- Email: [email protected]
Proteolysis and the G1-S transition: the SCF connection
36
Proteolysis and the GI-S transition: the SCF connection Wilhelm Krek Temporal control of ubiquitin-proteasome mediated protein degradation is critical for normal G 1 and S phase progression. Recent work has shown that central to the temporal control mechanism is a relationship between newly identified E3 ubiquitin protein ligases, designated SCFs (Skpl-cullin-F-box protein ligase complexes), which confer substrate specificity on ubiquitination reactions and the activities of protein kinases that phosphorylate substrates destined for destruction at specific sites, thereby converting them into preferred targets for ubiquitin modification catalyzed by SCFs. The constituents of SCFs are members of evolutionary conserved protein families. SCF-based ubiquitination pathways may play a key role in diverse biological processes, such as cell proliferation, differentiation and development.
Addresses Friedrich Miescher Institut, Maulbeerstrasse 66, CH-4058 Basel, Switzerland; e-mail: [email protected]
Current Opinionin Genetics& Development1998, 8:36-42
http:llbiomednet.co.lelecreflO959437XO0800036 © Current Biology Ltd ISSN 0959-437X
Abbreviations CDK cyclin-dependent protein kinase CKI
LRR SCF
CDK inhibitor protein leucine-richrepeat Skpl-cullin-F-box protein ligase complex
Introduction Ubiquitin-proteasome dependent protein degradation plays a fundamental role in the regulation of the eukaryotic cell cycle [1,2]. It is highly selective, precisely timed and allows an instant switch from one functional programme to another. During the cell cycle, protein degradation is required for the transition from G1 to S phase, sister chromatid separation in anaphase and the exit from mitosis [2]. Degradation of a protein via the ubiquitin-proteasome pathway involves the assembly of a ubiquitin chain on the substrate and the rapid degradation of the multiubiquitinated substrate by the 26S proteasome [3,4]. The conjugation of ubiquitin to proteins involves a series of well-defined enzymatic reactions. Ubiquitin is first activated via thioester formation by an ubiquitin-activating enzyme (El), which transfers ubiquitin to one member of a family of distinct ubiquitin-conjugating enzymes (E2s or UBCs), again forming a thioester. Certain E2s can transfer ubiquitin directly to substrates whereas others require the participation of a third component, termed E3 ubiquitin protein ligase [5]. E3 is the component of the ubiquitin conjugation system that is generally thought to be the most directly involved in substrate recognition. At the same
time, E3 components are the least well-understood factors involved in ubiquitin ligation. At present, a great deal of effort in cell cycle research is focused on elucidating the mechanisms by which the selectivity of protein destruction is achieved and the mechanisms by which proteolytic activity is limited to a specific cell cycle phase. Recent genetic and biochemical studies in Saccharomyces cerevisiae have led to the identification of a novel class of E3 ligases that function in combination with the E2 enzyme Cdc34 to promote the ubiquitination of key cell cycle regulatory proteins during the G1 and S phases of the cell cycle (Figure 1). The core components of these newly identified E3s are Skpl and Cdc53, which assemble with different F-box proteins, for example, Cdc4 or Grrl, into distinct protein complexes [6°,7,8",9"°,10°°]. The F-box protein subunits display selectivity in the recognition of potential ubiquitination targets. These units have been designated SCFs (Skpl-cullin-F-box protein ligase complexes) [9°°,10°°]. Structural and functional homologs of Skpl exist in evolutionarily diverse organisms [6°, 11,12]. Likewise, Cdc53 is a member of a large, evolutionarily conserved, multigenc family, referred to as the cullins [7,13"]. Finally, the F-box, which is thought to mediate binding to Skpl, is a conserved domain that is found in a large number of proteins [6°]. Related family members may undergo combinatorial interactions to generate a large number of SCFs, each potentially with a unique cellular function. A hallmark of known SCFs is that they recognize ubiquitination substrates in a phosphorylation-dependent manner. This review focuses on the role of Cdc34, SCFs and phosphorylation in the proteolytic pathways that govern the G1-S phase transition in the eukaryotic cell cycle. T h e i m p o r t a n c e of Cdc34 and phosphorylation for regulated proteolysis at t h e G 1 to S p h a s e transition Passage through the cell cycle requires the successive activation of different complexes between cyclin and cyclin-dependent protein kinase (CDK) [14,15], In mammalian cells, the cyclin-CDK complexes that are most critically involved in the decisions concerning growth and quiescence are D-type cyclins with their partners CDK4 and CDK6, and cyclins E and A with their partner CDK2 [14]. The activities of these CDK complexes are essential for driving cells through G 1 into S phase. Budding yeast contains functional homologs that act at similar stages of the cell cycle with the G 1 cyclins Clnl, Cln2, Cln3, ClbS and Clb6 sequentially activating the yeast CDK Cdc28 to promote S phase entry [16]. In general, CDK activity depends on the binding of cyclins, positive and negative regulatory phosphorylation events and the action of CDK
Proteolysis and the G1-S transition Krek
Figure 1 E3 ubiquitinligasecomplexes
Ubiquitination targets
&J
Sic1 Far1
scFCdc4
E2 [ ~
~
Cin2
scFGrrl Current
Opinionin Genetics & Development
A schematic diagram depicting the molecular composition of two distinct SCFs from S. cerevisiae and their role in the recognition and ubiquitination of targets. Cdc4 and Grrl are representatives of the F-box protein family [6°]. The nature of the F-box protein defines the repertoire of substrates targeted by the SOt=, The SCF Cdc4 recruits selectively phosphorylated Sic1 and Earl into the complex for ubiquitination by the E2 enzyme Cdc34 [9"',10°',29°]. The SCF Grrl displays affinity for phosphorylated CIn2 and not for Sicl or Earl [10°°]. It is thought that Cdc34 physically interacts with each of these SCFs [7,8°,9°',10"°]. E1 is a ubiquitin-activating enzyme required for ubiquitin activation and transfer onto the E2 Cdc34.
inhibitor proteins (CKIs) that through their association with either a CDK or a cyclin-CDK complex block function [17,18]. Recent work demonstrates that CDK activity is regulated by ubiquitin-mediated proteolysis. The first connection between cell cycle control and proteolysis was suggested by the discovery ten years ago that a ubiquitin-conjugating enzyme encoded by the CDC34 gene is required for G 1 to S phase progression in Saccharomyces cerevisiae [19]. In the absence of CDC34 function, cells exhibit high levels of Cln-Cdc28 kinase activity but fail to initiate DNA replication because the Clb5/6-Cdc28 kinases that are needed downstream for executing the onset of S phase are held in check by the CKI Sicl, whose degradation shortly before S phase is defective in CDC34 mutant strains [20,21]. These observations led to the proposal that Cdc34 degrades Sicl in late GI. In addition to CDC34, the destruction of Sicl also requires CLN-CDC28 function [22,23). The requirement for a G 1 cyclin-CDK dependent step in the degradation of Sicl suggests that phosphorylation may constitutes a recognition signal for ubiquitination by the Cdc34 pathway. Indeed, Sicl is a substrate of Cln-Cdc28 kinase and mutation of CDK consensus phosphorylation sites in Sicl reduces Sicl ubiquitination in vitro and stabilizes Sicl in vivo. In addition, Sicl mutants lacking CDK phosphorylation sites block DNA replication [24°]. Thus, the schedule of Sicl phosphorylation ensures that S phase is not initiated prior to the activation of Cln-Cdc28 at START (a point at which yeast cells commit to
37
the completion of a new cycle). Around that time, as sufficient Cln-Cdc28 complexes accumulate (and trigger phosphorylation of Sicl), the G I cyclins become phosphorylated by the bound Cdc28 kinase. This provokes the ubiquitination and degradation of G 1 cyclins [25,26"] and the rapid inactivation of their associated kinase activities as ceils initiate DNA replication. Different E2 enzymes, including Cdc34 [27] and Ubc9 [28], have been implicated in the ubiquitination of Clnl and Cln2. Phosphorylation also serves as a trigger for the degradation of the CKI Farl, a Cdc34 pathway substrate [29"]. Farl is required for a-factor (an antimitogen) induced cell cycle arrest and phosphorylation of Farl by Cln-Cdc28 kinase initiates the ubiquitination and proteasomal degradation of Farl. Elimination of Farl allows cells to resume cell-cycle progression after their release from (x-factor arrest [29°]. These observations highlight the importance of phosphorylation for the efficient degradation of cyclins and CKIs. In addition, Cdc34 is also required for the degradation of a number of other proteins, including the replication protein Cdc6 ]30,31] and the transcription factor Gcn4 [32]. It is not known whether phosphorylation of these proteins signals their degradation. Regardless, it appears that Cdc34 regulates various aspects of G 1 to S phase progression in S. cerevisiae. Two independent lines of experimental evidence indicate that Cdc34 plays an important role in cell-cycle regulation in all eukaryotic cells. First, a structural and functional human homolog of CDC34 was cloned and shown to complement the yeast defect [33]. Second, a dominant negative mutant of human CDC34, when added as recombinant protein to Xenopus interphase egg extracts, inhibits DNA replication [34"]. T h e requirement for CDC34 function in the initiation of DNA replication in Xenopus extracts may be directly linked to the failure to degrade Xicl, an inhibitor of cyclin E-CDK2. Although it has yet to be shown that CDC34 activity is critical for G 1 to S phase progression in animal cells, the evidence cited above suggests that CDC34 function is required for the onset of S phase in all eukaryotic cells. Not unexpectedly, phosphorylation also serves as a trigger for ubiquitin-mediated destruction of cyclins and CKIs in mammalian cells. Studies concerning the post-translational modifications of cyclin D1 [35°], cyclin E [36",37 °] and p27Kip I [38°,39"], demonstrate the importance of site-specific phosphorylation in rendering these proteins susceptible to the action of the ubiquitination machinery. In particular, the ability of D-type cyclins to act as growth factor sensors depends not only on their rapid induction by mitogens but also on their intrinsic instability, which ensures their abrupt degradation upon mitogen withdrawal. Elimination of cyclin D1 in response to mitogen withdrawal is mediated by ubiquitination and proteasomal degradation initiated by phosphorylation of cyclin D1 at a specific site [35°]. Interestingly, the protein
38
Oncogenes and cell proliferation
kinase that primes cyclin D1 for destruction may not be CDK4 or CDK6 itself, but rather an as yet uncharacterized protein kinasc.
