[Cell Cycle 3:7, e27-e29, EPUB Ahead of Print: http://www.landesbioscience.com/journals/cc/abstract.php?id=977; July 2004]; ©2004 Landes Bioscience
Don’t skip the G1 phase
Extra Views
How APC/CCdh1 Keeps SCFSkp2 in Check
Cell Cycle 2004; 3(7): http://www.landesbioscience.com/journals/cc/abstract.php?id=977
Once the issue is complete and page numbers have been assigned, the citation will
change accordingly.
KEY WORDS
Skp2, Cks1, p21, p27, APC/C, Cdh1, Cdc20,
Emi1, SCF, cell cycle
ibu
sci
ACKNOWLEDGEMENTS
No
tD
ist
r
This manuscript has been published online, prior to printing for Cell Cycle, Volume 3,
Issue 7. Definitive page numbers have not been assigned. The current citation is:
The cell cycle is controlled by a family of protein kinases known as cyclin-dependent
kinases or CDKs. Timely destruction of CDK subunits and their upstream regulators is an
essential means to ensure proper progression through the cell division cycle. Protein
degradation is irreversible, and thus constitutes a well suited mechanism to promote the
unidirectional passage from one cell cycle phase to the next.
In mammalian cells, the transition from G1 to S phase is controlled by the activity of
the CDK inhibitors (CKIs) p27 and, to a lesser extent, p21.1 Both CKIs act as negative
cell cycle regulators that prevent entry into S phase by inhibiting the activity of
Cdk2/Cyclin E, Cdk2/Cyclin A and likely other CDKs. Progression into S phase therefore necessitates the ubiquitin-mediated degradation of the inhibitory proteins. The
triggering signal is the CDK-mediated phosphorylation of p27 and p21, allowing the
SCFSkp2 (Skp1, Cul1, F box protein Skp2) ubiquitin ligase to specifically recognize the
two CKIs as substrates.2,3 SCFSkp2 subsequently catalyzes the polyubiquitination of p27
and p21, thus earmarking both proteins for destruction by the 26S proteasome.3-6 The
recognition and efficient ubiquitination of p27 and p21 requires the small accessory
protein Cks1 as an indispensable factor in the SCFSkp2 complex.3,7,8 Through its interaction
with Skp2, Cks1 may function as an allosteric effector that induces conformational
changes in the F box protein to increase its affinity for the substrate, or, more likely,
contributes to the binding of phosphorylated p27 by forming a bimolecular interface with
Skp2.9,10
As negative regulators of cell proliferation, p27 and p21 intrinsically possess tumor
suppressive properties. Hence, the elimination of these impediments to unrestrained
proliferation, particularly p27, is a hallmark of many types of cancer.11 Drastically reduced
p27 protein levels are frequently found in human malignancies and are often associated
with high aggressiveness and poor prognosis. As the antagonist of p27, Skp2 acts as an
oncogene, and Skp2 expression was found to inversely correlate with p27 in various carcinomas and lymphomas.11-13 Until recently, the investigation of Cks1 protein levels in
cancer has been hampered by the fact that specific antibodies (i.e., antibodies that do not
recognize the closely related Cks2 protein) have been unavailable. However, mRNA data
and the current limited immunohistochemical data indicate that in some types of cancer
overexpression of Cks1 is indeed found, correlating with low levels of p27.14,15 Although
more data are needed to evaluate the role of Cks1 in cancer development and progression,
it would not be surprising to find that Cks1 has oncogenic properties similar to Skp2 or
at least contributes to Skp2 tumorigenicity. This would highlight the need for tight regulation of both Skp2 and Cks1 to avoid unscheduled degradation of p27 and p21.
How are Skp2 and Cks1 regulated during an unperturbed cell cycle? It had been
previously shown that Skp2 mRNA is present in all phases of the cell cycle, displaying only
minor fluctuations.16 Skp2 protein, however, is absent in G0 and G1 cells, accumulates
during the G1-S transition and persists through S phase until mitosis. The discrepancy in
Skp2 levels between G0/G1 and S phase is due to differences in protein stability; Skp2
protein is very unstable in G0/G1, but upon entry into S phase it is stabilized significantly.
However, the mechanism that controls Skp2 stability had not been well understood. Our
study and that of Wei et al. have now provided evidence that the degradation of Skp2 in
Do
Received 05/12/04; Accepted 05/14/04
ce.
