the hydrophobic centre of the GBD–VCA globular domain (whichis shown in the figure). Thus, it seemed likely that a kink introduced bythe substitution of a proline residue in the mutated WASP would relievethe autoinhibition, effectively producing a constitutively active molecule.In agreement with this idea, when this mutation was introduced into dif-ferent constructs encompassing the autoinhibitory domains, the stabilityof the proteins was decreased because the domain was now unfolded.

In addition, the affinity of these proteins for Cdc42 was enhancedbecause the GBD domain was no longer buried. Finally, a construct con-taining the mutation and both domains was much more effective at stim-ulating the activity of the Arp2/3 complex in vitro, such that the additionof Cdc42 elicited no further stimulation.

The question now to be addressed is: how does constitutive activa-tion of WASP result in neutropenia? Devriendt and colleagues proposethat the constitutively activated WASP prevents normal signalling viaCdc42 and leads to higher cellular levels of F-actin. Because actin fila-ments and the cytoskeleton are involved in many aspects of T cell acti-vation, shape and motility, this might be the basis for the differenteffects of WAS and XLT on the one hand, and XLN on the other. Thus,WASP joins the small cohort of proteins in which different disorders canbe ascribed to activating and inactivating mutations.

ANGELA EGGLESTON

Stung by WASP

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purified from HeLa cell extracts. Strikinglysimilar conclusions about the critical role ofCks1 in targeting p27Kip1 for efficient degra-dation by SCFSkp2 were deduced by Sprucket al.2 based on experiments inspired by theabnormally small size of mice nullizygous(both alleles deleted) for Cks1, reflecting

the slow proliferation of Cks1-deficientcells that accumulated high levels ofp27Kip1. The mechanistic model thatemerges from these studies suggests thatCks1 associates with the F-box proteinSkp2 and probably confers an allostericchange in Skp2 that increases its affinity for

the p27Kip1 substrate that has been phos-phorylated by cyclin-E/A–CDK2 kinases.As a result, the Cks1–Skp2 interactionenables the efficient transfer of ubiquitin top27Kip1 by the multicomponent SCFSkp2

ubiquitin ligase (Fig. 1).The SCF complex comprises Skp1,

Cullin and F-box-protein subunits, togeth-er with the recently identified Roc1/Rbx1(refs 3–5). SCF acts as a ubiquitin ligase (orE3) downstream of the ubiquitin-activating(E1) and -conjugating (E2, in this caseUbc3) enzymes in the ubiquitin pathway;this pathway marks proteins for rapiddestruction by attaching to them chains ofsmall ubiquitin polypeptides that are subse-quently recognized by the proteasome3. Thespecificity of substrate recognition by SCFrelies on F-box proteins, whose wide spec-trum ensures their selectivity for diversesubstrates. The F-box proteins bind theSkp1 SCF component through their so-called F-box motif, while their variable C-terminal region interacts with the substratein a strictly phosphorylation-dependentmanner3. In vitro, the reconstituted ubiqui-tinylation reaction of the yeast CDKinhibitor Sic1 required ubiquitin, E1 and E2enzymes, the SCF subunits Cul1, Skp1 andRbx1, and the substrate-specific F-box pro-tein Cdc4 (ref. 5). However, similarattempts to reconstitute the ubiquitinyla-tion of the mammalian p27Kip1 have beenrepeatedly unsuccessful, even though thespecific F-box protein (Skp2) and the phos-phorylated residue of p27 required for itsrecognition by SCFSkp2 (T187) have beenidentified6. This indicated that, in case ofp27Kip1, the SCFSkp2 holoenzyme requires yetanother component, now identified byGanoth et al.1 and Spruck et al.2 as Cks1.How Cks1 might assist SCF in the efficientubiquitinylation of p27 is outlined in Fig. 1.

