Download the scf ubiquitin ligase: insights into a molecular machine

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THE SCF UBIQUITIN LIGASE:
INSIGHTS INTO A MOLECULAR
MACHINE
Timothy Cardozo and Michele Pagano
Abstract | Ubiquitin ligases are well suited to regulate molecular networks that operate on a posttranslational timescale. The F-box family of proteins — which are the substrate-recognition
components of the Skp1–Cul1–F-box-protein (SCF) ubiquitin ligase — are important players in
many mammalian functions. Here we explore a unifying and structurally detailed view of
SCF-mediated proteolytic control of cellular processes that has been revealed by recent studies.
CYCLIN-DEPENDENT KINASE
(CDK). A protein kinase that
controls cell-cycle progression in
all eukaryotes and requires
physical association with cyclins
to achieve full enzymatic
activity.
F-BOX PROTEIN
(FBP). A component of the
machinery for the ubiquitindependent degradation of
proteins. F-box proteins
recognize specific substrates
and, with the help of other
subunits of the E3 ubiquitin
ligase, deliver them to the E2
ubiquitin-conjugating enzyme.
Department of Pathology
and New York University
Cancer Institute, New York
University Medical Center,
550 First Avenue, MSB 599,
New York, New York 10016
USA.
Correspondence to M.P.
e-mail: michele.pagano@
med.nyu.edu
doi:10.1038/nrm1471
Fundamental cellular functions such as DNA replication, mitosis, DNA repair, transcription, cell differentiation and cell death are accomplished by large,
multi-protein and precisely regulated molecular
machines. Under most biological conditions, the driving forces of these machines — for example, the
CYCLIN-DEPENDENT KINASES (CDKS) in the eukaryotic cell cycle
— need to be humming constantly. Crucial modular
components must therefore lock into place or detach
and disappear in an instant to keep the machines from
spinning out of synchronization with the rest of the cell.
The cellular timescale of these active machines does not
lend itself to the appearance or disappearance of ratelimiting components by the processes of transcription
or translation. Instead, the disappearance of a component in a sudden and compartment-restricted manner
can be a finely tuneable brake (if the target component
is a catalyst) or a sensitive accelerator (if the target component is a brake or an inhibitory subunit). This type of
regulated disappearance can be achieved by the ubiquitin–proteasome system (BOX 1).
Recent findings have enhanced our appreciation of
this system, and here we will review our current understanding of an important ubiquitin ligase — the SCF
ligase — with a particular emphasis on the mammalian
enzymes. The SCF ligase relies on one of its components — the F-BOX PROTEIN (FBP) — for its specificity, and
the result is that the F-box family of proteins has
emerged as the effector class of proteins for a crucial,
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and therapeutically exploitable, step in the ubiquitinmediated degradation of many cell-cycle-regulatory
proteins and transcription factors.
The SCF ubiquitin ligase and the FBP family
E3 ubiquitin ligases (BOX 1) have been classified into three
groups: the single-subunit RING-FINGER type, the multisubunit RING-finger type and the HECT-domain type.
Most of the multi-subunit RING-finger type of E3 ligases
contain a cullin protein (Cul1–5, Cul7 and so on; so
named because they appeared to ‘cull’, or sort, substrates
for degradation)1. Many structural and functional details
have now been deciphered for the most well-characterized mammalian cullin-dependent ligase (CDL) — the
SCF (SKP1–CUL1–FBP) LIGASE. In this ligase, the cullin subunit
Cul1 functions as a molecular scaffold that simultaneously interacts at the amino terminus with the crucial
adaptor subunit Skp1 (S-phase-kinase-associated protein-1) and at the carboxyl terminus with a RING-finger
protein (Rbx1, which is also known as Roc1, or Roc2)
and a specific E2 enzyme or UBIQUITIN-CONJUGATING ENZYME
(UBC), such as Ubc3, Ubc4 or Ubc5. Skp1, in turn, binds to
one of many FBPs. Each FBP appears to be matched with
a discrete number of specific substrates through a protein–protein interaction domain.
The F-box domain. The FBPs are defined by the
presence within their sequence of the Skp1-binding
F-box domain. The first F-box gene — CCNF, which
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Box 1 | The ubiquitin–proteasome system
The cellular abundance of many key brakes or accelerators of cellular proliferation — a
large number of which are known to be oncoproteins or tumour suppressors — is
controlled by the ubiquitin–proteasome degradation system in a compartment-specific
and unidirectional manner108,55. The small soluble protein ubiquitin is transferred and
attached to these target substrate proteins by the sequential action of E1, E2 and E3
enzymes109,110. The E1 enzyme — of which there is only one in humans — activates the
entire cellular pool of ubiquitin molecules. E2 enzymes, or ubiquitin-conjugating
enzymes (UBCs), inherit the activated thiol-ester-bonded ubiquitin from the E1
enzyme. The specificity of the ubiquitylation is then provided by the E3 ligase enzyme,
which binds both the target substrate and the activated ubiquitin–E2 complex, and
ultimately completes the transfer of ubiquitin to the target. Several rounds of ubiquitin
conjugation can produce long chains of ubiquitin moieties
(polyubiquitylation), the first of which is covalently bound to the substrate. At this
point, the polyubiquitylated substrate is committed to association with, and unfolding
and degradation by, the 26S proteasome111, whereas monoubiquitylated proteins have
non-proteolyic fates112.
RING-FINGER
A protein-sequence motif
corresponding to a particular
folded protein domain that
binds Zn2+ through a four-point
arrangement of cysteine and
histidine amino acids. In the E3
ubiquitin ligases, this domain
seems to be responsible for
binding the E2 ubiquitinconjugating enzymes.
SCF UBIQUITIN LIGASE
A multisubunit ubiquitin ligase
that contains Skp1, a member of
the cullin family (Cul1), and an
F-box protein, as well as a RINGfinger-containing protein
(Roc1/Rbx1).
UBIQUITIN-CONJUGATING
ENZYME
(UBC). An enzyme (also known
as E2) that accepts ubiquitin
from a ubiquitin-activating
enzyme (E1) and, together with a
ubiquitin ligase (E3), transfers it
to a substrate protein.
β-PROPELLER
A compact structural domain, or
protein-folding pattern, in which
similarly sized β-sheets are
stacked and offset into a complete
cylinder, so that they resemble the
blades of a propeller.
740
encodes cyclin F — was discovered fortuitously in a
search for candidate genes at the polycystic kidney disease (PCKD) gene locus2. Although apparently unrelated to PCKD, the gene was notable for the presence of
a cyclin domain. Cyclin-F levels were later shown to
oscillate during the cell cycle to a similar extent as those
of cyclins A and B (REF. 3), but with a distinct peak in G2
and a trough in mitosis prior to that of cyclin B (REF. 4).
Most importantly, cyclin F was found to be a suppressor
of the yeast Cdc4 mutant that displays G1–S deficiency
and an inability to degrade Sic1, an inhibitor of Cdk1. A
search for other suppressors of the Cdc4 mutation led to
the discovery of yeast Skp1, the inactivation of which
resulted in cell-cycle arrest in both G1 (prior to S-phase
entry) and G2 (prior to mitosis). These arrests also coincided with the stabilization of key CDK regulators, and
Skp1 was shown to be required for the ubiquitinmediated proteolysis of these proteins. Skp1 was able
to bind to cyclin F, to Cdc4 and to many other proteins through a conserved 40-amino-acid domain.