*Correspondence to: Michele Pagano; Department of Pathology; MSB 599; New
York University School of Medicine; New York University Cancer Institute; 550 First
Avenue; New York, New York 10016 USA; Tel.: 212.263.5332; Fax: 212.263.5107;
Email: michele.pagano@med.nyu.edu
By keeping the levels of Skp2 and Cks1 low during G1 progression, APC/CCdh1
prevents unscheduled degradation of SCFSkp2 substrates and premature entry into S
phase. Thus, APC/CCdh1, a ubiquitin ligase involved in mitotic exit and maintenance of
G0/G1 phase, directly controls SCFSkp2, a ubiquitin ligase involved in the regulation of S
phase entry.
en
New York University School of Medicine; New York University Cancer Institute; New
York, New York USA
te
ABSTRACT
Tarig Bashir
Michele Pagano*
©
20
04
La
nd
es
Bio
We thank T. Cardozo for critically reading the
manuscript. We apologize to colleagues whose work
could not be mentioned due to space limitations.
Work in the Pagano lab is supported by grants
from the NIH (R01-CA76584 and R01-GM57587).
e27
Cell Cycle
2004; Vol. 3 Issue 7
HOW APC/CCDH1 KEEPS SCFSKP2 IN CHECK
G0/G1 is mediated by the APC/CCdh1 (anaphasepromoting complex/cyclosome in complex with
its activator Cdh1) ubiquitin ligase:17,18
ibu
te
1. Overexpression of Cdh1 (but not its homolog
Cdc20) in cells leads to a considerable destabilization of Skp2.
2. Depletion of Cdh1 by RNA interference
results in the stabilization and accumulation
of Skp2 in G0/G1.
3. Cdh1 interacts physically with Skp2.
4. APC/C supplemented with recombinant
Cdh1 is capable of ubiquitinating Skp2 in vitro.
©
20
04
La
nd
es
Bio
sci
en
ce.
Do
No
tD
ist
r
The APC/CCdh1-mediated degradation of
Skp2 is dependent on an N-terminal destruction
box (D box) motif (RXXL). Mutation of this
motif generates a stable Skp2 protein that is fully
active in G1 cells. Intriguingly, Skp2 mutants
harboring an inactive D box are still able to
interact with Cdh1, suggesting that the D box
sequence itself does not function as a landing
pad for the Cdh1 moiety of the APC/C. A more
detailed analysis of the Skp2 N-terminus
revealed that the actual binding site for Cdh1
resides within a stretch of 50 amino acids that is
1. The potential effects of defective Skp2/Cks1 degradation. Normal cell (left panel):
located approximately 40 amino acids downstream Figure Cdh1
APC/C
is activated in late M/early G1 phase and induces the degradation of the SCF subunits
from the D box (amino acids 45–94). In line Skp2 and Cks1. Consequently, the CKIs p27
and p21 accumulate, bind to CDK/cyclin complexes
with recent suggestions, the D box may thus and block their activity, thereby ensuring that G1 phase is established and maintained. During the
interact with components of the APC/C other G1-S transition, APC/CCdh1 is inactivated through association with Emi1 and CDK-mediated
than Cdh1, or it may function as a signalling inhibitory phosphorylation of Cdh1. Skp2 and Cks1 are stabilized, become part of an active
sequence that orients the multi-subunit APC/C SCFSkp2 complex and target p27 and p21 for degradation. CDK/cyclin complexes become active
and initiate progression into S phase. Cancer cell (right panel): In certain cancer cells APC/CCdh1
complex to the binding site.19
may be unable to interact with Skp2/Cks1 in G1 phase, hence the ubiquitination and subsequent
The oscillations and protein stabilities of degradation of both proteins is compromised. As a result, p27 and p21 are targeted for unschedCks1 during the cell cycle parallel those of uled degradation, rendering CDK/cyclin complexes active. The cell progresses faster into S phase
Skp2.17 Cks1 was also shown to bind Cdh1, and and DNA replication is prematurely initiated, which can be a cause of genetic instability.
overexpression or depletion of Cdh1 in cells have
an effect on Cks1 levels similar to Skp2. Although many findings mutant concomitantly stabilized endogenous Cks1 protein. It is thus
point to APC/CCdh1 as a direct regulator of Cks1 protein stability, we conceivable that the interaction between Skp2 and Cks1 shields the
were unable to ubiquitinate Cks1 in vitro using APC/CCdh1. This latter from ubiquitination and destruction, suggesting that the
leaves open the possibility that APC/CCdh1 does not target Cks1 regulation of Skp2 and Cks1 protein stability is closely linked. The
directly, but may instead trigger another, APC/CCdh1-dependent, APC/CCdh1-mediated elimination of Skp2 and Cks1 is therefore
ubiquitin ligase. Alternatively, an as yet to be identified factor neces- important to maintain the G1 state. In contrast, continuous aberrant
sary for Cks1 ubiquitination is missing in the purified preparation of removal of p27 and p21 is likely to be a cause of uncontrolled cell
APC/CCdh1.
