Phosphorylation of the human vitamin D receptor by protein kinase C

Biochemical and functional evaluation of the serine 51 recognition site

Jui-Cheng Hsieh, Peter W. Jurutka, Shigeo Nakajima, Michael A. Galligan, Carol A. Haussler, Yoshiko Shimizu, Nobuyoshi Shimizu, G Kerr Whitfield, Mark R Haussler

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Abstract

We have reported previously that the human vitamin D receptor (hVDR) is selectively phosphorylated by protein kinase C-β (PKC-β), in vitro, on a serine residue in the sequence RRS51MKRK, which is located between the two zinc fingers of hVDR and is potentially important to its transacting function (Hsieh, J.-C., Jurutka, P. W., Galligan, M. A., Terpening, C. M., Haussler, C. A., Samuels, D. S., Shimizu, Y., Shimizu, N., and Haussler, M. R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9315-9319). In the present experiments we evaluated this phosphorylation event using a series of hVDR mutants in which serine 51 or its flanking residues were modified. Alteration of serine 51 to a non-phosphorylatable residue resulted in an approximately 60% reduction in basal hVDR phosphorylation in intact cells but did not diminish 1,25-dihydroxyvitamin D3-stimulated phosphorylation. Such mutations also abolished subsequent phosphorylation of immunoprecipitated hVDR by purified PKC-β, in vitro, as did replacement of basic residues on either side of serine 51. Mutation of serine 51 to glycine (S51G) or to aspartic acid (S51D), as well as altering the basic residues flanking serine 51, abolished the interaction of hVDR with the vitamin D-responsive element (VDRE) as monitored by gel mobility shift analysis. Thus, we conclude that unmodified serine 51 and its surrounding basic residues are crucial not only for PKC-β substrate recognition but also for the optimal VDRE binding of native hVDR. In transactivation assays, S51G and S51D possessed only 35 and 10% of wild-type hVDR activity, respectively. Mutation of serine 51 to threonine (S51T) restored phosphorylation by PKC-β, in vitro, to about 40% of wild-type and transactivation to 45% of that of wild-type hVDR. Alteration of serine 51 to alanine, which is the residue in the corresponding position of the glucocorticoid, progesterone, mineralocorticoid, and androgen receptors, eliminated PKC-β phosphorylation but completely preserved the specific DNA binding activity and transactivation capacity of hVDR. Thus, phosphorylation of hVDR at serine 51 is not required for either VDRE binding or transactivation. Finally, incubation of Escherichia coli-expressed hVDR with PKC-β elicits marked phosphorylation of the receptor and significantly inhibits its ability to complex with the VDRE. We therefore speculate that posttranslational modification of hVDR at serine 51 may constitute a negative regulatory loop which could be operative when target cells are subject to PKC activation events.

Original languageEnglish (US)
Pages (from-to)15118-15126
Number of pages9
JournalJournal of Biological Chemistry
Volume268
Issue number20
StatePublished - Jul 15 1993

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Calcitriol Receptors
Phosphorylation
Serine
Protein Kinase C
Vitamin D
Transcriptional Activation
Glycine
Mutation
Mineralocorticoid Receptors
Calcitriol
Zinc Fingers
Glucocorticoid Receptors
Androgen Receptors
Electrophoretic Mobility Shift Assay
Progesterone Receptors
Threonine
Post Translational Protein Processing
Aspartic Acid
Alanine
Escherichia coli

ASJC Scopus subject areas

  • Biochemistry

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Phosphorylation of the human vitamin D receptor by protein kinase C : Biochemical and functional evaluation of the serine 51 recognition site. / Hsieh, Jui-Cheng; Jurutka, Peter W.; Nakajima, Shigeo; Galligan, Michael A.; Haussler, Carol A.; Shimizu, Yoshiko; Shimizu, Nobuyoshi; Whitfield, G Kerr; Haussler, Mark R.

In: Journal of Biological Chemistry, Vol. 268, No. 20, 15.07.1993, p. 15118-15126.

