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Endocrinology Vol. 143, No. 2 674-682
Copyright © 2002 by The Endocrine Society

PTH-CALCITONIN-VITAMIN D-BONE

PTH Induction of Transcriptional Activity of the cAMP Response Element-Binding Protein Requires the Serine 129 Site and Glycogen Synthase Kinase-3 Activity, But Not Casein Kinase II Sites

Darren R. Tyson1,2, John T. Swarthout2, Stephen C. Jefcoat and Nicola C. Partridge

Cell and Molecular Biology Program and Department of Pharmacological and Physiological Science, St. Louis University School of Medicine, St. Louis, Missouri 63104; and Department of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

Address all correspondence and requests for reprints to: Dr. Nicola C. Partridge, Department of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854. E-mail: partrinc{at}umdnj.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously shown that PTH induction of c-fos expression in the rat osteoblastic cell line UMR 106-01 requires the phosphorylation of cAMP response element-binding protein (CREB) at serine 133. Here we show that this event is not sufficient for induced transcriptional activity in UMR cells. Serine 129, but not the casein kinase II sites (serines 108, 111, 114, 117, and 121), also plays a role in the activation of CREB. First, by metabolically labeling an epitope-tagged CREB, we determined that, in addition to serine 133, other residues are phosphorylated in vivo. Using mutational analysis of a GAL4-CREB reporter system we demonstrate that serines 129 and 133 are both required for PTH-induced transcriptional activity, whereas the casein kinase II sites are not. Furthermore, PTH failed to induce transcriptional activity of GAL4-CREB in cells treated with genistein, a general tyrosine kinase inhibitor known to inhibit glycogen synthase kinase-3 (GSK-3) activity, or LiCl, the most specific GSK-3-inhibiting agent known, strongly implicating GSK-3ß in this process. Importantly, although genistein and LiCl each inhibit GSK-3ß activity, neither prevented the phosphorylation of serine 133 induced by PTH. Lastly, when serine 129 is replaced with a negatively charged aspartic acid, LiCl has no effect on the PTH-induced trans-activation of CREB. We propose that GSK-3ß phosphorylates CREB at serine 129 and thus is required for the increased transcriptional activity of CREB in response to PTH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PROTOTYPICAL MEMBER of the cAMP response element (CRE)-binding transcription factors is the CRE-binding protein (CREB) (1, 2, 3), which responds to increased intracellular levels of cAMP and calcium (reviewed in Refs. 4, 5, 6, 7, 8). We have shown that activation of c-fos by PTH is CREB dependent and that phosphorylation of CREB at serine 133 (S133) by PKA is required for this induction (9, 10). Furthermore, we have shown that 10 nM PTH maximally stimulates the rapid and transient increased expression of the immediate-early gene c-fos in the rat osteoblastic cell line UMR 106-01 (UMR cells) (11). The role of c-fos in bone remains elusive even though expression of its viral homolog, v-fos, by the FBJ and FBR murine sarcoma viruses was linked to osteocarcinogenesis as early as 1966 (12), and the viral oncoprotein v-fos is overexpressed in osteosarcomas (13). It has been shown that mice infected with the FBJ virus or transgenic mice overexpressing c-fos develop osteosarcomas in the same location as c-fos is normally expressed, specifically cartilage, bone, and teeth (14), suggesting a role for c-fos in mitogenesis and/or differentiation in the skeletal system. This effect is specific to Fos expression, as overexpression of other members of the activating protein-1 family (e.g. Jun and FosB) did not produce a higher incidence of osteosarcomas (15). Mice lacking a functional copy of the c-fos gene (fos-null mice) are viable, but develop osteopetrosis and have deficiencies in bone remodeling as a result of a lack of osteoclasts (16). In addition, a role for c-fos has been implicated in an osteogenic response to mechanical stress in osteocytes (17). Recent evidence suggests that induction of c-fos expression is required for the PTH-mediated increase in collagenase-3 expression in rat osteoblastic cells (18), which may be important for the initiation of bone resorption by osteoclasts. Thus, c-fos appears to have many pleiotropic and essential effects in bone.

The trans-activation of CREB by phosphorylation at S133 has been studied at great length in other systems and has been associated with a number of physiological functions, such as circadian rhythms (19) and long-term memory ( 20, 21). Although the phosphorylation of S133 has been the focus of most studies of CREB activation, other phosphorylation events have been shown to occur. Some members of the family of CREB-related proteins contain a consensus phosphorylation site for glycogen synthase kinase-3 (GSK-3), an enzyme originally characterized for its regulation of glycogen synthesis (22, 23). The GSK-3 consensus recognition sequence S-X-X-X-(P)S present in both CREB and CRE modulator protein {tau} (CREM{tau}) undergoes a hierarchical phosphorylation requiring phosphorylation of the C-terminal serine before phosphorylation of the N-terminal serine by GSK-3 (23). For CREB, phosphorylation of S133 (S117 of CREM{tau}) occurs first, enabling GSK-3 to phosphorylate S129. The ability of GSK-3 to phosphorylate both CREB and CREM{tau} was demonstrated in vitro and is dependent upon S133 phosphorylation (23, 24, 25).