Since it was originally noted in cyclin F, this domain
became known as the F-box and the F-box family of
proteins was born5. Cyclin F and Cdc4 were therefore
the first F-box proteins to be identified. A year later,
the different pieces of the puzzle came together when
it was shown that Cul1, Skp1 and Cdc4 form an E3
ligase that is required for Sic1 ubiquitylation, and this
complex was called the SCF ubiquitin ligase6,7.
Mammalian F-box-protein classification. The mammalian FBPs have been named according to the structural class of their substrate-binding domains 8,9
(TABLE 1), although some original names have been conserved for historical reasons. The substrate-binding
domain of the FBP almost invariably lies directly carboxy-terminal to the F-box domain in the sequence,
and no FBP seems to have more than one F-box domain
(according to the Pfam database; see the online links
box). One class of FBPs are known as FBWs (‘FB’ for
F-box and ‘W’ for WD-40 repeat domain); their substrate-binding domain is a β-PROPELLER structure that is
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found in many protein-binding contexts10 and seems to
recognize specific Ser/Thr phosphorylation (pS/pT)
consensus sequences: DpSGXXX(X)pS (where ‘X’ represents any amino acid) in Fbw1 (also known as β-Trcp1);
and a variable L[I/L/P][pS/pT]P sequence in Fbw7 and
Cdc4.
In the case of the FBLs (‘L’ for LEUCINE-RICH REPEAT
(LRR)), the domain is an arc-shaped α–β-repeat structure that is also found in many protein-binding contexts, including the extracellular-binding domain of
certain surface receptors11,12. In most cases, FBLs also
seem to involve substrate phosphorylation for their
interaction, but this does not seem to be a general phenomenon in LRR-containing proteins. In addition,
some substrates are required to be part of specific protein complexes to be recognized by the specific FBLs
and, at least in one case, recognition also requires an
accessory subunit (see below).
The third class of FBPs includes proteins that are
known as FBXs, and that do not have WD-40 repeats or
LRRs but often have different protein–protein interaction domains such as CASH (carbohydrate-interacting),
cyclin box, CH (calponin homology), TDL (Trafdomain-like), Sec7, zinc-finger and proline-rich
domains. Human FBXs have been identified that are
specific for N-glycan moieties13,14, and the FBP-related
Von-Hippel–Landau protein (VHL), which is a subunit
of a CDL2 E3 ligase, has a proclivity for a hydroxylated
HIF1α substrate. This indicates that, in addition to phosphorylation and glycosylation, the mode of binding
of FBPs to substrates could be through a wide range of
post-translational modifications such as methylation,
ribosylation, farnesylation and acetylation. If it is true
that FBPs are specific for these modifications, the implication is that SCF ligases are constructed to indirectly
sense the initial target-modifying activity — the kinase,
glycosylase, acetylase, ribosylase and so on — which
would be consistent with the view of SCFs functioning as
a brake and an accelerator of a molecular machine that is
driven by these activities.
F-box-protein functions
The FBPs Skp2, β-Trcp and Fbw7 target known substrates that implicate them in the control of cellular
proliferation (see below). The substrate for Fbx5, which
is also known as Emi1 (early mitotic inhibitor-1) is not
known. But, this protein is also central to cell proliferation because of its ability to inhibit, through means
other than targeted degradation, the critical M–G1
ubiquitin ligase — the ANAPHASE-PROMOTING COMPLEX/CYCLOSOME (APC/C) — during the S and G2 phases of the cell
cycle15,16. Evolutionary data (BOX 2) and data on the functions of several other human FBPs however, indicate
that they might be involved in a wide variety of organspecific functions (TABLE 1).
Mutations in Fbw4 (also known as dactylin in the
mouse) or its orthologues (such as Hagoromo in
zebrafish) result in embryonic patterning defects that
manifest as limb and digit dysplasia in the mouse and
stripe-pattern disorganization in the fish17,18. The corresponding human congenital malformation is known as
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Table 1 | Overview of mammalian F-box proteins and their known functions
Mammalian F-box protein (FBP)
name* (number of FBPs)
Aliases
Human approved
gene symbol‡
Main substrates
Comments
Fbw1
β-Trcp1
BTRC
Emi1, Cdc25a, Wee1,
β-catenin, IκB-family
members
Gene-knockout phenotype: defective
spermatogenesis, subtle mitotic defects,
centrosome overduplication
Fbw4
Dactylin
SHFM3
Unknown
Human split hand–foot malformation (SHFM) gene
FBXW6
Unknown
In addition to being part of an SCF ligase,
it also forms a complex with Skp1 and
Cul7; the latter interacts with SV-40 large T antigen
Cdc4,
Sel10
FBXW7
Cyclin E, Myc, Jun,
Notch-1 and -4
Gene-knockout phenotype: embryonic lethal at
E11, probably due to morphogenetic
cardiovascular defects. Mutations in ovarian and
breast cancer cell lines
Skp2
SKP2
p27, p21, p57, p130
Gene-knockout phenotype: hypoplasia in
most organs; endoreduplication;
centrosome overduplication; defect of
mitotic entry. Overexpressed in human tumours
Cyclin F
CCNF
Unknown
Function unknown. First-identified
mammalian FBP
FBXO2
Unknown
Recognizes N-glycans. ER-associated degradation
FBXO5
Unknown
Inhibitor of APC/C. Overexpressed in breast tumours
FBXO6
Unknown
Recognizes N-glycans
FBXO32
Unknown
Involved in skeletal muscle atrophy.
Higher expression in muscle cells
FBWs (17)
Fbw6
Fbw7
FBLs (22)
Fbl1
FBXs (39)
Fbx1
Fbx2
Fbx5
Emi1
Fbx6
Fbx32
Mafbx,
Atrogin1
*The nomenclature is based on that used in references 8–10. ‡The human approved gene symbol is that recommended by the HUGO Gene Nomenclature Committee
(HGNC). APC/C, anaphase-promoting complex/cyclosome; Cdc, cell division cycle; Cul7, cullin-7; E11, embryonic day 11; Emi1, early mitotic inhibitor-1; ER, endoplasmic
reticulum; IκB, inhibitor of nuclear factor (NF)κB; FBL, F-box and leucine-rich-repeat protein; FBW, F-box and WD40-domain protein; FBX, F-box-only protein;
SV40, simian virus-40.
LEUCINE-RICH REPEAT
(LRR). A protein-sequence motif
that contains regular occurrences
of the amino acid Leu, which are
present as tandem arrays in
certain proteins. The back-toback set of motifs was found to
correspond to a small
subdomain structure in the
protein that stacks next to
adjacent repeats to form a
parallel, β-sheet, arc-like
structure.
ANAPHASE-PROMOTING
COMPLEX/CYCLOSOME
(APC/C). Anaphase is the phase
of mitosis during which
condensed chromosomes
separate into sister chromatids
and move along the mitotic
spindles to opposite poles of the
cell. The APC/C is a multisubunit E3 ubiquitin ligase with
at least two alternative forms,
which are activated by two
different proteins (Cdc20 or
Cdh1) and are necessary for the
transition into anaphase, as well
as the exit from mitosis and the
maintenance of the G1 state.
split hand–foot malformation19,20. Fbw6 shows an
apparent interchangeability of an SCF key interface (see
below) in that this FBP assembles an SCF ligase with
either Cul1 or Cul7 (REF. 21). Cul7 was found to bind the
simian virus 40 large T antigen, and its targeted disruption in the mouse resulted in abnormal vascular development22. Interestingly, another component of this
CDL7 complex — Fap68 or glomulin — is the gene
product that is associated with human familial venous
malformations. CDL7Fbw6 therefore seems to have a role
in vascular morphogenesis.
