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

• Published in: The FEBS journal Vol. 281; no. 4; pp. 1159 - 1169
• Main Author: Mochida, Satoru
• Format: Journal Article
• Language: English
• Published: England 01.02.2014
• Subjects: Animals; cell cycle; Cell Cycle - physiology; cyclin‐dependent kinase; greatwall kinase; Peptides - metabolism; Phosphorylation; protein kinase A; Protein Phosphatase 2 - metabolism; protein phosphatase 2A; Xenopus laevis; greatwall kinase; cyclin-dependent kinase; protein kinase A; cell cycle; protein phosphatase 2A
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.12685

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. Mitosis requires both PP2A‐B55 inhibition and Cdk1 activation. This study reports that ENSA, an inhibitor of PP2A‐B55, is phosphorylated at two additional sites in addition to Ser67, the Greatwall site. These phosphorylations showed qualitatively/quantitatively different effects on ENSA's inhibition of PP2A‐B55. ENSA can be regulated by multiple cellular signals to tune PP2A‐B55 activity into several discrete levels.