CREM{tau} is phosphorylated on several residues in resting and stimulated cells, but these phosphorylation events have no discernible effects on the ability of CREM{tau} to activate transcription (24). Significantly, evidence supporting phosphorylation at the GSK-3 site on CREM{tau} in vivo or in vitro is lacking, and mutation of this site has no effect on CREM{tau}-mediated transcription (24). However, work by Fiol et al. (25) suggests that GSK-3 phosphorylation of S129 is a prerequisite for the enhanced transcriptional activity of CREB. Furthermore, these researchers showed that PKA- or forskolin-induced transcription from the somatostatin promoter required active GSK-3, implicating GSK-3 in the activation of CREB in response to increased levels of cAMP (25). Importantly, CREB has not been directly analyzed for phosphorylation at S129 in cultured cells.

Many different signaling cascades appear to be able to converge on CREB (8, 26), and activation of these signaling cascades by a variety of extracellular stimuli leads to differential phosphorylation of CREB, affecting its trans-activation potential. Additionally, although phosphorylation of CREB at S133 by PKA is required for the induction of c-fos expression by PTH (10), other phosphorylation events have not been ruled out. For these reasons we examined the potential phosphorylation events occurring on CREB in UMR cells with or without PTH treatment and the ability of PTH to activate CREB when S129 is unavailable for phosphorylation by GSK-3ß. Here, we present evidence that CREB is phosphorylated at casein kinase II (CKII) consensus sites in unstimulated UMR cells, but that these sites are unnecessary for the trans-activation of CREB under basal or PTH treatment conditions. Most importantly, we demonstrate that mutations of S129 and inhibition of GSK-3 can each alter the ability of PTH to induce the transcriptional activity of CREB.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids
The cDNA of rat CREB 341 from the second methionine (the third amino acid) through the stop codon was subcloned from pET15b-rCREB341 into pET30a+ (Novagen, Madison, WI). The vector was then modified by PCR to contain a FLAG epitope (LYKDDDDK) using the upstream primer 5'-CCAGATCTGGATTACAAGGACGACGAC and Pfu DNA polymerase (Stratagene, La Jolla, CA). The resultant plasmid is referred to as pET-TT-CREB, for triple-tagged CREB. The term triple tag is used because the amino terminus of the encoded protein contains three separate epitopes: a six-histidine tag for nonimmunological purification, an S-tag (a portion of ribonuclease S; Novagen) that can be used for nonimmunological detection, and a FLAG epitope to which highly specific antibodies are readily available (Fig. 1Go, A and B). For mammalian expression, the entire coding sequence was then subcloned into the XbaI and BamHI sites of pCDNA3 (Invitrogen, San Diego, CA). The reporter plasmid 5XG-luc was generated by amplifying the five copies of the GAL4-binding sites (upstream activating sequence) and the TATAA sequence from the 5XG-chloramphenicol acetyltransferase (5XG-CAT) vector (a gift from J. Chrivia, St. Louis University, St. Louis, MO) by PCR and subcloning into the pGL3-Basic vector (Promega Corp., Madison, WI). The cDNAs encoding GAL4-CREB{Delta}LZ and GAL4-CREB{Delta}LZ M1 (S133A) were subcloned from their respective plasmids [provided by M. Greenberg (27)] into pCDNA3 (Invitrogen). Other CREB mutants were generated using the QuickChange site-directed mutagenesis kit (Stratagene) and specific oligonucleotides according to the manufacturer’s protocol. All PCR-modified plasmids were verified by sequencing.



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  		Figure 1. Phosphate incorporation into CREB. A, Sequence of the triple tag. The His-tag, S-tag, and FLAG epitopes are overlined. Thrombin and enterokinase cleavage sites are indicated with arrowheads. B, Schematic diagram of TT-CREB showing the epitope tags (TT), glutamine-rich regions 1 and 2 (Q1 and Q2), the KID containing the sites of regulatory phosphorylation, the DNA-binding domain (DBD), and the leucine zipper motif (LZ). Data are modified from Ref. 5 . C, UMR cells were transiently transfected with TT-CREB and then metabolically labeled with [32P]orthophosphate for 6 h. Cells were left untreated (no PTH) or were treated with 10 nM PTH for 20 min, and TT-CREB was affinity purified and separated by 7% SDS-PAGE. The 32P-labeled TT-CREB bands were excised and digested with trypsin overnight. Tryptic fragments were separated on a one-dimensional isoelectric focusing gel, and the gel was dried and exposed to a phosphor screen for detection of 32P-labeled bands. As controls, recombinant TT-CREB was phosphorylated in vitro by PKA alone, PKA and GSK-3, or CKII, then affinity purified and processed as described for the in vivo TT-CREB samples. GSK-3 alone cannot be used to phosphorylate CREB, because it requires the phosphorylation of S133 for its action on S129. All bands were visualized using Storm detector and software (Molecular Dynamics, Inc.). These data indicate that CREB is phosphorylated in vivo in UMR cells in both the absence and presence of PTH at sites that colocalize with CREB phosphorylated in vitro by CKII, and S133 is phosphorylated in UMR cells only in response to PTH. The phosphorylation of S129 in UMR cells labeled in vivo cannot be specifically detected due to the comigration of bands containing the CKII sites. D, The sequences of rat CREB 341, human CREM{tau}, and rat ATF-1 were aligned manually to show the high homology in the KID. Serine and threonine residues that have been shown to be phosphorylated in vitro by various kinases are shown in bold. Numbers above the sequences refer to the residue number of rat CREB341. S133 has been shown to be phosphorylated by PKA, PKC, ribosomal S6 kinases (RSKs), and calcium/calmodulin-dependent kinases (CaMKs) as well as other kinases. Phosphorylation of CREB at S142 by CaMKII has been shown to be inhibitory (44 ).

