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3.3.1 Regulation of APC/C Activity
Once in mitosis, active Cdk1/cyclin-B phosphorylates core APC/C subunits, and
these modifications enhance its affinity for the coactivator Cdc20 (Kraft et al. 2003 ;
Rudner and Murray 2000 ; Shteinberg et al. 1999 ). Active APC/CCdc20 then targets
securin and cyclin-B for proteasomal destruction. Thus, Cdk1 triggers its own inacti-
vation by activating APC/CCdc20. Decreased cyclin-B levels, in turn, result in reduced
phosphorylation of the APC/C and, hence, in inactivation of APC/CCdc20. The destruc-
tion of the remaining cyclin-B is mediated by Cdh1 which, in contrast to Cdc20, binds
more efficiently to the APC/C in its dephosphorylated form (Kraft et al. 2003 ; Kramer
et al. 2000 ; Zachariae et al. 1998 ). Multisite phosphorylation events of the APC/C and
inhibitory phosphorylations on Cdc20 seem to be critical for the required time delay
between Cdk1/cyclin-B activation and the activation of the APC/CCdc20 (Labit et al.
2012 ; Yang and Ferrell 2013 ). To ensure that anaphase onset occurs only once all
chromosomes are correctly attached to spindle microtubules, a pathway referred to as
the spindle assembly checkpoint (SAC) prevents APC/C activation in the presence of
improperly attached chromosomes. Since this pathway does not seem to be active
during Xenopus early embryo development, we do not describe the mechanism of
SAC-mediated APC/C inhibition in detail, but refer to excellent review articles (Hauf
2013 ; Vleugel et al. 2012 ; Musacchio 2011 ; Musacchio and Salmon 2007 ; Lara-
Gonzalez et al. 2012 ). In brief, unattached kinetochores signal the formation of a
complex known as the mitotic checkpoint complex (MCC), which consists of Mad2,
Bub3, BubR1 and Cdc20, forming a strong inhibitor of APC/C-mediated poly-ubiq-
uitylation of cyclin-B and securin. Once chromosomes are correctly attached, this
checkpoint is turned off resulting in the activation of the APC/C, thereby targeting
securin and cyclin-B for destruction. Consequentially, cells separate sister chromatids
and exit M-phase. In interphase, APC/CCdh1 is kept inactive by early mitotic inhibitor
1 (Emi1), and this mechanism is important to stabilise cyclin-A, cyclin-B and gemi-
nin—a regulator of DNA replication. Destabilisation of these APC/C substrates in
Emi1-depleted cells induces DNA re-replication and prevents mitotic entry (Di Fiore
and Pines 2007 ; Machida and Dutta 2007 ). Emi1 levels are controlled in a cell cycle-
dependent manner. At the G1 to S transition, Emi1 is transcriptionally induced by the
E2F transcription factor and targeted for destruction shortly before NEBD via the E3
ligase SCFβ-TRCP (Skp1, Cullin, F-box protein) (Hansen et al. 2004 ; Hsu et al. 2002 ;
Moshe et al. 2004 ). SCFβ-TRCP-mediated ubiquitylation of Emi1 requires its prior phos-
phorylation by Polo-like kinase 1 (Plk1) and probably Cdk1. Xenopus Emi1-related
protein 1 (XErp1)/Emi2 represents another APC/C inhibitor which was initially iden-
tified based on its essential function in arresting mature oocytes in metaphase of meio-
sis II (Schmidt et al. 2005 ; Shoji et al. 2006 ; Tung et al. 2005 ). Upon fertilisation,
calcium- calmodulin-dependent kinase II (CaMKII) and Plk1 coordinate to target
XErp1 for SCFβ-TRCP-mediated degradation resulting in exit from meiosis and entry
into the first mitotic division (Liu and Maller 2005 ; Rauh et al. 2005 ). Notably, XErp1
reaccumulates during the prolonged first division of Xenopus embryos, and the pres-
ence of XErp1 during the subsequent fast divisions is critical for timely regulation of
APC/C activity (see below) (Tischer et al. 2012 ).
A. Heim et al.