proliferation and may ultimately contribute to the development of
Several control mechanisms ensure that the activity of APC/ cancer. The frequently observed overexpression of Skp2 in cancer
CCdh1 itself is restricted to late M and G0/G1 phase.20 The complex tissues has in some cases been attributed to gene amplification,23,24
is activated through the binding of dephosphorylated Cdh1 to APC/C however, other potential causes have not been investigated yet. There
in late M phase, and the continued absence of Cdk1 and Cdk2 is accumulating evidence that aberrant protein degradation of cell
kinase activity keeps APC/CCdh1 functional throughout G1 (or G0). cycle regulators is an important factor contributing to tumorigeneAt the G1-S transition APC/CCdh1 function is blocked through both sis,25 and this may hold true for Skp2 (and Cks1) as well (see Fig. 1).
the interaction of Cdh1 with the inhibitory protein Emi1 and Indeed, Skp2 stabilization has been observed in aggressive oral
CDK-mediated phosphorylation of Cdh1.21,22 The inactivation of cancers (Kudo Y, Pagano M, Takata T, unpublished results).
APC/CCdh1 permits the accumulation of Skp2 and Cks1, the assembly Increased protein levels of Skp2 in cancer cells may be a consequence
of an active SCFSkp2 complex and the targeting of p27 and p21 for of inactive Cdh1 (e.g., due to mutations or overexpression of Emi1)
or reduced Cdh1 expression. No data concerning the Cdh1 status in
ubiquitin-mediated degradation.
What are the consequences of defective Skp2 degradation in G1 cancer cells are available at present, but downregulation of Cdh1 has
cells? Our experiments have shown that a nondegradable Skp2 been shown to accelerate cell cycle progression, a potential cause of
mutant expressed at physiological levels in G1 cells promotes the genetic instability.17,18,26 It is also possible that alterations in other
unscheduled degradation of p27 and p21. As a consequence, these components of the APC/CCdh1 complex contribute to Skp2-Cks1
cells progressed significantly faster into S phase than control cells. In stabilization. Indeed, it has been shown that in human colon cancer
this context, it should be noted that the expression of the stable Skp2 cells two subunits of the APC/C, Apc6/Cdc16 and Apc8/Cdc23,
www.landesbioscience.com
Cell Cycle
e28
HOW APC/CCDH1 KEEPS SCFSKP2 IN CHECK
25. Bashir T, Pagano M. Aberrant ubiquitin-mediated proteolysis of cell cycle regulatory proteins and oncogenesis. Adv Cancer Res 2003; 88:101-44.
26. Sorensen CS, Lukas C, Kramer ER, Peters JM, Bartek J, Lukas J. Nonperiodic activity of
the human anaphase-promoting complex-Cdh1 ubiquitin ligase results in continuous
DNA synthesis uncoupled from mitosis. Mol Cell Biol 2000; 20:7613-23.
27. Wang Q, Moyret-Lalle C, Couzon F, Surbiguet-Clippe C, Saurin JC, Lorca T, et al.
Alterations of anaphase-promoting complex genes in human colon cancer cells. Oncogene
2003; 22:1486-90.
te
which are known to be key function elements in the complex, often
display inactivating mutations.27 Alternatively, Skp2 may harbor
mutations that obstruct any physical interaction between Skp2 and
APC/CCdh1. Conceivably, such mutations would primarily affect the
N-terminal D box or the Cdh1 binding site, rendering Skp2 refractory to APC/CCdh1-mediated ubiquitination and destruction.
Future studies will therefore have to unravel how APC/CCdh1 targets
Skp2 and Cks1 mechanistically to fully understand the role of defective Skp2/Cks1 degradation in cancer.
©
20
04
La
nd
es
Bio
sci
en
ce.
Do
No
tD
ist
r
ibu
References
1. Sherr CJ, Roberts JM. CDK inhibitors: Positive and negative regulators of G1-phase progression. Genes Dev 1999; 13:1501-12.
2. Montagnoli A, Fiore F, Eytan E, Carrano AC, Draetta GF, Hershko A, et al. Ubiquitination
of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation.
Genes Dev 1999; 13:1181-9.
3. Bornstein G, Bloom J, Sitry-Shevah D, Nakayama K, Pagano M, Hershko A. Role of the
SCFSkp2 ubiquitin ligase in the degradation of p21Cip1 in S phase. J Biol Chem 2003;
278:25752-7.
4. Carrano AC, Eytan E, Hershko A, Pagano M. Skp2 is required for ubiquitin-mediated
degradation of the Cdk inhibitor p27. Nat Cell Biol 1999; 1:193-9.
5. Sutterluty H, Chatelain E, Marti A, Wirbelauer C, Senften M, Muller U, et al. p45SKP2
promotes p27Kip1 degradation and induces S phase in quiescent cells. Nat Cell Biol 1999;
1:207-14.
