Biology Reference
In-Depth Information
regulation and does not alone provide a robust mechanism to ensure that cells do not
go backwards in the cell cycle. Cells have therefore evolved a second control
mechanism to provide directionality and ensure that events keep moving forwards,
that is the irreversible destruction of proteins (Murray
2004
). Cyclins are the
archetypal example of proteins that accumulate and then are abruptly destroyed in a
cell cycle-dependent manner and these determine the oscillations in Cdk activity
(Glotzer et al.
1991
). However, many other proteins are similarly destroyed at spe-
cific times when their continued presence would be detrimental to further cell cycle
progression. Most cell cycle-dependent protein degradation is mediated by the 26S
proteasome. However, recognition by the proteasome requires tagging of the sub-
strate protein with polyubiquitin chains, a process that involves an E1 ubiquitin
activating enzyme, an E2 ubiquitin conjugating enzyme, and an E3 ubiquitin ligase
(Hershko and Ciechanover
1998
). It is the E3 ligase that determines substrate
specificity and there are two RING-family E3 ligases in particular that regulate cell
cycle transitions, namely the anaphase-promoting complex/cyclosome (APC/C),
and the Skp1/Cullin/F-box (SCF) protein (Skaar and Pagano
2009
).
The APC/C is a large multisubunit complex, containing at least 15 different protein
components (Peters
2006
). Its catalytic core consists of a cullin subunit, Apc2, and a
RING-H2 protein, Apc11. Together, these facilitate the transfer of ubiquitin from the
E2 enzyme onto the substrate. However, substrate recognition by the APC/C also
requires one of two APC/C co-activator proteins, Cdc20 or Cdh1, which directs the
APC/C to specific substrates at defined points in the cell cycle. The substrates them-
selves have particular amino acid sequence motifs, such as a D-box or KEN-box, which
enable the APC/C-co-activator complexes to identify them. The APC/C was originally
discovered for its role in the degradation of Cyclin B that promotes the transition from
metaphase to anaphase (Glotzer et al.
1991
). However, it is also required at this time for
the proteolysis of securin that triggers sister chromatid separation. In fact, the APC/C
degrades many cell cycle control proteins, including kinases, such as the Aurora
kinases, Polo-like kinases (Plks), and NIMA-related kinases (Neks), along with pro-
teins involved in regulating mitotic spindle formation and DNA replication.
Importantly, the activity of the APC/C is cell cycle regulated with high activity
from mitosis to late G1 and low activity from S-phase to late G2. Activity during
early mitosis, from prophase to metaphase, is associated with APC/C
Cdc20
, whereas
activity from anaphase to late G1 is associated with APC/C
Cdh1
. In addition to
co-activator binding, APC/C activity is controlled by phosphorylation and inhibitor
proteins, such as Emi1 or the mitotic checkpoint proteins, Mad2, and BubR1. The
fact that both co-activators and inhibitors of the APC/C are themselves subject to cell
cycle-dependent degradation adds a further layer of control to the system.
In contrast to the APC/C, the SCF is active throughout the cell cycle, but its
ability to ubiquitylate its substrates depends on their post-translational modifica-
tion, which only occurs at specific times in the cell cycle (Cardozo and Pagano
2004
). Substrate modification allows recognition by an F-box protein, of which
about 70 have been identified in humans, with Skp2, Fbxw7, and b-TrCP having
the most well-defined roles in regulating the cell cycle. In addition to an F-box
protein, the SCF also consists of a cullin subunit, Cul1, a RING-H2 protein, Hrt/
