95(Fig. 3.5) (Gharbi-Ayachi et al. 2010 ; Mochida et al. 2010 ). Initially, Arpp19 was
identified as a cAMP-dependent neuronal phosphoprotein, and Ensa as endogenous
ligand for neuronal sulfonylurea receptors (Girault et al. 1988 ; Virsolvy-Vergine
et al. 1992 ). As shown by the teams of Lorca and Hunt, phosphorylation of Ensa/
Arpp19 by Cdk1-activated Gwl turns them into high-affinity inhibitors of PP2A-B55.
Thus, the identification of Ensa/Arpp19 as Gwl substrates finally provided the
molecular explanation for how Cdk1/cyclin-B activation at mitotic entry results in
the inactivation of the antagonising phosphatase PP2A-B55.
3.5.1.4 The Reactivation of PP2A-B55 at Exit from M-Phase
Exit from M-phase requires the reactivation of PP2A-B55 to reset the mitotic phos-
phorylation state to interphase. Inactivation of the Gwl-Ensa/Arpp19 module after
Cdk1/cyclin-B inactivation sets the timing for the reactivation of PP2A-B55 at
mitotic exit. The importance of the Gwl-Ensa/Arpp19 module for the correct tem-
poral order during exit from M-phase has been shown recently by mathematical
modelling and experiments using the spindle midzone protein PRC1 as model sub-
strate, i.e. uncoupling PP2A-B55 inhibition from Cdk1/cyclin-B activity results in
impaired dephosphorylation of PRC1 resulting in premature onset of cytokinesis
(Cundell et al. 2013 ). Studies addressing the underlying mechanism for Gwl-Ensa/
Arpp19 inactivation revealed that Gwl-phosphorylated Ensa/Arpp19 is not only an
inhibitor of PP2A-B55 but also a substrate. However, compared to other mitotic
substrates, phosphorylated Ensa/Arpp19 seems to have a very high affinity for
PP2A-B55, but its dephosphorylation by PP2A-B55 is very slow (Williams et al.
2014 ). Based on these findings, the authors proposed a model of ‘inhibition by
unfair competition’: during early mitosis, most PP2A-B55 holoenzymes are occu-
pied with Ensa/Arpp19, and the few molecules of Ensa/Arpp19 that get dephos-
phorylated will be replenished by active Gwl and will rebind to PP2A-B55. At exit
from M-phase, Gwl gets inactivated and consequently, the pool of phosphorylated
Ensa/Arpp19 decreases, allowing PP2A-B55 to focus on other mitotic substrates.
An independent study, however, identified RNA polymerase II carboxy-terminal
domain phosphatase Fcp1 as the Ensa/Arpp19 phosphatase (Hegarat et al. 2014 ).
While further studies are required to clarify the contributions of PP2A-B55 and
Fcp1, it is beyond dispute that PP2A-B55 reactivation requires the dephosphoryla-
tion, i.e. inactivation, of both Ensa/Arpp19 and Gwl itself. Studies from the group
that identified Fcp1 as the Ensa/Arpp19 phosphatase suggest that PP2A-B55 liber-
ates itself from inhibition by dephosphorylation of Gwl (Hegarat et al. 2014 ).
3.5.1.5 Additional Mechanisms Regulating PP2A-B55 and Ensa/Arpp19
Arpp19 and Ensa are not only phosphorylated by Gwl but also by Cdk1 and protein
kinase A (PKA) (Mochida et al. 2010 ). The phosphorylation by Cdk1 seems to have
an activating effect on Arpp19, thereby enabling a Gwl-independent mode of
PP2A-B55 inhibition in mitosis. Yet, the physiological relevance of this mechanism
3 Regulation of Cell Division