 
Metabolic labeling and immunoprecipitation
UMR cells were grown as previously described (28). Cells were seeded into 100-mm dishes and allowed to recover overnight at 37 C in 5% CO2. Cells were then transiently transfected with 4 µg pCDNA3-TT-CREB/100-mm dish using LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s instructions and were allowed to grow to confluence. The cells were washed once with phosphate-free MEM to remove free phosphate, followed by addition of 4 ml phosphate-free MEM containing 600 µCi [32P]orthophosphate (NEN Life Science Products, Boston, MA) to each dish. Cells were incubated in the presence of [32P]orthophosphate for 6 h. Vehicle or 10 nM PTH was added directly to the labeling medium, and cells were incubated for another 20 min. Cells were then washed twice with 5 ml wash buffer [50 mM Tris-HCl (pH 7.3), 10 mM sodium pyrophosphate, 10 mM EDTA, and 5 mM EGTA], scraped into 1 ml of the same buffer, and transferred into 1.5-ml microcentrifuge tubes. Cells were pelleted by centrifugation at 12,000 x g for 30 sec, resuspended in 100 µl wash buffer, and boiled for 10 min. Cell debris was then pelleted by centrifugation at 12,000 x g for 2 min. Large 32P-labeled protein aggregates were removed from the lysates using a spin filter with a mol wt cut-off of 100,000. Retentates were discarded, and eluates were transferred to new tubes. Five hundred microliters of Tris-buffered saline containing 0.1% Tween 20 and 50 µl anti-FLAG M2 affinity gel (Kodak, Rochester, NY) were added to each lysate, and each was incubated with rocking at room temperature for 1 h to form immune complexes. The affinity gel was washed four or five times with Tris-buffered saline containing 0.1% Tween 20, then TT-CREB was separated from the agarose beads by adding 50 µl H2O and 50 µl 3x SDS sample buffer (150 mM Tris-HCl, 15% SDS, 50 mM ß-mercaptoethanol, 50% glycerol, 0.1% bromophenol blue) and boiling for 3 min. Agarose beads were pelleted by centrifugation, and the supernatant fraction was passed through a 0.2-µm pore size spin filter (Amicon, Beverly, MA) to remove any remaining agarose.

Tryptic phosphopeptide analysis
Immunoprecipitated, metabolically labeled TT-CREB and recombinant TT-CREB which had been phosphorylated in vitro with PKA alone, PKA and GSK-3, or CKII (all from New England Biolabs, Inc.) were separated by 7.5% SDS-PAGE. The gel containing 32P-labeled proteins was dried between cellulose paper, and 32P-labeled bands were detected either using a phosphor screen and a STORM detector (Molecular Dynamics, Inc., Sunnyvale, CA) or by autoradiography. Bands containing 32P-labeled proteins were excised from the dried gel and digested for 2 h with 25 µg trypsin (Roche Molecular Biochemicals, Indianapolis, IN) in 50 mM NH4HCO3, pH 8.8, then overnight with an additional 25 µg trypsin. Trypsinized peptide fragments were dried and resuspended in 50 mM NH4HCO3, pH 8.8, then separated by isoelectric focusing using precast gels (Bio-Rad Laboratories, Inc., Richmond, CA; pH range, 3–10) according to the manufacturer’s instructions.

Reporter assays
UMR 106-01 cells were seeded into six-well plates and transiently transfected using Lipofectamine. Each well contained 100 ng CAT or luciferase reporter plasmid and 100 ng of each cotransfected expression vector and was normalized to contain 500 ng total plasmid DNA using the empty pCDNA3 vector. Some transfections using the CAT reporter plasmid also contained 1 ng pGL3-Control (Promega Corp.) to normalize transfection efficiency. Cells were serum-starved for 24 h, then treated with various agents and incubated for 16 h. Lysates were prepared using 100 µl reporter lysis buffer (Promega Corp.). Lysates containing luciferase were analyzed immediately for luciferase activity using the luciferase assay reagent (Promega Corp.) and an OptoComp II luminometer (MGM Instruments, Hamden, CT). Levels of CAT present in lysates were analyzed by incubating 40 µl of each lysate with 5 µl Fast-CAT Green substrate (Molecular Probes, Inc.) and 5 µl 9 mM acetyl coenzyme A for 1 h at 37 C, and further processing was performed according to the manufacturer’s instructions. Fluorescent spots were detected and quantitated using a STORM scanning detector (Molecular Dynamics, Inc.).

Immunoprecipitation and GSK-3ß assays
Measurement of endogenous GSK-3ß kinase activity (with or without prior treatment with LiCl) was performed by modification of a previously described method (29). Briefly, UMR cells were washed twice in 1x PBS, and proteins were solubilized in lysis buffer [50 mM HEPES (pH 7.5), 10 mM NaCl, 1 mM EDTA, 5 mM EGTA, 50 mM NaF, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 0.1% Tween 20, and 10% glycerol] supplemented with a mixture of protease inhibitors (10 µg/ml aprotinin, leupeptin, and pepstatin). GSK-3ß was immunoprecipitated from whole cell lysates (200 µg total protein) with 5 µg anti-GSK-3ß rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and 30 µl protein A/G Sepharose with rocking overnight at 4 C. The immunoprecipitants were washed twice with lysis buffer and twice with GSK-3ß kinase buffer [20 mM HEPES (pH 7.2), 10 mM MgCl2, 10 mM MnCl2, 1 mM dithiothreitol, 0.2 mM EGTA, and 5 µM ATP]. GSK-3ß kinase assays were performed at 30 C for 15 min in 20 µl GSK-3ß kinase buffer supplemented with 3 µg recombinant Tau (Calbiochem) and 5 µCi [{gamma}-32]ATP for each reaction. Adding 3x SDS sample buffer and boiling for 5 min terminated the reaction. The entire reaction was then resolved on 12% SDS-PAGE, dried, and exposed to a PhosphorImager plate or x-ray film. To determine the effect of lithium chloride on GSK-3ß activity, cells were treated with increasing concentrations of LiCl (1–50 mM) for 1 h before lysis. As a positive control 5 U GSK-3ß enzyme (New England Biolabs, Inc.) were added to kinase buffer with or without 10 mM LiCl for 20 min before addition of substrate, and then GSK-3ß activity was assayed.