Cyclin E is expressed periodically, peaking at G1-S, a time at which maximal levels of cyclin E - C D K 2 activity are induced, and then disappears as S phase progresses [40,41]. T h e timely disappearance of cyclin E protein in S phase is initiated by the phosphorylation of cyclin E in cis by the bound CDK2 itself, which triggers its degradation [36",37"]. Because cyclin E promotes its own turnover by activating the enzyme that signals its destruction, it assures an appropriate temporal window for the activation of its CDK partner.
The CKI p27Kip 1 is also degraded by the ubiquitinproteasome pathway [42]. Cells treated with antimitogenic factors reversibly arrest in late G1 and this arrest correlates with the inhibition of cyclin E - C D K 2 by p27Kipl; however, cyclin E - C D K 2 is also able to reverse the inhibitory effect of p27Kip 1 by phosphorylating it at a specific site, which signals the ubiquitination and proteasomal degradation of the bound CKI [38",39"]. This creates a conundrum: how does cyclin E - C D K 2 inactivate p27Kip 1, if the latter is tightly bound to the enzyme and inhibits its catalytic activity? It has been proposed that p27Kip 1 can bind cyclin E - C D K 2 in a noninhibitory mode, under which conditions cyclin E-CDK2 phosphorylates p27Kip 1, thereby signals its elimination by the ubiquitin-proteasome pathway, or in an inhibitory mode, under which conditions p27Kip I converts the enzyme into a catalytically inactive form. Whether the transition from a noninhibitory mode to an inhibitory mode is regulated in vivo is not known. Finally, in recent reports, it was shown that low levels of p27Kip I correlate with poor prognosis of human breast and colon carcinomas [43-46] and it was suggested that this may bc due to enhanced p27Kipl degradation [45]. These observations hint at the possibility that the p27Kip I destruction pathway may be deregulated in certain human tumors.
T h e view cmerging from the above cited work is that the timing of substrate ubiquitination is controlled by the activity of distinct protein kinases, which prime selected targets for destruction by site-specific phosphorylation; however, differential regulation of the stability of ubiquitin-proteasome pathway substratcs may be achieved through controls other then direct substrate phosphorylation. For example, recent studies have shown that binding of the rctinoblastoma gcne product (pRB) to the transcription factor E2F-1 in G 1 protects the latter from degradation [47"-49"]. Increased stability of E 2 F - I - p R B complexes may contribute to the maintenance of active transcriptional repression, a key event for normal G1 exit control.
Selective protein ubiquitination catalyzed by SCFs T h e issue that must now be resolved is the identification of the key components of the ubiquitination system that are involved in the recognition and targeting of only those proteins that bear a specific degradation signal and whose degradation is desired. As outlined in the introduction, cells can choose from a large repertoire of SCFs to accomplish substrate specificity over ubiquitination reactions (Figure 1). T h e first SCF has been identified in budding yeast and is composed of Skpl, Cdc53 and the F-box protein Cdc4; hence, it has been designated SCF Cdc4. It functions as a Sicl E3 ubiquitin protein ligase [9",10"]. Specifically, purified SCF Cdc4 binds phosphorylated Sicl (but not phosphorylated Cln2) and when supplemented with purified Cdc34, an E1 and ubiquitin promotes muhiubiquitination of Sicl in vitro. Thus, the SCF Cdc4 satisfies the criteria of a functional E3 ligase; namel.~, it binds an E2 (Cdc34), an established ubiquitination substtate (Sicl) and it facilitates multiubiquitination of the bound substrate. T h e F-box protein Cdc4 contains eight WD-40 repeats. In fact, many of the known F-box proteins contain in addition to their F-box other recognizable sequence motifs such as WD-40 repeats or leucine-rich repeats (LRRs), implicated in protein-protein interactions. Mutants of Cdc4 that lack the three most carboxy-terminal WD-40 repeats fail to bind phosphorylated Sicl in vitro, suggesting that this domain might serve as a substrate-recruiting domain [10"]. Interestingly, the SCF Cdc4 also targets phosphorylated Farl for ubiquitination [29"]. Thus, the S e E Cdc4 is not monospecific and temporal control of SCFCdc4-catalyzed ubiquitination is a direct reflection of the activities of the protein kinases, which phosphorylate Farl and Sicl at the relevant sites to permit SCF targeting. T h e F-box containing proteins - - Grrl and M e t 3 0 - - a r e structurally and functionally distinct from Cdc4 but like Cdc4, Grrl associates with Skpl and Cdc53. This complex, however, displays binding specificity towards phosphorylated Cln2 and not Sicl [8",10"]. Thus, the identity of the F-box protein subunit determines the repertoire of substrates targeted by these complexes (Figure 1). A purified SCF (,rrl does not support multiubiquitination of phosphorylated Cln2 in vitro, suggesting that proper functioning of the SCF Grrl requires additional signals; for example, it has been shown that S k p l - G r r l complex formation is regulated by glucose levels [50]. Further analyses of controls that are normally imposed on SCFs will provide important clues for unraveling this issue. T h e F-box protein Met30 has been implicated in the regulation of Met4, a transcription factor required for methionine biosynthetic gene expression [51]. It is possible that Met30 functions in a ubiquitination pathway
Proteolysis and the G1-S transition Krek
to degrade Met4. Finally, the Popl gene product of fission yeast, a close relative of budding yeast Cdc4, appears to bc a critical determinant for the maintenance of genome ploidy and is required for ubiquitin-proteasome mediated degradation of the CKI Ruml and the S phase regulator Cdcl8 [52"]. Taken together, these examples suggest a critical function for F-box proteins in diverse cellular processes in these yeasts.
Figure 2
ScFCdc4
SCFP45SKP2
39
F-box protein essential for S phase entry and p19 SKP1, a structural and functional homolog of Skpl [12], associate with human C U L l and CDC34 in vivo [54"]. The p19SKP1-CULl-p45 SKP2 complex portrayed in Figure 2 may serve as an SCF, guiding human CDC34 to potential ubiquitination targets that have yet to be identified. Interestingl,~; the expression of p45 SKp2 is cell-cycle regulated, peaking in S phase and declining as cells progress through M phase. In contrast, p19 sKpl, C U L l and CDC34 are expressed throughout the cell cycle ([54"]; F Reymond, W Krek, unpublished data). Thus, p19SKP1-CULl-p45 SKP2 complex formation is governed, at least in part, by the S-phase specific accumulation of the p45 SKI'2 F-box protein subunit.
Figure 3
E2
S. cerevisiae
E2
Signals ('e -,
hi]
Human Current Opinion in Genetics & Development
A schematic drawing that illustrates the subunit composition of an established Cdc34-SCF complex from S. cerevisiae and a human S-phase promoting complex. The architecture of these complexes is hypothetical because the exact nature of the protein-protein interactions depicted here have yet to be defined. Human p19 sKP1 is a structural and functional homolog of budding yeast Skpl. Likewise, human CULl and S. cerevisiae Cdc53 are members of the cullin family, p45 SKP2 contains an F-box and seven LRRs and associates directly with the S-phase cyclin A-CDK2. Each complex is found in association with the ubiquitin-conjugating enzyme CDC34. In analogy to budding yeast, the pl 9SKPI-CUL1-p45 SKP2 complex may function as an SCF to recruit specific substrates for ubiquitination by CDC34. Because p45 sKP2 function is required for S-phase entry, the SCFP 45SKP2 may catalyze the ubiquitination of one or more proteins that negatively regulate S-phase progression.
The role(s) that Skpl and Cdc53 play in these complexes have yet to be precisely defined. Genetic evidence suggests that both Skpl and Cdc53 are essential for ubiquitin-mediated proteolysis of a number of cell-cycle regulatory" proteins [6",8"]. A nematode homolog of CDC53, termed cul-1, is required for cell cycle exit [13"]. Biochemical experiments in yeast suggest that Skpl functions to promote Cdc4-substrate interaction whereas Cdc53 links the Skpl-Cdc4 complex to the E2 Cdc34 [9"',10"]. According to this model, Skpl would promote the assembly of an SCE the subsequent binding of a substrate and ultimately destruction. Whether this holds for other SCFs remains to be determined. In addition to its role in GI-S phase control, Skpl is an essential component of the kinetochore complex CBF3 [11,53]. Recent experiments suggest that protein complexes of similar molecular composition to budding yeast SCFs may also exist in mammalian cells (Figure 2). In particular, two cyclin A-CDK2 associated proteins, p45 SKp2, an
',, ~) T
eP",
=>7:-: Protein kinases
z~= Signals
26S proteasome
O Ubiquitio Current Opinion in Genetics & Development
A model pathway explaining the connection between SCF-catalyzed substrate ubiquitination and protein kinase signaling pathways. Ubiquitination of different SCF substrates is regulated by distinct protein kinases, which may act as recipients of positive and negative signals that converge on them. These signals determine whether or not a substrate is phosphorylated and ultimately recognized by a relevant E2-SCF complex, which in turn, triggers ubiquitination of the bound substrate. Multiubiquitinated substrates are subsequently degraded by the 26S proteasome. Similarly, physiological signals may also impinge on SCFs themselves. In principle, the modulation of SCF activity could be accomplished, for example, by regulating the expression, subcellular localization and/or phosphorylation state of SCF components.