Why are these two reports so surpris-ing? Cks proteins were discovered in themid 1980s as subunits that interactedtightly with cyclin–CDK complexes7.Naturally, the search for their role(s) in cellcycle regulation focused on potential func-tion(s) of Cks proteins in controlling CDKactivity. However, with the exception ofSaccharomyces cerevisiae Cln2/Cln3–Cdk1,no other tested cyclin–CDK complexesrequire Cks/Suc1 proteins for their activity,nor are they inhibited by interacting withthese proteins. In fact, immobilized Cks(the famous ‘Suc1 beads’) have been com-monly used to purify active cyclin–CDKcomplexes. The crystal structure of Cksrevealed a stretch of potentially phosphate-binding basic residues contiguous withtheir CDK-interaction domain, and led to aproposal that Cks might facilitate substraterecognition by cyclin–CDK holoenzymesand thus promote full-scale (multiphos-phate) phosphorylation of key cell cycleregulators7. Indeed, phosphorylation ofCdc25 phosphatase, Myt1 and Wee1 kinases

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a

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Figure 1 Requirements for productive ubiquitinylation of p27. a, In the absence of Cks1,the otherwise completely assembled SCFSkp2 ubiquitin (Ub) ligase does not efficiently rec-ognize p27, even though all other components required for the ubiquitinylation reaction(E1, Ubc3, ubiquitin) are not limiting and p27 is phosphorylated on threonine 187 bycyclin-E/A–Cdk2. The major obstacle appears to be the conformation of the Skp2 sub-strate-binding domain, which is not compatible with docking the SCFSkp2 holoenzyme intophosphorylated p27. b, Cks1 promotes allosteric changes in the Skp2 F-box protein thatare necessary and sufficient for a phosphorylation-dependent recognition and subse-quent ubiquitinylation of p27. This function of Cks1 is strictly dependent on its directbinding to Skp2. To trigger measurable ubiquitinylation of p27, Cks1 does not requireassociation with cyclin–CDK complexes, nor it is dependent on CDK activity providedthat the phosphorylation of p27 on threonine 187 have been accomplished. c, Two otherfeatures of Cks1 might help to increase the velocity of the SCFSkp2-dependent ubiquitiny-lation of p27. First, Cks1 might facilitate the recruitment of phosphorylated p27 intoSCFSkp2 ubiquitin ligase and/or stabilize a Skp2–p27 interaction owing to its ability tobind phosphate residues (Thr187 in case of p27 (ref. 1)). Second, a simultaneous inter-action (if it can be proved) of Cks1 with Skp2 and cyclin–CDK complexes might con-tribute to the correct positioning of p27 bound to SCFSkp2 and thereby further promotethe ubiquitinylation reaction. This scenario seems to be compatible with the somewhatgreater ability of the wild type than the CDK-binding-deficient mutant Cks1 to facilitatethe polyubiquitinylation of p27 in vitro2.

© 2001 Macmillan Magazines Ltd

(all involved in regulation of entry intomitosis), and the Cdc27 subunit of theAPC–cyclosome ubiquitin ligase (essentialfor initiating sister chromatid separationat the metaphase–anaphase transition)were shown to be stimulated by the Cksproteins8–10. However, at least in some cases,similar effects on cell cycle progression wereachieved by either excess or depletion of theCks proteins7,9, leaving the puzzle of the

exact mechanistic role of the Cks proteinsin cell cycle control unsolved.

Most, if not all, of the Cks functions pos-tulated to date were discovered in yeast or ina cell-free system recapitulating earlyembryonic cycles of Xenopus laevis, and verylittle is known about the mammalian Cksproteins. Also, the bulk of available evidencepointed to mitotic role(s) of Cks7–10, includ-ing the recent report on Cks-facilitated

recruitment of ubiquitinylated B-typecyclins to the proteasome11 for their timelydestruction, necessary for exit from mitosis.Given such focus on emerging mitotic rolesof Cks proteins and their generally acceptedlink with the direct binding of Cks tocyclin–CDK complexes, it comes as a bigsurprise that, when a role for Cks1 in mam-malian cells is finally found, it turns out toregulate exclusively the G1–S transition andto operate independently of any directinteraction of Cks1 with cyclin–CDK com-plexes1,2. Perhaps paradoxically, the newdiscovery revealed that the long-soughteffect of Cks1 on cyclin–CDK activity doesexist but is only indirect, brought about bystimulating the destruction of p27Kip1, apowerful inhibitor of cyclin-E/A–Cdk2complexes, which are essential for DNAreplication in all vertebrate cells. As yetanother surprise, the role of Cks1 in pro-moting p27 destruction cannot be substi-tuted by its close relative Cks2 (refs 1,2),despite the very high homology of the twoproteins and redundancy in all their previ-ously suggested functions.

