PMID- 8392065
OWN - NLM
STAT- MEDLINE
DCOM- 19930812
LR  - 20210212
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 268
IP  - 20
DP  - 1993 Jul 15
TI  - Phosphorylation of the human vitamin D receptor by protein kinase C. Biochemical 
      and functional evaluation of the serine 51 recognition site.
PG  - 15118-26
AB  - We have reported previously that the human vitamin D receptor (hVDR) is 
      selectively phosphorylated by protein kinase C-beta (PKC-beta), 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-beta, 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-beta 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-beta, 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, mineral-ocorticoid, and androgen receptors, 
      eliminated PKC-beta 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-beta 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.
FAU - Hsieh, J C
AU  - Hsieh JC
AD  - Department of Biochemistry, University of Arizona Health Sciences Center, Tucson 
      85724.
FAU - Jurutka, P W
AU  - Jurutka PW
FAU - Nakajima, S
AU  - Nakajima S
FAU - Galligan, M A
AU  - Galligan MA
FAU - Haussler, C A
AU  - Haussler CA
FAU - Shimizu, Y
AU  - Shimizu Y
FAU - Shimizu, N
AU  - Shimizu N
FAU - Whitfield, G K
AU  - Whitfield GK
FAU - Haussler, M R
AU  - Haussler MR
LA  - eng
GR  - AR-15781/AR/NIAMS NIH HHS/United States
GR  - DK-33351/DK/NIDDK NIH HHS/United States
GR  - GM-24375/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Receptors, Calcitriol)
RN  - 0 (Receptors, Steroid)
RN  - 452VLY9402 (Serine)
RN  - 9007-49-2 (DNA)
RN  - EC 2.7.11.13 (Protein Kinase C)
RN  - FXC9231JVH (Calcitriol)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Base Sequence
MH  - Calcitriol/metabolism
MH  - Catalysis
MH  - Cell Line
MH  - DNA/metabolism
MH  - Down-Regulation
MH  - Enhancer Elements, Genetic
MH  - Escherichia coli
MH  - Haplorhini
MH  - Humans
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - Phosphorylation
MH  - Protein Kinase C/*metabolism
MH  - Receptors, Calcitriol
MH  - Receptors, Steroid/genetics/*metabolism
MH  - Sequence Homology, Amino Acid
MH  - Serine/*metabolism
MH  - Transcriptional Activation
EDAT- 1993/07/15 00:00
MHDA- 1993/07/15 00:01
CRDT- 1993/07/15 00:00
PHST- 1993/07/15 00:00 [pubmed]
PHST- 1993/07/15 00:01 [medline]
PHST- 1993/07/15 00:00 [entrez]
AID - S0021-9258(18)82445-0 [pii]
PST - ppublish
SO  - J Biol Chem. 1993 Jul 15;268(20):15118-26.