Data on other FBPs are still preliminary. Fbx2 and
Fbx6 are part of SCF ligases that recognize N-glycan
moieties on endoplasmic-reticulum proteins that are
translocated to the cytoplasm for degradation13,14.
Expression of Fbx3 is increased in activated or proliferating joint epithelium (synovium) from patients with
rheumatoid arthritis23. A heat-shock protein, α-crystallin, which is implicated in the pathogenesis of
desmin-related myopathy, interacts with Fbx4 in a
manner that is dependent on the mitotic-specific phosphorylation of certain serines in α-crystallin24. This
indicates that α-crystallin might be a substrate or a
cofactor of Fbx4. Fbx32 (also known as Mafbx or
Atrogin1) is the product of one of only two genes that
are upregulated in a panel of rat models of muscle atrophy25–27. Deficiency of this protein conferred resistance
to muscle atrophy and the gene is induced by the Foxo
family of transcription factors.
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How SCF ubiquitin ligases modulate CDKs
Despite the expected diversity of substrate targets,
multi-subunit E3 ubiquitin ligases are all thought to use
a similar mechanism, with the diversity of their molecular-machine substrates determining the diversity of
their cellular functions. The prototype of the mechanistic signature for the E3 ubiquitin ligases is the SPINDLE
CHECKPOINT in mitosis, in which the onset of anaphase is
arrested globally in the cell by a signal that emanates
from the KINETOCHORE28. Elegant studies in yeast showed
that Mad and Bub, the key proteins that control the
spindle checkpoint, function by sequestering Cdc20, a
key activator of APC/C29,30. Once it is released by the
appropriate signal, which indicates that spindles have
achieved the appropriate tension and that metaphase
chromosomes have lined up in the proper fashion,
APC/C
induces the ubiquitin-mediated proteolytic
degradation of securin, a key inhibitor of sister-chromatid separation. Such a compartment-restricted ‘hairtrigger’ point cannot rely on transcription or translation
for its execution, and eukaryotic biology has evolved an
elegant solution to this need: at the prescribed time on
the post-translational timescale, a readily activatable E3
ubiquitin ligase (the APC/C) degrades a key component
(securin) that is functioning as a brake for its target
molecular machine .
Mitosis and the DNA-synthesis phase (S phase)
require exceptional fidelity and precision to correctly manage the massive DNA-replication and
CDC20
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Box 2 | Genomics and evolution
Saccharomyces cerevisiae (~11)
Fungi (~50)
Caenorhabditis briggsae/elegans (~75/350)
Nematoda (~425)
Metazoa (~600)
Fruitfly (~30)
Arthropoda (~50)
Chordata (~300)
Mouse (73)
Human (78)
Eukaryota (~1,600)
Viruses (~20)
Archaea (~0)
Bacteria (~1)
Cyanobacteria (~0)
Synechocystis PCC (~0)
Rice spp. (~300)
Arabidopsis thaliana (~500–600)
Green plants (~1,000)
Plastid group (~1,000)
Other eukaryotes (<10)
The distribution of genes that encode F-box proteins (FBPs) in several genomes is
evident from genomic analysis (see figure), with several mammalian FBPs having
important orthologues in lower organisms113. The figure shows the taxonomic
distribution of FBPs from the InterPro database (see the online links box). Outer circles
represent distal branches of the evolutionary tree (that is, genus and species; as opposed
to the inner circles, which represent order and kingdom). The true number of FBPs for
any organism is consistently less than that found in InterPro. There are actually 78 FBPs
in humans and 73 in mice as opposed to the higher number that can be found in
InterPro, and the other species numbers have been interpolated accordingly. The
evolutionary distribution of the FBPs mirrors the mechanistic role of the interchangeable
FBP in the otherwise invariant SCF (Skp1–Cul1–FBP) ubiquitin ligase. For any given
species, there are very few Cul1 (cullin-1) and Skp1 (S-phase-kinase-associated protein-1)
proteins and a correspondingly larger number of FBPs (1 Skp1 and 78 FBPs in humans
versus 10–20 Skp1 proteins and 350 FBPs in Caenorhabditis elegans114,115, for example).
Individual plant cells and their resident compartment-restricted molecular machines
might be more transparently positioned to respond immediately to important
environmental signals such as light116. Indeed, plants — such as Arabidopsis thaliana with
its 500–600 genes that encode FBPs and 20 Skp1-like genes117,118 — show a comparatively
larger increase in the number of FBPs over metazoans. Interestingly, although it was
previously thought that no true FBPs existed in viruses or bacteria, several are now present
in the InterPro and Pfam databases (see the online links box). This indicates a picture of
FBP restriction to higher eukaryotic functions with rare adaptation by bacteria via gene
transfer, and similarly rare pathogenic exploitation by viruses.
SPINDLE CHECKPOINT
The molecular process that
specifically controls the assembly
of the kinetochore on the
chromosomal centromere and
the timing of kinetochore
dissociation. Dissociation
involves the movement of the
kinetochores, along with their
attached sister chromatids, to
opposite poles of the mitotic
spindle during anaphase.
KINETOCHORE
The complicated protein
assembly that links the
specialized areas of condensed
chromosomes that are known as
centromeres to the microtubulebased mitotic spindle.
742
chromosome-segregation machines. In addition, these
two important molecular machines of the cell cycle are
necessarily interrelated — there is a clear need for inhibition of the synthetic machinery while mitosis is occurring, and vice versa. Finally, the behaviour of complex
systems such as that of the cell cycle provides a fundamental paradox: a cyclic process has difficulty in producing checkpoints, and a system that produces
checkpoints has trouble in maintaining repetitive
cycles31. Clearly, the biologically diverse contexts of the
cell cycle in higher eukaryotes (for example, embryogenesis, organ renewal and wound healing) require
the ability to engage both endless repetitive cycles and
checkpoints to ensure the synchronization of the cycle
with surrounding cellular and tissue-based processes. A
perspective that combines what we know about the
activity of CDKs in the cell cycle and what we now know
about SCF-complex function hints at how the cell
resolves this paradox.
G1 phase. p21 and p27 are key inhibitors of both Cdk1
(REFS 32,33) and Cdk2 (REF. 33). Seen through the prism of
CDK-activity profiles in the cell cycle (BOX 3), the finding
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that SCFSkp2 is responsible for the ubiquitin-mediated
degradation of p27 (REFS 34–38) and p21 (REFS 39,40) during
the G1–S checkpoint reveals the role of this SCF ligase.
First, it executes the transition to S-phase by degrading
these CDK inhibitors; and second, it maintains Cdk1 and
Cdk2 activities by keeping the environment clear of p21
and p27 later in the cell cycle (BOX 3 figure, parts a and b).
Interestingly, the other main cell-cycle E3 ubiquitin ligase
— APC/C — completes the picture.APC/CCdh1 maintains
the attenuation of both Cdk1 and Cdk2 during G1 by
simultaneously targeting Skp2 (REFS 41,42) and its cofactor
Cks1 for degradation, and continuing its degradation of
cyclins A and B, which was initiated during mitosis.
Cyclin A binds to both Cdk2 and Cdk1, and cyclin B activates only Cdk1. The first action of APC/CCdh1 therefore
preserves the inhibition of Cdk1 and Cdk2 by p27 and
p21, and the second keeps the levels of cyclins A and B
low during G1. Additionally, APC/CCdh1 degrades the
key Cdk1-activating phosphatase Cdc25a during this
period43. The conditional inactivation of the APC/C
subunit Apc2 in quiescent hepatocytes causes unscheduled cell-cycle re-entry, possibly by these convergent
mechanisms44.