The two studies on p27 degradation alsoprovide the first example of the productiveubiquitinylation of a mammalian cell cycleregulatory target by SCF reconstituted frompurified components. As well as encourag-ing future attempts to assemble other com-plex molecular machines in vitro, the dataraise a number of conceptually importantquestions. For instance, exactly how Cks1potentiates Skp2 to recognize p27 efficientlyremains unclear, yet the recently solved crys-tal structure of Skp2 (ref. 12) suggests apotential mechanism for Cks1-dependentremodelling of the substrate binding face ofSkp2. The structure shows a carboxy-ter-minal tail domain that folds back into theconcave presumptive substrate-bindingdomain, composed of leucine-rich repeats12,potentially blocking access to substratessuch as p27. The function of Cks1 might beto alter the structure of Skp2 so that thisdomain is displaced, allowing substratebinding.

The question of whether the function ofCks1 in p27 protein turnover in vivo is alsoentirely independent of its binding tocyclin–CDKs has not been directlyaddressed. The ability of cyclin A to interactwith Skp2 indicates that at least somecyclin–CDK complexes might help torecruit Cks1 to SCFSkp2. This issue couldbest be addressed by genetically knocking-in a mutant Cks1 whose interaction with CDK is selectively disrupted2, or byreconstituting Cks1–/– cells with the CDK-binding-deficient Cks1 to see whether allproliferative effects, as well as p27’s half-life,could be rescued to normal values2.

It would also be informative to examinewhether the ubiquitinylation of any otherSCF targets requires auxiliary factors anal-ogous to Cks1, or whether this scenario

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Figure 2 Deregulation of p27 protein turnover contributes to tumorigenesis. a, In normaltissue, initiation of DNA replication strictly requires the S-phase-promoting cyclin-E–Cdk2activity. Depending on its abundance, p27 can either inhibit cyclin-E–Cdk2 or becomephosphorylated and destined for rapid destruction. In the presence of a growth-inhibitorysignal, stabilization of p27 allows its accumulation to levels sufficiently high to changethe equilibrium towards cyclin-E–Cdk2 inhibition, thereby preventing unscheduled S-phase entry. b, In cancerous tissue, aberrant amplification or stabilization of cyclin E (i),overexpression of Skp2 (ii) and haploinsufficiency (such as by promoter methylation15) ofp27 (iii) contribute to accelerated degradation and prevent the accumulation of p27.This might lead to the induction of unscheduled DNA replication by uninhibited cyclin-E–Cdk2, even under otherwise growth-restraining conditions. Whether or not deregula-tion of Cks1 itself contributes to oncogenesis is a major challenge for future research.Ub, ubiquitin.

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highlights an exceptionally tight control ofp27, owing to its central role in protectingmammalian cells from excessive prolifera-tion. To appreciate the biological implica-tions of these new results fully1,2, oneshould remember that the vast majority ofour cells are not proliferating at any giventime, but rather exist in the G0 phaseknown as quiescence. The CDK inhibitorp27Kip1 is a key regulator of both entry intoand maintenance of the G0 state, and micelacking p27Kip1 show gigantism withorganomegaly and a cancer-prone pheno-type, which is consistent with the acceptedroles of p27Kip1 in embryonic development,tissue homeostasis and prevention ofmalignant growth6.

Better mechanistic insight into p27degradation might therefore help us tounderstand better the complex process ofmultistep tumorigenesis. In fact, aggressivehuman tumours contain high p27-specificdegradation activity, and the absence ofp27Kip1 is a powerful prognostic marker ofpoor survival in patients with breast,oesophageal, lung and colorectal carcino-mas6. Skp2 is overexpressed in some tumourtypes, such as lymphomas, and correlateswith the grade of malignancy and inverselywith p27 levels13. Moreover, many tumour

types have elevated levels of cyclin E, whoseactivity is essential for phosphorylation-dependent p27 degradation. Thus, analo-gous to other critical cell-growth regulatorycascades, deregulation of each of the com-ponents in the cyclin-E/Cdk2–p27–Skp2interplay might contribute to oncogenesis(Fig. 2). Whereas cyclin E and Skp2 qualifyas proto-oncogenes, p27Kip1 is a tumoursuppressor whose cancer-promoting down-modulation6 reflects diverse aberrations(Fig. 2), including the unorthodox haploin-sufficiency14,15, again documenting the

strict requirement in mammalian cells for adelicately balanced abundance of this CDKinhibitor. Given its role in promoting p27degradation1,2, it is tempting to speculatethat Cks1 might turn out to be a novelproto-oncogene, a prediction that could betested by screening human tumour materialfor gene amplification and/or overexpres-sion of Cks1.Jiri Bartek and Jiri Lukas are in the Department ofCell Cycle and Cancer, Institute of Cancer Biology,Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmarke-mail: bartek@biobase.dk

1. Ganoth, D. et al. Nature Cell Biol. 3, 321–324 (2001).

2. Spruck, C. et al. Mol. Cell (in the press).

3. Krek, W. Curr. Opin. Genet. Dev. 8, 36–42 (1998).

4. Ohta, T., Michel, J. J., Schottelius, A. J. & Xiong, Y. Mol. Cell 3,

535–541 (1999).

