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Phosphorylation of immunopurified rat liver glucocorticoid receptor by the catalytic subunit of cAMP-dependent protein kinase

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Abstract

We have examined phosphorylation of the rat liver glucocorticoid receptor (GR) and GR-associated protein kinase (PK) activity in the immunopurified receptor preparations. Affinity labeling of hepatic cytosol with [3H]dexamethasone 21-mesylate showed a covalent association of the steroid with a 94 kDa protein. GR was immunopurified with antireceptor monoclonal antibody BuGR2 (Gametchu & Harrison, Endocrinology 114: 274–279, 1984) to near homogeneity. A 23° C incubation of the immunoprecipitated protein A-Sepharose adsorbed GR with [γ-32P]ATP, Mg2+ and the catalytic subunit of cAMP-dependent PK (cAMP-PK) from bovine heart, led to an incorporation of radioactivity in the 94 kDa protein. Phosphorylation of GR was not evident in the absence of the added kinase. Of the radioinert nucleotides (ATP, GTP, UTP or CTP) tested, only ATP successfully competed with [γ-32P]ATP demonstrating a nucleotide specific requirement for the phosphorylation of GR. Other divalent cations, such as Mn2+ or Ca2+, could not be substituted for Mg2+ during the phosphorylation reaction. Phosphorylation of GR was sensitive to the presence of the protein kinase inhibitor, H-8, an isoquinoline sulfonamide derivative. In addition, the incorporation of radioactivity into GR was both time- and temperature-dependent. The phosphorylation of GR by cAMP-PK was independent of the presence of hsp-90 and transformation state of the receptor. The results of this study demonstrate that GR is an effective substrate for action of cAMP-PK and that the immunopurified protein A-Sepharose adsorbed GR lacks intrinsic kinase activity but can be conveniently used for the characterization of the phosphorylation reaction in the presence of an exogenous kinase.

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Abbreviations

BUGR2:

anti-GR monoclonal antibody

cAMP-PK:

cAMP-dependent protein kinase

DMSO:

dimethyl sulfoxide

EDTA:

ethylenediamine tetra acetic acid

GR:

glucocorticoid receptor

H-8:

Isoquinoline sulfonamide derivative

hsp-90:

90 kDa heat-shock protein

PMSF:

phenylmethylsulfonyl fluoride

PR:

progesterone receptor

NaF:

sodium fluoride

SDS:

sodium dodecyl sulfate

SDS-PAGE:

SDS-polyacrylamide gel electrophoresis

SR:

steroid receptor

TA:

triamcinolone acetonide

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Author notes

  1. Taraneh Haske

    Present address: Parke Davis, 2800 Plymouth Road, 48104, Ann Arbour, MI

  2. Makoto Nakao

    Present address: Department of Internal Medicine, Aisenbashi Hospital, Osaka, Japan

Authors and Affiliations

  1. Department of Biological Sciences and the Institute for Biochemistry and Biotechnology, Oakland University, 48309-4401, Rochester, MI, USA

    Taraneh Haske, Makoto Nakao & V. K. Moudgil

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  1. Taraneh Haske
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  2. Makoto Nakao
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  3. V. K. Moudgil
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Haske, T., Nakao, M. & Moudgil, V.K. Phosphorylation of immunopurified rat liver glucocorticoid receptor by the catalytic subunit of cAMP-dependent protein kinase. Mol Cell Biochem 132, 163–171 (1994). https://doi.org/10.1007/BF00926925

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  • DOI: https://doi.org/10.1007/BF00926925

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