Immunoblotting
UMR cells were washed twice in PBS and then lysed in ice-cold lysis buffer [20 mM Tris-HCl (pH 8.0), 10% glycerol, 1% Triton X-100, 2 mM EDTA, 50 mM ß-glycerophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluoride, and 10 µg/ml aprotinin, leupeptin, and pepstatin]. Monolayers were scraped into 1.5-ml Eppendorf tubes and incubated on ice for 30 min. The lysates were cleared by centrifugation (12,000 x g, 10 min, 4 C). The protein contents of the supernatants were determined using the Bradford reagent (Bio-Rad Laboratories, Inc.) at 595 nm. Cell lysates containing 50 µg total protein in lysis buffer were boiled for 5 min with 2x SDS sample buffer, centrifuged (12,000 x g, 3 min), and placed on ice. SDS-PAGE was performed with 6% and 12% stacking and resolving gels, respectively. The proteins were transferred electrophoretically to a polyvinylidene difluoride membrane at 100 V for 1 h. After blocking the membrane in Tween-Tris-buffered saline (0.1% Tween 20, 138 mM NaCl, 5 mM KCl, and 25 mM Tris-HCl, pH 8.0) containing 5% (wt/vol) nonfat dry milk, membranes were probed with the appropriate antibodies (diluted 1:1,000), followed by incubation with horseradish peroxidase-conjugated goat antirabbit secondary antibody (diluted 1:10,000). The antigen-antibody complexes were detected by the ECL kit and Hyperfilm ECL from Amersham Pharmacia Biotech (Arlington Heights, IL).

Statistical analysis
Data represented with bar graphs are representative experiments performed in triplicate, with bars representing means, and lines indicating the SEM. To determine differences among the samples, one-way ANOVA with a post-hoc analysis of multiple comparisons using the pairwise Student-Newman-Keuls method was performed. Analysis was performed using SigmaStat 2.0 for Windows (SPSS, Inc., Chicago, IL). All other data are shown as a representative of at least three separate experiments with similar results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Metabolic labeling and analysis of 32P incorporation into TT-CREB
To examine the phosphorylation events occurring on CREB, UMR 106-01 cells were transiently transfected with epitope-tagged CREB (TT-CREB; Fig. 1BGo) that could easily be purified from cells. Cells were then metabolically labeled with [32P]orthophosphate and treated with vehicle or PTH, and TT-CREB was immunoprecipitated with anti-FLAG antibodies and separated by SDS-PAGE. The 32P-labeled TT-CREB proteins were then excised from the gel, individually digested with trypsin, and separated by isoelectric focusing. The major 32P-containing peptide fragments from in vivo labeled UMR cells correspond to fragments generated from TT-CREB labeled in vitro by CKII (S129/CKII; Fig. 1CGo), suggesting that CREB is phosphorylated in both untreated and PTH-treated UMR on the consensus CKII sites. In addition, PTH-treated cells demonstrated incorporation of 32P into trypsin fragments that comigrate with fragments produced by TT-CREB phosphorylated in vitro by PKA (S133; Fig. 1CGo); this confirms our earlier reports of CREB phosphorylation at S133 in response to PTH (9, 10). A relatively lower incorporation of 32P into TT-CREB at S133 was observed vs. incorporation into CKII sites (Fig. 1CGo). This is because there are five CKII sites phosphorylated with or without PTH, accounting for the 3-fold higher level of phosphorylation. The 32P-labeled bands detected on the lower portion of the gel in Fig. 1CGo are probably more completely digested fragments, as some trypsin digests of CKII-labeled TT-CREB produced stronger bands in this region. However, the possibility exists that these bands represent different phosphate-containing peptide fragments, such as peptides containing S156, which has been shown to be phosphorylated by CKII in vitro and is phosphorylated in vivo on the analogous residue on CREM{tau} (24). Unfortunately, the methods employed here were not sufficient to separate peptides containing S129 from peptides containing the CKII acceptor sites. To assess phosphorylation by GSK-3ß, it was necessary to add PKA. This is because the GSK-3 consensus recognition sequence in CREB is S-X-X-X-(P)S and undergoes a hierarchical phosphorylation requiring phosphorylation of the C-terminal serine before phosphorylation of the N-terminal serine. After digestion with trypsin, it is apparent that GSK-3ß phosphorylated TT-CREB (Fig. 1CGo, lane 4).