Temporal control of SCF action by cell-cycle dependent synthesis of selected F-box proteins would be reminiscent of the sequential activation of distinct CDKs by the time-dependent synthesis of their cognate cyclin subunits. Another level of control is provided by the p45SKp2-bound cyclin A-CDK2. Mutants of p45 SKP2, defective in cyclin A-CDK2 binding, fail to bind the p19 SKpl subunit [54°]. Thus, cyclin A-CDK2 may provide a critical regulatory function. Taken together, these observations suggest first that the p19SKPI-CULl-p45SKl'Z S-phase promoting complex may function, in analogy to budding yeast, as an S-phase specific SCF in human cells and second
40
Oncogenes and cell proliferation
that the timing of SCF-catalyzed ubiquitination may be subject to many levels of regulation besides substrate phosphorylation. Finally, the abundance of p45 SKP2 is dramatically increased in a variety of transformed cells ([12]; A Marti, E Chatelain, W Krek, unpublished data). Whether deregulated expression of p45 SKp2 contributes to deregulated entry, into S phase remains to be determined. In light of the work cited above, it is tempting to speculate that increased abundance of p45 SKP2 could result in unscheduled proteolysis of certain growth inhibitory proteins. Database searches revealed that Elongin A, a subunit of the transcription elongation factor Elongin SIII, which activates transcriptional elongation by RNA polymerase II, contains an F-box [6"] . Elongin A binds Elongins B and C [55], which are related to ubiquitin and p19 SKP1, respectively [6"]. Interestingly, the yon Hippel-Lindau tumor suppressor (VHL) competes with Elongin A for Elongin B and C binding [56,57]. The VHL-Elongin B-Elongin C complex also contains CUL2 [58]. Although these observations are intriguing, at this stage it appears premature to propose a role for Elongin subunits and VHL in ubiquitin-mediated proteolysis. Finally, there is the F-box protein cyclin F, the first F-box protein identified and from which the name of this family is derived. T h e abundance of cyclin F is cell-cycle regulated, peaking around S-G2 [59]. In vitro, cyclin F can bind S k p l [6"]. An interesting question is now whether cyclin F functions as a 'true' cyclin or as a core element of an SCF.
Conclusions Ubiquitin-mediated proteolytic pathways play a crucial role in the regulation of cell-cycle progression. The challenge for these pathways is to identify and destroy from a large pool of proteins only those whose degradation is desired. Although a detailed molecular picture is not yet at hand, the prevailing concept emerging is that a cell can choose from a large pool of SCFs to accomplish this task. SCF-catalyzed substrate ubiquitination can be modulated by temporally regulated substrate phosphorylation and/or by regulating the activity of the proteins that are part of the ubiquitin conjugation system itself (Figure 3). The discovery of this novel class of E3 ligase complexes brings us a considerable step closer to a better understanding of the regulatory circuits that control the transition from GI to S phase in all eukaryotic cells. There are, however, major elements still missing. First, what is the precise mechanistic role of the core components of SCFs? Second, how are they regulated? Third, how is SCF-controlled ubiquitination linked to the subsequent destruction of proteins by the proteasome? Fourth, what are the substrates of SCF-linked proteolytic pathways and what is the significance of their degradation for cell-cycle control? Finally, are SCF-based pathways involved in other biological processes, such as immune responses and developmental decisions. The centrality of SCF-controlled ubiquitination in cell-cycle control is
only beginning to emerge and studies of the biology of SCF function have just started. It would not come as a surprise if SCF-catalyzed ubiquitination pathways were found to be deregulated in human malignancies. The first indications are already on the horizon.
Acknowledgements 1 would like thank B Amati, B Hemmings, F Hofmann, U Mtiller, M Peter and G Thomas for insightful comments on the manscript. I am likcwise indebted to W Harper and R Deshaies for providing manuscripts prior to publication. I also thank all members of my laborato O, for helpfid discussion. This work was supported in part by Novartis and by grants from the Swiss National Science Foundation, Swiss Cancer [,eague and the Erwin Schrtidinger Society (Austria).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • =-
of special interest of outstanding interest
1.
Deshaies RJ: Phosphorylation and proteolysis: partners in the regulation of cell division in the budding yeast. Curr Opin Genet Dev 1997, 7:7-16.
2.
King RW, Deshaies R.I, Peters JM, Kirschner MW: How proteolysis drives the cell cycle. Science 1996, 274:1652-1659.
3.
Ciechanover A: The ubiquitin-proteasome proteolytic pathway. Ceil 1994, 79:13-21.
4.
Hochstrasser M: Ubiquitin-dependent protein degradation. Annu Rev Genet 1996, 30:405-439.
5.
Scheffner M, Huibregtse JM, Vierstra RD, Howley PM: The HPV16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Ceil 1993, 75:495-505.
6. •
Bai C, Sen P, Hofmann K, Ma L, Goebl M, Harper JW, Elledge S J: SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Ceil 1996, 86:263-274. In this paper, SKP1 is identified as a suppressor of a cdc4 ts mutant. Skpl binds to Cdc4 and other F-box containing proteins from different organisms, including human cyclin F and p45 SKP2. Skpl is required for ubiquitin-mediated proteolysis of CIn2, CIb5 and Sic1. Different skpl ts mutants arrest cells in G 1 or in mitosis, suggesting that Skpl may function at different times in the cell cycle. The mitotic arrest phenotype of certain Skpl mutants may be due to the participation of Skpl in the kinetochore complex CBF3 [11,52"]. 7.
Mathias N, Johnson SL, Winey M, Adams AE, Goetsch L, Pringle JR, Byers B, Goebl MG: Cdc53p acts in concert with Cdc4p and Cdc34p to control the Gl-to-S-phase transition and identifies a conserved family of proteins. Mol Ceil Biol 1996, 16:66346643.
8. •
Witlems AR, Lanker S, Patton EE, Craig KL, Nason TF, Mathias N, Kobayashi R, Wittenberg C, Tyers M: Cdc53 targets phosphorylated G t cyclins for degradation by the ubiquitin proteolytic pathway. Ceil 1996, 86:453-463. This paper shows that CDC53 targets phosphorylated CIn2 for Cdc34-dependent ubiquitination. Cdc53 is a tightly bound subunit of GIn2-Cdc28. It associates selectively with phosphorylated CIn2 and also with Cdc34. These results suggest that Cdc53 has the properties of an E3 ligase. 9. •*
Feldman RM, Correll CC, Kaplan KB, Deshaies RJ: A complex of Cdc4p, Skplp, and Cdc53/Cullin catalyzes ubiquitination of the phosphorylated CDK inhibitor Sic1 p. Ceil 1997, 91:221-230. See annotation [10"]. 10. •.
Skowyra D, Craig KL, Tyers M, Elledge S J, Harper JW: F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Ceil 1997, 91:209-219. A very elegant and convincing study showing that seven purified components, an El, ubiquitin, Cdc34, Cdc4, Cdc53, Skpl and CIn-Cdc28 kinase, are necessary and sufficient to promote the multiubiquitination of the CDK inhibitor Sic1 in vitro. This work provides conclusive evidence that a complex composed of Skpl, Cdc53 and the F-box protein Cdc4 can function as a Sic1 E3 ligase. These studies illustrate the importance of F-box proteins in providing substrate specificity to ubiquitination reactions [9"'].
Proteolysis and the G1-S transition Krek
11.
Connelly C, Hieter P: Budding yeast SKP1 encodes an evolutionarily conserved kinetochore protein required for cell cycle progression. Cell 1996, 86:275-265.
12.
Zhang H, Kobayashi R, Galaktionov K, Beach D: plgSkpl and p45Skp2 are essential elements of the cyclin A-CDK2 S phase kinase. Cell 1995, 82:915-925.
41
32.
Kornitzer D, Raboy B, Kutka RG, Fink GR: Regulated degradation of the transcription factor Gcn4. EMBO J 1994, 13:6021-6030.
33.
Plon SE, Leppig KA, Do HN, Groudine M: Cloning of the human homolog of the CDC34 cell cycle gene by complementation in yeast. Proc Nat/Acad Sci USA 1993, 90:10484-10488.
Kipreos ET, Lander LE, Wing JP, He WW, Hedgecock EM: cul-1 is • required for cell cycle exit in C. elegans and identifies a novel gene family. Ceil 1996, 85:829-839. A nematode gene, cul-1, which is required to limit cell divisions during embryonic development, is found to be closely related to CDC53. Database searches reveal that cul-1 and CDC53 are members of a multigene family, referred to as the cullins, which are conserved throughout evolution. Humans have at least six cullins.
Yew PR, Kirschner MW: Proteolysis and DNA replication: the CDC34 requirement in the Xenopus egg cell cycle. Science 1997, 277:1672-1676. CDK-dependent initiation of DNA replication in Xenopus eggs requires a CDC34 homolog. Dominant negative mutants of human CDC34 block the degradation of a Xenopus CDK inhibitor, thereby preventing initiation of DNA replication. Evidence is provided that CDC34 functions in a large molecular size complex. These data provide the best evidence to date that CDC34 function is also required in higher eukaryotes.
14.
35.
13.
Cherr CJ: G1 phase progression: cycling on cue. Cell 1994, 79:551-555.
15.
Hunter "1",Pines J: Cyclins and cancer. I1. Cyclin D and CDK inhibitors come of age. Ceil 1994, 79:573-582.
16.
NasmythK: Control of the yeast cell cycle by the Cdc28 protein kinase. Curt Opin Cell Bio/1993, 5:166-1 79.
1 "7.
Morgan DO: Principles of CDK regulation. Nature 1995, 374:131-134.
18.
Nigg EA: Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle. Bioessays 1995, 17:471-480.
19.
Goebl MG, Yochem J, Jentsch S, McGrath JP, Varshavsky A, Byers B: The yeast call cycle gene CDC34 encodes a ubiquitinconjugating enzyme. Science 1986, 241:1331-1335.
20.
Mendenhall MD: An inhibitor of p34cdc28 protein kinase activity from S. cerevisiae. Science 1993, 259:216-219.
21.
Schwob E, Bohm T, Mendenhall MD, Nasmyth K: The B-type cyclin kinase inhibitor p40SIC1 controls the Gt to S transition in S. cerevisiee. Cell 1994, 79:233-244.
22.
Schneider BL, Yang QH, Futcher AB: Linkage of replication to start by the Cdk inhibitor Sic1. Science 1996, 272:560-562.
23.
TyersM: The cyclin-dependent kinase inhibitor p40SIC1 imposes the requirement for CIn G1 cyclin function at start. Proc Nat/Acad Sci USA 1996, 93:7772-7776.
24. •
VermaR, Annan RS, Huddleston MJ, Cart SA, Reynard G, Deshaies RJ: Phosphorylation of Siclp by G1 CDK required for its degradation and entry into S phase. Science 1997, 278:455460. Mutations of several CDK consensus phosphorylation sites in Sic1 yields a mutant that can no longer be ubiquitinated in vitro. Expression of this mutant in vivo results in a stable protein that causes a G 1 arrest. 25.
Yaglom J, Linskens MH, Sadis S, Rubin DM, Futcher B, Finley D: p34Cdc28-mediated control of CIn3 cyclin degradation. Mo/ Ceil Bio11995, 15:731-741.
