(1) The APC Dephosphorylation by PP2A^Cdc55 

Cdc28–Clb2 phosphorylates the APC subunits Cdc16, Cdc23, and Cdc27 upon spindle damage conditions to activate APC [40,41]. The phospho-null mutants for these proteins [42] and the inactivation of Cdc28 [43] impaired APC/C^Cdc20 activity. Conversely, PP2A^Cdc55 counteracts the Cdk1 phosphorylation of the APC/C subunit Cdc16 [26,43], keeping the spindle checkpoint assembly (SAC) active until the cell is prepared for anaphase. Tight balance between Cdc28–Clbs and PP2A^Cdc55 activities is important for the adaptation to the spindle checkpoint [43].

(2) The Regulation of the Cohesin Cleavage by PP2A^Cdc55 

 PP2A^Cdc55 is also required for the proper temporal initiation of meiotic events [62]. Similar to mitosis, PP2A^Cdc55 also regulates the FEAR pathway during meiosis [63,64]. PP2A^Cdc55 dephosphorylates Net1 and promotes Cdc14 release from the nucleolus, preventing precocious exit from meiosis I. In addition, PP2A^Cdc55 is required for reductional chromosome segregation in the absence of recombination independently of its role in the FEAR pathway [65].   

(4) MEN (SIN) Regulation by PP2A

Cdc14 activation and release during anaphase is mediated by two parallel pathways: the FEAR and the mitotic exit network (MEN) [66]. MEN (also known as the Hippo pathway in higher eukaryotes) is a GTPase-driven signaling cascade  (spindle pole body, SPB in yeast) that regulates mitotic exit, enables the control of the spindle orientation, and promotes cytokinesis in budding yeast.

The core of the MEN cascade consists of two serine/threonine kinases, Cdc15 (PAK kinase in higher eukaryotes) and Dbf2-Mob1 (LATS kinase in higher eukaryotes). They are activated in mid–late anaphase to maintain Cdc14 released from the nucleolus and promote its full activation [67,68,69]. During an unperturbed cell cycle, the inhibitor complex Bub2/Bfa1 keeps MEN inactive. PP2A^Cdc55 contributes to keep Bub2/Bfa1 active by dephosphorylating Bfa1 in metaphase [70]. When cells reach anaphase with a correct aligned mitotic spindle, Cdc5 phosphorylates Bfa1 and inactivates the Bub2/Bfa1 GAP activity [71,72]. The anaphase-specific inactivation of PP2A^Cdc55 also contributes to increase the Cdc5-dependent Bfa1 phosphorylation and promotes the activation of MEN. Therefore, PP2A^Cdc55 not only facilitates the FEAR-dependent Cdc14 release in early anaphase but also contributes to alleviation of the MEN inhibitory signal imposed by Bfa1/Bub2.

In addition, most of the MEN proteins are regulated by phosphorylation, making MEN activity restrained by Cdk1 and stimulated by the action of the opposing phosphatases, Cdc14 and PP2A^Cdc55 (Figure 3). Cdk1 restrains MEN activity through Cdc15 and Mob1 phosphorylation [76]. At anaphase, Cdc15 is dephosphorylated by the FEAR-released Cdc14 facilitating its activation [77,78,79,80,81]. Mob1 dephosphorylation at late anaphase is necessary for Dbf2-Mob1 activation. Abrupt Cdk1 inactivation and Cdc14 release from the nucleolus contribute to Mob1 dephosphorylation in late anaphase [76]. In addition, PP2A^Cdc55 also dephosphorylates Mob1 protein [70]. At anaphase onset, PP2A^Cdc55 downregulation facilitates Cdk1-dependent phosphorylation of Mob1, contributing to Dbf2–Mob1 inhibition. During exit from mitosis, PP2A^Cdc55 reactivation could promote Mob1 dephosphorylation supporting Dbf2–Mob1 activation.

Figure 2. Multiple roles of PP2A regulating MEN and SIN pathways. The cartoon shows the different PP2A holoenzymes and their substrates regulating MEN during mitosis in S. cerevisiae and SIN in S. pombe during septation. 

Although PP2A^Cdc55 is the main PP2A regulating mitotic exit, PP2A^Rts1 was also described to regulate MEN upon activation of the spindle position checkpoint (SPOC). The activation and functionality of SPOC depend on the ability of Bub2/Bfa1 to inhibit MEN. Phosphorylation of Bfa1 by Kin4 prevents the Cdc5-dependent phosphorylation of Bfa1, keeping MEN inactive [72,82,85,86]. PP2A^Rts1 phosphatase is a SPOC component acting upstream of Kin4. PP2A^Rts1 dephosphorylates Kin4, regulating the association of Kin4 to the SPBs, and thereby restraining MEN activity [87]. 

The MEN pathway is closely related to the septation initiation network (SIN) in Schizosaccharomyces pombe and the Hippo pathway in mammals. Their most conserved role is the regulation of cytokinesis. At the core of the SIN pathway, Sid1-Cdc14 is the PAK-like kinase (Cdc15) and Sid2-Mob1 is the LATS-kinase (Dbf2-Mob1). Two additional kinases, the polo-like Plo1 and the Ste20-family Cdc7 are also part of the SIN pathway. Mutations of the PP2A regulatory subunits (Pab1 and Par1) and the major catalytic subunit Ppa2 rescue conditional SIN mutants [9,10,90], suggesting that PP2A inhibits SIN signaling (Figure 2). PP2A^Pab1 inhibits the SIN pathway [9] and, similar to S. cerevisiae, it has been proposed that the main candidate to be the target of PP2A^Pab1 is the GAP Byr4-Cdc16 (Bub2/Bfa1 in S. cerevisiae) [9]. The other PP2A holoenzyme, PP2A^Par1/2, regulates the localization of the Cdc7 kinase inhibiting SIN, in order to avoid multiple rounds of septation [10,90,91].

Finally, PP2A^Pab1 and PP2A^Par1 are also regulated by the PP1-like phosphatase Dis2. PP1 binds to and activates PP2A^Pab1 through a conserved RVXF motif present in Pab1, the B55 subunit. Active PP2A^Pab1 dephosphorylates Par1 and promotes PP1 recruitment, which in turn further activates PP2A^Par1 phosphatase. In this way, PP1-induced activation of both PP2A^B55 and PP2A^B56 coordinates mitotic progression and exit from mitosis [92]