Functional analysis of CREB mutations
As sites other than S133 appear to be phosphorylated in UMR cells, we examined the functional consequence of mutating the potential sites of phosphorylation. To accomplish this we transiently transfected an expression plasmid encoding the cDNA of a chimeric GAL4-CREB protein that has had the leucine zipper domain removed together with a reporter vector containing five copies of the GAL4 upstream activating sequence (GAL4-binding site; Fig. 2Go). This system has been used by many other groups and allows for the analysis of mutations on the transcriptional activity of CREB without the complicating effects of endogenous CREB (reviewed in Refs. 8, 26).



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  		Figure 2. Vectors used for analysis of CREB transcriptional activity. A chimeric protein of the DNA-binding domain of GAL4 (amino acids 1–148) and CREB was made that is devoid of the CREB dimerization motif, its leucine zipper domain, to eliminate heterodimerization with endogenous CREB (GAL4-CREB{Delta}LZ). This protein can bind to the five GAL4-binding sites (upstream activating sequence) of the regulatory region of the promoter/reporter vectors. The promoter/reporter vectors will express the bacterial CAT (5XG-CAT) or firefly luciferase (5XG-luc) genes only in the presence of a functional transcriptional activation domain, which is lacking from the GAL4 DNA-binding domain and is supplied by functional CREB.

 
When the promoter/reporter plasmid containing the binding sites for GAL4 was transiently transfected into UMR cells alone, CAT activity was negligible in both control and PTH-treated cells (Fig. 3AGo, none). When wild-type GAL4-CREB expression vector was cotransfected with the reporter plasmid, PTH treatment induced a 7-fold increase in CAT activity vs. untreated cells (Fig. 3AGo, WT). As expected, when S133 was mutated to an alanine (S133A) the PTH inducibility was completely abolished (Fig. 3AGo, S133A). As Fiol et al. (25) had previously described a critical role for the GSK-3 site on CREB (S129), we chose to mutate this site to an alanine (S129A) while leaving S133 unchanged. As with the S133A mutant, S129A also completely abolished the PTH response (Fig. 3AGo, S129A). The inability of PTH to trans-activate CREB was not due to the prevention of S133 phosphorylation, as recombinant CREB, which has the S129A mutation, could still be phosphorylated by PKA at S133 in vitro (not shown). The possibility that the S129A mutation alters the conformation of CREB in such a way as to prevent the interaction of phosphoserine 133 with important factors cannot be ruled out; however, this mutation does not appear to affect the basal transcriptional activity of CREB (compare S129A to none in Fig. 3AGo), suggesting that the protein is at least expressed and localized to the nucleus.



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  		Figure 3. PTH induction of CREB-mediated transcription. A, UMR cells were seeded into six-well plates and transfected with the reporter plasmid 5XG-CAT alone (none) or with a GAL4-CREB{Delta}LZ expression plasmid (WT, S133A or S129A). Cells were then either left untreated ({blacksquare}) or were treated with 10 nM PTH ({square}) overnight. CAT activity was determined as described in Materials and Methods. These data provide strong evidence for a role of both S129 and S133 in the activation of CREB. B, The various CKII sites were mutated individually in the GAL4-CREB{Delta}LZ expression vector and analyzed as described in A. None of these mutations demonstrated a substantial effect on CREB transcriptional activity. C, As S114A and S117A in B appeared to slightly inhibit the response, the double mutant S114/117A was created and analyzed as in A and B. Additionally, the remaining CKII site serines were mutated to simulate phosphorylation by substituting negatively charged aspartic acid residues (S108D, S111D, and S121D) and analyzed as described for the others. None of these mutants significantly altered the ability of CREB to activate transcription. D, A representation of all of the mutations within the KID of CREB that were analyzed in these experiments is shown. All experiments were repeated at least twice, and a single experiment performed in triplicate is shown in each panel.

 
As CREB appears to be phosphorylated in vivo in UMR cells at the CKII sites we examined their role by mutating each of these sites individually. As shown in Fig. 3BGo, none of the CKII point mutants demonstrated significant inhibition of the PTH-induced CAT activity. As S114A and S117A seemed to slightly inhibit the PTH response, we created a double mutant S114/S117A to determine whether the two mutations combined could significantly inhibit the PTH response. Also, as S108A, S111A, and S121A had no effect or perhaps slightly enhanced the PTH response, we made serine to aspartic acid mutants to attempt to mimic a phosphorylated state (S108D, S111D, and S121D). As shown in Fig. 3CGo, none of these mutants produced any significant effect on PTH inducibility. Thus, it appears that phosphorylation of the CKII sites within the kinase-inducible domain (KID; Fig. 1DGo) is not functionally required for PTH action; therefore, we have not investigated these sites further, instead choosing to investigate the role of S129 in mediating the response of CREB to cellular stimulation by PTH.

Genistein dose-dependently inhibits GAL4-CREB- mediated transcription
The activity of both isoforms of GSK-3 is thought to be strongly dependent on phosphorylation of tyrosine residues (30, 31); therefore, inhibiting tyrosine phosphorylation would also inhibit GSK-3 activity. Genistein is a general tyrosine kinase inhibitor that has been used extensively to inhibit receptor tyrosine kinase activity; it has also been used previously to inhibit GSK-3{alpha} activity (32). Furthermore, we demonstrate that GSK-3 activity was reduced by 60% when immunoprecipitated and assayed from cells treated with genistein vs. untreated cells (Fig. 4AGo). Thus, genistein was employed in these studies to determine whether tyrosine phosphorylation is important for the PTH induction of CREB-mediated transcription. As shown in Fig. 4BGo, genistein inhibited the PTH induction of CREB-mediated transcription in a dose-dependent manner. The effect of genistein was not due to the inhibition of the phosphorylation of CREB at S133, as even in the presence of 100 µM genistein PTH still induces the phosphorylation of CREB at S133 (Fig. 4CGo).