26. •
LankerS, ValdMeso MH, Wittenberg C: Rapid degradation of the G1 cyclin CIn2 induced by CDK-dependent phosphorylation. Science 1996, 271:1597-1601. Mutations of several CDK consensus phosphorylation sites in CIn2 yields a mutant that can no longer be phosphorylated by Cdc28 in vitro. The mutant is several-fold more stable then its wild-type counterpart and expression of this mutant in vivo results in a shortened G t phase. 27.
Deshaies RJ, Chau V, Kirschner M: Ubiquitination of the Gt cyclin CIn2p by a Cdc34p-dependent pathway. EMBO J 1995, 14:303-312.
28.
Blondel M, Mann C: G2 cyclins are required for the degradation of Gt cyclins in yeasL Nature 1996, 384:279-282.
Henchoz S, Chi Y, Catarin B, Herskowitz I, Deshaies R, Peter M: Phosphorylation and ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor Farlp in budding yeast. Genes Dev 199?, 11:3046-3060. A Far1 mutant is identified that is able to induce cell cycle arrest in the absence of co-factor. Cells that overexpress this mutant arrest in G1 with low CIn-Cdc28 kinase activity. Whereas wild-type Far1 is readily ubiquitinated in a Cdc34-dependent manner, the mutant is not. This defect is linked to the failure of CIn-Cdc28 kinase to phosphorylate the mutant Far1 protein.
34. •
Diehl JA, Zindy F, Sherr CJ: Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin-proteasome pathway. Genes Dev 1997, 11:957-972. This paper shows that cyclin D1 is phosphorylated in vivo on Thr286. Mutation of this site to alanine prevents this phosphorylation, inhibits multiubiquitination and markedly stabilizes the protein. The kinase responsible for Thr286 phosphorylation appears to be an as yet unidentified kinase. Phosphorylation drives ubiquitination and proteasomal degradation of cyclin D1 in response to serum withdrawal. •
36. •
ClurmanBE, Sheaff R.I, Thress K, Groudine M, Roberts JM: Turnover of cyclin E by the ubiquitin-proteasome pathway is regulated by cdk2 binding and cyclin phosphorylation. Genes Dev 1996, 10:1979-1990. See annotation [37*]. 37.
Won KA, Reed SI: Activation of cyclin E/CDK2 is coupled to site-specific autophosphorylation and ubJquitin-dependent degradation of cyclin E EMBO J 1996, 15:4182-4193. This paper, together with [36"], presents evidence that cyclin E is degraded by the ubiquitin-proteasome system and that this degradation is regulated by phosphorylation. The cyclin E-bound CDK2 initiates this degradation by phosphorylating cyclin E at a specific site. Phosphorylation causes cyclin E to disassemble from CDK2, resulting in the ubiquitination and degradation of free cyclin E. Interestingly, the binding of CDK2 to cyclin E appears to protect the latter from degradation. •
36.
Sheaff RJ, Groudine M, Gordon M, Roberts JM, Clurman BE: Cyclin E-CDK2 is a regulator of p27Kip1. Genes Dev 1997, 11:1464-1478. See annotation [39"]. •
39. •
Vlach J, Hennecke S, Amati B: Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27KIP1. EMBO J 1997, 16:5334-5344. This paper, together with [38 °] shows that the CDK inhibitor p27Kip1 is a substrate of cyclin E-CDK2. Phosphorylation of p2?KiP1 by cyclin E-CDK2 promotes its ubiquitination and proteasomal degradation. Elimination of p27 =s required for cells to transit from G 1 into S phase. The authors provide a model that explains how p27Kip1 can be an inhibitor and substrate of cyclin E-CDK2 at the same time. 40.
Dulic V, Lees E, Reed Sh Association of human cyclin E with a periodic G1-S phase protein kinase. Science 1992, 257:19561961.
41.
Koff A, Giordano A, Desai D, Yamashita K, Harper JW, Elledge S, Nishimoto T, Morgan DO, Franza BR, Roberts JM: Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle. Science 1992, 257:1689-1694.
42.
Pagano M, Tam SW, Theodoras AM, Beer-Romero P, Del Sal G, Chau V, Yew PR, Draetta GF, Rolfe M: Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p2Z Science 1995, 269:682685.
43.
Fredersdorf S, Burns J, Milne AM, Packham G, Fallis L, Gillett CE, Royds JA, Peston D, Hall PA, Hanby AM, Barnes DM et aL: High level expression of p27KJP1 and cyclin D1 in some human breast cancer cells: inverse correlation between the expression of p27KiP1 and degree of malignancy in human breast and colorectal cancers. Proc Nat/Acad Sci USA 1997, 94:6380-5385.
44.
Catzavelos C, Bhattacharya N, Ung YC, Wilson JA, Roncari L, Sandhu C, Shaw P, Yeger H, Morava-Protzner I, Kapusta Let aL: Decreased levels of the cell cycle inhibitor p27Km1 protein: prognostic implications in primary breast cancer. Nat Med 1997, 3:227-230.
45.
Loda M, Cukor B, Tam SW, Lavin P, Fiorentino M, Draetta GF, Milburn-Jessup J, Pagano M: Increased proteasome-dependent
29. •
30.
PiattiS, Bohm T, Cocker JH, Diffley JF, Nasmyth K: Activation of S-phase-promoting CDKs in late G1 defines a =point of no return" after which Cdc6 synthesis cannot promote DNA replication in yeast. Genes Dev 1996, 10:1516-1531.
31.
DruryLS, Perkins G, Diffley JFX: The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast. EMBO J 1997, 16:5966-5976.
42
Oncogenes and cell proliferation
degradation of the cyclin-dependent kinase inhibitor p27 in aggessive colorectal carcinomas. Nat Med 1997, 3:231-234. 46.
Porter PL, Malone KE, Heagerty PJ, Alexander GM, Gatti LA, Firpo EJ, Daling JR, Roberts JM: Expression of the cell cycle regulators p27 Kip1 and cyclin E alone and in combination, correlate with survival in young breast cancer patients. Nat Med 1997, 3:222-226.
47. •
Hateboer G, Kerkhoven RM, Shvarts A, Bernards R, Beijersbergen RL: Degradation of E2F by the ubiquitin-proteasome pathway: regulation by retinoblastoma family proteins and adenovirus transforming proteins. Genes Dev 1996, 10:2960-2970. See annotation [48°]. 48. •
HofmannF, Martelli F, Livingston DM, Wang Z: The retinoblastoma gene product protects E2F-1 from degradation by the ubiquitin-proteasome pathway. Genes Dev 1996, 10:2949-2459. This paper, together with [47°,49"], establishes that members of the E2F transcription factor family are unstable proteins and are targeted for degradation by the ubiquitin-proteasome pathway. Moreover, it is shown that degradation of E2Fs is regulated by retinoblastoma family proteins. 49. •
Campanero MR, Flemington EK: Regulation of E2F through ubiquitin-proteasome-dependent degradation: stabilization by the pRB tumor suppressor protein. Proc Nat/Acad Sci USA 1997, 94:2221-2226. See annotation [48"]. 50.
Li FN, Johnston M: Grrl of Saccharomyces cerevisiae is connected to the ubiquitin proteolysis machinery through Skpl: coupling glucose sensing to gone expression and the cell cycle. EMBO J 1997, 16:5629-5638.
51.
ThomasD, Kuras L, Barbey R, Cherest H, Blaiseau PL, SurdinKerjan Y: Met30p, a yeast transcriptional inhibitor that responds to S-adenosylmethionine, is an essential protein with WD-40 repeats. Mo/ Ceil Bio/1995, 15:6526-6534.
52. •
KominamiK, Toda T: Fission yeast WD-repeat protein pop1 regulates genome ploidy through ubiquitin-proteasomemediated degradation of the CDK inhibitor Rum1 and the S-phase initiator Cdc18. Genes Dev 1997, 11:1546-1560. A gone is identified that is important for the maintenance of ploidy in fission yeast. The gone, pop1, is highly related to the gone for budding yeast CDC4, an F-box protein. In pop1 mutants, Cdc18 and Rum1 are not ubiquitinated
and accumulate to high levels. Pop1 contains an F-box motif and associates with Cdc18, suggesting that Pop1 functions as a recognition factor in a ubiquitination pathway. 53.
StemmannO, Lechner J: The Saccharomyces cerevisiae kinetochore contains a cyclin-cdk complexing homolog as identified by in vitro reconstitution. EMBO J 1996, 15:36113620.
54. •
LisztwanJ, Marti A, Suetterluety H, Gstaiger M, Wirbelauer C, Krek W: Association of human CUL-1 and ubiquitin-conjugating enzyme CDC34 with the F-box protein p45SKP2: evidence for evolutionary conservation in the subunit composition of the CDC34-SCF pathway. EMBO J 1998, 17:368-383. This paper shows that human CUL-1, a member of the cullin family associates with p45SKP2, p19 sKP1 and cyclin A-CDK2 in vivo and that these components assemble into a large multiprotein complex. Since the molecular composition of this complex closely resembles budding yeast SCF-type E3 ligase complexes, it was proposed that the requirement for p45SKP2's function for S phase progression may reflect a requirement for ubiquitination of selected celt cycle regulatory proteins by this potential E3 ligase complex. In addition, this report shows that formation of this complex is governed, in part, by the S phase-specific expression of p45 SKP2 and the p45SKP2-bound cyclin A-CDK2. 55.
Aso T, Haque D, Barstead RJ, Conaway RC, Conaway JW: The inducible elongin A elongation activation domain: structure, function and interaction with the elongin BC complex. EMBO J 1996, 15:5557-5566.
56.
Kibel A, Iliopoulos O, DeCaprio JA, Kaolin WG: Binding of the yon Hippel Lindau tumor suppressor protein to elongin B and C. Science 1995, 269:1444-1446.
5?.
D u a nRD, Pause A, Burgess WH, Aso T, Chen DYT, Garrett KP, Conaway RC, Conaway JW, Linehan M, Klausner RD: Inhibition of transcription elongation by the VHL tumor suppressor protein. Science 1995, 269:1402-1406.
58.
PauseA, Lee S, Worrell RA, Chen DY, Burgess WH, Linehan WM, Klausner RD: The yon HippeI-Lindau tumor-suppressor gone product forms a stable complex with human CUL-2, a member of the Cdc53 family of proteins. Proc Natl Acad Sci USA 1997, 94:2156-2161.
59.
Bai C, Richman R, Elledge SJ: Human cyclin F. EMBO J 1994, 13:6087-6098.
