Greatwall-phosphorylated Endosulfine is both an inhibitor and a substrate of PP2A-B55 heterotrimers

Abstract

During M phase, Endosulfine (Endos) family proteins are phosphorylated by Greatwall kinase (Gwl), and the resultant pEndos inhibits the phosphatase PP2A-B55, which would otherwise prematurely reverse many CDK-driven phosphorylations. We show here that PP2A-B55 is the enzyme responsible for dephosphorylating pEndos during M phase exit. The kinetic parameters for PP2A-B55’s action on pEndos are orders of magnitude lower than those for CDK-phosphorylated substrates, suggesting a simple model for PP2A-B55 regulation that we call inhibition by unfair competition. As the name suggests, during M phase PP2A-B55’s attention is diverted to pEndos, which binds much more avidly and is dephosphorylated more slowly than other substrates. When Gwl is inactivated during the M phase-to-interphase transition, the dynamic balance changes: pEndos dephosphorylated by PP2A-B55 cannot be replaced, so the phosphatase can refocus its attention on CDK-phosphorylated substrates. This mechanism explains simultaneously how PP2A-B55 and Gwl together regulate pEndos, and how pEndos controls PP2A-B55.DOI: http://dx.doi.org/10.7554/eLife.01695.001

Protein phosphatase 1 is essential for Greatwall inactivation at mitotic exit

“…PP2A-B55 has also been suggested to dephosphorylate Gwl at a Cdk1 site (T193 in Xenopus) in the activation loop [14]. However, PP2A-B55 is unlikely to be the phosphatase that initiates Gwl inactivation at mitotic exit because it is inhibited in this situation by the product of Gwl, that is, phosphorylated Arpp19 (pArpp19) which is present in molar excess over PP2A-B55 [15]. Such a system would be futile with PP2A-B55 not being able to inactivate Gwl as long as pArpp19 levels are high and pArpp19 levels remaining high as long as Gwl is active.…”

“…The concomitant re-activation of PP2A-B55 depends on the dephosphorylation of both Arpp19, to relieve its inhibitory effect on PP2A-B55, and Gwl, to stop the replenishment of phosphorylated Arpp19. The identity of the phosphatase dephosphorylating Arpp19 remains controversial with Fcp1 and PP2A-B55 being potential candidates [1,14,15]. According to Williams et al (2014), Arpp19 is not only an inhibitor of PP2A-B55, but also a substrate that binds extremely tightly to PP2A-B55 and is dephosphorylated at a very low rate.…”

“…The identity of the phosphatase dephosphorylating Arpp19 remains controversial with Fcp1 and PP2A-B55 being potential candidates [1,14,15]. According to Williams et al (2014), Arpp19 is not only an inhibitor of PP2A-B55, but also a substrate that binds extremely tightly to PP2A-B55 and is dephosphorylated at a very low rate. Based on these findings, an "inhibition by unfair competition" model has been postulated according to which phosphorylated Arpp19 inhibits PP2A-B55 by blocking access of other substrates while being slowly dephosphorylated.…”

The Biochemistry of Mitosis

“…The drop in cyclin -Cdk activity levels in anaphase indirectly reactivates PP2A complexes because Greatwall kinase depends on cyclin -Cdk for its activity. As Greatwall activity declines, so does the rate at which phospho-ENSA is generated, thereby shifting the balance toward PP2A dephosphorylating and inactivating ENSA (Cundell et al 2013;Williams et al 2014). Reactivation of PP1, PP2A, and other phosphatases in a spatially and temporally coordinated fashion reshapes the cell to its interphase architecture and resets it to begin the next cycle of DNA replication, or to choose the alternative fates of quiescence or differentiation.…”

“…It was a biochemical study using Xenopus extracts that underlined the importance of controlling phosphatases for mitotic entry. Mochida et al showed that activating cyclin BCdk kinase was not sufficient to drive cells into mitosis; a member of the PP2A phosphatase family that antagonized cyclin B-Cdk1 had to be inactivated too (Mochida et al 2009 Williams et al 2014). Phospho-ENSA is then slowly dephosphorylated by PP2A, which allows PP2A to reactivate once cyclin -Cdk and Greatwall kinase activity decline later in mitosis (Williams et al 2014).…”