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

eLife digest

The most dramatic stage of the cell division cycle is M phase, when the cell splits into two genetically identical daughter cells. If this process goes wrong, the cell might die, so cells employ a complicated regulatory process to ensure that M phase begins and ends at the right time.

As with many biological processes, regulation of the cell cycle depends on the activation and inhibition of a range of enzymes. Enzymes act as biological catalysts, binding target molecules (substrates) to active sites so that chemical reactions can take place. However, the activity of the enzyme can be shut down if a different type of molecule, called a competitive inhibitor, binds to the active site.

For M phase to proceed, an enzyme called M phase promoting factor adds phosphate groups to hundreds of target proteins. At the end of M phase, a different enzyme, called PP2A-B55, removes these phosphate groups. Cells can enter M phase because an inhibitor called Endosulfine blocks the active site of the PP2A-B55 enzyme. However, the cells need to unblock the PP2A-B55 enzyme at the end of M phase. Williams et al. have now established the mechanism behind this unblocking of the PP2A-B55 enzyme.

One basis for this mechanism is that Endosulfine works as an inhibitor only when it is phosphorylated (contains a phosphate group). Throughout M phase, a plentiful supply of newly phosphorylated Endosulfine inhibitor molecules binds very tightly to the active sites of the PP2A-B55 enzyme molecules, blocking the enzyme’s more loosely binding substrates from accessing the active sites. The second basis for this mechanism is that PP2A-B55 can also slowly remove the phosphate groups from Endosulfine molecules bound at the active site. In other words, phosphorylated Endosulfine works as an inhibitor only because it is really a substrate with special properties. It binds very tightly to the active site, where it is destroyed very slowly. For this reason, Williams et al. have named the process inhibition by unfair competition.

In the final stages of M phase, the cell cannot produce any more phosphorylated Endosulfine molecules, and the PP2A-B55 enzyme can then destroy all the existing inhibitors. Even though this reaction is relatively slow, it is still achieved within a couple of minutes. After its active site is no longer blocked, the PP2A-B55 enzyme is then free to remove the phosphate groups from the target proteins, and M phase can come to an end.

DOI: http://dx.doi.org/10.7554/eLife.01695.002