Proteolysis and the GI-S transition: the SCF connection Wilhelm Krek Temporal control of ubiquitin-proteasome mediated protein degradation is critical for normal G 1 and S phase progression. Recent work has shown that central to the temporal control mechanism is a relationship between newly identified E3 ubiquitin protein ligases, designated SCFs (Skpl-cullin-F-box protein ligase complexes), which confer substrate specificity on ubiquitination reactions and the activities of protein kinases that phosphorylate substrates destined for destruction at specific sites, thereby converting them into preferred targets for ubiquitin modification catalyzed by SCFs. The constituents of SCFs are members of evolutionary conserved protein families. SCF-based ubiquitination pathways may play a key role in diverse biological processes, such as cell proliferation, differentiation and development.
Addresses Friedrich Miescher Institut, Maulbeerstrasse 66, CH-4058 Basel, Switzerland; e-mail: [email protected]
Current Opinionin Genetics& Development1998, 8:36-42
http:llbiomednet.co.lelecreflO959437XO0800036 © Current Biology Ltd ISSN 0959-437X
Abbreviations CDK cyclin-dependent protein kinase CKI
LRR SCF
CDK inhibitor protein leucine-richrepeat Skpl-cullin-F-box protein ligase complex
Introduction Ubiquitin-proteasome dependent protein degradation plays a fundamental role in the regulation of the eukaryotic cell cycle [1,2]. It is highly selective, precisely timed and allows an instant switch from one functional programme to another. During the cell cycle, protein degradation is required for the transition from G1 to S phase, sister chromatid separation in anaphase and the exit from mitosis [2]. Degradation of a protein via the ubiquitin-proteasome pathway involves the assembly of a ubiquitin chain on the substrate and the rapid degradation of the multiubiquitinated substrate by the 26S proteasome [3,4]. The conjugation of ubiquitin to proteins involves a series of well-defined enzymatic reactions. Ubiquitin is first activated via thioester formation by an ubiquitin-activating enzyme (El), which transfers ubiquitin to one member of a family of distinct ubiquitin-conjugating enzymes (E2s or UBCs), again forming a thioester. Certain E2s can transfer ubiquitin directly to substrates whereas others require the participation of a third component, termed E3 ubiquitin protein ligase [5]. E3 is the component of the ubiquitin conjugation system that is generally thought to be the most directly involved in substrate recognition. At the same
time, E3 components are the least well-understood factors involved in ubiquitin ligation. At present, a great deal of effort in cell cycle research is focused on elucidating the mechanisms by which the selectivity of protein destruction is achieved and the mechanisms by which proteolytic activity is limited to a specific cell cycle phase. Recent genetic and biochemical studies in Saccharomyces cerevisiae have led to the identification of a novel class of E3 ligases that function in combination with the E2 enzyme Cdc34 to promote the ubiquitination of key cell cycle regulatory proteins during the G1 and S phases of the cell cycle (Figure 1). The core components of these newly identified E3s are Skpl and Cdc53, which assemble with different F-box proteins, for example, Cdc4 or Grrl, into distinct protein complexes [6°,7,8",9"°,10°°]. The F-box protein subunits display selectivity in the recognition of potential ubiquitination targets. These units have been designated SCFs (Skpl-cullin-F-box protein ligase complexes) [9°°,10°°]. Structural and functional homologs of Skpl exist in evolutionarily diverse organisms [6°, 11,12]. Likewise, Cdc53 is a member of a large, evolutionarily conserved, multigenc family, referred to as the cullins [7,13"]. Finally, the F-box, which is thought to mediate binding to Skpl, is a conserved domain that is found in a large number of proteins [6°]. Related family members may undergo combinatorial interactions to generate a large number of SCFs, each potentially with a unique cellular function. A hallmark of known SCFs is that they recognize ubiquitination substrates in a phosphorylation-dependent manner. This review focuses on the role of Cdc34, SCFs and phosphorylation in the proteolytic pathways that govern the G1-S phase transition in the eukaryotic cell cycle. T h e i m p o r t a n c e of Cdc34 and phosphorylation for regulated proteolysis at t h e G 1 to S p h a s e transition Passage through the cell cycle requires the successive activation of different complexes between cyclin and cyclin-dependent protein kinase (CDK) [14,15], In mammalian cells, the cyclin-CDK complexes that are most critically involved in the decisions concerning growth and quiescence are D-type cyclins with their partners CDK4 and CDK6, and cyclins E and A with their partner CDK2 [14]. The activities of these CDK complexes are essential for driving cells through G 1 into S phase. Budding yeast contains functional homologs that act at similar stages of the cell cycle with the G 1 cyclins Clnl, Cln2, Cln3, ClbS and Clb6 sequentially activating the yeast CDK Cdc28 to promote S phase entry [16]. In general, CDK activity depends on the binding of cyclins, positive and negative regulatory phosphorylation events and the action of CDK
Proteolysis and the G1-S transition Krek
Figure 1 E3 ubiquitinligasecomplexes
Ubiquitination targets
&J
Sic1 Far1
scFCdc4
E2 [ ~
~
Cin2
scFGrrl Current
Opinionin Genetics & Development
A schematic diagram depicting the molecular composition of two distinct SCFs from S. cerevisiae and their role in the recognition and ubiquitination of targets. Cdc4 and Grrl are representatives of the F-box protein family [6°]. The nature of the F-box protein defines the repertoire of substrates targeted by the SOt=, The SCF Cdc4 recruits selectively phosphorylated Sic1 and Earl into the complex for ubiquitination by the E2 enzyme Cdc34 [9"',10°',29°]. The SCF Grrl displays affinity for phosphorylated CIn2 and not for Sicl or Earl [10°°]. It is thought that Cdc34 physically interacts with each of these SCFs [7,8°,9°',10"°]. E1 is a ubiquitin-activating enzyme required for ubiquitin activation and transfer onto the E2 Cdc34.
inhibitor proteins (CKIs) that through their association with either a CDK or a cyclin-CDK complex block function [17,18]. Recent work demonstrates that CDK activity is regulated by ubiquitin-mediated proteolysis. The first connection between cell cycle control and proteolysis was suggested by the discovery ten years ago that a ubiquitin-conjugating enzyme encoded by the CDC34 gene is required for G 1 to S phase progression in Saccharomyces cerevisiae [19]. In the absence of CDC34 function, cells exhibit high levels of Cln-Cdc28 kinase activity but fail to initiate DNA replication because the Clb5/6-Cdc28 kinases that are needed downstream for executing the onset of S phase are held in check by the CKI Sicl, whose degradation shortly before S phase is defective in CDC34 mutant strains [20,21]. These observations led to the proposal that Cdc34 degrades Sicl in late GI. In addition to CDC34, the destruction of Sicl also requires CLN-CDC28 function [22,23). The requirement for a G 1 cyclin-CDK dependent step in the degradation of Sicl suggests that phosphorylation may constitutes a recognition signal for ubiquitination by the Cdc34 pathway. Indeed, Sicl is a substrate of Cln-Cdc28 kinase and mutation of CDK consensus phosphorylation sites in Sicl reduces Sicl ubiquitination in vitro and stabilizes Sicl in vivo. In addition, Sicl mutants lacking CDK phosphorylation sites block DNA replication [24°]. Thus, the schedule of Sicl phosphorylation ensures that S phase is not initiated prior to the activation of Cln-Cdc28 at START (a point at which yeast cells commit to
37
the completion of a new cycle). Around that time, as sufficient Cln-Cdc28 complexes accumulate (and trigger phosphorylation of Sicl), the G I cyclins become phosphorylated by the bound Cdc28 kinase. This provokes the ubiquitination and degradation of G 1 cyclins [25,26"] and the rapid inactivation of their associated kinase activities as ceils initiate DNA replication. Different E2 enzymes, including Cdc34 [27] and Ubc9 [28], have been implicated in the ubiquitination of Clnl and Cln2. Phosphorylation also serves as a trigger for the degradation of the CKI Farl, a Cdc34 pathway substrate [29"]. Farl is required for a-factor (an antimitogen) induced cell cycle arrest and phosphorylation of Farl by Cln-Cdc28 kinase initiates the ubiquitination and proteasomal degradation of Farl. Elimination of Farl allows cells to resume cell-cycle progression after their release from (x-factor arrest [29°]. These observations highlight the importance of phosphorylation for the efficient degradation of cyclins and CKIs. In addition, Cdc34 is also required for the degradation of a number of other proteins, including the replication protein Cdc6 ]30,31] and the transcription factor Gcn4 [32]. It is not known whether phosphorylation of these proteins signals their degradation. Regardless, it appears that Cdc34 regulates various aspects of G 1 to S phase progression in S. cerevisiae. Two independent lines of experimental evidence indicate that Cdc34 plays an important role in cell-cycle regulation in all eukaryotic cells. First, a structural and functional human homolog of CDC34 was cloned and shown to complement the yeast defect [33]. Second, a dominant negative mutant of human CDC34, when added as recombinant protein to Xenopus interphase egg extracts, inhibits DNA replication [34"]. T h e requirement for CDC34 function in the initiation of DNA replication in Xenopus extracts may be directly linked to the failure to degrade Xicl, an inhibitor of cyclin E-CDK2. Although it has yet to be shown that CDC34 activity is critical for G 1 to S phase progression in animal cells, the evidence cited above suggests that CDC34 function is required for the onset of S phase in all eukaryotic cells. Not unexpectedly, phosphorylation also serves as a trigger for ubiquitin-mediated destruction of cyclins and CKIs in mammalian cells. Studies concerning the post-translational modifications of cyclin D1 [35°], cyclin E [36",37 °] and p27Kip I [38°,39"], demonstrate the importance of site-specific phosphorylation in rendering these proteins susceptible to the action of the ubiquitination machinery. In particular, the ability of D-type cyclins to act as growth factor sensors depends not only on their rapid induction by mitogens but also on their intrinsic instability, which ensures their abrupt degradation upon mitogen withdrawal. Elimination of cyclin D1 in response to mitogen withdrawal is mediated by ubiquitination and proteasomal degradation initiated by phosphorylation of cyclin D1 at a specific site [35°]. Interestingly, the protein
38
Oncogenes and cell proliferation
kinase that primes cyclin D1 for destruction may not be CDK4 or CDK6 itself, but rather an as yet uncharacterized protein kinasc.