G1–S, S and G2 phases. A switch in the regulation of
Cdk1 and Cdk2 that involves two other FBPs occurs
during the G1–S transition and in the S and G2 phases
(BOX 3 figure, part b). First, the FBP Emi1 accumulates as
a result of transcription that is induced by the transcription factor E2F(REF. 15; which also induces transcription
of the genes that encode cyclins A, B and E). The accumulated Emi1 inhibits APC/CCdh1 by a non-proteolytic
mechanism45. Interestingly, this simultaneously releases
Skp2 and cyclins A and B from APC/CCdh1-mediated
destabilization. This begs the question: how is Cdk1
attenuated when its activating cyclin subunits are both
stabilized and transcribed? This is where the second FBP
comes in. For its full activity, Cdk1 requires the activity of
the Cdc25a phosphatase that counteracts an inactivating
phosphorylation by the kinase Wee1. SCFβ-Trcp has
recently been shown to degrade phosphorylated Cdc25a
in the S and G2 phases46,47. So, a balanced picture
emerges that explains the maintenance of Cdk2 activity
and Cdk1 attenuation. The activities of Cdk2–cyclin-E
and Cdk2–cyclin-A are maintained by the action of
SCFSkp2 on p21 and p27 and that of Emi1 on APC/CCdh1;
and low Cdk1 activity is maintained by SCFβ-Trcp, despite
the loss of cyclin-A and -B destabilization by APC/CCdh1
and p21 and p27 degradation through SCFSkp2. Cdk2 also
helps in inactivating APC/CCdh1 by phosphorylating
Cdh1 to inhibit its binding to APC/C30. This places Emi1
and Cdk2 both upstream and downstream of APC/CCdh1,
which indicates that some unknown factor (X? in BOX 3
figure, part b) initiates a loop during this period of the
cell cycle. Alternatively, Emi1 and Cdk2 might seem to
be both upstream and downstream of APC/C if they
are part of a constitutive feedback loop, only one arm
of which (for example, only the inhibition of Cdh1 by
Cdk2, and not the destabilization of cyclins A and B
by Cdh1) is active enough to be apparent under
experimental conditions. The ‘quiet’ arm of the loop
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Box 3 | CDK activity profiles
a G1 phase
b G1–S, S and G2 phase
p27
APC/CCdh1
Emi1
Cyclin E
Cdk1 and
Cdk2
SCFSkp2
p27
p21
APC/CCdh1
X?
Cyclins
A and B
Cdc25a
Cdk2
SCFSkp2
Cdk1
p21
Cyclin A
Cdk1
Cyclin B
SCFβ-Trcp
d Mitotic progression and exit
SCFFbw7
SCFSkp2
Securin
APC/CCdc20
Cyclins
A and B
Cyclin E
Cdk2
p27
Separase
Emi1
Wee1
c G2–M phase
Cohesins
SCFβ-Trcp
Cdc25a
Emi1
Cdk1
APC/CCdh1
Cyclins
A and B
SCFβ-Trcp
Cdc25a
p21
Cdk1
Wee1
Cyclin-dependent kinases (CDKs) represent both the engine and the pacemaker of the cell cycle. It is now clear that
proteolysis is a significant force that imposes the required checks and balances on this engine. During G1, Cdk1 and
Cdk2 need to be idle to avoid premature DNA synthesis and mitosis (see figure, panel a). Starting at the G1–S checkpoint
— the point at which DNA replication and centrosome duplication begins — Cdk2 markedly increases its activity (see
figure, panel b). The activity of Cdk2, and therefore the phosphorylation of its substrates, builds to a peak through the
DNA-synthesis and centrosome-duplication phase into G2, at which point the activity of Cdk2 is attenuated again. The
coincidence of peaking Cdk2 activity and peaking activity of the DNA-replication and centrosome-duplication
machinery indicates that Cdk2 somehow releases and maintains the nuclear environment that is necessary for the
synthetic machinery to function. Although Cdk2 seems to be the optimal agent for this purpose, it is not absolutely
required. Low Cdk1 activity and/or the action of other CDKs contribute to these processes, and can accomplish them in
the absence of Cdk2, at least under normal cellular conditions3.
At around the same time that Cdk2 activity attenuates in G2, Cdk1 markedly increases its activity, building the unique
mitotic cellular environment of its phosphorylated downstream substrates (see figure, panel c). Anaphase is sharply
demarcated by a sudden decline of Cdk1 activity, and both Cdk1 and Cdk2 are maintained in their inactive states until
the next G1–S transition (see figure, panel d). See main text for more details. X? represents an unknown factor (see main
text). Blue boxes signify activated forms of the respective proteins. APC/C, anaphase-promoting complex/cyclosome;
Cdc25a, cell-division cycle-25a; Emi1, early mitotic inhibitor-1; SCF, Skp1–Cul1–F-box-protein; Skp, S-phase-kinaseassociated protein.
might emerge periodically in vivo without the need for
an initiating factor.
APC/CCDC20
According to the convention for
multi-subunit E3 ligases, the
presumed activator, or substratetargeting, subunit is shown in
superscript text. So, the E3 ligase
that is formed by the APC/C
subunits and Cdc20 is written as
shown, and the SCF ligase
formed by Skp1, Cul1, Roc1 and
the F-box protein Skp2 is known
as SCFSkp2.
G2–M transition. Interpreting the states of CDK activity
at the G2–M transition through the E3 ligases that control them is similarly revealing (BOX 3 figure, part c).
Cdk2 attenuation is now maintained by SCFFbw7-mediated degradation of its necessary partner cyclin E
(REFS 48–50), although some Cdk2 activity probably
remains thanks to the presence of cyclin A. Cdk1 activity
is now maintained by the continued inhibition of
APC/CCdh1, as well as APC/CCdc20, by Emi1; and by the
renewed availability of Cdc25a because SCFβ-Trcp actively
degrades it only in S and G2 phase. SCFβ-Trcp continues
to have a role in this phase, however, through its
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
induced degradation of the Cdk1 inhibitor Wee1 (REF. 51).
Notably, this is also the point at which Tome1 — an
antagonist of Wee1 that contains an F-box-like domain
— appears, further promoting Cdk1 activity52.
Mitosis. In late mitosis, both CDKs are attenuated. The
block on APC/C is removed by yet another activity of
SCFβ-Trcp — the ubiquitin-mediated degradation of
Emi1 (REFS 53,54). APC/CCdc20 is therefore released and, in
addition to its anaphase-promoting induction of the
degradation of securin, actively induces the degradation
of cyclins A and B, thereby quenching Cdk1 activity
(BOX 3 figure, part d). Interestingly, Cdk1 activates
APC/CCdc20 by phosphorylating several APC/C subunits,
thereby contributing to its own attenuation30.
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This model emphasizes the contribution of E3 ligases
to the regulation of CDKs. There are, of course, crucial
contributions by CDK transcription, translation and
translocation mechanisms (for reviews, see REFS 3,55),
which were omitted to focus on how the SCF complexes
work together with the cascade of kinases that execute
the cell cycle. The phosphorylation sites through
which the SCF complexes are primed and targeted are
frequently created by kinases — both CDKs and nonCDKs — which are themselves indirectly affected by the
resulting degradation in a feedback loop. The requirement of SCF for the phosphorylation of its substrates
indicates that part of how these E3 ligases contribute to
the execution of these checkpoints is to sense the activity
of these kinases indirectly. For example, p27 ubiquitylation by SCFSkp2, which ultimately results in the activation
of Cdk1 and Cdk2, requires the CDK-dependent phosphorylation of its carboxyl terminus56–58. Cyclin E is
targeted for ubiquitylation by SCFFbw7 through its phosphorylation at several sites by Cdk2 and glycogensynthase kinase 3β (GSK3β)48–50,59. Cdc25a is primed
through its phosphorylation by Chk1, and its β-Trcpbinding motif is phosphorylated by an as-yet-unidentified kinase46,47. The Wee1 kinase is phosphorylated by a
Polo-like kinase-1 (Plk1) after priming by Cdk1-dependent phosphorylation, this forms an auto-amplification
loop that connects these three central mitotic kinases
(Wee1, Plk1 and Cdk1)51. Similarly, phosphorylation by
Plk1 and Cdk1 targets Emi1 to β-Trcp60.