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  		Figure 4. Genistein dose dependently inhibits PTH-induced CREB-mediated transcription. A, Cells were untreated or treated with 100 µM genistein for 30 min at 37 C then GSK-3ß activity was assessed. Genistein inhibited the activity of GSK-3 by approximately 60%. B, GAL4-CREB{Delta}LZ, 5XG-CAT, and pGL3-Control were cotransfected into UMR cells, then the cells were either left alone (none) or were pretreated with vehicle (V) or increasing doses of genistein (numbers indicate micromolar concentration of genistein) before treatment with 10 nM PTH. Cell lysates were analyzed for CAT activity and normalized to luciferase activity [relative light units (RLU)]. {blacksquare}, No PTH treatment; {square}, treatment with PTH. Genistein dose-dependently inhibited CREB-mediated transcription induced by PTH. C, UMR cells were pretreated with 100 µM genistein for 30 min before treatment with or without 10 nM PTH for 15 min. Cell lysates were analyzed for phosphorylation of CREB at S133 by immunoblotting with an antibody that recognizes CREB phosphorylated at S133 (pCREB) or an antibody that recognizes CREB whether it is phosphorylated or not (CREB). Genistein has no effect on the PTH-induced phosphorylation of CREB at S133. Bands were visualized by enhanced chemiluminescence exposure to autoradiography film (Kodak) and were digitally reproduced using a Microtek Scanmaker E6 scanner. The digital image was manipulated using Adobe Illustrator 7.0.

 
Inhibition of GSK-3ß by LiCl inhibits PTH-induced CREB trans-activation
GSK-3 is thought to be constitutively active in resting cells (reviewed in Ref. 33), and we confirm this in osteoblastic cells (Figs. 4Go and 5Go). Therefore, it is necessary to inhibit its activity to determine its role in the regulation of the transcriptional activity of CREB. Although the use of genistein in Fig. 4Go indicates that tyrosine phosphorylation is required for CREB trans-activation by PTH, it does not specifically implicate GSK-3. To help confirm a role for GSK-3 in this system we used lithium chloride (Fig. 5Go, A–C), which has been shown to inhibit GSK-3 activity both in vivo (34, 35) and in vitro (36). Previously, GSK-3ß was found to be inhibited by LiCl with 50% inhibition (IC50) at 2 mM and was inhibited more than 80% at 10 mM (37). To assess the ability of LiCl to inhibit GSK-3ß activity in UMR cells, kinase assays were performed using recombinant Tau as a substrate (29). UMR cells were serum-starved for 24 h, then LiCl was added 1 h before performing the assays. GSK-3ß was dose-dependently inhibited by LiCl with an IC50 of approximately 5 mM (Fig. 5CGo). As a positive control, 5 U GSK-3ß enzyme was incubated with or without 10 mM LiCl for 20 min, and activity was assessed. Phosphorylation of Tau protein by GSK-3ß was reduced by about 78% in the presence of LiCl (Fig. 5BGo).



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  		Figure 5. LiCl dose dependently inhibits GSK-3ß activity. UMR cells were seeded into six-well plates and serum-starved for 24 h. LiCl was added to some of the wells (as indicated) 1 h before GSK-3 assays. A, The upper panels show the relative incorporation of phosphate into Tau protein ([32P]Tau), and the lower panels demonstrate the level of GSK-3ß protein (GSK-3ß) by Western blot present in the assay of UMR cells treated with or without 10 mM LiCl. The results are from one representative experiment and indicate that GSK-3ß activity in UMR cells is significantly inhibited by 10 mM LiCl. B, Five units of GSK-3ß enzyme was added to kinase buffer with or without 10 mM LiCl for 20 min before addition of substrate and then GSK-3ß activity assayed to demonstrate that LiCl directly inhibits GSK-3ß activity in vitro. C, UMR cells were seeded into six-well plates and serum-starved for 24 h. LiCl was added 1 h before GSK-3 assays. The graph represents relative GSK-3ß activity from one representative experiment of the UMR cells in triplicate, with error bars depicting the SEM. Activity was assessed by incorporation of radiolabeled phosphate into Tau protein quantitated by PhosphorImager (Molecular Dynamics, Inc.). These results indicate that GSK-3ß in UMR cells is dose dependently inhibited by LiCl with an IC50 of approximately 5 mM.

 
Having shown that LiCl inhibits GSK-3ß activity in UMR cells, we next examined the ability of LiCl to prevent the transcriptional activity of CREB induced by PTH. As shown in Fig. 6AGo, the ability of PTH to stimulate the transcriptional activity of CREB was significantly inhibited (P <= 0.001) by approximately 74% after pretreatment of cells with 10 mM LiCl. We were concerned that treatment of cells with LiCl may prevent transcriptional activation of CREB either by inhibiting PKA and thus preventing phosphorylation of CREB at S133 or by inhibiting some other cellular process important for CREB trans-activation. However, CREB was still phosphorylated even in cells treated with 50 mM LiCl (Fig. 6BGo), a concentration shown to completely inhibit CREB trans-activation by PTH (Fig. 6AGo). Importantly, it has been shown previously that PKA has full activity toward kemptide even in the presence of 20 mM LiCl (36, 37).



