The mouse glucocorticoid receptor DNA-binding domain is not phosphorylated invivo

Benjamin, Wieke S. van der Weijden; Hendry, William J.; Harrison, Robert W.

doi:10.1016/0006-291x(90)90900-8

Cited by 7 publications

(3 citation statements)

References 23 publications

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“…This label was localized to the steroid-binding domain in receptors from WEHI-7 cells (146), and to the DNA-binding domain (isolated as ã 16-kDa tryptic fragment with the BuGR antiglucocorticoid receptor antibodies) in receptors from L fibroblasts (145) and hepatoma cells (143). In a separate study, the radioactivity associated with the DNA-binding fragment was ascribed to an impurity of similar molecular mass (149). Recently we have found that a highly purified tryptic fragment containing the steroid-binding domain is not phosphorylated (147), indicating that the earlier result from our laboratory on labeling of this domain (146) may have been due to a contaminant.…”

Section: Location Of Phosphorylated Sitesmentioning

confidence: 92%

Phosphorylation of Steroid Hormone Receptors*

1992

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Many observations with intact cells as well as cell-free systems suggest that receptors of the steroid hormone superfamily, along with other transcription factors, are regulated by phosphorylation. All receptors that have been analyzed carefully so far have turned out to be phosphoproteins. They are basally phosphorylated in the absence of ligand, and in many cases become hyperphosphorylated in the presence of hormone or other agonists, and sometimes of antagonists. Several studies indicate that hyperphosphorylation of receptors follows activation, and may require nuclear binding of the receptor. Serines are the predominant phosphorylated residues detected in receptors, with minor amounts of threonine. Tyrosine phosphorylation of the estrogen receptor is a subject of controversy. With various receptors, evidence has been found for phosphorylation in vivo of the N-terminal, hormone-binding, and DNA-binding domains, as well as of the hinge region. All but one of the phosphorylated sites identified in progesterone and glucocorticoid receptors by phosphopeptide mapping and sequencing are in the N-terminal domain; one is in the hinge region. Even after hormone treatment most of those sites are only partly phosphorylated, which means that several subpopulations of receptors, characterized by different states of phosphorylation and potentially different biological activities, must coexist. The majority of identified phosphorylated sites lie in consensus sequences for the PDPK. Many parallels can be discerned between phosphorylation of receptors and of other transcription factors. For example, several transcription factors become hyperphosphorylated on stimulation, and much indirect evidence points to regulation of both receptors and transcription factors by kinases and phosphatases, with cycling between different phosphorylated states. Functions of receptors that are regulated by phosphorylation are only beginning to be investigated. With transcription factors a substantial body of information is already available, and functions that appear to be thus regulated include dimerization, interactions with other proteins, binding to DNA, nuclear-cytoplasmic localization, and transcriptional activity. These and other functions may be found to be regulated by phosphorylation of receptors.

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Section: Location Of Phosphorylated Sitesmentioning

confidence: 92%

Phosphorylation of Steroid Hormone Receptors*

1992

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“…Indeed, we have demonstrated previously that this BuGR-adsorbed 15 kDa fragment is fully competent in binding DNA [20]. Thus, we must disagree with van der Weijden Benjamin et al [19] who concluded that the mouse GR DNA-binding domain is not phosphorylated in vivo. However, it is clear that the 15 kDa tryptic fragment is not a major site for phosphorylation, as we estimate that this fragment contains only ~ 5% of the total receptor phosphate.…”

Section: Resultsmentioning

confidence: 67%

“…1) is phosphorylated in GR isolated from mouse L cells [10] and rat hepatoma cells [11]. These reports have been challenged by van der Weijden Benjamin et al [19] who concluded from a study of GR phosphorylation in cultured AtT-20 cells that the 15 kDa phosphorylated tryptic fragment is similar in size to the DNAbinding tryptic fragment containing the BuGR epitope (C375-K505) but is not the DNAbinding, BuGR-reactive fragment itself. The data of Fig.…”

Section: Resultsmentioning

confidence: 99%

Localization of the ∼12 kDa Mr discrepancy in gel migration of the mouse glucocorticoid receptor to the major phosphorylated cyanogen bromide fragment in the transactivating domain

Hutchison

Dalman

Hoeck

et al. 1993

The Journal of Steroid Biochemistry and Molecular Biology

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The intact wild-type mouse glucocorticoid receptor has a theoretical molecular weight of approximately 96 kDa based on amino acid sequence, but on SDS-polyacrylamide gel electrophoresis it migrates as a protein of approximately 98 kDa. It is not known where the unusual primary structure or covalent modification responsible for this anomalous migration is located within the amino acid chain. In the course of examining the pattern of fragmentation of 32P-labeled glucocorticoid receptors from Chinese hamster ovary (CHO) cells containing amplified mouse receptor cDNA, we have found a localized region in the amino-terminal half of the receptor that accounts for this anomalous behavior. Cyanogen bromide treatment of the intact receptor produces a 23.4 kDa (theoretical) fragment consisting of residues 108-324 and containing all of the identified phosphorylated serines within the receptor. We find that the only large resolvable 32P-labeled receptor fragment produced after complete cyanogen bromide cleavage of intact receptors migrates with an apparent molecular weight of approximately 35 kDa. Because the apparent difference between the theoretical and the experimentally observed molecular weights of this cyanogen bromide fragment is essentially the same as the difference between the theoretical and experimental molecular weights of the intact mouse glucocorticoid receptor, we propose that some feature lying within this fragment accounts for slower migration. Although the existence of an additional phosphorylation site lying within the 15 kDa tryptic receptor fragment containing the DNA-binding domain has been contested, we also demonstrate that this fragment of the mouse glucocorticoid receptor is phosphorylated in vivo upon incubation of CHO cells in growth medium containing [32P]orthophosphate.

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