In summary, Skp2 is an activator of both Cdk1 and
Cdk2; Fbw7 is an inhibitor of Cdk2; and β-Trcp contributes by turning Cdk1 off during the S and G2
phases, turning it on at the G2–M transition, and finally
turning it off again at the end of mitosis. These three
FBPs also regulate important transcription factors:
E2F1 and Myc are regulated by Skp2; Myc, Jun, and
Notch-1 and -4 are regulated by Fbw7; and β-catenin
and nuclear factor κB (NFκB) are regulated by β-Trcp1.
In turn, these transcription factors, among several other
functions, control CDK activities by inducing or suppressing the transcription of genes that encode certain CDK subunits and their regulators. Notably, SCF
ligases and APC/C control each other: in G1,
APC/C Cdh1 induces the degradation of Skp2 and
Cks1, thereby maintaining the G1 state; and in early
mitosis SCFβ-Trcp activates APC/CCdc20 by inducing the
degradation of Emi1. So, a picture emerges of waves
of different SCF ligases that instantaneously modify
components of the CDK regulatory network to maintain, activate or attenuate these drivers of the cell
cycle, apparently for the purpose of establishing
checkpoints in an endless cycle.
Structural picture of the SCF ubiquitin ligase
ALLOSTERIC SITE
A site on an enzyme, which, on
binding of a modulator, causes
the protein to undergo a
conformational change that
might alter the catalytic or
binding properties of the
enzyme.
744
Why is the SCF ligase constructed the way it is? The
mechanism of most known protein enzymes involves
the specific binding of a protein to a chemical substrate
such that the substrate is positioned and orientated relative to a key catalytic atom or residue. The current collection of structural information on the SCF ligases
indicates a similar mechanism, but, interestingly, this
| SEPTEMBER 2004 | VOLUME 5
mechanism seems to operate at the macromolecular
level of protein subunits rather than residues and secondary-structure elements. So, instead of the transfer of
a small chemical group (for example, a phosphate group
in a kinase), an entire protein (ubiquitin) is transferred.
Instead of a key catalytic residue, as in common
acid–base catalysis, there is an activated protein subunit
— the activated E2 enzyme. Instead of a substratebinding subdomain, there is an entire specific substrate-binding protein (the FBP). And, instead of the
orientation of the donor and acceptor groups, as well as
ALLOSTERIC shifts, being encoded by the interrelationships
of a single folded protein chain, these are encoded in the
SCF ligase by the structural characteristics of the cullin
protein and the FBP and by their respective protein surfaces and interfaces.
Alternative mechanisms are also possible. These
include the possibility that SCF might have oligomeric
forms, that modifications occur at the cullin protein,
and that the binding of certain SCF factors to both the
amino and carboxyl termini of the cullin protein regulate
its activity (reviewed in REF. 61). This latter observation,
in particular, indicates that the mechanism of ubiquitin
ligation proceeds through a significant cooperative
structural change in the ligase from one end to the
other. Other possibilities are that substrate unfolding or
SCF oligomerization are required to bring the substrate
and ubiquitin together — perhaps also with other
unknown intermediate molecules — for ligation to
occur. However, as structural information is available
only for the SCF complex and not for possible cooperating factors, we will explore here the model that
depicts the SCF ligase as a kind of ‘super-enzyme’ with
an active-site-like ‘hot zone’ and briefly discuss alternative models thereafter.
Crystal structures have been published of: the
SCFSkp2(F-box only) complex (which contains only the F-box
domain of Skp2), the complete Skp1–Skp2 complex, the
Skp1–β-Trcp complex, the Skp1–Cdc4 complex, and
related CDL and E2-enzyme complexes62–68. The common structures are remarkably consistent; they enable
reliable three-dimensional models of complete SCF
ligases to be built from the different structures, and
allow the differences between them to be interpreted
with respect to their mechanism of action. The models
show that different protein subunits fit together into a
single rigid C-shaped superstructure with a distance of
around 59 Å between the E2 enzyme on one end and
the substrate-binding surface of the FBP on the other.
The mechanistic model that is indicated by the structures is that the 59-Å space — into which the substrate
might float due to its affinity for the FBP-binding
domain — is presumed to be a kind of hot zone, or
super-sized active site, within which a ubiquitin molecule is likely to attach as soon as an appropriate acceptor
atom is in reach. This is not unlike classic enzyme catalysis except on a larger molecular scale. This hot zone is
encoded by the identities, stoichiometry and features of
the SCF subunits. Interestingly, this space is reasonably
well conserved in the structurally divergent single-subunit HECT ubiquitin ligases69,70, which indicates that
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Aa
Ab
B
Helix 8
Skp2
VHL
'Safety belt'
Ac
Ad
Helix 7
Homology
Elongin C
Skp1
Elongin B
Figure 1 | The interface between the F-box domain of Skp2 and Skp1. Aa | Backbone ribbon depiction of the F-box domain
of Skp2 (S-phase-kinase-associated protein-2) with consensus conserved residues highlighted: hydrophobic residues (yellow); Pro
residue (red); positively-charged residues (green). Ab | Electrostatic surface of the Skp1-binding face of the F-box domain of Skp2 in
the same orientation as in Aa, pictured through a skeletal ribbon representation of the Skp1 molecule. Ac | Electrostatic surface of
the F-box-binding face in Skp1 with a skeletal ribbon representation of the F-box domain from Skp2. This view is rotated 180o
around the vertical axis with respect to Aa and Ab. Ad | A ‘docked’ picture of the two electrostatic surfaces of Skp2 and Skp1 in the
same orientation as in Ac. B | Comparison of the equivalent interfaces of the F-box domain of Skp2 with Skp1 and the cyclindependent-ligase-2–Von-Hippel–Landau-protein (CDL2VHL) complex. There is a structural homology between elongin C and the
central domain of Skp1 (see boxed areas). However, helices 7 and 8 in Skp1 contribute to the orientation of the F-box domain and
its attached protein-binding domain with Skp2. By contrast, no interaction is seen in this area (indicated by a dashed line) in the
CDL2VHL complex. The segment of Skp2 that is referred to as a ‘safety belt’ might pin down bound substrates into the concave
surface of the leucine-rich repeat. Part B of the figure is modified with permission from REF. 64 © (2002) Macmillan Magazines Ltd.
many E3 ligases might use the same mechanism, but
that the localized properties and critical interfaces in the
super-enzyme can sometimes be accomplished within a
single protein chain.
