Regulation of α–endosulfine, an inhibitor of protein phosphatase 2 A , by multisite phosphorylation

Progression into M  phase requires inhibition of heterotrimeric PP 2 A containing the regulatory B 55 subunit ( PP 2A– B 55) as well as the activation of cyclin‐dependent kinase 1 ( C dk1). α–endosulfine ( ENSA )/cyclic AMP ‐regulated 19 kDa phosphoprotein ( ARPP –19) family proteins phosphorylated at S 67 by G reatwall kinase bind and inhibit PP 2 A – B 55. This study shows that endogenous kinases phosphorylate not only S 67 but also two additional sites in ENSA ( T 28 and S 109) with different kinetics at different cell‐cycle stages in X enopus laevis intact cells and cell‐free egg extracts. When assayed in vitro , these phosphorylations had qualitatively and/or quantitatively different effects on inhibition of PP 2 A – B 55 by ENSA . Structural analyses revealed that the most‐conserved middle region of ENSA containing S 67 physically interacts with PP 2 A – B 55 at the interface of the B 55 and C subunits, where the catalytic centre of PP 2 A is located. As non‐phosphorylated ENSA has an intrinsic potential for PP 2 A – B 55 inhibition, these three phosphorylations differentially affect physical interaction of the middle region of ENSA with PP 2 A – B 55. These results suggest that the two additional phosphorylation sites together with S 67 allow ENSA to function as a ‘stepwise tuner’ for PP 2 A – B 55, which may be regulated by multiple cellular signals, rather than a simple ‘on/off’ switch.