Abstract
PP2A-B55 is one of the major phosphatases regulating cell division. Despite its importance for temporal control during mitotic exit, how B55 substrates are recognized and differentially dephosphorylated is unclear. Using phosphoproteomics combined with kinetic modeling to extract B55-dependent rate constants, we have systematically identified B55 substrates and assigned their temporal order in mitotic exit. These substrates share a bipartite polybasic recognition determinant (BPR) flanking a Cdk1 phosphorylation site. Experiments and modeling show that dephosphorylation rate is encoded into B55 substrates, including its inhibitor ENSA, by cooperative action of basic residues within the BPR. A complementary acidic surface on B55 decodes this signal, supporting a cooperative electrostatic mechanism for substrate selection. A further level of specificity is encoded into B55 substrates because B55 displays selectivity for phosphothreonine. These simple biochemical properties, combined with feedback control of B55 activity by the phosphoserine-containing substrate/inhibitor ENSA, can help explain the temporal sequence of events during exit from mitosis.