96
has not been fully addressed (Mochida 2014 ; Okumura et al. 2014 ). In contrast,
studies in immature Xenopus laevis oocytes revealed that phosphorylation of
Arpp19/Ensa by PKA has an inhibitory effect (Mochida 2014 ; Dupre et al. 2014 ).
Yet, if this mechanism also applies to mitotic divisions remains to be investigated.
A recent study suggested that phosphorylation of B55α affects its ability to assem-
ble as a PP2A holoenzyme (Schmitz et al. 2010 ). The identified phosphorylation
site matched the Cdk1 consensus motif suggesting that high Cdk1 activity in mitosis
might interfere with the assembly of the PP2A-B55α holoenzyme. However, future
studies are required to clarify the physiological relevance and consequence of this
event in detail. In accordance with this, there is evidence from fission yeast for a
direct inactivation of B55 by mitotic kinases, and this is counteracted by the phos-
phatase PP1, which binds and reactivates B55 after anaphase onset (Grallert et al.
2015 ).
3.5.2 The PP2A-B ́56 Phosphatase
Several mitotic functions of PP2A holoenzymes with a regulatory B ́56 subunit
have been revealed since the discovery of this protein class in 1995 (McCright and
Virshup 1995 ). The best understood function of PP2A-B ́56 in mitosis is its role in
the stabilisation of kinetochore-microtubule attachments and the protection of cen-
tromeric cohesion during prophase (Kitajima et al. 2006 ; Suijkerbuijk et al. 2012 ;
Foley et al. 2011 ; Nijenhuis et al. 2014 ; Xu et al. 2014 ). While both functions are
critical for the fidelity of chromosome segregation (Kitajima et al. 2006 ; Foley et al.
2011 ; Nijenhuis et al. 2014 ; Xu et al. 2014 ), in this book chapter we focus on the
role of B ́56 as an element of the DNA damage-responsive checkpoint which acts
on the Cdk1 amplification loop and its role in controlling the APC/C (Fig. 3.6). To
prevent M-phase entry in the presence of DNA damage, Cdc25 is phosphorylated at
serine-287 (Xenopus laevis Cdc25; serine-216 in human Cdc25C). Several kinases
are known to phosphorylate S287, e.g. the DNA damage-activated kinases Chk1
and Chk2 as well as the kinases c-TAK1 and PKA (Furnari et al. 1997 ; Duckworth
et al. 2002 ; Peng et al. 1998 ; Zeng et al. 1998 ; Kumagai et al. 1998 ; Sanchez et al.
1997 ), and this phosphorylation promotes binding of the inhibitory protein 14-3-3
(Peng et al. 1997 ). 14-3-3 binding to Cdc25 is weakened by Cdk2-dependent phos-
phorylation of Cdc25 at a different site (Guadagno and Newport 1996 ; Margolis
et al. 2006a). Phosphorylation of B ́56δ by DNA damage checkpoint kinases leads
to an increased incorporation of this subunit into PP2A holoenzymes, which antago-
nises the phosphorylation of Cdc25 by Cdk2 and thereby prevents the dissociation
of 14-3-3 from Cdc25 (Margolis et al. 2006a). Thus, in the presence of DNA dam-
age, checkpoint kinases keep Cdc25 inactive by stimulating the formation of a com-
plex between Cdc25 and 14-3-3 in a PP2A-B ́56δ-dependent manner. Once DNA
defects are repaired, this mechanism (see below for more details) fades resulting in
Cdc25 activation and entry into M-phase (Fig. 3.6).
A. Heim et al.