Research output: Contribution to journalArticle

Hsieh, Jui-Cheng ; Jurutka, Peter W. ; Nakajima, Shigeo ; Galligan, Michael A. ; Haussler, Carol A. ; Shimizu, Yoshiko ; Shimizu, Nobuyoshi ; Whitfield, G Kerr ; Haussler, Mark R. / Phosphorylation of the human vitamin D receptor by protein kinase C : Biochemical and functional evaluation of the serine 51 recognition site. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 20. pp. 15118-15126.
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title = "Phosphorylation of the human vitamin D receptor by protein kinase C: Biochemical and functional evaluation of the serine 51 recognition site",
abstract = "We have reported previously that the human vitamin D receptor (hVDR) is selectively phosphorylated by protein kinase C-β (PKC-β), in vitro, on a serine residue in the sequence RRS51MKRK, which is located between the two zinc fingers of hVDR and is potentially important to its transacting function (Hsieh, J.-C., Jurutka, P. W., Galligan, M. A., Terpening, C. M., Haussler, C. A., Samuels, D. S., Shimizu, Y., Shimizu, N., and Haussler, M. R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9315-9319). In the present experiments we evaluated this phosphorylation event using a series of hVDR mutants in which serine 51 or its flanking residues were modified. Alteration of serine 51 to a non-phosphorylatable residue resulted in an approximately 60{\%} reduction in basal hVDR phosphorylation in intact cells but did not diminish 1,25-dihydroxyvitamin D3-stimulated phosphorylation. Such mutations also abolished subsequent phosphorylation of immunoprecipitated hVDR by purified PKC-β, in vitro, as did replacement of basic residues on either side of serine 51. Mutation of serine 51 to glycine (S51G) or to aspartic acid (S51D), as well as altering the basic residues flanking serine 51, abolished the interaction of hVDR with the vitamin D-responsive element (VDRE) as monitored by gel mobility shift analysis. Thus, we conclude that unmodified serine 51 and its surrounding basic residues are crucial not only for PKC-β substrate recognition but also for the optimal VDRE binding of native hVDR. In transactivation assays, S51G and S51D possessed only 35 and 10{\%} of wild-type hVDR activity, respectively. Mutation of serine 51 to threonine (S51T) restored phosphorylation by PKC-β, in vitro, to about 40{\%} of wild-type and transactivation to 45{\%} of that of wild-type hVDR. Alteration of serine 51 to alanine, which is the residue in the corresponding position of the glucocorticoid, progesterone, mineralocorticoid, and androgen receptors, eliminated PKC-β phosphorylation but completely preserved the specific DNA binding activity and transactivation capacity of hVDR. Thus, phosphorylation of hVDR at serine 51 is not required for either VDRE binding or transactivation. Finally, incubation of Escherichia coli-expressed hVDR with PKC-β elicits marked phosphorylation of the receptor and significantly inhibits its ability to complex with the VDRE. We therefore speculate that posttranslational modification of hVDR at serine 51 may constitute a negative regulatory loop which could be operative when target cells are subject to PKC activation events.",
author = "Jui-Cheng Hsieh and Jurutka, {Peter W.} and Shigeo Nakajima and Galligan, {Michael A.} and Haussler, {Carol A.} and Yoshiko Shimizu and Nobuyoshi Shimizu and Whitfield, {G Kerr} and Haussler, {Mark R}",
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T1 - Phosphorylation of the human vitamin D receptor by protein kinase C

T2 - Biochemical and functional evaluation of the serine 51 recognition site

AU - Hsieh, Jui-Cheng

AU - Jurutka, Peter W.

AU - Nakajima, Shigeo

AU - Galligan, Michael A.

AU - Haussler, Carol A.