Of all the localized properties that are indicated by
these structures, one of the most important is the
rigidity of the central portion of the superstructure, as
it has the purpose of correctly positioning the donor
and acceptor entities in space. The central portion of
the SCF ligase was shown to consist primarily of the
cullin protein, Skp1, the F-box domain and possibly
the intervening segment (linker) between the F-box
domain and the substrate-binding (WD-40, LRR and
so on) domain of the FBP. The introduction of flexible
linkers that eliminate the rigidity of the Cul1 scaffold
inactivates SCFSkp2 without affecting its ability to bind
the substrate or the E2 enzyme67. Likewise, mutations
that were designed to affect the rigid coupling between
the F-box domain and substrate-binding domains in
Cdc4 disrupted its function in vivo71. What is seen in
the structure of the SCF ligase might apply to other E3
ligases such as CDL2VHL. VHL residues that are important for the relative arrangement, and probably also
the rigidity, of its α and β domains — which bind the
elongin-C adaptor and the Hif1-α substrate, respectively — are frequently mutated in the VHL cancerpredisposition syndrome65. Also, mutations in Cbl,
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
another E3 ligase, that affect the rigid linkage between
the E2-binding and substrate-binding domains abolished its function without significantly affecting its
ability to bind either substrate or E2 (REF. 72).
Because there are only a few E2-ubiquitin donors
and many target substrates, another critical protein
interface is found at the transition from the conserved
E2-binding site on one side of the rigid central portion, to the diversified substrate-binding FBP on the
other side. In SCF, this transition point is between
Skp1 and the F-box domain. The F-box domain is
therefore like an anchor that can bring a wide variety
of substrates to the SCF ligase. Accordingly, the structural manifestations of the distinctive F-box-domain
sequence patterns are nonspecific folding and proteinsurface features (FIG. 1Aa). The F-box domain has a
compact trihelical conformation that forms an interlocked heterodimer with a broad, shallow pocket in
Skp1. The structure shows that the strictly conserved
portions of the sequence motif correspond to key folding features. This includes the signature Pro residue,
which precisely breaks the first helix of the domain,
and the only conserved charged residue — a Lys —
that seems to enforce the turn between helices 2 and 3.
Other key residues form a conserved hydrophobic surface that is continuous with the folding core of the
domain and that binds to Skp1 (FIG. 1Ab).
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The remainder of the F-box-domain surface is electronegative to ensure that the correct orientation will be
achieved by repulsion of the largely electronegative surface that surrounds the hydrophobic target pocket of
Skp1. This produces the tight interlocked complex
(FIG. 1Ac,Ad). Even slight disturbances in the orientation
of this key transition point might be amplified at the
distal protein-binding surface of the FBP — with functional consequences. For example, mutation of a Skp1
residue that is important for the precise arrangement of
the Skp1–Skp2 interface was shown to disrupt Skp1
function in yeast without abolishing Skp1–Skp2
binding 64.
Although the core F-box-domain–Skp1 interaction
is similar in the crystal structures of Skp1–Skp2,
Skp1–β-Trcp and Skp1–Cdc4, a carboxy-terminal βstrand of Skp2 seems to rigidify the spatial position of the
LRR in Skp2 by participating in the core F-boxdomain–Skp1 complex, whereas the same region of
Skp1–Cdc4 and Skp1–β-Trcp comprises only Skp1 and
F-box-domain atoms. Remote elements in the components of different SCF complexes, such as the carboxyl terminus in Skp2, might therefore contribute different pieces
to this core interface structure. The cullin component of
the complex might also contribute, and, indeed, Cul1 and
probably Cul7 (REF. 22) are implicated in this interface.
Again, this theme might be a general one in the
E3-ligase family, as a subdomain of Skp1 and elongin C
of CDL2VHL are structurally similar. The α-domain of
VHL is structurally equivalent to the FBP and interacts
with elongin C in the same orientation as the FBP does
with Skp1 (FIG. 1b). When the structures of these two
complexes are compared, however, equivalent portions
of Skp1 are missing. These include Skp1 helices 7 and 8,
which are notably less conserved in Skp1 homologues,
and might be provided by other proteins such as the
cullins, or might not be necessary. Finally, in the recently
described CDL3, the equivalent of the Skp1–FBP interface is entirely within a single polypeptide chain73–76. So,
although the ‘jigsaw puzzle’ of this interface is constructed in many different ways, these might all just be
different solutions to the common problem of rigidly
positioning the distal substrate in the hot zone, at least in
the structurally supported model of SCF function.
Versatile F-box proteins
DEGRON
A protein element, usually a
sequence motif, that targets the
protein for proteolytic
degradation.
746
Has nature designed FBPs to be flexible enough on their
substrate-binding end to bind biophysically diverse substrates — for example, those intercalated into complex
molecular machines — while concurrently achieving a
convergence to the common jigsaw-puzzle interface
with Skp1? The FBP recognition sites of SCFβ-Trcp and
SCFCdc4 consist of phosphorylated protein loops (seen as
peptides in the crystal structures) that tether substrates,
such as β-catenin and cyclin E, that have high affinity for
the WD-40 domains of their respective SCFs. β-catenin
is a globular soluble oncoprotein that is phosphorylated
on its FBP-recognition site (‘DEGRON’) by GSK3β77–80. The
spacing between this degron tether and the key ubiquitin-accepting Lys residue is stringent and seems to be a
mechanistic determinant for some β-Trcp substrates
| SEPTEMBER 2004 | VOLUME 5
such as β-catenin and IκB66 (inhibitor of NFκB).
Furthermore, this type of substrate–FBP interaction
seems characteristic for WD-40 domains, with the key
FBW residues and the substrate peptide clustering at
the top of the narrow channel in the centre of the cylindrical β-Trcp WD-domain. The Skp1–F-box-domain
interaction is similar in both SCFs. So, according to the
model whereby the substrate is stably tethered to the
WD-40 domain, an acceptor Lys is present in a predictable location in space and the F-box domain is
appropriately docked in Skp1, all that seems to be
required to position the acceptor Lys is a relatively simple, rigid, linker region. This consists primarily of a
rigid helix, between the F-box domain and the WD-40
domain (FIG. 2a).
Other β-Trcp substrates, such as Cdc25a, which
contains a variant phospho-degron, show that the
structural arrangement of their FBP-recognition sites
and ubiquitin-acceptor groups is not overly stringent.
Therefore, various SCF substrates, which might have
different, but closely related, arrangements of these
sites might bind FBPs that are similarly constructed
(that is, FBPs that comprise a rigid helix that links the
F-box domain and the substrate-binding domain).
Indeed, a yeast substrate of SCFCdc4 — Sic1 — is a very
different type of substrate compared with β-catenin
and cyclin E: it is a disordered protein with several lowaffinity sites for interaction with, and ubiquitylation
by, its FBP. The most efficient ubiquitylation sites of
Sic1 are on its amino terminus where several acceptor
Lys residues are found. Mutation of these Lys residues
abolishes polyubiquitylation of Sic1 if it is in complex
with Cdk1, but not if it is free81. The implication is that
Cdc4, although restrained by the fact that it has only
one binding site for free Sic1, can position a ubiquitinacceptor Lys from one of the many different binding
sites on free Sic1 into the correct spatial region. Free
Sic1 might ‘dance’ on the single Sic1-binding site on
Cdc4 — thereby bringing potential acceptor Lys
residues into the hot zone, and possibly increasing the
likelihood of this occurrence with each ubiquitin addition82. When in complex with Cdk1, Sic1 might be less
structurally flexible and, therefore, more easily positioned through a particular phosphorylation site by
the SCF ligase, although substantial segments might
still be disordered. It remains to be seen whether the
first of these intriguing observations — the regulated
degradation of a free disordered protein — occurs in
the cell. But this versatility might be a signature feature
of the SCF-ligase mechanism that is indicated by these
structures: all the substrates simply require the positioning of acceptor Lys residues in the hot zone, and
the FBP has evolved to achieve this task.