6. Tsvetkov LM, Yeh KH, Lee SJ, Sun H, Zhang H. p27Kip1 ubiquitination and degradation
is regulated by the SCFSkp2 complex through phosphorylated Thr187 in p27. Curr Biol
1999; 9:661-4.
7. Ganoth D, Bornstein G, Ko TK, Larsen B, Tyers M, Pagano M, et al. The cell-cycle regulatory protein Cks1 is required for SCF(Skp2)-mediated ubiquitinylation of p27. Nat Cell
Biol 2001; 3:321-4.
8. Spruck CH, Strohmaier H, Watson M, Smith AP, Ryan A, Krek W, et al. A CDK-independent function of mammalian Cks1: Targeting of SCF(Skp2) to the CDK inhibitor
p27Kip1. Mol Cell 2001; 7:639-50.
9. Sitry D, Seeliger MA, Ko TK, Ganoth D, Breward SE, Itzhaki LS, et al.Three different
binding sites of Cks1 are required for p27-ubiquitin ligation. J Biol Chem 2002;
277:42233-40.
10. Seeliger MA, Breward SE, Friedler A, Schon A, Itzhaki LS. Cooperative organization in a
macromolecular complex. Nat Struct Biol 2003; 10:718-24.
11. Bloom J, Pagano M. Deregulated degradation of the cdk inhibitor p27 and malignant
transformation. Semin Cancer Biol 2003; 13:41-7.
12. Philipp-Staheli J, Payne SR, Kemp CJ. p27Kip1: Regulation and function of a haploinsufficient tumor suppressor and its misregulation in cancer. Exp Cell Res 2001; 264:148-68.
13. Pagano M, Benmaamar R. When protein destruction runs amok, malignancy is on the
loose. Cancer Cell 2003; 4:251-6.
14. Masuda TA, Inoue H, Nishida K, Sonoda H, Yoshikawa Y, Kakeji Y, et al. Cyclin-dependent kinase subunit 1 gene expression is associated with poor prognosis in gastric carcinoma. Clin Cancer Res 2003; 9:5693-8.
15. Shapira M, Ben-Izhak O, Bishara B, Futerman B, Minkov I, Krausz MM, et al. Alterations
in the expression of the cell cycle regulatory protein cyclin kinase subunit 1 in colorectal
carcinoma. Cancer 2004; 100:1615-21.
16. Wirbelauer C, Sutterluty H, Blondel M, Gstaiger M, Peter M, Reymond F, et al. The F-box
protein Skp2 is a ubiquitylation target of a Cul1-based core ubiquitin ligase complex:
Evidence for a role of Cul1 in the suppression of Skp2 expression in quiescent fibroblasts.
EMBO J 2000; 19:5362-75.
17. Bashir T, Dorrello NV, Amador V, Guardavaccaro D, Pagano M. Control of the
SCFSkp2-Cks1 ubiquitin ligase by the APC/CCdh1 ubiquitin ligase. Nature 2004; 428:190-3.
18. Wei W, Ayad NG, Wan Y, Zhang GJ, Kirschner MW, Kaelin Jr WG. Degradation of the
SCF component Skp2 in cell-cycle phase G1 by the anaphase-promoting complex. Nature
2004; 428:194-8.
19. Yamano H, Gannon J, Mahbubani H, Hunt T. Cell cycle-regulated recognition of the
destruction box of cyclin B by the APC/C in Xenopus egg extracts. Mol Cell 2004;
13:137-47.
20. Harper JW, Burton JL, Solomon MJ. The anaphase-promoting complex: It’s not just for
mitosis any more. Genes Dev 2002; 16:2179-206.
21. Hsu JY, Reimann JD, Sorensen CS, Lukas J, Jackson PK. E2F-dependent accumulation of
hEmi1 regulates S phase entry by inhibiting APCCdh1. Nat Cell Biol 2002; 4:358-66.
22. Lukas C, Sorensen CS, Kramer E, Santoni-Rugiu E, Lindeneg C, Peters JM, et al.
Accumulation of cyclin B1 requires E2F and cyclin A-dependent rearrangement of the
anaphase-promoting complex. Nature 1999; 401:815-18.
23. Yokoi S, Yasui K, Saito-Ohara F, Koshikawa K, Iizasa T, Fujisawa T, et al. A novel target
gene, SKP2, within the 5p13 amplicon that is frequently detected in small cell lung cancers.
Am J Pathol 2002; 161:207-16.
24. Zhu CQ, Blackhall FH, Pintilie M, Iyengar P, Liu N, Ho J, et al. Skp2 gene copy number
aberrations are common in nonsmall cell lung carcinoma, and its overexpression in tumors
with ras mutation is a poor prognostic marker. Clin Cancer Res 2004; 10:1984-91.
e29
Cell Cycle
2004; Vol. 3 Issue 7