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RACK1 regulates PP2A‐mediated de‐phosphorylation of threonine 38 of the human nuclear xenobiotic receptor CAR
Author(s) -
Mutoh Shingo,
Osabe Makoto,
Moore Rickie,
Vinal Kellie,
Sueyoshi Tatsuya,
Negishi Masahiko
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.585.11
Subject(s) - phosphorylation , protein phosphatase 2 , chemistry , immunoprecipitation , microbiology and biotechnology , threonine , kinase , serine , biochemistry , phosphatase , biology , gene
Before activation by therapeutics, the human nuclear xenobiotic receptor hCAR is sequestered in the cytoplasm, by phosphorylating threonine 38 (T38) within its DNA binding domain. Phenobarbital, the classic hCAR activator, de‐phosphorylates T38 to translocate hCAR into the nucleus (Mutoh, et al., J. Biol. Chem . in press). Given that T 38 is the target of de‐phopshorylation, we are now investigating the molecular mechanism by which T38 can be de‐phosphorylated. Subsequently, r eceptor for a ctivated C k inase 1 (RACK1) has been identified as a protein that specifically interacts with the phosphorylated form of hCAR (T38D mutant of hCAR); by employing yeast two hybrid screening and co‐immunoprecipitation assays. T38 of recombinant hCAR protein was in vitro phosphorylated by pure protein kinase C and was used as the substrate for an in vitro de‐phosphorylation assay by pure protein phosphatase 2A (PP2A). PP2A was then found to de‐phosphorylate T38 in the presence of RACK1. To substantiate these roles for RACK1 and PP2A, whole cell extracts, prepared from Huh7 cells treated with siRNA RACK1 and siRNA PP2Ac, were incubated with recombinant hCAR proteins as the enzyme sources for phosphorylation. Both knock downs of RACK1 and of PP2Ac resulted in an increased phosphorylation of T38. These results indicate that PP2A de‐phosphorylates T38 and RACK1 regulates this PP2A‐mediated de‐phosphorylation.