The 1,25-dihydroxy-vitamin D3 receptor is phosphorylated in response to 1,25-dihydroxy-vitamin D3 and 22-oxacalcitriol in rat osteoblasts, and by casein kinase II, in vitro

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Abstract

We analyzed the endogenous nuclear 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) receptor (VDR) in rat osteosarcoma (ROS 17/2.8) cells and present biochemical evidence that it is a phosphoprotein. When ROS 17/2.8 cells are labeled metabolically with [35S]methionine, treatment with 10-8 M 1,25(OH)2D3 elicits a decrease in the electrophoretic mobility of immunoprecipitated VDR in denaturing polyacrylamide gels, a property characteristic of phosphorylated proteins. Similar labeling of cells with [32P] orthophosphate results in a rapid (≤30 min), 1,25(OH)2D3-dependent incorporation of 32P into a 54-kDa VDR species that comigrates with the slower migrating receptor species extracted from [35S]methionine-labeled ROS 17/2.8 cells that have been exposed to 1,25(OH)2D3. Alkaline phosphatase treatment of immunoprecipitated VDR from 1,25(OH)2D3-treated cells converts the form of the VDR with reduced mobility to the faster migrating species present in 1,25(OH)2D3-deficient cells. Incubation of ROS 17/2.8 cells with the non-hypercalcemic 1,25(OH)2D3 analog, 22-oxacalcitriol (OCT), produces a level of VDR phosphorylation similar to that elicited by 1,25(OH)2D3 treatment. Transient transfection of osteosarcoma cells with a reporter vector containing a vitamin D responsive element derived from the rat osteocalcin gene yields equivalent transcriptional activation in the presence of either 1,25(OH)2D3 or OCT. Further experiments performed at various 1,25 (OH)2D3 concentrations to assess the relationship between receptor phosphorylation and transcriptional activity in intact cells showed a positive correlation between these two parameters, indicating that the 1,25(OH)2D3 hormone stimulates VDR phosphorylation and transcriptional activation in parallel. Finally, highly purified casein kinase II (CK-II) phosphorylates the VDR in a 1,25(OH)2D3-independent, in vitro reaction. Comparison of the migration in denaturing gel electrophoresis of in vivo and in vitro phosphorylated VDR reveals a different electrophoretic mobility of the CK-II-phosphorylated receptor, suggesting that CK-II-mediated phosphorylation may be distinct from that occurring in response to 1,25(OH)2D3 or OCT. We speculate that hormone-dependent phosphorylation of the VDR may be required for transcriptional activation, while hormone-independent, CK-II-mediated phosphorylation may play a role in the modulation of receptor activity.

Original languageEnglish (US)
Pages (from-to)8184-8192
Number of pages9
JournalBiochemistry
Volume32
Issue number32
StatePublished - 1993

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Casein Kinase II
Calcitriol Receptors
Phosphorylation
Osteoblasts
Rats
Electrophoretic mobility
Chemical activation
Hormones
Methionine
Transcriptional Activation
Osteosarcoma
Phosphoproteins
Osteocalcin
Electrophoresis
Vitamin D
Labeling
Alkaline Phosphatase
maxacalcitol
dihydroxy-vitamin D3
In Vitro Techniques

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{89a608a70781435f906d92927abd9ffa,
title = "The 1,25-dihydroxy-vitamin D3 receptor is phosphorylated in response to 1,25-dihydroxy-vitamin D3 and 22-oxacalcitriol in rat osteoblasts, and by casein kinase II, in vitro",
abstract = "We analyzed the endogenous nuclear 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) receptor (VDR) in rat osteosarcoma (ROS 17/2.8) cells and present biochemical evidence that it is a phosphoprotein. When ROS 17/2.8 cells are labeled metabolically with [35S]methionine, treatment with 10-8 M 1,25(OH)2D3 elicits a decrease in the electrophoretic mobility of immunoprecipitated VDR in denaturing polyacrylamide gels, a property characteristic of phosphorylated proteins. Similar labeling of cells with [32P] orthophosphate results in a rapid (≤30 min), 1,25(OH)2D3-dependent incorporation of 32P into a 54-kDa VDR species that comigrates with the slower migrating receptor species extracted from [35S]methionine-labeled ROS 17/2.8 cells that have been exposed to 1,25(OH)2D3. Alkaline phosphatase treatment of immunoprecipitated VDR from 1,25(OH)2D3-treated cells converts the form of the VDR with reduced mobility to the faster migrating species present in 1,25(OH)2D3-deficient cells. Incubation of ROS 17/2.8 cells with the non-hypercalcemic 1,25(OH)2D3 analog, 22-oxacalcitriol (OCT), produces a level of VDR phosphorylation similar to that elicited by 1,25(OH)2D3 treatment. Transient transfection of osteosarcoma cells with a reporter vector containing a vitamin D responsive element derived from the rat osteocalcin gene yields equivalent transcriptional activation in the presence of either 1,25(OH)2D3 or OCT. Further experiments performed at various 1,25 (OH)2D3 concentrations to assess the relationship between receptor phosphorylation and transcriptional activity in intact cells showed a positive correlation between these two parameters, indicating that the 1,25(OH)2D3 hormone stimulates VDR phosphorylation and transcriptional activation in parallel. Finally, highly purified casein kinase II (CK-II) phosphorylates the VDR in a 1,25(OH)2D3-independent, in vitro reaction. Comparison of the migration in denaturing gel electrophoresis of in vivo and in vitro phosphorylated VDR reveals a different electrophoretic mobility of the CK-II-phosphorylated receptor, suggesting that CK-II-mediated phosphorylation may be distinct from that occurring in response to 1,25(OH)2D3 or OCT. We speculate that hormone-dependent phosphorylation of the VDR may be required for transcriptional activation, while hormone-independent, CK-II-mediated phosphorylation may play a role in the modulation of receptor activity.",
author = "Haussler, {Mark R}",
year = "1993",
language = "English (US)",
volume = "32",
pages = "8184--8192",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "32",