5. Kamura, T. et al. Science 284, 657–661 (1999).

6. Slingerland, J. & Pagano, M. J. Cell Physiol. 183, 10–17 (2000).

7. Pines, J. Curr. Biol. 11, 1399–1402 (1996).

8. Patra, D., Wang, S. X., Kumagai, A. & Dunphy, W. G. J. Biol.

Chem. 274, 36839–36842 (1999).

9. Patra, D. D. & Dunphy, W. G. Genes Dev. 12, 2549–2559 (1998).

10. Shteinberg, M. H. et al. Biochem. Biophys. Res. Commun. 257,

12–18 (1999).

11. Kaiser, P. et al. Genes Dev. 13, 1190–1202 (1999).

12. Schulman, B. A. et al. Nature 408, 381–386 (2000).

13. Latres, E. et al. Proc. Natl Acad. Sci. USA 98, 2515–2520 (2001).

14. Fero, M. L., Randel, E., Gurley, K. E., Roberts, J. M. & Kemp, C.

J. Nature 396, 177–180 (1998).

15. Worm, J. et al. Oncogene 19, 5111–5115 (2000).

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Arnold Greenberg died on 11 February 2001, aged 59, of cancer.Should I emphasize his scientific achievements? These consistently

revolved around cell-mediated cytotoxicity and cell death, notably his1973 discovery, while working as a PhD student in Ivan Roitt’s labora-tory, of cytotoxic ‘null’ lymphoid cells (later known as NK cells anddescribed independently at about the same time in Stockholm and atthe NIH). In 1992, he described the rat serine esterase fragmentin, alsoknown as Granzyme B, and subsequently studied the role of this mole-cule together with perforin in one of the pathways of lymphoid cell-medi-ated cell death. Starting in 1994, he published reports of the evolution-arily fascinating role of cell cycle molecules such as CDC2 in cell death,a chapter that is probably far from closed, and, more recently, studiesof the role of the Bcl-2 family member BNIP3 in caspase-independentcell death. More generally, he contributed to several important facets ofthe molecular mechanism(s) of normal and tumour cell death.

Should I recall how much he did for the development of research inhis home town, Winnipeg, where he returned after training in the UnitedStates and England? He worked there for many years, until the very lastdays of his life, and successfully built up the Manitoba Institute of CellBiology there, which now includes 13 research teams working mostlyon cancer and cell death, perhaps the professional achievement ofwhich he was the most proud. He was an excellent ambassador forCanadian science and the University of Manitoba, and, more generally,he provided inspiration to Canadian (and other) scientists. His contribu-tions were recognized, in particular, through the award of the CSICinader Award and of a Canadian Research Chair.

No. What I really want to stress is that he was a warm and generousperson. He expressed genuine and spontaneous interest in the workand achievements of students, which they highly appreciated, and in thework of more senior colleagues, which he was able to discuss knowl-

edgeably but tactfully. Not only was he a professional scientist but alsohe loved his fellow people. It was in Arnold’s and his wife Faye’s flatbehind the harbour in Cassis (during a sabbatical stay in SouthernFrance) that I discovered Arnold’s talent for cooking, and how much heloved to share the results of cooking, as well as science, with hisfriends. I also discovered how much he liked discussing literature andart, history and politics, and his own experiences and pleasures, suchas once hiking around a Mediterranean island, which clearly had beenquite important to both Faye and him. He was also lucid and determinedthroughout his final illness and worked until the day before he died. Heis survived and missed by his wife, Faye Hellner, by his children andgrandchildren, and by friends all over the world.

PIERRE GOLSTEINCentre d’immunologie, INSERM-CNRS de Marseille-LUMINY,

Marseille, France;golstein@ciml.univ-mrs.fr

Obituary Arnold Greenberg

Given its role in promoting

p27 degradation, it is

tempting to speculate that

Cks1 might turn out to be a

novel proto-oncogene, a

prediction that could be

tested by screening human

tumour material.

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