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  		Figure 6. LiCl inhibits PTH-induced CREB-mediated transcription, but not PTH-induced phosphorylation of CREB at S133. A, UMR cells were cotransfected with GAL4-CREB{Delta}LZ and p5XG-Luc, serum-starved for 24 h, and then treated with vehicle or various concentrations of LiCl before treatment with 10 nM PTH. Cell lysates were analyzed for luciferase activity, and the graph represents relative luciferase activity from one representative experiment in triplicate. Error bars represent the SEM. These data show that the PTH induction of CREB-mediated transcription can be dose dependently inhibited by LiCl. B, UMR cells were seeded into six-well plates, serum-starved for 24 h, then treated with vehicle or increasing concentrations of LiCl for 1 h. Cells were then treated with 10 mM PTH as indicated, and cell lysates were analyzed for phosphorylation of CREB as described in Fig. 4Go. This shows that PTH-induced phosphorylation of S133 is not affected by LiCl.

 
To determine whether the effects of LiCl were mediated through GSK-3 (and specifically at S129), we mimicked a phosphoserine at S129 by replacing it with a negatively charged aspartic acid residue (S129D). UMR cells were transfected with pGAL4-CREB{Delta}LZ S129D and p5XG-Luc, pretreated with or without LiCl, and treated with or without PTH. As shown in Fig. 7Go, PTH significantly stimulated transcriptional activity of GAL4-CREB{Delta}LZ S129D in the absence (P < 0.001) or presence of 10 mM LiCl (P < 0.001). These results demonstrate that the negatively charged Asp residue is sufficient to overcome the inhibition of CREB transcriptional activity by LiCl and provides evidence for the requirement of a negative charge at this position. Taken together, these data suggest that LiCl specifically inhibits GSK-3ß, thereby preventing the phosphorylation of CREB at S129 and inhibiting the transcriptional activity of CREB.



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  		Figure 7. Mutation of CREB S129 to aspartic acid does not inhibit its activation and prevents the inhibition by LiCl. UMR cells were cotransfected with GAL4-CREB{Delta}LZ containing a mutant in which the GSK-3 site serine was mutated to simulate phosphorylation by substituting a negatively charged aspartic acid residue, GAL4-CREB{Delta}LZ S129D, along with the reporter plasmid p5XG-luc. The cells were serum-starved for 24 h, then either left untreated or treated with 25 mM LiCl for 1 h before treatment with or without 10 nM PTH. Cell lysates were analyzed for luciferase activity, and the graph represents relative luciferase activity from one representative experiment performed in triplicate. Error bars represent the SEM. Below is a depiction of the mutation within the CREB KID. This clearly shows that the negative charge at position 129 (S129D) is sufficient to overcome the inhibition of CREB transcriptional activity by LiCl, indicating that GSK-3ß phosphorylation of S129 is necessary for full trans-activation of CREB by PTH.

 
Various agents have no effect on GAL4-CREB-mediated transcription
As PTH can activate several signaling cascades that could lead to tyrosine kinase activation (38, 39), and because genistein is a general tyrosine kinase inhibitor, there is the possibility that genistein affected other pathways important for regulation of the transcriptional activity of CREB. To investigate this further, several agents were used in conjunction with the GAL4-CREB{Delta}LZ system that could help identify potential roles of tyrosine kinases; however, none of these agents had a significant effect on the response to PTH (Fig. 8Go). The ß{gamma}-subunits of the heterotrimeric G proteins have been previously shown to activate tyrosine kinases (40, 41). To determine whether the ß{gamma}-subunits of the G proteins associating with the PTH receptor are involved in the activation of CREB transcriptional activity the {alpha}-subunit of transducin was cotransfected with the GAL4-CREB components (Fig. 8Go, G{alpha}T). The {alpha}-subunit of transducin acts as an inhibitor of ß{gamma}-mediated responses by binding to ß{gamma}-subunits and preventing their action. Also, PP1, a Src-like tyrosine kinase inhibitor, was used to determine whether the effects of genistein were mediated through the inhibition of these enzymes. Finally, a dominant negative inhibitor of Ras, N17Ras, was employed. As shown in Fig. 8Go, none of these agents had a significant effect on the PTH induction of transcription mediated by GAL4-CREB.



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  		Figure 8. Various inhibitors have no effect on PTH induction of CREB-mediated transcription. The GAL4-CREB{Delta}LZ system plasmids were cotransfected into UMR cells alone (for control and PP1 treatment) or with expression plasmids for dominant negative Ras (N17Ras) or {alpha}-subunit of transducin (G{alpha}T) then treated with or without 10 nM PTH. PP1 pretreatment at the concentrations indicated were performed for 30 min before PTH treatment. A single representative experiment performed in triplicate is depicted, and error bars represent the SEM. None of these inhibitors affected the ability of PTH to induce CREB-mediated transcription, indicating that these other pathways are not involved in PTH’s regulation of CREB.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The regulation of CREB transcriptional activity has been studied for many years, ever since it was first purified in 1987 (42). Subsequently, much of the focus of the regulatory mechanisms of CREB have revolved around the phosphorylation of CREB on S133. The dogma has been that for CREB to become trans-activated only the single phosphorylation event on S133 is necessary. Even though Fiol et al. (25) described the requirement of GSK-3 phosphorylation of S129 for the trans-activation of CREB, very few studies addressed this possibility. Furthermore, other phosphorylation events have been demonstrated to occur on CREB/activating transcription factor family members, but very little is known about their functional significance. As many different pathways have been shown to cause the phosphorylation of CREB at S133, it is entirely possible these same pathways induce multiple phosphorylation events on CREB to promote transcription. A similar rationale lead de Groot et al. (24) to examine the phosphorylation of CREM{tau} both in vitro and in vivo. Although there is high homology between CREB and CREM{tau}, the phosphorylation of CREB and subsequent activation of its transcriptional activity may be regulated quite differently.

