Regulation of Type 1 Protein Phosphatase/Inhibitor-2 Complex by Glycogen Synthase Kinase-3β in Intact Cells

Abstract

Inhibitor 2 (I-2) is a ubiquitous regulator of type 1 protein phosphatase (PP1). Previous in vitro studies suggested that its inhibitory activity towards PP1 is regulated by phosphorylation at Thr72 by glycogen synthase kinase-3β (GSK-3β), and at Ser86, Serl20, and Serl2l by casein kinase 2 (CK2). Here we report that GSK-3β expressed in COS-7 cells phosphorylates wild-type I-2 but not an I-2 mutant carrying a T to A substitution at residue 72, showing that GSK-3β phosphorylates I-2 at T72 in vivo as well. Co-immunoprecipitation study demonstrated that HA-GSK-3β and I-2-FLAG co-exist in a same complex in the intact cells, but they do not bind directly. It is noteworthy that co-expression of Myc-PP1C significantly increased co-precipitation of HA-GSK-3β with I-2-FLAG, showing a complex formation of HA-GSK-3β/Myc-PP1C/I-2-FLAG in vivo. Further studies using a GSK-3β kinase-dead mutant and LiCl, an inhibitor of GSK-3β, showed that the enzyme activity of GSK-3β is required for co-precipitation. IP-Western study using several I-2 mutants substituted at phosphorylation sites (T72, S86, S120, and S121) suggested that phosphorylation of I-2 by CK2 is also involved in enhancement of association between GSK-3β and I-2 in vivo. This study is the first demonstration that GSK-3β associates with PP1C/I-2 complex and phosphorylates I-2 at T72 in the intact cells.