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TY - JOUR

T1 - The 1,25-dihydroxy-vitamin D3 receptor is phosphorylated in response to 1,25-dihydroxy-vitamin D3 and 22-oxacalcitriol in rat osteoblasts, and by casein kinase II, in vitro

AU - Haussler, Mark R

PY - 1993

Y1 - 1993

N2 - We analyzed the endogenous nuclear 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) receptor (VDR) in rat osteosarcoma (ROS 17/2.8) cells and present biochemical evidence that it is a phosphoprotein. When ROS 17/2.8 cells are labeled metabolically with [35S]methionine, treatment with 10-8 M 1,25(OH)2D3 elicits a decrease in the electrophoretic mobility of immunoprecipitated VDR in denaturing polyacrylamide gels, a property characteristic of phosphorylated proteins. Similar labeling of cells with [32P] orthophosphate results in a rapid (≤30 min), 1,25(OH)2D3-dependent incorporation of 32P into a 54-kDa VDR species that comigrates with the slower migrating receptor species extracted from [35S]methionine-labeled ROS 17/2.8 cells that have been exposed to 1,25(OH)2D3. Alkaline phosphatase treatment of immunoprecipitated VDR from 1,25(OH)2D3-treated cells converts the form of the VDR with reduced mobility to the faster migrating species present in 1,25(OH)2D3-deficient cells. Incubation of ROS 17/2.8 cells with the non-hypercalcemic 1,25(OH)2D3 analog, 22-oxacalcitriol (OCT), produces a level of VDR phosphorylation similar to that elicited by 1,25(OH)2D3 treatment. Transient transfection of osteosarcoma cells with a reporter vector containing a vitamin D responsive element derived from the rat osteocalcin gene yields equivalent transcriptional activation in the presence of either 1,25(OH)2D3 or OCT. Further experiments performed at various 1,25 (OH)2D3 concentrations to assess the relationship between receptor phosphorylation and transcriptional activity in intact cells showed a positive correlation between these two parameters, indicating that the 1,25(OH)2D3 hormone stimulates VDR phosphorylation and transcriptional activation in parallel. Finally, highly purified casein kinase II (CK-II) phosphorylates the VDR in a 1,25(OH)2D3-independent, in vitro reaction. Comparison of the migration in denaturing gel electrophoresis of in vivo and in vitro phosphorylated VDR reveals a different electrophoretic mobility of the CK-II-phosphorylated receptor, suggesting that CK-II-mediated phosphorylation may be distinct from that occurring in response to 1,25(OH)2D3 or OCT. We speculate that hormone-dependent phosphorylation of the VDR may be required for transcriptional activation, while hormone-independent, CK-II-mediated phosphorylation may play a role in the modulation of receptor activity.

AB - We analyzed the endogenous nuclear 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) receptor (VDR) in rat osteosarcoma (ROS 17/2.8) cells and present biochemical evidence that it is a phosphoprotein. When ROS 17/2.8 cells are labeled metabolically with [35S]methionine, treatment with 10-8 M 1,25(OH)2D3 elicits a decrease in the electrophoretic mobility of immunoprecipitated VDR in denaturing polyacrylamide gels, a property characteristic of phosphorylated proteins. Similar labeling of cells with [32P] orthophosphate results in a rapid (≤30 min), 1,25(OH)2D3-dependent incorporation of 32P into a 54-kDa VDR species that comigrates with the slower migrating receptor species extracted from [35S]methionine-labeled ROS 17/2.8 cells that have been exposed to 1,25(OH)2D3. Alkaline phosphatase treatment of immunoprecipitated VDR from 1,25(OH)2D3-treated cells converts the form of the VDR with reduced mobility to the faster migrating species present in 1,25(OH)2D3-deficient cells. Incubation of ROS 17/2.8 cells with the non-hypercalcemic 1,25(OH)2D3 analog, 22-oxacalcitriol (OCT), produces a level of VDR phosphorylation similar to that elicited by 1,25(OH)2D3 treatment. Transient transfection of osteosarcoma cells with a reporter vector containing a vitamin D responsive element derived from the rat osteocalcin gene yields equivalent transcriptional activation in the presence of either 1,25(OH)2D3 or OCT. Further experiments performed at various 1,25 (OH)2D3 concentrations to assess the relationship between receptor phosphorylation and transcriptional activity in intact cells showed a positive correlation between these two parameters, indicating that the 1,25(OH)2D3 hormone stimulates VDR phosphorylation and transcriptional activation in parallel. Finally, highly purified casein kinase II (CK-II) phosphorylates the VDR in a 1,25(OH)2D3-independent, in vitro reaction. Comparison of the migration in denaturing gel electrophoresis of in vivo and in vitro phosphorylated VDR reveals a different electrophoretic mobility of the CK-II-phosphorylated receptor, suggesting that CK-II-mediated phosphorylation may be distinct from that occurring in response to 1,25(OH)2D3 or OCT. We speculate that hormone-dependent phosphorylation of the VDR may be required for transcriptional activation, while hormone-independent, CK-II-mediated phosphorylation may play a role in the modulation of receptor activity.

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M3 - Article

VL - 32

SP - 8184

EP - 8192

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

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