Here we show that CREB is phosphorylated in vivo in rat osteoblastic cells with or without PTH treatment. The sites that are phosphorylated in vivo, before stimulation of the cells, appear to be the same sites that can be phosphorylated by CKII in vitro. The exact sites have not been determined, and we could not differentiate phosphorylation of CKII sites from phosphorylation on S129. However, because GSK-3 is active in unstimulated cells (this report and Ref. 33), and GSK-3 phosphorylates S129 (23, 25), the results favor phosphorylation of S129. Nevertheless, the main conclusion that can be drawn from this part of our study is that in addition to S133, other sites on CREB are phosphorylated in UMR cells in both unstimulated and PTH-treated cells.

As shown here for CREB, the CKII phosphorylation sites on CREM{tau} have been found to be phosphorylated in vivo (24). Interestingly, the phosphorylation of these residues enhances the ability of CREM{tau} to bind DNA (24). Even though the DNA-binding activity of CREB may be regulated by phosphorylation of the CKII sites, these sites do not appear to be involved in the regulation of CREB trans-activation by PTH. First, the levels of phosphorylation of CREB on the CKII sites in untreated vs. PTH-treated cells do not appear to change significantly. Also, mutating these sites does not appear to affect the ability of PTH to induce CREB-mediated transcription. Furthermore, although the GAL4-CREB{Delta}LZ system removes a potential regulatory step, that of DNA binding, work performed previously in this laboratory demonstrated that CREB can bind to the CRE in the c-fos promoter equally well in unstimulated cells as in cells treated with PTH (9). Taken together, these data argue against a role for CKII in the regulation of CREB-mediated transcription by PTH.

Even though the phosphorylation of CREB by CKII does not appear to be regulated by PTH, this does not rule out an important role for the phosphorylation of CREB at these sites. It is possible that the phosphorylation of CREB at these sites can be regulated in some manner that subsequently alters its binding to the CRE. The binding of CREB and CREM{tau} does appear to be modulated by phosphorylation at these sites when assayed in vitro (24), and a recent study suggests that phosphorylation of the CKII sites is regulated in a cell cycle-dependent manner (43). Studies employing the GAL4-CREB{Delta}LZ system cannot address the possibility of CKII site phosphorylation events affecting DNA binding, because the DNA binding is controlled by the portion of the chimeric protein encoded by GAL4.

Although CKII sites do not appear to be important for the regulation of CREB trans-activation, S129 appears to be very important for this activity. In these studies we do not provide direct evidence that S129 of CREB is being phosphorylated in response to PTH or by GSK-3ß; however, we present several findings that suggest that CREB is, in fact, phosphorylated at S129 by GSK-3 and that this event is required for the transcriptional activity of CREB. First, the S129A mutant of CREB cannot be trans-activated in response to PTH, whereas none of the CKII site mutants produced this effect. Next, both genistein and LiCl inhibited GSK-3 activity and inhibited the PTH-mediated trans-activation of CREB without affecting the phosphorylation of CREB at S133. And, most significantly, when S129 was replaced with the negatively charged aspartic acid residue (S129D), LiCl was unable to inhibit the trans-activation of CREB induced by PTH. This strongly suggests that a negative charge at position 129, i.e. phosphoserine, is required for the transcriptional activity of CREB and that LiCl inhibits this event, presumably by inhibiting GSK-3 activity. Taken together, the evidence presented here suggests that, at least in the rat osteoblastic UMR cell line, the PTH-dependent increase in CREB transcriptional activity requires the phosphorylation of CREB at S129 by GSK-3. A schematic diagram of our proposed model is shown in Fig. 9Go.



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  		Figure 9. Model of PTH-induced increase in c-fos expression mediated by CREB. PTH activation of its receptor and release of the {alpha}-subunit of the heterotrimeric G protein-coupled receptor ({alpha}s) leads to the activation of adenylyl cyclase (AC) and an increase in intracellular concentrations of cAMP. The increase in cAMP causes release of the regulatory subunits (R) from the catalytic subunits (C) of PKA, allowing it to translocate into the nucleus and phosphorylate CREB at S133. The phosphorylation of CREB at S133 creates the consensus recognition motif of GSK-3 and leads to the phosphorylation of CREB at S129 by this enzyme. CREB dually phosphorylated at S129 and S133 can activate transcription of target genes such as c-fos.

 


   Footnotes
 
This work was supported in part by NIH Grant DK-48109.

1 Present address: Department of Physiology and Biophysics, University of California, Irvine, California 92697-4560. Back

2 D.R.T. and J.T.S. contributed equally to this manuscript. Back

Abbreviations: CAT, Chloramphenicol acetyltransferase; CKII, casein kinase II; CRE, cAMP response element; CREB, cAMP response element-binding protein; CREM{tau}, CRE modulator protein {tau}; GSK-3, glycogen synthase kinase-3; KID, kinase-inducible domain; S133, serine 133; TT-, triple-tagged.

Received May 11, 2001.

Accepted for publication October 10, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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