J. Biochem, 2003, Vol. 133, No. 2 165-171
© 2003 Japanese Biochemical Society
BIOCHEMISTRY |
Regulation of Type 1 Protein Phosphatase/Inhibitor-2 Complex by Glycogen Synthase Kinase-3ß in Intact Cells
1 Division of Biochemical Oncology and Immunology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815; 2 Department of Biochemistry II, Nagoya University School of Medicine, 65 Tsurumai-machi, Showa-ku, Nagoya 466-0065; and 3 Departments of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551
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-3ß (GSK-3ß), and at Ser86, Ser120, and Ser121 by casein kinase 2 (CK2). Here we report that GSK-3ß 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-3ß phosphorylates I-2 at T72 in vivo as well. Co-immunoprecipitation study demonstrated that HA-GSK-3ß 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-3ß with I-2-FLAG, showing a complex formation of HA-GSK-3ß/Myc-PP1C / I-2-FLAG in vivo. Further studies using a GSK-3ß kinase–dead mutant and LiCl, an inhibitor of GSK-3ß, showed that the enzyme activity of GSK-3ß 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-3ß and I-2 in vivo. This study is the first demonstration that GSK-3ß associates with PP1C/I-2 complex and phosphorylates I-2 at T72 in the intact cells.
+ To whom correspondence should be addressed. Tel: +81-11-706-7541, Fax: +81-11-706-7541, E-mail: hshima{at}imm.hokudai.ac.jp
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