Cyclin E is expressed periodically, peaking at G1-S, a time at which maximal levels of cyclin E - C D K 2 activity are induced, and then disappears as S phase progresses [40,41]. T h e timely disappearance of cyclin E protein in S phase is initiated by the phosphorylation of cyclin E in cis by the bound CDK2 itself, which triggers its degradation [36",37"]. Because cyclin E promotes its own turnover by activating the enzyme that signals its destruction, it assures an appropriate temporal window for the activation of its CDK partner.
The CKI p27Kip 1 is also degraded by the ubiquitinproteasome pathway [42]. Cells treated with antimitogenic factors reversibly arrest in late G1 and this arrest correlates with the inhibition of cyclin E - C D K 2 by p27Kipl; however, cyclin E - C D K 2 is also able to reverse the inhibitory effect of p27Kip 1 by phosphorylating it at a specific site, which signals the ubiquitination and proteasomal degradation of the bound CKI [38",39"]. This creates a conundrum: how does cyclin E - C D K 2 inactivate p27Kip 1, if the latter is tightly bound to the enzyme and inhibits its catalytic activity? It has been proposed that p27Kip 1 can bind cyclin E - C D K 2 in a noninhibitory mode, under which conditions cyclin E-CDK2 phosphorylates p27Kip 1, thereby signals its elimination by the ubiquitin-proteasome pathway, or in an inhibitory mode, under which conditions p27Kip I converts the enzyme into a catalytically inactive form. Whether the transition from a noninhibitory mode to an inhibitory mode is regulated in vivo is not known. Finally, in recent reports, it was shown that low levels of p27Kip I correlate with poor prognosis of human breast and colon carcinomas [43-46] and it was suggested that this may bc due to enhanced p27Kipl degradation [45]. These observations hint at the possibility that the p27Kip I destruction pathway may be deregulated in certain human tumors.
T h e view cmerging from the above cited work is that the timing of substrate ubiquitination is controlled by the activity of distinct protein kinases, which prime selected targets for destruction by site-specific phosphorylation; however, differential regulation of the stability of ubiquitin-proteasome pathway substratcs may be achieved through controls other then direct substrate phosphorylation. For example, recent studies have shown that binding of the rctinoblastoma gcne product (pRB) to the transcription factor E2F-1 in G 1 protects the latter from degradation [47"-49"]. Increased stability of E 2 F - I - p R B complexes may contribute to the maintenance of active transcriptional repression, a key event for normal G1 exit control.
Selective protein ubiquitination catalyzed by SCFs T h e issue that must now be resolved is the identification of the key components of the ubiquitination system that are involved in the recognition and targeting of only those proteins that bear a specific degradation signal and whose degradation is desired. As outlined in the introduction, cells can choose from a large repertoire of SCFs to accomplish substrate specificity over ubiquitination reactions (Figure 1). T h e first SCF has been identified in budding yeast and is composed of Skpl, Cdc53 and the F-box protein Cdc4; hence, it has been designated SCF Cdc4. It functions as a Sicl E3 ubiquitin protein ligase [9",10"]. Specifically, purified SCF Cdc4 binds phosphorylated Sicl (but not phosphorylated Cln2) and when supplemented with purified Cdc34, an E1 and ubiquitin promotes muhiubiquitination of Sicl in vitro. Thus, the SCF Cdc4 satisfies the criteria of a functional E3 ligase; namel.~, it binds an E2 (Cdc34), an established ubiquitination substtate (Sicl) and it facilitates multiubiquitination of the bound substrate. T h e F-box protein Cdc4 contains eight WD-40 repeats. In fact, many of the known F-box proteins contain in addition to their F-box other recognizable sequence motifs such as WD-40 repeats or leucine-rich repeats (LRRs), implicated in protein-protein interactions. Mutants of Cdc4 that lack the three most carboxy-terminal WD-40 repeats fail to bind phosphorylated Sicl in vitro, suggesting that this domain might serve as a substrate-recruiting domain [10"]. Interestingly, the SCF Cdc4 also targets phosphorylated Farl for ubiquitination [29"]. Thus, the S e E Cdc4 is not monospecific and temporal control of SCFCdc4-catalyzed ubiquitination is a direct reflection of the activities of the protein kinases, which phosphorylate Farl and Sicl at the relevant sites to permit SCF targeting. T h e F-box containing proteins - - Grrl and M e t 3 0 - - a r e structurally and functionally distinct from Cdc4 but like Cdc4, Grrl associates with Skpl and Cdc53. This complex, however, displays binding specificity towards phosphorylated Cln2 and not Sicl [8",10"]. Thus, the identity of the F-box protein subunit determines the repertoire of substrates targeted by these complexes (Figure 1). A purified SCF (,rrl does not support multiubiquitination of phosphorylated Cln2 in vitro, suggesting that proper functioning of the SCF Grrl requires additional signals; for example, it has been shown that S k p l - G r r l complex formation is regulated by glucose levels [50]. Further analyses of controls that are normally imposed on SCFs will provide important clues for unraveling this issue. T h e F-box protein Met30 has been implicated in the regulation of Met4, a transcription factor required for methionine biosynthetic gene expression [51]. It is possible that Met30 functions in a ubiquitination pathway
Proteolysis and the G1-S transition Krek
to degrade Met4. Finally, the Popl gene product of fission yeast, a close relative of budding yeast Cdc4, appears to bc a critical determinant for the maintenance of genome ploidy and is required for ubiquitin-proteasome mediated degradation of the CKI Ruml and the S phase regulator Cdcl8 [52"]. Taken together, these examples suggest a critical function for F-box proteins in diverse cellular processes in these yeasts.
Figure 2
ScFCdc4
SCFP45SKP2
39
F-box protein essential for S phase entry and p19 SKP1, a structural and functional homolog of Skpl [12], associate with human C U L l and CDC34 in vivo [54"]. The p19SKP1-CULl-p45 SKP2 complex portrayed in Figure 2 may serve as an SCF, guiding human CDC34 to potential ubiquitination targets that have yet to be identified. Interestingl,~; the expression of p45 SKp2 is cell-cycle regulated, peaking in S phase and declining as cells progress through M phase. In contrast, p19 sKpl, C U L l and CDC34 are expressed throughout the cell cycle ([54"]; F Reymond, W Krek, unpublished data). Thus, p19SKP1-CULl-p45 SKP2 complex formation is governed, at least in part, by the S-phase specific accumulation of the p45 SKI'2 F-box protein subunit.
Figure 3
E2
S. cerevisiae
E2
Signals ('e -,
hi]
Human Current Opinion in Genetics & Development
A schematic drawing that illustrates the subunit composition of an established Cdc34-SCF complex from S. cerevisiae and a human S-phase promoting complex. The architecture of these complexes is hypothetical because the exact nature of the protein-protein interactions depicted here have yet to be defined. Human p19 sKP1 is a structural and functional homolog of budding yeast Skpl. Likewise, human CULl and S. cerevisiae Cdc53 are members of the cullin family, p45 SKP2 contains an F-box and seven LRRs and associates directly with the S-phase cyclin A-CDK2. Each complex is found in association with the ubiquitin-conjugating enzyme CDC34. In analogy to budding yeast, the pl 9SKPI-CUL1-p45 SKP2 complex may function as an SCF to recruit specific substrates for ubiquitination by CDC34. Because p45 sKP2 function is required for S-phase entry, the SCFP 45SKP2 may catalyze the ubiquitination of one or more proteins that negatively regulate S-phase progression.
The role(s) that Skpl and Cdc53 play in these complexes have yet to be precisely defined. Genetic evidence suggests that both Skpl and Cdc53 are essential for ubiquitin-mediated proteolysis of a number of cell-cycle regulatory" proteins [6",8"]. A nematode homolog of CDC53, termed cul-1, is required for cell cycle exit [13"]. Biochemical experiments in yeast suggest that Skpl functions to promote Cdc4-substrate interaction whereas Cdc53 links the Skpl-Cdc4 complex to the E2 Cdc34 [9"',10"]. According to this model, Skpl would promote the assembly of an SCE the subsequent binding of a substrate and ultimately destruction. Whether this holds for other SCFs remains to be determined. In addition to its role in GI-S phase control, Skpl is an essential component of the kinetochore complex CBF3 [11,53]. Recent experiments suggest that protein complexes of similar molecular composition to budding yeast SCFs may also exist in mammalian cells (Figure 2). In particular, two cyclin A-CDK2 associated proteins, p45 SKp2, an
',, ~) T
eP",
=>7:-: Protein kinases
z~= Signals
26S proteasome
O Ubiquitio Current Opinion in Genetics & Development
A model pathway explaining the connection between SCF-catalyzed substrate ubiquitination and protein kinase signaling pathways. Ubiquitination of different SCF substrates is regulated by distinct protein kinases, which may act as recipients of positive and negative signals that converge on them. These signals determine whether or not a substrate is phosphorylated and ultimately recognized by a relevant E2-SCF complex, which in turn, triggers ubiquitination of the bound substrate. Multiubiquitinated substrates are subsequently degraded by the 26S proteasome. Similarly, physiological signals may also impinge on SCFs themselves. In principle, the modulation of SCF activity could be accomplished, for example, by regulating the expression, subcellular localization and/or phosphorylation state of SCF components.
Temporal control of SCF action by cell-cycle dependent synthesis of selected F-box proteins would be reminiscent of the sequential activation of distinct CDKs by the time-dependent synthesis of their cognate cyclin subunits. Another level of control is provided by the p45SKp2-bound cyclin A-CDK2. Mutants of p45 SKP2, defective in cyclin A-CDK2 binding, fail to bind the p19 SKpl subunit [54°]. Thus, cyclin A-CDK2 may provide a critical regulatory function. Taken together, these observations suggest first that the p19SKPI-CULl-p45SKl'Z S-phase promoting complex may function, in analogy to budding yeast, as an S-phase specific SCF in human cells and second
40
Oncogenes and cell proliferation
that the timing of SCF-catalyzed ubiquitination may be subject to many levels of regulation besides substrate phosphorylation. Finally, the abundance of p45 SKP2 is dramatically increased in a variety of transformed cells ([12]; A Marti, E Chatelain, W Krek, unpublished data). Whether deregulated expression of p45 SKp2 contributes to deregulated entry, into S phase remains to be determined. In light of the work cited above, it is tempting to speculate that increased abundance of p45 SKP2 could result in unscheduled proteolysis of certain growth inhibitory proteins. Database searches revealed that Elongin A, a subunit of the transcription elongation factor Elongin SIII, which activates transcriptional elongation by RNA polymerase II, contains an F-box [6"] . Elongin A binds Elongins B and C [55], which are related to ubiquitin and p19 SKP1, respectively [6"]. Interestingly, the yon Hippel-Lindau tumor suppressor (VHL) competes with Elongin A for Elongin B and C binding [56,57]. The VHL-Elongin B-Elongin C complex also contains CUL2 [58]. Although these observations are intriguing, at this stage it appears premature to propose a role for Elongin subunits and VHL in ubiquitin-mediated proteolysis. Finally, there is the F-box protein cyclin F, the first F-box protein identified and from which the name of this family is derived. T h e abundance of cyclin F is cell-cycle regulated, peaking around S-G2 [59]. In vitro, cyclin F can bind S k p l [6"]. An interesting question is now whether cyclin F functions as a 'true' cyclin or as a core element of an SCF.
