Regulation of type 1 protein phosphatase/inhibitor-2 complex by glycogen synthase kinase-3beta in intact cells.
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-3beta (GSK-3beta), and at Ser86, Ser120, and Ser121 by casein kinase 2 (CK2). Here we report that GSK-3beta 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-3beta phosphorylates I-2 at T72 in vivo as well. Co-immunoprecipitation study demonstrated that HA-GSK-3beta 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-3beta with I-2-FLAG, showing a complex formation of HA-GSK-3beta/Myc-PP1C / I-2-FLAG in vivo. Further studies using a GSK-3beta kinase-dead mutant and LiCl, an inhibitor of GSK-3beta, showed that the enzyme activity of GSK-3beta 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-3beta and I-2 in vivo. This study is the first demonstration that GSK-3beta associates with PP1C/I-2 complex and phosphorylates I-2 at T72 in the intact cells.
Sakashita
Gyosuke
G
Division of Biochemical Oncology and Immunology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan.
Shima
Hiroshi
H
Komatsu
Masakazu
M
Urano
Takeshi
T
Kikuchi
Akira
A
Kikuchi
Kunimi
K
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D054645:Protein Phosphatase 1; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D014162:Transfection; D000097847:Protein Phosphatase Inhibitory Proteins
D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D011506:Proteins; D011994:Recombinant Proteins; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins; C000605506:GSK3B protein, human