993.6.2 PIPs Regulating PP1 Activity
Some PIPs do not confer substrate selectivity, but act as inhibitors of the PP1 cata-
lytic subunit by masking its active site. Two well-characterised examples of inhibi-
tory PIPs are the heat-stable proteins Inhibitor-1 and Inhibitor-2, both of which play
important cell cycle regulatory roles (Huang and Glinsmann 1976 ). Inhibitor-1 is
phosphorylated by the cAMP-activated kinase PKA, which turns it into a potent
inhibitor of PP1. In early mitosis, PP1 is phosphorylated and inactivated by Cdk1
(see below), and it was proposed that PKA-phosphorylated Inhibitor-1 helps to pre-
vent PP1 from precocious autodephosphorylation and activation. After Cdk1 is
inactivated at the metaphase-to-anaphase transition, PP1 can dephosphorylate both
Inhibitor-1 and itself to mediate dephosphorylations of mitotic substrate proteins as
cells exit mitosis (Wu et al. 2009 ). Inhibitor-2 was initially identified as a heat-
stable unfolded protein that inhibits the activity of PP1 towards its substrate glyco-
gen phosphorylase a (Huang and Glinsmann 1976 ). However, its function is more
complex in that Inhibitor-2 is also critical for the correct folding of nascent PP1
polypeptides (Alessi et al. 1993 ). Properly folded PP1 is further kept inactive by
Inhibitor-2 and phosphorylation of Inhibitor-2 results in the release of active PP1.
Thus, by acting as an inhibitory chaperone, Inhibitor-2 provides a cytosolic reser-
voir of PP1 that can be activated on demand. Several kinases including GSK3β,
Cdk1 and MAPK show in vitro activity towards Inhibitor-2, but their contributions
to Inhibitor-2 phosphorylation in cells are not fully understood (Li et al. 2006 ;
Puntoni and Villa-Moruzzi 1995 ; Leach et al. 2003 ). In cells, Inhibitor-2 localises to
centrosomes, mitotic spindle, midzone and midbody where it seems to be important
to balance the interplay between PP1 and Aurora-B activity (Leach et al. 2003 ;
Wang et al. 2008 ). Interfering with the activity of Inhibitor-2 results in errors in
chromosome segregation and cytokinesis (Wang et al. 2008 ). Notably, apart from its
role in modulating the activity of PP1, Inhibitor-2 is directly involved in the regula-
tion of the kinase Aurora-A (Shindo et al. 1998 ; Kufer et al. 2002 ; Marumoto et al.
2003 ). Binding of Inhibitor-2 to Aurora-A, with a domain different to the PP1-
binding domain, allosterically activates Aurora-A in vitro, and both proteins seem
to form a complex in cells (Satinover et al. 2004 ).
3.6.3 The Role of PP1 in Regulating the Cell Cycle
As mentioned earlier (see Sect. 3.5.2), the Cdk1-activating phosphatase Cdc25 is inacti-
vated in interphase or after DNA damage by phosphorylation of S287 (in Xenopus, S216 in
humans), which results in the recruitment of the 14-3-3 protein. At the onset of mitosis,
PP1 contributes to the activation of Cdc25 by directly dephosphorylating S287 (Margolis
et al. 2003 ). Once activated, the autoamplification loop starts where Cdc25 dephosphory-
lates and activates Cdk1/cyclin-B, which in turn phosphorylates Cdc25 at multiple sites
resulting in enhanced recruitment of PP1 and increased enzymatic activity of Cdc25
(Margolis et al. 2006b). Thus, in this case PP1—in contrast to PP2A-B ́56δ—has a
3 Regulation of Cell Division