Conclusions Ubiquitin-mediated proteolytic pathways play a crucial role in the regulation of cell-cycle progression. The challenge for these pathways is to identify and destroy from a large pool of proteins only those whose degradation is desired. Although a detailed molecular picture is not yet at hand, the prevailing concept emerging is that a cell can choose from a large pool of SCFs to accomplish this task. SCF-catalyzed substrate ubiquitination can be modulated by temporally regulated substrate phosphorylation and/or by regulating the activity of the proteins that are part of the ubiquitin conjugation system itself (Figure 3). The discovery of this novel class of E3 ligase complexes brings us a considerable step closer to a better understanding of the regulatory circuits that control the transition from GI to S phase in all eukaryotic cells. There are, however, major elements still missing. First, what is the precise mechanistic role of the core components of SCFs? Second, how are they regulated? Third, how is SCF-controlled ubiquitination linked to the subsequent destruction of proteins by the proteasome? Fourth, what are the substrates of SCF-linked proteolytic pathways and what is the significance of their degradation for cell-cycle control? Finally, are SCF-based pathways involved in other biological processes, such as immune responses and developmental decisions. The centrality of SCF-controlled ubiquitination in cell-cycle control is
only beginning to emerge and studies of the biology of SCF function have just started. It would not come as a surprise if SCF-catalyzed ubiquitination pathways were found to be deregulated in human malignancies. The first indications are already on the horizon.
Acknowledgements 1 would like thank B Amati, B Hemmings, F Hofmann, U Mtiller, M Peter and G Thomas for insightful comments on the manscript. I am likcwise indebted to W Harper and R Deshaies for providing manuscripts prior to publication. I also thank all members of my laborato O, for helpfid discussion. This work was supported in part by Novartis and by grants from the Swiss National Science Foundation, Swiss Cancer [,eague and the Erwin Schrtidinger Society (Austria).
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • =-
of special interest of outstanding interest
1.
Deshaies RJ: Phosphorylation and proteolysis: partners in the regulation of cell division in the budding yeast. Curr Opin Genet Dev 1997, 7:7-16.
2.
King RW, Deshaies R.I, Peters JM, Kirschner MW: How proteolysis drives the cell cycle. Science 1996, 274:1652-1659.
3.
Ciechanover A: The ubiquitin-proteasome proteolytic pathway. Ceil 1994, 79:13-21.
4.
Hochstrasser M: Ubiquitin-dependent protein degradation. Annu Rev Genet 1996, 30:405-439.
5.
Scheffner M, Huibregtse JM, Vierstra RD, Howley PM: The HPV16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Ceil 1993, 75:495-505.
6. •
Bai C, Sen P, Hofmann K, Ma L, Goebl M, Harper JW, Elledge S J: SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Ceil 1996, 86:263-274. In this paper, SKP1 is identified as a suppressor of a cdc4 ts mutant. Skpl binds to Cdc4 and other F-box containing proteins from different organisms, including human cyclin F and p45 SKP2. Skpl is required for ubiquitin-mediated proteolysis of CIn2, CIb5 and Sic1. Different skpl ts mutants arrest cells in G 1 or in mitosis, suggesting that Skpl may function at different times in the cell cycle. The mitotic arrest phenotype of certain Skpl mutants may be due to the participation of Skpl in the kinetochore complex CBF3 [11,52"]. 7.
Mathias N, Johnson SL, Winey M, Adams AE, Goetsch L, Pringle JR, Byers B, Goebl MG: Cdc53p acts in concert with Cdc4p and Cdc34p to control the Gl-to-S-phase transition and identifies a conserved family of proteins. Mol Ceil Biol 1996, 16:66346643.
8. •
Witlems AR, Lanker S, Patton EE, Craig KL, Nason TF, Mathias N, Kobayashi R, Wittenberg C, Tyers M: Cdc53 targets phosphorylated G t cyclins for degradation by the ubiquitin proteolytic pathway. Ceil 1996, 86:453-463. This paper shows that CDC53 targets phosphorylated CIn2 for Cdc34-dependent ubiquitination. Cdc53 is a tightly bound subunit of GIn2-Cdc28. It associates selectively with phosphorylated CIn2 and also with Cdc34. These results suggest that Cdc53 has the properties of an E3 ligase. 9. •*
Feldman RM, Correll CC, Kaplan KB, Deshaies RJ: A complex of Cdc4p, Skplp, and Cdc53/Cullin catalyzes ubiquitination of the phosphorylated CDK inhibitor Sic1 p. Ceil 1997, 91:221-230. See annotation [10"]. 10. •.
Skowyra D, Craig KL, Tyers M, Elledge S J, Harper JW: F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Ceil 1997, 91:209-219. A very elegant and convincing study showing that seven purified components, an El, ubiquitin, Cdc34, Cdc4, Cdc53, Skpl and CIn-Cdc28 kinase, are necessary and sufficient to promote the multiubiquitination of the CDK inhibitor Sic1 in vitro. This work provides conclusive evidence that a complex composed of Skpl, Cdc53 and the F-box protein Cdc4 can function as a Sic1 E3 ligase. These studies illustrate the importance of F-box proteins in providing substrate specificity to ubiquitination reactions [9"'].
Proteolysis and the G1-S transition Krek
11.
Connelly C, Hieter P: Budding yeast SKP1 encodes an evolutionarily conserved kinetochore protein required for cell cycle progression. Cell 1996, 86:275-265.
12.
Zhang H, Kobayashi R, Galaktionov K, Beach D: plgSkpl and p45Skp2 are essential elements of the cyclin A-CDK2 S phase kinase. Cell 1995, 82:915-925.
41
32.
Kornitzer D, Raboy B, Kutka RG, Fink GR: Regulated degradation of the transcription factor Gcn4. EMBO J 1994, 13:6021-6030.
33.
Plon SE, Leppig KA, Do HN, Groudine M: Cloning of the human homolog of the CDC34 cell cycle gene by complementation in yeast. Proc Nat/Acad Sci USA 1993, 90:10484-10488.
Kipreos ET, Lander LE, Wing JP, He WW, Hedgecock EM: cul-1 is • required for cell cycle exit in C. elegans and identifies a novel gene family. Ceil 1996, 85:829-839. A nematode gene, cul-1, which is required to limit cell divisions during embryonic development, is found to be closely related to CDC53. Database searches reveal that cul-1 and CDC53 are members of a multigene family, referred to as the cullins, which are conserved throughout evolution. Humans have at least six cullins.
Yew PR, Kirschner MW: Proteolysis and DNA replication: the CDC34 requirement in the Xenopus egg cell cycle. Science 1997, 277:1672-1676. CDK-dependent initiation of DNA replication in Xenopus eggs requires a CDC34 homolog. Dominant negative mutants of human CDC34 block the degradation of a Xenopus CDK inhibitor, thereby preventing initiation of DNA replication. Evidence is provided that CDC34 functions in a large molecular size complex. These data provide the best evidence to date that CDC34 function is also required in higher eukaryotes.
14.
35.
13.
Cherr CJ: G1 phase progression: cycling on cue. Cell 1994, 79:551-555.
15.
Hunter "1",Pines J: Cyclins and cancer. I1. Cyclin D and CDK inhibitors come of age. Ceil 1994, 79:573-582.
16.
NasmythK: Control of the yeast cell cycle by the Cdc28 protein kinase. Curt Opin Cell Bio/1993, 5:166-1 79.
1 "7.
Morgan DO: Principles of CDK regulation. Nature 1995, 374:131-134.
18.
Nigg EA: Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle. Bioessays 1995, 17:471-480.
19.
Goebl MG, Yochem J, Jentsch S, McGrath JP, Varshavsky A, Byers B: The yeast call cycle gene CDC34 encodes a ubiquitinconjugating enzyme. Science 1986, 241:1331-1335.
20.
Mendenhall MD: An inhibitor of p34cdc28 protein kinase activity from S. cerevisiae. Science 1993, 259:216-219.
21.
Schwob E, Bohm T, Mendenhall MD, Nasmyth K: The B-type cyclin kinase inhibitor p40SIC1 controls the Gt to S transition in S. cerevisiee. Cell 1994, 79:233-244.
22.
Schneider BL, Yang QH, Futcher AB: Linkage of replication to start by the Cdk inhibitor Sic1. Science 1996, 272:560-562.
23.
TyersM: The cyclin-dependent kinase inhibitor p40SIC1 imposes the requirement for CIn G1 cyclin function at start. Proc Nat/Acad Sci USA 1996, 93:7772-7776.
24. •
VermaR, Annan RS, Huddleston MJ, Cart SA, Reynard G, Deshaies RJ: Phosphorylation of Siclp by G1 CDK required for its degradation and entry into S phase. Science 1997, 278:455460. Mutations of several CDK consensus phosphorylation sites in Sic1 yields a mutant that can no longer be ubiquitinated in vitro. Expression of this mutant in vivo results in a stable protein that causes a G 1 arrest. 25.
Yaglom J, Linskens MH, Sadis S, Rubin DM, Futcher B, Finley D: p34Cdc28-mediated control of CIn3 cyclin degradation. Mo/ Ceil Bio11995, 15:731-741.