PHOSPHORECOGNITION seems to function in a fundamentally different manner in FBLs compared with the
FBWs, and might vary from FBL to FBL as required by
the substrate. The mode of FBL–substrate binding is not
yet known — there are two FBLs with known substrates
(human Skp2 and yeast Grr1), but only Skp2 has been
crystallographically resolved. Several three-dimensional
structures of non-FBP LRRs have been determined
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a
UBC
β-Trcp
59 Å
Ub
Substrateinteraction
domain
Linker
Rbx1
F-box
domain
Skp1
Cul1
b
Cdk2
p27
UBC
59 Å
Cks1
Ub
Skp2
Rbx1
Cul1
Skp1
Figure 2 | Models of the complete SCF-ligase complexes with their substrate targets.
a | A model of the Skp1–Cul1–F-box-protein (SCF)β-Trcp complex bound to the phosphorylated
destruction-box peptide of β-catenin, which is shown in atom-coloured, space-filling depiction
(carbon, grey; nitrogen, blue; oxygen, red). Each protein subunit is labelled by name in the same
colour as its stucture. The location of the ~59-Å space between the predicted ubiquitin site and
the F-box substrate-binding interface (the ‘hot zone’) is indicated. Adapted from REF. 66.
b | Equivalent view of SCFSkp2 next to a model of the Cks1–Cdk2–p27 complex (where Cks1
stands for Cdc28-protein-kinase regulatory subunit-1 and Cdk2 stands for cyclin-dependent
kinase-2), which was produced by superimposition of the CDK domains from the Cks1–Cdk2
structure and the p27–Cdk2–cyclin-A structure. In the underlying crystallographic structure, p27
is a fragment of the protein amino-terminal to residue Thr187. A dashed red line indicates how the
backbone up to residue Thr187 might extend in the complex to the Cks1–Skp2 bimolecular
interface, possibly forming a circular complex. Adapted from REF. 67. Cul1, cullin-1; Rbx1,
RING-box protein-1; Skp, S-phase-kinase-associated protein; Ub, ubiquitin; UBC, ubiquitinconjugating enzyme (or E2 ubiquitin ligase).
PHOSPHORECOGNITION
The process by which a specific
binding site on one protein
recognizes a specifically
constructed site on another
protein that has been posttranslationally modified by the
addition of a phosphate group to
amenable side chains such as Ser
or Thr.
experimentally, however, and many of them contain
their ligands in the complex, which bind in the concave
surface of the LRR arc11,12. If enough repeats are present,
LRRs might form an almost complete circle that
encloses a small protein ligand. The substrates of yeast
Grr1, Cln1 and Cln2 are multi-phosphorylated in a
non-sequence-specific manner for their recognition83,
and the phospho-interaction surface of yeast Grr1 was
predicted to be in this concave surface where a patch of
mutation-sensitive cationic residues lie84.
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
The prototype human FBL, Skp2, also requires
phosphorylation of at least one of its substrates on a
specific residue that is part of a CDK phosphorylationconsensus motif (residue Thr187 in p27). The interaction of Skp2 with phosphorylated p27 also requires the
association of Skp2 with Cks1 (REFS 85,86). The binding
site on SCF that binds Thr187-phosphorylated p27 is
thought to be a bimolecular site that is formed by Skp2
and Cks1 (REF. 87). This is consistent with the fact that
either Cks1 or Skp2 alone bind Thr187-phosphorylated
p27 poorly — as both proteins normally participate in
the interaction. An Asp residue in Skp2 (see space-filling
group in FIG. 2b) that is critical for the interaction of
Skp2 with Cks1 is located at the edge of the concave surface of Skp2 (REF. 88). Taken together, these data indicate
the possibility that at least one portion of p27 and/or
Cks1 binds in the LRR concave surface of Skp2, as
shown in the known structures of LRR–ligand complexes. The interaction between the phosphorylated
Thr187 residue of p27 and Cks1 probably occurs at that
same LRR concave site, but these proteins might also
interact at another site (that is, there are two binding
determinants). Indeed, unphosphorylated p27 was
shown to bind to Skp2 with low affinity 89. The loosely
packed carboxy-terminal strand that is seen in the concave LRR surface of the Skp2 structure (FIG. 1b) might
therefore serve as a kind of molecular ‘safety belt’ for the
substrate-binding determinants: the amino terminus of
this strand originates from the LRR tip and its carboxyl
terminus is locked back into the jigsaw-puzzle interface of
the F-box domain. So, if Skp2 represents FBLs in general,
post-translationally modified substrates might bind in
relatively diverse ways, and the binding might be rigidly
coupled to the Skp1–F-box-domain interface, through
features like the safety-belt loop, just as in the FBWs.
Studies on p27 degradation by Skp2 showed that it
must be complexed to a CDK to undergo Skp2-mediated degradation. Three well-established Skp2 substrates — p27, p21 and p57 — are thought to be
unstructured, and partly disordered, free proteins that
become ordered on association with their target complex90–92. At least in the case of p21 and p27, protein
folding after complex formation is required for SCFSkp2
to ligate ubiquitin40, 57,93. In other studies, the maximal
affinity for p27 was recorded when the complete complex, including Cdk2 bound to Cks1, was formed87, 94.
The structures of the CDKs can be taken from the
crystal structures of the Cks1–Cdk2 complex (REF. 95),
and the Cdk2–cyclin-A complex bound to a fragment of
p27 that is amino-terminal to Thr187 (REF. 96), and can
be superimposed to create a model of the
Cks1–Cdk2–p27 complex (FIG. 2b). This model shows
that p27 and Cks1 bind on opposite faces of Cdk2 that
are separated by a distance that is almost equal to the
length of Skp2 — although approximately 100 residues
of p27 that are not present in the structure could easily
make up the distance. In addition, the superimposition
of the Skp2 LRR onto the β-Trcp WD-40 domain in the
complete model clearly places the concave surface of the
LRR (where p27 is expected to bind) some distance away
from the equivalent location of the WD-repeat-bound
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phosphorylated peptide (compare FIG. 2a,b). A model
that was indicated by the recent structure of the carbohydrate-binding Fbx2 domain proposes that the substrate is bound in an equivalent region of space97. Does
Skp2 bind the Cdk–p27–Cks1 complex en bloc such
that the appropriate residues of p27 are placed into
the same position as the substrates in the WD-40 and
Fbx2 structures? The anticipated crystal structure of
the Skp2–Cks1–p27 complex should clarify these
Box 4 | Drug design at protein interfaces
a
b
Chk1 SH2 domain
β-Trcp WD-40 domain
Some drugs that are already available target protein–protein interfaces119. Other studies
have established that protein interfaces can be targeted by high-throughput screening
with small molecules120–124 — notably, an Mdm2–p53 interaction inhibitor has been
identified125. In addition, there are many examples of point mutations that disrupt
protein interactions. Clearly, a small molecule that disrupts the position of one or two key
residues might be equally effective. Nevertheless, there has historically been skepticism
regarding targeting protein interfaces for drug discovery126,127.