AU - Shimizu, Yoshiko

AU - Shimizu, Nobuyoshi

AU - Whitfield, G Kerr

AU - Haussler, Mark R

PY - 1993/7/15

Y1 - 1993/7/15

N2 - We have reported previously that the human vitamin D receptor (hVDR) is selectively phosphorylated by protein kinase C-β (PKC-β), in vitro, on a serine residue in the sequence RRS51MKRK, which is located between the two zinc fingers of hVDR and is potentially important to its transacting function (Hsieh, J.-C., Jurutka, P. W., Galligan, M. A., Terpening, C. M., Haussler, C. A., Samuels, D. S., Shimizu, Y., Shimizu, N., and Haussler, M. R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9315-9319). In the present experiments we evaluated this phosphorylation event using a series of hVDR mutants in which serine 51 or its flanking residues were modified. Alteration of serine 51 to a non-phosphorylatable residue resulted in an approximately 60% reduction in basal hVDR phosphorylation in intact cells but did not diminish 1,25-dihydroxyvitamin D3-stimulated phosphorylation. Such mutations also abolished subsequent phosphorylation of immunoprecipitated hVDR by purified PKC-β, in vitro, as did replacement of basic residues on either side of serine 51. Mutation of serine 51 to glycine (S51G) or to aspartic acid (S51D), as well as altering the basic residues flanking serine 51, abolished the interaction of hVDR with the vitamin D-responsive element (VDRE) as monitored by gel mobility shift analysis. Thus, we conclude that unmodified serine 51 and its surrounding basic residues are crucial not only for PKC-β substrate recognition but also for the optimal VDRE binding of native hVDR. In transactivation assays, S51G and S51D possessed only 35 and 10% of wild-type hVDR activity, respectively. Mutation of serine 51 to threonine (S51T) restored phosphorylation by PKC-β, in vitro, to about 40% of wild-type and transactivation to 45% of that of wild-type hVDR. Alteration of serine 51 to alanine, which is the residue in the corresponding position of the glucocorticoid, progesterone, mineralocorticoid, and androgen receptors, eliminated PKC-β phosphorylation but completely preserved the specific DNA binding activity and transactivation capacity of hVDR. Thus, phosphorylation of hVDR at serine 51 is not required for either VDRE binding or transactivation. Finally, incubation of Escherichia coli-expressed hVDR with PKC-β elicits marked phosphorylation of the receptor and significantly inhibits its ability to complex with the VDRE. We therefore speculate that posttranslational modification of hVDR at serine 51 may constitute a negative regulatory loop which could be operative when target cells are subject to PKC activation events.

AB - We have reported previously that the human vitamin D receptor (hVDR) is selectively phosphorylated by protein kinase C-β (PKC-β), in vitro, on a serine residue in the sequence RRS51MKRK, which is located between the two zinc fingers of hVDR and is potentially important to its transacting function (Hsieh, J.-C., Jurutka, P. W., Galligan, M. A., Terpening, C. M., Haussler, C. A., Samuels, D. S., Shimizu, Y., Shimizu, N., and Haussler, M. R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9315-9319). In the present experiments we evaluated this phosphorylation event using a series of hVDR mutants in which serine 51 or its flanking residues were modified. Alteration of serine 51 to a non-phosphorylatable residue resulted in an approximately 60% reduction in basal hVDR phosphorylation in intact cells but did not diminish 1,25-dihydroxyvitamin D3-stimulated phosphorylation. Such mutations also abolished subsequent phosphorylation of immunoprecipitated hVDR by purified PKC-β, in vitro, as did replacement of basic residues on either side of serine 51. Mutation of serine 51 to glycine (S51G) or to aspartic acid (S51D), as well as altering the basic residues flanking serine 51, abolished the interaction of hVDR with the vitamin D-responsive element (VDRE) as monitored by gel mobility shift analysis. Thus, we conclude that unmodified serine 51 and its surrounding basic residues are crucial not only for PKC-β substrate recognition but also for the optimal VDRE binding of native hVDR. In transactivation assays, S51G and S51D possessed only 35 and 10% of wild-type hVDR activity, respectively. Mutation of serine 51 to threonine (S51T) restored phosphorylation by PKC-β, in vitro, to about 40% of wild-type and transactivation to 45% of that of wild-type hVDR. Alteration of serine 51 to alanine, which is the residue in the corresponding position of the glucocorticoid, progesterone, mineralocorticoid, and androgen receptors, eliminated PKC-β phosphorylation but completely preserved the specific DNA binding activity and transactivation capacity of hVDR. Thus, phosphorylation of hVDR at serine 51 is not required for either VDRE binding or transactivation. Finally, incubation of Escherichia coli-expressed hVDR with PKC-β elicits marked phosphorylation of the receptor and significantly inhibits its ability to complex with the VDRE. We therefore speculate that posttranslational modification of hVDR at serine 51 may constitute a negative regulatory loop which could be operative when target cells are subject to PKC activation events.

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