26. •
LankerS, ValdMeso MH, Wittenberg C: Rapid degradation of the G1 cyclin CIn2 induced by CDK-dependent phosphorylation. Science 1996, 271:1597-1601. Mutations of several CDK consensus phosphorylation sites in CIn2 yields a mutant that can no longer be phosphorylated by Cdc28 in vitro. The mutant is several-fold more stable then its wild-type counterpart and expression of this mutant in vivo results in a shortened G t phase. 27.
Deshaies RJ, Chau V, Kirschner M: Ubiquitination of the Gt cyclin CIn2p by a Cdc34p-dependent pathway. EMBO J 1995, 14:303-312.
28.
Blondel M, Mann C: G2 cyclins are required for the degradation of Gt cyclins in yeasL Nature 1996, 384:279-282.
Henchoz S, Chi Y, Catarin B, Herskowitz I, Deshaies R, Peter M: Phosphorylation and ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor Farlp in budding yeast. Genes Dev 199?, 11:3046-3060. A Far1 mutant is identified that is able to induce cell cycle arrest in the absence of co-factor. Cells that overexpress this mutant arrest in G1 with low CIn-Cdc28 kinase activity. Whereas wild-type Far1 is readily ubiquitinated in a Cdc34-dependent manner, the mutant is not. This defect is linked to the failure of CIn-Cdc28 kinase to phosphorylate the mutant Far1 protein.
34. •
Diehl JA, Zindy F, Sherr CJ: Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin-proteasome pathway. Genes Dev 1997, 11:957-972. This paper shows that cyclin D1 is phosphorylated in vivo on Thr286. Mutation of this site to alanine prevents this phosphorylation, inhibits multiubiquitination and markedly stabilizes the protein. The kinase responsible for Thr286 phosphorylation appears to be an as yet unidentified kinase. Phosphorylation drives ubiquitination and proteasomal degradation of cyclin D1 in response to serum withdrawal. •
36. •
ClurmanBE, Sheaff R.I, Thress K, Groudine M, Roberts JM: Turnover of cyclin E by the ubiquitin-proteasome pathway is regulated by cdk2 binding and cyclin phosphorylation. Genes Dev 1996, 10:1979-1990. See annotation [37*]. 37.
Won KA, Reed SI: Activation of cyclin E/CDK2 is coupled to site-specific autophosphorylation and ubJquitin-dependent degradation of cyclin E EMBO J 1996, 15:4182-4193. This paper, together with [36"], presents evidence that cyclin E is degraded by the ubiquitin-proteasome system and that this degradation is regulated by phosphorylation. The cyclin E-bound CDK2 initiates this degradation by phosphorylating cyclin E at a specific site. Phosphorylation causes cyclin E to disassemble from CDK2, resulting in the ubiquitination and degradation of free cyclin E. Interestingly, the binding of CDK2 to cyclin E appears to protect the latter from degradation. •
36.
Sheaff RJ, Groudine M, Gordon M, Roberts JM, Clurman BE: Cyclin E-CDK2 is a regulator of p27Kip1. Genes Dev 1997, 11:1464-1478. See annotation [39"]. •
39. •
Vlach J, Hennecke S, Amati B: Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27KIP1. EMBO J 1997, 16:5334-5344. This paper, together with [38 °] shows that the CDK inhibitor p27Kip1 is a substrate of cyclin E-CDK2. Phosphorylation of p2?KiP1 by cyclin E-CDK2 promotes its ubiquitination and proteasomal degradation. Elimination of p27 =s required for cells to transit from G 1 into S phase. The authors provide a model that explains how p27Kip1 can be an inhibitor and substrate of cyclin E-CDK2 at the same time. 40.
Dulic V, Lees E, Reed Sh Association of human cyclin E with a periodic G1-S phase protein kinase. Science 1992, 257:19561961.
41.
Koff A, Giordano A, Desai D, Yamashita K, Harper JW, Elledge S, Nishimoto T, Morgan DO, Franza BR, Roberts JM: Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle. Science 1992, 257:1689-1694.
42.
Pagano M, Tam SW, Theodoras AM, Beer-Romero P, Del Sal G, Chau V, Yew PR, Draetta GF, Rolfe M: Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p2Z Science 1995, 269:682685.
43.
Fredersdorf S, Burns J, Milne AM, Packham G, Fallis L, Gillett CE, Royds JA, Peston D, Hall PA, Hanby AM, Barnes DM et aL: High level expression of p27KJP1 and cyclin D1 in some human breast cancer cells: inverse correlation between the expression of p27KiP1 and degree of malignancy in human breast and colorectal cancers. Proc Nat/Acad Sci USA 1997, 94:6380-5385.
44.
Catzavelos C, Bhattacharya N, Ung YC, Wilson JA, Roncari L, Sandhu C, Shaw P, Yeger H, Morava-Protzner I, Kapusta Let aL: Decreased levels of the cell cycle inhibitor p27Km1 protein: prognostic implications in primary breast cancer. Nat Med 1997, 3:227-230.
45.
Loda M, Cukor B, Tam SW, Lavin P, Fiorentino M, Draetta GF, Milburn-Jessup J, Pagano M: Increased proteasome-dependent
29. •
30.
PiattiS, Bohm T, Cocker JH, Diffley JF, Nasmyth K: Activation of S-phase-promoting CDKs in late G1 defines a =point of no return" after which Cdc6 synthesis cannot promote DNA replication in yeast. Genes Dev 1996, 10:1516-1531.
31.
DruryLS, Perkins G, Diffley JFX: The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast. EMBO J 1997, 16:5966-5976.
42
Oncogenes and cell proliferation
degradation of the cyclin-dependent kinase inhibitor p27 in aggessive colorectal carcinomas. Nat Med 1997, 3:231-234. 46.
Porter PL, Malone KE, Heagerty PJ, Alexander GM, Gatti LA, Firpo EJ, Daling JR, Roberts JM: Expression of the cell cycle regulators p27 Kip1 and cyclin E alone and in combination, correlate with survival in young breast cancer patients. Nat Med 1997, 3:222-226.
47. •
Hateboer G, Kerkhoven RM, Shvarts A, Bernards R, Beijersbergen RL: Degradation of E2F by the ubiquitin-proteasome pathway: regulation by retinoblastoma family proteins and adenovirus transforming proteins. Genes Dev 1996, 10:2960-2970. See annotation [48°]. 48. •
HofmannF, Martelli F, Livingston DM, Wang Z: The retinoblastoma gene product protects E2F-1 from degradation by the ubiquitin-proteasome pathway. Genes Dev 1996, 10:2949-2459. This paper, together with [47°,49"], establishes that members of the E2F transcription factor family are unstable proteins and are targeted for degradation by the ubiquitin-proteasome pathway. Moreover, it is shown that degradation of E2Fs is regulated by retinoblastoma family proteins. 49. •
Campanero MR, Flemington EK: Regulation of E2F through ubiquitin-proteasome-dependent degradation: stabilization by the pRB tumor suppressor protein. Proc Nat/Acad Sci USA 1997, 94:2221-2226. See annotation [48"]. 50.
Li FN, Johnston M: Grrl of Saccharomyces cerevisiae is connected to the ubiquitin proteolysis machinery through Skpl: coupling glucose sensing to gone expression and the cell cycle. EMBO J 1997, 16:5629-5638.
51.
ThomasD, Kuras L, Barbey R, Cherest H, Blaiseau PL, SurdinKerjan Y: Met30p, a yeast transcriptional inhibitor that responds to S-adenosylmethionine, is an essential protein with WD-40 repeats. Mo/ Ceil Bio/1995, 15:6526-6534.
52. •
KominamiK, Toda T: Fission yeast WD-repeat protein pop1 regulates genome ploidy through ubiquitin-proteasomemediated degradation of the CDK inhibitor Rum1 and the S-phase initiator Cdc18. Genes Dev 1997, 11:1546-1560. A gone is identified that is important for the maintenance of ploidy in fission yeast. The gone, pop1, is highly related to the gone for budding yeast CDC4, an F-box protein. In pop1 mutants, Cdc18 and Rum1 are not ubiquitinated
and accumulate to high levels. Pop1 contains an F-box motif and associates with Cdc18, suggesting that Pop1 functions as a recognition factor in a ubiquitination pathway. 53.
StemmannO, Lechner J: The Saccharomyces cerevisiae kinetochore contains a cyclin-cdk complexing homolog as identified by in vitro reconstitution. EMBO J 1996, 15:36113620.
54. •
LisztwanJ, Marti A, Suetterluety H, Gstaiger M, Wirbelauer C, Krek W: Association of human CUL-1 and ubiquitin-conjugating enzyme CDC34 with the F-box protein p45SKP2: evidence for evolutionary conservation in the subunit composition of the CDC34-SCF pathway. EMBO J 1998, 17:368-383. This paper shows that human CUL-1, a member of the cullin family associates with p45SKP2, p19 sKP1 and cyclin A-CDK2 in vivo and that these components assemble into a large multiprotein complex. Since the molecular composition of this complex closely resembles budding yeast SCF-type E3 ligase complexes, it was proposed that the requirement for p45SKP2's function for S phase progression may reflect a requirement for ubiquitination of selected celt cycle regulatory proteins by this potential E3 ligase complex. In addition, this report shows that formation of this complex is governed, in part, by the S phase-specific expression of p45 SKP2 and the p45SKP2-bound cyclin A-CDK2. 55.
Aso T, Haque D, Barstead RJ, Conaway RC, Conaway JW: The inducible elongin A elongation activation domain: structure, function and interaction with the elongin BC complex. EMBO J 1996, 15:5557-5566.
56.
Kibel A, Iliopoulos O, DeCaprio JA, Kaolin WG: Binding of the yon Hippel Lindau tumor suppressor protein to elongin B and C. Science 1995, 269:1444-1446.
5?.
D u a nRD, Pause A, Burgess WH, Aso T, Chen DYT, Garrett KP, Conaway RC, Conaway JW, Linehan M, Klausner RD: Inhibition of transcription elongation by the VHL tumor suppressor protein. Science 1995, 269:1402-1406.
58.
PauseA, Lee S, Worrell RA, Chen DY, Burgess WH, Linehan WM, Klausner RD: The yon HippeI-Lindau tumor-suppressor gone product forms a stable complex with human CUL-2, a member of the Cdc53 family of proteins. Proc Natl Acad Sci USA 1997, 94:2156-2161.
59.
Bai C, Richman R, Elledge SJ: Human cyclin F. EMBO J 1994, 13:6087-6098.