Regulation of α-endosulfine, an Inhibitor of Protein Phosphatase 2A, by Multisite Phosphorylation

Progression into M phase requires inhibition of heterotrimeric PP2A containing the regulatory B55 subunit (PP2A-B55) as well as the activation of cyclin-dependent kinase 1 (Cdk1). α-endosulfine (ENSA)/cyclic AMP-regulated 19 kDa phosphoprotein (ARPP-19) family proteins phosphorylated at S67 by Greatwall kinase bind and inhibit PP2A-B55. This study shows that endogenous kinases phosphorylate not only S67 but also two additional sites in ENSA (T28 and S109) with different kinetics at different cell-cycle stages in Xenopus laevis intact cells and cell-free egg extracts. When assayed in vitro, these phosphorylations had qualitatively and/or quantitatively different effects on inhibition of PP2A-B55 by ENSA. Structural analyses revealed that the most-conserved middle region of ENSA containing S67 physically interacts with PP2A-B55 at the interface of the B55 and C subunits, where the catalytic centre of PP2A is located. As non-phosphorylated ENSA has an intrinsic potential for PP2A-B55 inhibition, these three phosphorylations differentially affect physical interaction of the middle region of ENSA with PP2A-B55. These results suggest that the two additional phosphorylation sites together with S67 allow ENSA to function as a 'stepwise tuner' for PP2A-B55, which may be regulated by multiple cellular signals, rather than a simple 'on/off' switch.