Much of this skepticism arose from the failure to find compounds that inhibit
SH2-domain interactions despite intense efforts.Although point mutations were identified
that disrupted the interaction surfaces of SH2 domains128, the search for drugs that could
do the same failed. The three-dimensional structure of the targeted SH2 surface shows why:
it is a flat protein surface with no pocket for a small molecule to bind (see figure part a, top
panel). The secret might be that a combination of two factors is required for a small
molecule to disrupt a protein interface: the compound must interact with a key residue and
that key residue must be near or inside a protein cavity of suitable size and character to bind
a drug-like compound. This combination of requirements might divide protein interfaces
into those that are not, and more importantly, those that are susceptible to targeted drug
discovery. The three-dimensional backbone structures of the SH2 domain from Chk1
(checkpoint kinase-1; see figure part a, top panel) and the β-Trcp WD-40 domain (see
figure part b, top panel) show that these two interfaces are indeed shaped quite differently,
with the latter surface appearing to be more suitable for drug discovery. In each structure,
the largest drug-binding pocket at the protein-binding interface was found using the
software program ICM PocketFinder (Molsoft Limited Liability Corporation, La Jolla,
California, USA) and is displayed as a grey geometric object. The volumes of these pockets
were calculated and plotted on a graph of volume (X axis) versus area (Y-axis; see the lower
panels). The central region of the graph that corresponds to the volume/area distribution of
most known drugs is coloured purple. The volume and area of the largest pocket on the
surface of the SH2 domain (red square) falls outside of this region, whereas that formed by
the central channel of the WD-40 domain in β-Trcp (red square) falls well within it.
748
| SEPTEMBER 2004 | VOLUME 5
interactions. But, the existing structures indicate an
explanation for why free p21 and p27 (and Sic1) would
not be efficiently ubiquitylated in the absence of complex formation with a CDK, as only the complex places
its ubiquitin-attachment target location optimally into
the hot zone. Interestingly, there is no reason why a
high-affinity FBP–substrate interaction, in conjunction
with the productive location of a non-Lys ubiquitinacceptor atom, might not result in non-Lys ubiquitylation in this model. Indeed, amino-terminal ubiquitylation of p21 has been shown98. So, the current data
indicate that Skp2 uses a complicated combination of its
surfaces and intramolecular interactions to accomplish
the targeting of tricky, intrinsically disordered, posttranslationally modified proteins, even while they are
intimately entwined with their constituent molecular
machines. The design of related SCF ligases might therefore be suited to operation in molecular-machinery-rich
environments such as the CDK-driven cell-cycle.
The structural information on the SCF ligase illustrates the details of only one possible model of SCF
function. Certain findings, however, hint at complementary or alternative models. For example, numerous FBPs
have amino-terminal domains of unknown function,
which were not present in any crystal structure; but in
β-Trcp, for example, this domain is a homodimerization
determinant99. These findings raise the possibility that
oligomerization of the SCF complex is required for the
substrate and ubiquitin to come together for ligation
and ubiquitin-chain elongation to occur. Furthermore,
kinetic studies indicate that E2–E3 interactions and E2
oligomerization might be significant determinants of
polyubiquitin-chain formation100,101. The E2-binding
site is not precisely known and seems to be strongly
influenced by the addition of a Nedd8 moiety to the
cullin near the E2-binding site102. The ubiquitin-like
Nedd8 is removed for regulatory purposes by the COP9
61
SIGNALOSOME . Some SCF-active co-factors (for example,
Cand1) bind to both the amino terminus (where Nedd8
is attached) and the carboxyl terminus of the cullin protein to regulate its activity (reviewed in REF. 61). This last
observation, in particular, indicates the existence of
intermediate forms of the SCF complex that are associated with cycles of Nedd8 ligation and removal in conjunction with Cand1–Cul1 binding cycles. Although
such intermediates would be part of a model of SCF
function that is alternative to the hot zone model,
Cand1 might also turn out to provide a functional link
between the E2-binding end of the SCF and the FBP
end, without disturbing the rigid orientation that is seen
in the crystal structures. Indeed, Nedd8 modification of
the SCF dissociates Cand1 from Cul1, thereby allowing
the Skp1–FBP complex to bind Cul1. Finally, the autoubiquitylation of FBPs that is observed in yeast103,104 and
mammals105,106 would also favour the hot zone model, as
the FBP is itself in the hot zone.
Applied engineering of SCF ligases
Evidence for the pathogenic involvement of SCF ligases
in cancer and other proliferative processes continues
to accumulate107. If these enzymes are truly a class of
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©2004 Nature Publishing Group
REVIEWS
COP9 SIGNALOSOME
An eight-subunit protein
complex that regulates protein
ubiquitylation and turnover in a
variety of developmental and
physiological contexts.
Extensively characterized in
plants but fundamental to all
eukaryotes, this complex posttranslationally modifies the
cullin subunit of E3-ubiquitin
ligases by cleaving off the
covalently coupled peptide,
Nedd8.
several E3 ligases that function as the checkpoint
enablers of the cell cycle, this rich compendium of convergent structural and biological data might serve as an
important asset for targeted drug discovery. An endogenous small-molecule-binding active site of the target is
usually the focus of targeted drug discovery. The target
site might be approximated by its activity in a scaleable
assay in high-throughput screening, or it might be used
in full atomic detail, as occurs in rational drug discovery.
These active sites tend to be small, deep, rigid pockets in
the three-dimensional structure of the protein. The SCF
represents an unusual challenge in this regard: how can
a small chemical compound competitively inhibit the
large, protein-sized ‘active site’ of this unique enzyme?
In theory, compounds that function as inhibitors
could be designed to compete for binding the phosphorecognition sites of the FBP, as well as to allosteric
pockets on this super-enzyme that affect its rigid
angles of orientation. In both cases, the targeting of
protein interfaces will be necessary — but, fortunately, some protein surfaces of the SCF have a valuable combination of features (BOX 4). The top of the
narrow central channel of the doughnut-shaped
WD-40 domain, for example, is also the site of the
key residues that are involved in binding the phosphorylated peptide motif ( BOX 4, panel b). Several
solvent cavities and pockets of unknown function can
be found in some of the linker regions and at the E2
end of the cullin protein of the SCF structural models.
The volume of these pockets is sufficient to accommodate small molecules that would be of an average size for
known drugs. The targeted, structure-based discovery of
small-molecule binders to some of these regions might
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Acknowledgments
We thank T. Hunter and N. Watanabe for communicating results
prior to publication. We apologize to colleagues whose work could
not be mentioned due to space limitations. M.P. is grateful to T. M.
Thor for continuous support. Molecular graphics were produced by
ICM software (Molsoft Limited Liability Corporation, La Jolla,
California, USA). Work in the Pagano laboratory is supported by
grants from the National Institutes of Health.
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
Competing interests statement
The authors declare that they have no competing financial interests.
Online links
DATABASES
The following terms in this article are linked online to:
Entrez: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi
CCNF
InterPro: http://www.ebi.ac.uk/interpro/
F-box domain | HECT domain | LRR | RING-finger domain
OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM
split hand–foot malformation
Saccharomyces genome database:
http://www.yeastgenome.org/
Cdc4 | Cdc20 | Sic1
Swiss-Prot: http://us.expasy.org/sprot/
Cul1 | Fbw1 | Fbw4 | Fbw7 | Fbx2 | Fbx3 | Fbx4 | Fbx5 |
Fbx6 | Fbx32 | Nedd8 | p21 | p27 | Rbx1 | Skp1 | Skp2 | Ubc3 |
VHL
FURTHER INFORMATION
HGNC F-box gene family nomenclature
http://www.gene.ucl.ac.uk/nomenclature/genefamily/
FBX.shtml
Pfam database:
http://pfam.wustl.edu/cgi-bin/getdesc?name=F-box
InterPro database:
http://www.ebi.ac.uk/interpro/
Michele Pagano’s laboratory:
http://www.med.nyu.edu/Path/Pagano/
Access to this links box is available online.
VOLUME 5 | SEPTEMBER 2004 | 7 5 1
©2004 Nature Publishing Group